MINISTÉRIO DA SAÚDE FUNDAÇÃO OSWALDO CRUZ INSTITUTO OSWALDO CRUZ Mestrado em Biologia Parasitária ESTUDO DO EFEITO DE MUTAÇÕES NO GENE DO CANAL DE SÓDIO DE AEDES AEGYPTI: DISTRIBUIÇÃO EM POPULAÇÕES NATURAIS, CORRELAÇÃO COM RESISTÊNCIA A PIRETROIDE E CUSTO EVOLUTIVO LUIZ PAULO DE BRITO OLIVEIRA SOUZA Rio de Janeiro Março de 2014 INSTITUTO OSWALDO CRUZ Programa de Pós-Graduação em Biologia Parasitária LUIZ PAULO DE BRITO OLIVEIRA SOUZA ESTUDO DO EFEITO DE MUTAÇÕES NO GENE DO CANAL DE SÓDIO DE AEDES AEGYPTI: DISTRIBUIÇÃO EM POPULAÇÕES NATURAIS, CORRELAÇÃO COM RESISTÊNCIA A PIRETROIDE E CUSTO EVOLUTIVO Dissertação apresentada ao Instituto Oswaldo Cruz como parte dos requisitos para obtenção do título de Mestre em Biologia Parasitária Orientador: Prof. Dr. Ademir de Jesus Martins Júnior RIO DE JANEIRO Março de 2014 ii Ficha catalográfica elaborada pela Biblioteca de Ciências Biomédicas/ ICICT / FIOCRUZ - RJ S729 Souza, Luiz Paulo de Brito Oliveira Estudo do efeito de mutações no gene do canal de sódio de aedes aegypti: distribuição em populações naturais, correlação com resistência a piretroide e custo evolutivo / Luiz Paulo de Brito Oliveira Souza. – Rio de Janeiro, 2014. xv, 96 f.: il. ; 30 cm. Dissertação (Mestrado) – Instituto Oswaldo Cruz, Pós-Graduação em Biologia Parasitária, 2014. Bibliografia: f. 90-95 1. Knockdown resistance (kdr). 2. Aedes aegypti. 3. Resistência a inseticidas. I. Título. CDD 616.91852 INSTITUTO OSWALDO CRUZ Programa de Pós-Graduação em Biologia Parasitária LUIZ PAULO DE BRITO OLIVEIRA SOUZA ESTUDO DO EFEITO DE MUTAÇÕES NO GENE DO CANAL DE SÓDIO DE AEDES AEGYPTI: DISTRIBUIÇÃO EM POPULAÇÕES NATURAIS, CORRELAÇÃO COM RESISTÊNCIA A PIRETROIDE E CUSTO EVOLUTIVO ORIENTADOR: Prof. Dr. Ademir de Jesus Martins Júnior Aprovada em: 31 / 03 / 2014 EXAMINADORES: Prof. Dr. Rafael Maciel de Freitas – Presidente (Fundação Oswaldo Cruz - IOC) Prof. Dr. Mario Antonio Navarro da Silva - (Universidade Federal do Paraná) Prof. Dr. Renata Schama Lellis - (Fundação Oswaldo Cruz - IOC) Prof. Dr. Patricia Hessab Alvarenga - (Universidade Federal do Rio de Janeiro) Prof. Dr. Moacyr Alvim Horta Barbosa da Silva - (Fundação Getúlio Vargas) Rio de Janeiro, 31 de março de 2014 Anexar a cópia da Ata que será entregue pela SEAC já assinada. iv Aos meus pais, sempre... E a todos os personagens de livros que me fizeram sorrir ou chorar no ônibus. v AGRADECIMENTOS Como não poderia ser diferente, minha gratidão eterna é primeiramente aos meus pais. A eles devo a base de toda a minha vida bem como o pilar principal em que hoje me ergo. Aprendi a ter bom humor, humildade, ser grato ao que tenho e ver a felicidade nas coisas simples da vida com meu pai, Paulo Jorge, e com a minha mãe, Helena, aprendi a ser sério quando necessário, a dosar o orgulho, a buscar sempre mais, a ter e exigir respeito e a ser decidido na vida. Embora tenham me ensinado valores que muitas vezes pareçam díspares, nunca testemunhei uma ofensa entre eles em toda a minha vida e atribuo a este fato junto ao carinho e amor recebido à ternura e paz interna que desenvolvi para lidar com as adversidades do mundo. Aos dois, meu eterno e mais verdadeiro “eu te amo”. Agradeço a minha grande família, em especial a minha tia Marlene, tia por sangue, mãe por dedicação, avó por mimos, irmã pelo companheirismo e pai pela garra. Se há uma mulher que realmente representa um anjo na terra, eu sou sobrinho dela. Graças a ela também posso agradecer muito aos meus primos, Matheus, Marcos Paulo e a minha também linda afilhada Maryanna. Aos meus irmãos, Leandro e Leonardo. Aos meus primos Filipe, Xandinho e Kauã. As minhas sobrinhas, Nauane e Laura, pelo apoio simplesmente no fato de vierem ao mundo no ano em que a matriarca da nossa família nos deixou e a ela, minha avó Neuza, deixo um beijo especial do seu eterno “Guirim”. Agradeço aos meus tios, tias, primos e primas de sangue ou de consideração, e mesmo aos que já fizeram parte do meu heredograma em algum momento. As minhas cunhadas, Gisele e Isabela. A minha madrinha Nilda. Aos tantos e inúmeros mascotes que me fizeram companhia e ainda farão ao longo da minha vida, mesmo que em pensamentos. Quando o aprender está fora dos livros, eu o encontro em pessoas especiais a quem chamo de amigos. Estes não são muitos, mas são os melhores, alguns antigos e outros novos, alguns reciclados e outros que de tão gasto é difícil acreditar que durem tanto. Agradeço em especial a Ana Brigida, Wagner, Vanessa, Carol, Camila e Everton. Aos amigos de não tão longa data assim, mas já de várias histórias, ao Jonas e aos moradores e agregados da casa da Suzana: Ferrerez, Gabriel, Popó, Lopes, Vinícius, Guilherme, Sapão, Rose e um tal de Israel... e ao Yoda. Bem como no pessoal, no profissional também tenho muitos a quem devo agradecer. Primeiramente ao tutor que se tornou meu orientador e amigo, Dr. vi Ademir Martins. Agradeço pela paciência, sabedoria, atenção e ajuda ao longo deste percurso e enquanto houver pesquisa em suas mãos, sei que a ciência no Brasil prosperará. Agradeço imensamente a oportunidade dada a mim pela Dra. Denise Valle e pelo Dr. José Bento de poder ingressar no LAFICAVE e compreender o sentido do trabalho em equipe. Agradeço aos mosqueteiros da primeira geração, Priscila, Diogo, Thiago, Luana Farnesi, Nathalia, e aos da segunda geração Helena, Suellen, Felipe, Aline, Letícia, Taíza, Monique e Íngrid. Aos sempre mosqueteiros fixos ou temporários André, Gabi, Luana Carrara, Raquel, Luciana, Adriana, Sandrine, Gilberto, Mariana, Simone, Renata, Gustavo, Grazi, Anna Paula, Ignez, Sandro e Cynara, e em especial, com muito carinho, à Jutta, a quem devo uma visão de mundo maravilhosa sobre tudo na vida. Não menos especial é meu agradecimento a Raquel Souza e Thais Chouin pela determinação e esforço conjunto em entrar no mestrado, rir e chorar nessa montanha russa e sobreviver a estes dois anos mais intensos da vida de um acadêmico, obrigado meninas. Obrigado aos colegas da turma do mestrado e aos de disciplinas feitas nesta e em outras instituições. Agradeço ao Dr. Rafael Maciel de Freitas, pela revisão da dissertação e pela amizade, e também ao LATHEMA pela parceria nos trabalhos. Também com carinho especial ao Laboratório de Biologia Molecular de Insetos, agradeço a todos e mais ainda a Dra. Alejandra Saori, a Dra. Rafaela Bruno e ao Dr. Alexandre Peixoto, que em pouquíssimo tempo de convívio me ensinou muito e que deixa saudades imensas no coração de todos que o conheceram. Agradeço à Coordenação de Aperfeiçoamento de pessoal de Nível Superior – CAPES, ao Instituto Oswaldo Cruz, ao Instituto de Biologia do Exército e a todos que fazem da Fundação Oswaldo Cruz referência em ensino e pesquisa. vii “A mente que se abre a uma nova ideia jamais voltará ao seu tamanho original.” Albert Einstein viii INSTITUTO OSWALDO CRUZ RESUMO ESTUDO DO EFEITO DE MUTAÇÕES NO GENE DO CANAL DE SÓDIO DE AEDES AEGYPTI: DISTRIBUIÇÃO EM POPULAÇÕES NATURAIS, CORRELAÇÃO COM RESISTÊNCIA A PIRETROIDE E CUSTO EVOLUTIVO DISSERTAÇÃO DE MESTRADO EM BIOLOGIA PARASITÁRIA Luiz Paulo de Brito Oliveira Souza Aedes aegypti é o principal vetor de dengue. Uma das formas mais adotadas de controle é o uso de inseticidas, notadamente da classe dos piretroides, contra o mosquito no estágio alado. Estes compostos são prediletos, pois apresentam um menor impacto ambiental e conferem atuação rápida no sistema nervoso dos insetos, conhecida como efeito knockdown. O intenso uso de inseticidas tem selecionado populações de mosquitos resistentes em todo o mundo. Uma das principais formas fisiológicas de resistência é a alteração no canal de sódio regulado por voltagem (NaV), sítio alvo dos piretroides. Substituições de aminoácido no NaV, que conferem resistência ao efeito knockdown, são chamadas de mutações kdr (knockdown resistance). Em Ae. aegypti, há pelo menos duas mutações kdr descritas na América Latina: Val1016Ile e Phe1534Cys, respectivamente, nos domínios IIS6 e IIIS6 do NaV. Nesta dissertação, estudamos a distribuição das mutações kdr em populações naturais de Ae. aegypti do Brasil. Para tanto, genotipamos cerca de 30 indivíduos de cada uma de 30 populações para os sítios 1016 e 1534, via PCRs alelo-específicas. Considerando ambos os sítios para configuração alélica de um único locus, identificamos três alelos: NaVS (selvagem), NaVR1 (mutante no sítio 1534) e NaVR2 (mutante em ambos os sítios). O alelo kdr NaVR1 encontrou-se distribuído em todas as regiões do Brasil, porém o NaVR2 apenas na região centro-sul, ou em baixa frequência em algumas localidades do norte-nordeste, com exceção do estado de Roraima. Análise de amostras de quatro localidades, em um espaço de uma década, indicaram que estes alelos estão aumentando rapidamente de frequência no país. Em um dos trabalhos, avaliamos o custo evolutivo da mutação kdr NaVR2. Para tanto, isolamos uma linhagem homozigota para a mutação em ambos os sítos 1016 e 1534, a partir de uma população natural. Em seguida, o alelo mutante foi inserido em um background genético padrão de vigor e susceptibilidade, através de retrocruzamentos com a cepa Rockefeller (Rock). Esta linhagem, chamada de Rock-kdr, levou mais tempo no desenvolvimento larvar, teve aumento na atividade locomotora e produziu menor número de ovos, comparada à cepa Rock. Ensaios de caixas de populações ix mostraram que a frequência do alelo mutante diminuiu consideravelmente em 15 gerações, em ambiente livre de inseticida, corroborando a hipótese de efeito colateral da resistência. Finalmente, apresentamos uma série de evidências da ocorrência de duplicação gênica no NaV de Ae. aegypti. A presença da mutação Ile1011Met sempre em heterozigose foi a primeira sugestão do fenômeno. O sequenciamento da região IIS6 do NaV de mosquitos individuais revelou ainda a presença de três haplótipos, os quais sugeriram a configuração de um alelo duplicado. Experimentos com cruzamentos de parentais com genótipos conhecidos para o sítio 1011 corroboraram nossa hipótese de duplicação. Molecularmente, ensaios de avaliação do número de cópias gênicas por PCR em tempo real sugeriram a amplificação de cinco vezes mais cópias do fragmento na linhagem de laboratório em comparação com Rock. No conjunto, estes trabalhos contribuem para o entendimento da dinâmica da resistência a inseticida, podendo ser utilizados em estratégias do controle do vetor de dengue. x INSTITUTO OSWALDO CRUZ STUDIES OF THE EFFECTS OF MUTATIONS IN VOLTAGE GATED SODIUM CHANNEL OF AEDES AEGYPTI: DISTRIBUTION IN NATURAL POPULATIONS, CORRELATION WITH RESISTANCE TO PYRETHROID AND ADAPTIVE COST ABSTRACT MASTER DISSERTATION IN PARASITOLOGY Luiz Paulo de Brito Oliveira Souza Aedes aegypti is the main dengue vector. One of the most adopted tool of control is the use of insecticides, notedly pyrethroids, against adult mosquitoes. These compounds are preferred, since they present a lower environmental impact and act rapidly in the nervous system of the insects, known as the knockdown effect. The intense use of insecticides has been selecting resistant mosquito populations all over the world. One of the principal physiological aspects selected for resistance is alteration in the voltage gated sodium channel (NaV), target site of pyrethroids. Aminoacid substitutions in the NaV, which confer resistance to the knockdown effect, are referred to as kdr mutations (knockdown resistant). In Ae. aegypti, there are at least two kdr mutations described in Latin America: Val1016Ile and Phe1534Cys, respectively, in the IIS6 and IIIS6 NaV domains. In the present dissertation, we studied the distribution of kdr mutations in Ae. aegypti natural populations from Brazil. To do so, we genotyped the 1016 and 1534 sites, through allele-specific PCRs, of around 30 individuals from 30 populations each. Taking into account both sites for the allelic configuration in one unique locus, we identified three alleles: NaVS (wild-type), NaVR1 (1534 mutant) and NaVR2 (1016 and 1534 mutant). The NaVR1 kdr allele was distributed in all Brazilian regions, and the NaVR2 only in the cetral-south, or under low frequencies in some northern-eastern localities, with exception of Roraima State. Analisys of samples from four localities, in time space of a decade, indicated that the frequencies of these alleles are incresing rapidly in the country. In the following paper, we evaluated the fitness cost of NaVR1 kdr mutation. In order to acomplish this, we isolated an homozygous lineage for the mutation in both 1016 and 1534 sites, starting from a natural population. Following, the mutant allele was inserted into a genetic background gold-standard for vigour and susceptibility, through retro crosses with Rockefeller (Rock) strain. This lineage, called Rock-kdr, developed longer, had and increase in the locomotor activity and produced smaller number of eggs, compared to Rock. Population-cages assays showed that the mutant allele frequency considerably diminished over 15 generations under an environmental free of insecticide, corroborating the hypothesis of side effects hitchhiked by the resistance. Finally, we presented a series of evidence that the occurrence of gene duplication in Ae. aegypti NaV. The presence of the mutation Ile1011Met always in heterozygosis was the first suggestion for the phenomena. Sequencing of the IIS6 NaV region of individual mosquitoes showed the presence of three haplotypes, of which suggested the configuration of one duplicated allele. Crosses experiments with parental with known genotypes for the 1011 site corroborated the hypothesis of gene duplication. Molecularly, copy number variation assays based on real-time PCR suggested the amplification of five times more copies of the evaluated NaV fragment in the laboratory lineage than in Rock. Summing all, the results here presented contribute to understand the insecticide resistance dynamics, which may be applied for dengue vector control strategies. xi ÍNDICE 1. Introdução 7 1.1 Dengue 7 1.2 Aedes aegypti 12 1.3 Controle químico de Aedes aegypti 14 1.4 Resistência a piretroides 17 1.5 Mutações kdr 19 1.6 Duplicação gênica e resistência a 21 inseticidas 1.7 Custo evolutivo da resistência 22 2. Objetivos 24 3. Apresentação dos capítulos 25 4. Capítulo I 26 5. Capítulo II 38 6. Capítulo III 53 7. Discussão 69 Anexo I 74 Anexo II 75 Anexo III 76 8. Perspectivas 77 9. Conclusões 78 10. Referências bilbiográficas 75 xii ÍNDICE DE FIGURAS Figura 1 - Ciclos do vírus dengue.. ..................................................................................... 17! Figura 2 - Distribuição global de risco de dengue de acordo com a Organização Mundial de Saúde. . 18! Figura 3 - Número de casos de dengue, de óbitos em decorrência da doença e incidência dos sorotipos virais no Brasil, entre os anos de 1995 e 2012. ......................................................... 19! Figura 4 - Taxa de incidência de dengue por Unidade Federativa do Brasil - 1982 a 2008. ............. 19! Figura 5 - Média anual do número de casos de dengue entre 2004 e 2010 nos países mais endêmicos. ................................................................................................................................... 20! Figura 6 - Possíveis mecanismos de resistência metabólica a inseticidas. .................................. 27! Figura 7 – Esquema do Canal de sódio regulado por voltagem (NaV) com seus seis segmentos hidrofóbicos (S1-S6) para cada domínio (I-IV). ...................................................................... 28! xiii INDICE DE TABELAS Tabela 1 - Quantitativo de dengue no Brasil entre os anos de 2010 e 2013, semana epidemiológica 1 a 42................................................................................................................................ 21! Tabela 2 - Compostos e formulações recomendados pela Organização Mundial de Saúde para o controle de larvas de mosquitos. ........................................................................................ 25! Tabela 3 - Compostos e formulações recomendados pela Organização Mundial de Saúde para o controle de mosquitos, via tratamento espacial. ..................................................................... 26! Tabela 4 - Mutações kdr observadas no gene do canal de sódio regulado por voltagem em Ae. aegypti. ........................................................................................................................ 29! xiv LISTA DE SIGLAS E ABREVIATURAS Abreviaturas AaNaV AaNaV Ace-1 ace-1 Asp (D) Bti CB CYP Cys (C) DC DDT FHD GABA Gly (G) GST His (H) IGR Ile (I) Kdr L1, L2, L3 e L4 Leu (L) Met (M) MFO MoReNAa MS NaV NaV NaVR1 NaVR2 OC OMS OP OPAS Phe (F) PI PNCD Pro (P) Rdl Ser (S) SVS Tyr (Y) UF Val (V) Significado Canal de sódio regulado por voltagem de Aedes aegypti Gene de canal de sódio regulado por voltagem de Aedes aegypti Acetilcolinesterase Gene da acetilcolinesterase Aspartato ou ácido aspártico Bacillus thuringiensis variedade israelenses Carbamato Família gênica de P450 Cisteína Dengue Clássica Dicloro-difenil-tricloroetano Febre Hemorrágico do dengue do inglês: Gamma-AminoButyric Acid; Ácido gama-aminobutírico Glicina Glutationa-S-transferase Histidina do inglês: insect growth regulator; Regulador de hormônio juvenil Isoleucina do inglês: knockdown resistance Estágios larvais Leucina Metionina Monoxigenase de função mista; Monoxigenase de função múltipla; também referida como P450 Rede Nacional de Monitoramento da Resistencia de Aedes aegypti a Inseticidas Ministério da Saúde Canal de sódio regulado por voltagem Gene do canal de sódio regulado por voltagem Alelo contendo mutação apenas no sítio 1534 Alelo contendo a mutação nos sítio 1016 e 1534 Organoclorado Organização Mundial de Saúde (WHO – World Health Organization) Organofosforado Organização Pan-Americana de Saúde Fenilalanina Piretroide Programa Nacional de Controle de Dengue Prolina Gene codificante do canal receptor de GABA Serina Secretaria de Vigilancia Sanitária Tirosina Unidade Federativa Valina xv 1. INTRODUÇÃO 1.1 Dengue A dengue é um dos principais problemas que acometem a saúde pública em diversas regiões do planeta. É caracterizada como uma doença viral, febril e aguda, transmitida ao homem por meio da picada do mosquito Aedes aegypti fêmea infectada por um dos quatro sorotipos do vírus DENV, gênero Flavivirus, descritos até o momento (DENV-I, DENV-II, DENV-III e DENV-IV). Estes quatro sorotipos circulantes entre humanos evoluíram independentemente de cepas ancestrais silvestres que ocorriam em primatas não-humanos. A partir do estabelecimento de populações humanas próximas a áreas silvestres, amplas e densas o suficiente para transmissão contínua entre mosquitos e humanos, os vírus ancestrais evoluíram para as formas circulantes no ciclo humano (Vasilakis et al. 2011). No momento discute-se a descrição de um quinto sorotipo, observado em amostra de soro de um paciente de dengue grave, de surto no Borneo, em 2007 (Normile 2013). A Figura 1 mostra os ambientes onde ocorrem os ciclos silvestres e humanos e os mosquitos envolvidos. Apesar da dengue muitas vezes ser caracterizada como assintomática ou com sintomas brandos que podem ser confundidos com qualquer outra virose, em geral é dividida em dois quadros clínicos distintos de acordo com a sua sintomatologia: a forma clássica chamada de febre do dengue e a forma hemorrágica chamada de dengue hemorrágico (Febre Hemorrágico do Dengue – FHD). A sintomatologia clínica registra casos de cefaleia, febre, mialgias (dores musculares), prostração e artralgias (dores nas articulações). No entanto, todos estes sintomas podem sofrer graus de variação que se estabilizam entre a forma mais branda e a forma mais severa da doença. A FHD é decorrente de uma série de fatores relacionados à idade do enfermo, que é geralmente baixa, sorotipo do vírus, estado imunológico do paciente e carga viral. Todos estes são agravantes para o paciente e podem, em um estágio mais acentuado, levá-lo ao quadro denominado de síndrome de choque do dengue (SCD) (Forattini 2002). Uma vez infectado, o paciente desenvolve imunidade cruzada parcial e apenas por um curto espaço de tempo para alguns sorotipos (Adams et al. 2006). Este fato dificulta o desenvolvimento de uma vacina e permite que populações já expostas a um dos sorotipos tornem-se a ser infectadas por outro. Vale uma ressalva que, a partir deste ano de 2014, o Brasil adotará a classificação de casos de dengue utilizada pela Organização Mundial de Saúde: dengue, dengue com sinais de alarme e dengue grave. Os novos casos a partir de então serão notificados ao Sinan (Sistema de Informação de Agravos de Notificação) nestas formas (Brasil/MS 2014). 16 Aedes aegypti subsp. aegypti (trópicos) Aedes albopictus (trópicos) Aedes polynesiensis (Polinésia) Aedes luteocephalus (Áfria ocidental) Aedes furcifer (África ocidental) Aedes niveus spp. (Sudeste asiático) Aedes furcifer (África ocidental) Aedes albopictus (Sudeste asiático) Zona de emergência Ciclo silvestre Áreas rurais Ciclo urbano Figura 1 - Ciclos do vírus dengue. Os sorotipos virais do DENV circulam em dois ciclos de transmissão ecológica e evolutivamente distintos: silvestre e humano. No primeiro, primatas nãohumanos e mosquitos Aedes arborícolas estão envolvidos em focos de transmissão na África ocidental e na Malásia. As espécies domésticas Aedes aegypti e peridomésticas Aedes albopictus estão envolvidas no ciclo humano, em diversos tipos de ambientes tropicais e subtropicais. Neste ciclo, os humanos são os únicos reservatórios e amplificadores virais, caracterizando um perfil único entre as arboviroses (Hanley & Weaver 2008) apud (Vasilakis et al. 2011). Figura adaptada de (Vasilakis et al. 2011) A dengue faz parte de um grupo de doenças transmitidas por artrópodes que recebem a denominação comum de “arboviroses”, abreviação do inglês “arthropod borne virus” (Forattini 2002). Estima-se que cerca de 2,5 bilhões de pessoas no mundo correm o risco de contrair a doença e que, a cada ano, pelo menos 50 milhões de casos sejam contabilizados. Destes, 550 mil resultam em internações graves e 20 mil em óbitos (WHO 2009). As áreas sob risco de transmissão de dengue, segundo a compilação mais recente da Organização Mundial de Saúde, estão apresentadas na Figura 2. Fluxo migratório de pessoas, saneamento básico inadequado, mudanças climáticas e aumento da urbanização são fatores que afetam a eficiência dos programas de controle de dengue em diversos países tropicais e subtropicais. E embora o desenvolvimento de uma vacina seja vital para o fortalecimento destes programas, políticas sócio-educativas devem atuar em conjunto nas estratégias de controle já estabelecidas para que, de fato, a doença possa vir a ser controlada (Tapia-Conyer et al. 2012). 17 Susceptibilidade à transmissão de dengue Alta susceptibilidade Baixa susceptibilidade Não suscpetíveis ou não endêmicas Figura 2 - Distribuição global de risco de dengue de acordo com a Organização Mundial de Saúde. Figura adaptada de Simmons et al 2012. A história das epidemias de dengue nas Américas é bem retratada na revisão publicada por (Dick et al. 2012), onde a história das epidemias no continente é resumida em quatro fases distintas: introdução da dengue nas Américas, plano continental de erradicação do Ae. aegypti, reinfestação do mosquito e aumento na dispersão e circulação do vírus dengue. Abrangendo desde o registro dos primeiros possíveis surtos no continente em 1635, no Panamá, até o aumento na distribuição do Ae. aegypti e dos sorotipos circulantes pelos outros países no ano de 2010, esta compilação de dados aponta que das cinco maiores epidemias ocorridas nas Américas, o Brasil esteve entre os países com maior número de casos em quatro delas: 1998, 2002, 2009 e 2010 (Dick et al. 2012). A problemática da dengue no Brasil se intensificou a partir da década de 1980, quando a doença passou a ser endêmica no país. Consolidada como uma doença reemergente, a dengue atingiu todos os Estados brasileiros e desde então, nenhuma unidade federativa conseguiu erradicar a doença. A distribuição geográfica se manteve e atualmente encontra-se disseminada nos 26 Estados do país e também no Distrito Federal (BRASIL/MS 2003). A Figura 3 mostra o número de casos, de óbito em decorrência da doença entre os anos de 2001 e 2012, e a incidência dos sorotipos virais. A evolução da incidência de dengue por Unidade Federativa pode ser visualizada na Figura 4. No ano de 2010, o Brasil notificou quase 1 milhão de casos da doença, chegando a quase 1,5 milhão em 2013 (Brasil/MS 2014). De acordo com a OMS, o Brasil teve o maior número médio de casos de dengue por ano de 2004 a 2010, entre os 30 países mais endêmicos (Figura 5) (OMS 2012). 18 600 800000 500 700000 600000 400 500000 300 400000 300000 200 200000 100 100000 12 11 20 10 20 09 20 08 20 07 20 06 20 05 20 04 20 03 20 02 20 01 20 00 20 99 20 98 19 19 19 19 19 97 0 96 0 número de óbitos em decorrência de dengue 700 900000 95 número de casos de dengue confirmados 1000000 ano casos de dengue óbitos DENV 1 + 2 DENV 1 + 2 + 3 DENV 1 + 2 + 3 + 4 Figura 3 - NúmeroSource: de casos de dengue, de óbitos em decorrência da doença e incidência dos PAHO /Health Surveillance andeDisease sorotipos virais no1995-2000: Brasil, entre os anos de 1995 2012.Prevention and Control / Communicable Diseases / Dengue Fontes: 1995-2000: OPAS/ /Health Surveillance Disease Prevention and Control / Communicable Diseases / Dengue 20012012: Brazil/ Ministryand of Health/ Sinanweb/ Dengue 2001-2012: Brasil/MS/ Sinan/DENGUE - Notificações registradas no Sistema de Informação de Agravos de Notificação Sinan/ Dengue Figura 4 - Taxa de incidência de dengue por Unidade Federativa do Brasil - 1982 a 2008. Figura adaptada de (Catão 2011). 19 Figura 5 - Média anual do número de casos de dengue entre 2004 e 2010 nos países mais endêmicos. Figura adaptada de (OMS 2012). Um balanço dos casos notificados de dengue no país nos anos de 2010 e 2013, revela que, embora tenha ocorrido uma diminuição significativa de casos na maioria dos Estados da região norte e nordeste, a situação agravou-se em todas as demais regiões que compreendem o centro-sul brasileiro. A Tabela 1 apresenta o número de casos notificados, casos graves e óbitos, por região e por Estado, naquele período. 20 Tabela 1 - Quantitativo de dengue no Brasil entre os anos de 2010 e 2013, semana epidemiológica 1 a 42. UF Casos Notificados Casos Graves Óbitos 2010 2013 2010 2013 2010 2013 RO 18,670 9,365 351 28 18 3 AC 26,217 2,577 56 4 5 0 AM 4,921 16,858 238 96 6 9 RR 7,373 849 275 1 5 0 PA 11,346 8,682 357 37 17 10 AP 2,878 1,667 11 7 3 2 TO 8,449 8,669 50 17 4 4 Norte 79,854 48,667 1,338 190 58 28 MA 5,184 3,586 192 36 4 12 PI 6,615 4,664 115 19 7 1 CE 15,854 32,039 169 159 13 54 RN 6,302 16,035 238 102 7 8 PB 5,833 13,050 90 92 5 14 PE 33,177 8,650 1074 42 24 19 AL 45,449 8,935 450 16 21 4 SE 564 745 34 5 0 3 BA 41,803 61,974 974 125 33 21 Nordeste 160,781 149,678 3,336 596 114 136 MG 212,157 435,828 1,367 360 83 116 ES 22,835 66,874 1,468 1,686 13 23 RJ 26,800 212,933 2,437 1,207 41 48 SP 205,796 220,865 2,897 428 140 72 Sudeste 467,588 936,500 8,169 3,681 277 259 PR 36,645 69,444 184 224 13 24 SC 180 370 1 1 0 0 RS 3,633 485 52 1 0 0 Sul 40,458 70,299 237 226 13 24 MS 62,489 81,741 1792 695 42 34 MT 33,550 34,012 875 99 51 27 GO 95,527 140,399 997 1,063 78 58 DF 14,840 15,621 41 16 5 7 Centro-Oeste 206,406 271,773 3,705 1,873 176 126 BRASIL 955,087 1,476,917 16,785 6,566 638 573 Fonte: (Saúde 2013) 1.2 Aedes aegypti Pelo que se conhece até o momento, a transmissão do vírus da dengue nas Américas se dá exclusivamente pela picada de fêmeas de Aedes (Stegomya) aegypti infectadas com um dos quatro sorotipos descritos até o presente. Um segundo potencial vetor é o Ae. albopictus, também presente no continente americano, mas que até o momento não tem sido associado à veiculação do vírus em 21 ambientes naturais. Como visto na Figura 1, outras espécies pertencentes ao mesmo gênero são potenciais vetores de dengue, como o caso do Aedes polynesiensis na Ásia (Braga & Valle 2007a). Além do vírus da dengue, o Ae. aegypti também é o principal vetor de duas outras arboviroses que acometem os seres humanos: febre amarela urbana e o chikungunya (Braga & Valle 2007c; Powell & Tabachnick 2013). Ae. aegypti é um inseto da ordem dos dípteras, família Culicidae, tribo Aedini. Pertence ao gênero Aedes, que contém 44 subgêneros, e entre eles o Stegomya, e900 espécies de mosquitos são agrupadas neste gênero (Forattini 2002). Quanto à biologia, o Ae. aegypti é ativo durante o dia, principalmente nas primeiras horas da manhã e ao final do entardecer. Como todos os dípteros, seu desenvolvimento é holometabólico, ou seja, passa por metamorfoses completas, sendo seus estágios iniciais obrigatoriamente dentro da água, limpa e parada, seguida por quatro estágios larvares (L1, L2, L3 e L4), um estágio de pupa e o último estágio, adulto, ocorre em ambiente terrestre. Em média, o Ae. aegypti vive em torno de 30 dias em sua fase adulta, alimentando-se de seiva vegetal. As fêmeas, no entanto, possuem hábito hematofágico, pois necessitam do sangue para maturação dos ovos. Uma única inseminação é suficiente para fecundar todos os ovos que a fêmea venha a produzir ao longo de sua vida (Consoli & Lourenço-de-Oliveira 1994). O Ae. aegypti é um mosquito exótico que teve sua introdução no Brasil provavelmente através de embarcações vindas da África e acabou por se adaptar bem às condições climáticas do país, tornando-se parte da fauna culicídica (Braga & Valle 2007c). De hábitos extremamente antropofílico, muito provavelmente por conta do aumento da população humana e invasão em seu ambiente natural, o Ae. aegypti se adaptou bem ao convívio humano onde conseguiu fonte de alimentação sanguínea abundante e também locais de oviposição, em decorrência da necessidade humana de estocar água (Powell & Tabachnick 2013). Posteriormente passou a ser bem adaptado aos ambientes urbanos e suburbanos, sendo, atualmente, sua distribuição quase cosmopolita, com destaque para as regiões tropicais e subtropicais. No Brasil, encontra-se atualmente disseminado pelos 26 Estados, bem como no Distrito Federal (MS/ Brasil, 2014), refletindo a ocorrência de dengue por todo o país, como visto acima. Durante a metade do século XX, intensas campanhas anti-febre amarela resultaram na erradicação do Ae. aegypti do Brasil e de várias regiões das Américas, certificados pela Organização Pan-Americana de Saúde (OPAS). Contudo, em 1976 o vetor foi reintroduzido no Brasil a partir de populações remanescentes, provavelmente, vindas da América central e Guiana Francesa (Braga & Valle 2007a). Desta forma, com a sua reintrodução, desde a década de 1980, surtos epidêmicos periódicos de dengue têm sido observados em várias regiões do país. 22 1.3 Controle químico de Aedes aegypti De forma geral, o controle de populações de mosquito pode ser classificado como mecânico, biológico ou químico. Mais recentemente, novas metodologias têm permitido a implementação de alternativas como mosquitos transgênicos e mosquitos infectados com bactérias do gênero Wolbachia. A primeira visa a supressão de populações naturais (Speranca & Capurro 2007; Harris et al. 2011), já a segunda pretende substituir populações por uma sem competência vetorial para o DENV (Moreira et al. 2009; Walker et al. 2011). O controle mecânico é uma das formas mais eficientes e menos impactantes ao meio-ambiente a curto e longo prazos, consistindo na remoção de criadouros artificiais e instalação de barreiras que inviabilizem a continuação do ciclo de vida do mosquito. Citam-se como principais exemplos a drenagem de áreas alagadas e a correta vedação de reservatórios de água. O controle biológico consiste na utilização de predadores e organismos entomopatogênicos (ou seus derivados) para o controle populacional, como por exemplo, o uso da endotoxina liberada pelo Bacillus thuringiensis israelensis (Bti) no controle de larvas. Finalmente, o controle químico é feito através da utilização de compostos inseticidas contra as formas larvais e adultas, apresentando geralmente efeitos contra os sistemas nervoso ou endócrino dos insetos (Braga & Valle 2007b) Desde a epidemia de 1986, resultante da introdução do vírus DENV-I no país, a principal forma de combate ao Ae. aegypti tem sido o uso de inseticidas neurotóxicos tanto para as formas imaturas, chamados de larvicidas, quanto para os adultos, chamados de adulticidas. De modo geral, estes compostos interagem com moléculas do sistema nervoso central do inseto, levando-o à morte. Muitos inseticidas foram desenvolvidos ao longo do século passado, mas os que são utilizados por recomendação da OMS são resumidos principalmente a quatro classes principais: organoclorados, organofosforados, carbamatos e piretroides (Braga & Valle 2007b). Os organoclorados são compostos químicos que possuem em sua estrutura carbono, hidrogênio e cloro. Dentro do grupo dos principais organoclorados encontra-se o DDT (dicloro-difeniltricloroetano), inseticida responsável pelo controle das principais pragas que aflingiram o século XX (Braga & Valle 2007b). Seu estudo como potencial inseticida que poderia substituir o piretro na erradicação da febre tifoide e malária foi descoberto pelo suíço Paul Hermann Müller e em 1948 rendeu-lhe o prêmio Nobel de Medicina (D’Amatos et al 2002). Com seu uso intenso em várias campanhas de erradicação de doenças transmitidas por vetores, como malária, febre amarela e pediculose, não tardou muito a aparecerem os primeiros registros de insetos resistentes ao composto. Os primeiros trabalhos descrevendo a resistência de mosquitos ao DDT datam da década de 1950 (Busvine 1951; Georgopoulos 1954; Pampana 1954; Trapido 1954). Contudo, mesmo com o surgimento da resistência, a suspeita de seu potencial cancerígeno, alta toxicidade e seus efeitos 23 negativos sobre o ambiente foram os fatores que mais contribuíram para que o DDT fosse banido em vários países (Egan 1966; D’Amatos et al 2002). Atualmente, a OMS recomenda o uso de DDT para borrifação contra anofelinos, em situações específicas de países africanos com altos níveis de malária (Hougard et al. 2003). Com a proibição do uso do DDT, outros inseticidas neurotóxicos ganharam destaque sobre as recomendações da OMS. Os organofosforados e carbamatos foram os primeiros inseticidas para o controle de mosquitos vetores, como alternativa ao DDT (Casida 1980). Atuam na fenda sináptica, inibindo a atuação da acetilcolinesterase, enzima responsável pela degradação do neurotransmissor acetilcolina, durante a propagação do impulso nervoso. Com a inibição da acetilcolinesterase pelos inseticidas, a acetilcolina não é degrada em seus produtos, colina e ácido acético, e com isso permanece ligada aos respectivos receptores nos neurônios pós-sinápticos, tornando o impulso nervoso contínuo. Este efeito leva a um quadro de propagação irregular do impulso nervoso que acomete a atividade de vários órgãos do inseto onde a enzima atuaria, como por exemplo, no sistema nervoso central e nas glândulas controladas pelo sistema nervoso autônomo, dependentes do sistema nervoso parassimpático (Fukuto 1990). Embora os organoclorados, os organofosforados e os carbamatos tenham ajudado significativamente no controle de pragas agrícolas e urbanas entre a metade do século XX e início da década de 1980, os programas de controle de insetos restringiram em muito o seu uso por conta dos impactos ambientais, tempo de persistência e efeitos tóxicos sobre outros animais incluindo o homem (Casida 1980). Historicamente, os inseticidas são classificados em três gerações, sendo os elementos químicos comumente tóxicos para a maioria dos seres vivos os representantes da primeira geração, por exemplo, enxofre, arsênio, chumbo e cádmio. Os inseticidas da terceira geração são relacionados com alterações no sistema endócrino dos insetos e atuam em sua maior parte sob o efeito dos hormônios, como por exemplo, os reguladores de hormônio juvenil (IGR, do inglês insect growth regulator). Já os da segunda geração representam os inseticidas químicos sintéticos, dos quais destacam-se os organoclorados, os organofosforados, carbamatos e piretroides. Este último apresentou menores impactos ao meio ambiente e são menos tóxicos, sendo, por conta disso, preferencialmente adotados pelos programas de saúde pública. Os piretroides compõem a classe de inseticida sintéticos análogos à piretrina, substância extraída de plantas do gênero Chrysanthemum, família Asteraceae (Casida 1980). São comumente subdivididos em dois grupos, tipo I e tipo II, que diferem quanto à toxicidade e estabilidade química, sendo baixas para os do tipo I e altas para os do tipo II. Além disso, piretroides do tipo II apresentam correlação positiva com a temperatura, ou seja, são mais eficientes em temperaturas mais elevadas, o que, em teoria, facilita a sua aplicação em países de clima tropical (Soderlund 2012). Tal qual o DDT, atuam nos axônios dos neurônios, via interação com as moléculas do canal de sódio regulado por 24 voltagem (NaV), mantendo-os em sua conformação aberta por mais tempo, o que leva a uma propagação contínua do impulso nervoso. Em consequência disto, o inseto sofre repetidas contrações involuntárias, seguidas de paralisia e morte, efeito comumente referido como knockdown (Martins & Valle 2012). Os piretroides são menos tóxicos e mais facilmente aplicáveis que as demais classes de inseticidas e, como provocam o efeito knockdown, matam rapidamente o inseto. Estes são os principais fatores que tem feito dos piretroides a classe de inseticida mais utilizada não somente contra insetos de importância médica, mas também contra pragas da agricultura e pecuária (Beaty & Marquardt 1996). O início do uso de piretroides pelo Programa Nacional do Controle de Dengue (PNCD) se deu em escala nacional a partir do ano 2000 e, desde então, tem sido crescente a utilização deste inseticida no controle de Ae. aegypti apenas contra a forma adulta, por meio de aplicações espaciais (Braga et al. 2004). Contudo, o intenso uso desta classe de inseticida tem selecionado rapidamente populações de Ae. aegypti resistentes por todo o país (da-Cunha et al. 2005; Montella et al. 2007; Martins et al. 2009a; Martins et al. 2009b;). Vale destacar que a resistência aos organofosforados no Brasil somente começou a ser detectada três décadas após a intensificação de sua aplicação contra larvas e adultos do vetor (Lima et al. 2003; Montella et al. 2007). As Tabelas 2 e 3 mostram os inseticidas recomendados para uso em saúde pública. Nota-se que para uso dentro de casa apenas piretroides são recomendados. Além disso, esta é também a única classe permitida para impregnação em cortinas, redes de cama e roupas (WHOPES 2006). Tabela 2 - Compostos e formulações recomendados pela Organização Mundial de Saúde para o controle de larvas de mosquitos. Larvicida Bacillus thringiensis israelensis Chlorpyrifos EC Diflubenzuron DT, GR, WP Novaluron EC Pyriproxyfen GR Fenthion EC Pirimiphos-methyl EC Temephos EC, GR Spinosad DT, EC, GR, SC Classe BL OP BU BU JH OP OP OP SP BL-larvicida bacteriano, BU-benzoilureas, JH-análogos de hormônio juvenil, OP-organofosforados, SP-espinosinas DT-tablete de aplicação direta, GR-grânulado, EC-concentrado emulsionável, WG#grânulo+dispersível+em+água,+WP#pó+molhável Atualizado em outubro de 2013. Fonte: (WHOPES 2014b) 25 Tabela 3 - Compostos e formulações recomendados pela Organização Mundial de Saúde para o controle de mosquitos, via tratamento espacial. Composto e formulação Deltametrina UL Deltametrina EW Lambda-cialotrina EC Malathion EW e UL Permetrina + s-bioaletrina + Piperonil butóxido EW d-d, trans-cifenotrina EC Classe química PI PI PI OP domicílio X X PI X X PI peridomicílio X X X X X OP-organofosforados, PI-piretróides EC-concentrado emulsionável, EW-emulsão, UL-ultra-baixo volume (UBV) Atualizado em julho de 2012. Fonte: (WHOPES 2014a) 1.4 Resistência a piretroides Resistência é definida como capacidade de um organismo ou população em tolerar um composto em dose normalmente letal para a maioria dos outros organismos da sua própria espécie. No caso dos insetos, é a capacidade de resistir a uma dose do inseticida que em condições normais, levaria ao óbito (Beaty & Marquardt 1996; Braga & Valle 2007b) Os principais mecanismos fisiológicos selecionados para resistência a inseticidas podem ser classificados como ‘resistência metabólica’ e ‘resistência do sítio-alvo’. A primeira refere-se a um incremento na capacidade de detoxificação dos inseticidas, seja por uma maior produção de enzimas detoxificantes, seja pelo aumento de sua atividade específica (Figura 6). Estão envolvidas enzimas das super-famílias das Esterases, Glutationa S-transferases (GST) e Monoxigenases de função mista (MFO ou P450) (Hemingway et al. 2004). Estas enzimas fazem parte de super-famílias gênicas, cada qual composta de dezenas de genes, resultado de duplicações gênicas e mutações ao longo da evolução (Montella et al. 2012; Ranson et al. 2002). Com isto há diversos genes destas famílias que são específicos de determinadas espécies, com potencial de serem selecionados para resistência de forma espécie-específica. Outra classe que tem ganhado destaque quanto à capacidade de atuar na detoxificação de inseticidas e cujo número de cópias gênicas parece ter relação direta com diferentes graus de resistência é a da família dos transportadores ABC (Dermauwa & Leeuwen, 2014). 26 Apesar de algumas destas enzimas atuarem de modo inespecífico sobre vários inseticidas no processo de detoxificação (Hemingway & Ranson 2000; Kumar et al. 2002), entre as enzimas detoxificantes, os genes de MFO ou P450 (famílias CYP) merecem destaque na resistência a piretroides em Ae. aegypti. Esta constatação tem sido possível a partir da comparação entre os transcriptomas de populações resistentes e susceptíveis a piretroides (microarrays contendo genes relacionados à detoxificação de inseticidas, os detoxchips) (Poupardin et al. 2008; Strode et al. 2008; Marcombe et al. 2009; Bingham et al. 2011; Strode et al. 2012; S Bariami et al. 2012; SaavedraRodriguez et al. 2013) . Figura 6 - Possíveis mecanismos de resistência metabólica a inseticidas. Quadrados laranjas representam os genes, novelos em vermelho as proteínas e novelo azul a proteína mutante. Em A e B mais enzimas são produzidas, já em C uma modificação estrutural torna a enzima mais eficiente na detoxificação. Fonte: figura de autoria de André Torres (modificada). A resistência do sítio alvo caracteriza-se por mudanças na estrutura da molécula-alvo do inseticida, resultando em perda de sensibilidade ao composto (Hemingway et al. 2004). Os alvos dos inseticidas neurotóxicos desempenham função primordial na fisiologia da propagação do impulso nervoso e, além disso, são estruturas muito conservadas ao longo da evolução. Desta forma, poucas alterações estruturais são permissivas sem comprometimento da viabilidade do inseto (ffrenchConstant et al. 1998). Há mutações conhecidas relacionadas à resistência nos genes ace-1 e rdl, codificantes respectivamente da acetilcolicenesterase (alvo de organofosforados e carbamatos) e receptor de GABA (alvo do organoclorado dieldrin), em sítios conservados entre diferentes espécies de insetos (Ang et al. 2013; Asih et al. 2012; Domingues et al. 2013; Remnant et al. 2013a; Wondji et al. 2011). Similarmente, a resistência ao efeito knockdown dos piretroides e DDT acima descrita, é proporcionada por mutações no gene do canal de sódio regulado por voltagem (NaV). Com isto, 27 alterações relacionadas a este tipo de resistência são conhecidas como mutações kdr (do inglês, knockdown resistance) (Soderlund & Knipple 2003). 1.5 Mutações kdr Estruturalmente, o NaV é uma proteína composta de quatro domínios homólogos (I-IV), cada um destes constituído por seis subunidades em alfa-hélice transmembranares (S1-S6) e um loop entre os segmentos S5 e S6 (Figura 7). Mutações pontuais no segmento S6 dos domínios I, II e III têm sido associadas à redução da sensibilidade do NaV a piretroides (Soderlund 2008). Domínios extracelular intracelular Figura 7 – Esquema do Canal de sódio regulado por voltagem (NaV) com seus seis segmentos hidrofóbicos (S1-S6) para cada domínio (I-IV). Em azul, segmentos S4, responsáveis pela percepção de alteração da voltagem e, em verde, segmento S6, responsáveis pela cinética do poro. Adaptado de (Martins & Valle 2012). Em particular, uma mutação específica – a substituição Leu/Phe no segmento 6 do domínio II, códon 1014 (Leu1014Phe) - foi primeiramente observada em Musca domestica (Smith et al. 1997; Williamson et al. 1996) e em seguida, em sítio homólogo em uma série de insetos vetores e mesmo em pragas da agricultura (Brun-Barale et al. 2005; Donnelly et al. 2009; Soderlund & Knipple 2003). Leu1014Phe vem sendo, portanto, referida como a mutação kdr clássica. Com a descoberta da relação entre mutação no gene codificante do NaV e resistência a piretroides e DDT, diversas espécies de insetos tiveram partes deste gene sequenciadas na tentativa de se determinar um marcador molecular para resistência, identificando-se outras mutações além da clássica. Uma extensa revisão dos sítios mutantes observados no NaV de várias espécies foi recentemente apresentada por Rinkevich et al. 28 (2013). A Tabela 4 apresenta uma adaptação da compilação indicando as diversas mutações descritas no NaV de Ae. aegypti. Tabela 4 - Mutações kdr observadas no gene do canal de sódio regulado por voltagem em Ae. aegypti. Espécie Mutação Ser989Pro Ile1011Met Ile1011Val Val1016Gly Aedes aegypti Val1016Ile Aedes albopictus Val1016Ile Val1016Ile + Phe1534Cys Asp1763Tyr Phe1534Cys Referências Srisawat et al 2010 Brengues et al 2003; Rajatileka et al 2008; Martins et al 2009a; Lima et al 2011 Saavedra-Rodriguez et al 2007 Brengues et al 2003; Rajatileka et al 2008; Rajatileka et al 2008; Chang et al 2009; Srisawat et al 2010; Lin et al 2013 Saavedra-Rodriguez et al 2007; Martins et al 2009a; Harris et al 2010; Lima et al 2011; Marcombe et al 2012; Marcombe et al 2013; Aponte et al 2013 Martins et al 2009b Harris et al 2010; Yanola et al 2011; Stenhouse et al 2013; Seixas et al 2013; Linss et al 2014 Chang et al 2009; Lin et al 2013 Kasai et al 2011 Além de Phe, foram também encontrados Ser, His e Cys substituindo a Leu em sítio homólogo ao 1014 de M. domestica em mosquitos dos gêneros Anopheles (Martinez-Torres et al. 1998) e Culex (Chandre et al. 1998). Em Ae. aegypti, porém, não foi observada qualquer substituição naquele sítio. Isto ocorre provavelmente porque, nesta espécie, seriam necessárias duas substituições nucleotídicas no mesmo códon para resultar na substituição Leu/Phe, ao invés de uma única alteração, como na maioria dos insetos. Contudo, outras mutações em sítios próximos foram encontradas em Ae. aegypti: sítio 1011 (Ile/Met ou Val) e 1016 (Val/ Ile ou Gly), referidas como responsáveis pelo fenótipo kdr em Ae. aegypti (Brengues et al. 2003; Harris et al. 2010a; Lima et al. 2011; Martins et al. 2009a; Martins et al. 2009c; Saavedra-Rodriguez et al. 2007). Além destas, outra mutação no domínio III-S6, sítio 1534 (Phe1534Cys), foi recentemente classificada como mutação kdr no vetor (Harris et al. 2010a, 2010b; Yanola et al. 2010). No Brasil, foram detectadas as mutações Ile1011Met e Val1016Ile, com forte indicação de participação desta última na resistência a piretroides (Martins et al. 2009b; Martins et al. 2009d). A detecção desta mutação em populações naturais do país foi incorporada às atividades de rotina para o diagnóstico dos mecanismos de resistência pela Rede Nacional de Monitoramento da Resistência de Aedes aegypti a Inseticidas (Rede MoReNAa), a fim de subsidiar o Programa Nacional de Controle de Dengue (PNCD) na utilização racional de inseticidas. Tem-se observado que a mutação kdr Val1016Ile está se espalhando e aumentando de frequência de forma muito acelerada no país, tal qual ocorreu no México (Garcia et al. 2009). Nas Regiões Norte/ Nordeste, contudo, embora existam populações 29 resistentes a piretroides, a frequência da mutação Val1016Ile, quando encontrada, era baixa (Martins et al. 2009a). Para estas localidades seria, portanto, importante investigar a possível ocorrência da mutação Phe1534Cys, já observada na região do Caribe (Harris et al. 2010). 1.6 Duplicação gênica De modo geral, a duplicação de um gene é um evento que resulta em uma cópia excedente, que pode ser livre de pressão de seleção e que permite o rápido acúmulo de mutações no genoma do organismo (Bass & Field 2011). Além disto, a duplicação gênica cria condições mais permissivas à ocorrência de mutações que seriam deletérias recessivas, caso presentes em cópia única (Kimura & King 1979). É um evento relativamente comum quando se observa a grande diversidade de vida no planeta e, indubitavelmente, é a base da evolução dos genomas de todos os organismos. Classicamente, os eventos de duplicações gênicas antecedem aos eventos de diversificação funcional e permite que as cópias sofram divergência sem que a sua função primordial seja alterada (Force et al. 1999). Ao longo da evolução, as duplicações podem seguir diferentes rumos, como: 1) subfuncionalização, onde ambas as cópias podem sofrer alterações desde que a expressão gênica não se altere; 2) neofuncionalização, onde uma cópia mantém sua expressão enquanto a outra adquire uma nova função; e 3) degeneração ou perda de função adquirida por uma das cópias, onde esta região do genoma passa a não ser transcrita e fica livre para sofrer mutações e alterações que não comprometem em quase nada o organismo. Este último evento é responsável pela formação dos chamados pseudogenes, ou seja, genes que perderam sua função (Bass & Field 2011). O termo amplificação se aplica a eventos que produziram mais de uma cópia do gene original. Já quando dois genes são duplicados juntos ou sofrem mais de uma duplicação, trata-se respectivamente de coduplicação e co-amplificação (Force et al. 1999). Apesar das diversas possibilidades de ocorrerem duplicações nos genomas, tais eventos não são muito frequentes em genes de atuação direta no sistema nervoso. Tal qual mutações não sinônimas, duplicação de genes de neurotransmissores, receptores de GABA e canais iônicos são raras (ffrench-Constant et al. 1998). Como visto acima, os genes envolvidos com a resistência metabólica possuem uma grande diversidade de genes parálogos, resultantes de duplicações gênicas (Ranson et al. 2002). Desta forma, é provável que alterações numéricas ou mesmo estruturais nestes genes sejam mais favoravelmente selecionadas para a resistência, com menores efeitos colaterais ao organismo, do que modificações nas moléculas-alvo do inseticida. Até recentemente já haviam sido identificados 26, 49 e 160 genes, respectivamente, de GST, esterases e P450 em Ae. aegypti (Strode et al. 2008). Amplificação gênica foi observada no gene de P450 CYP9J26 de Ae. aegypti, contendo 30 cerca de sete vezes mais cópias em duas populações resistentes comparadas a uma linhagem controle (Bariami et al. 2012). Aedes aegypti apresenta uma abundância de genes de enzimas detoxificantes, resultado de uma expansão via duplicações e amplificações gênicas, um fato intrigante no estudo da fisiologia molecular dos culicídeos (Strode et al. 2008). Como anteriormente apresentado, mutações pontuais no gene ace-1, codificante da enzima acetilcolinesterase, são amplamente reportadas por fornecer resistência a organofosforados. Contudo, foi visto que mosquitos do gênero Culex portadores da mutação Gly119Ser possuíam atividade reduzida da acetilcolinesterase, prejudicando-lhes em aspectos de sua tabela de vida (Labbe et al. 2007a; Weill et al. 2003). Com a utilização contínua de organofosforados, foram selecionadas populações com duplicação no gene ace-1, de forma que uma das cópias manteve-se inalterada e a outra apresentava a mutação. O alelo duplicado aumentou de frequência, sobrepondo-se ao mutante não-duplicado nas populações naturais, sugerindo que a duplicação propiciou a resistência, sem efeitos colaterais (Labbe et al. 2007a). Tal qual o ace-1, os insetos só apresentam um NaV (Zakon 2012). Eventos de duplicação recente neste gene, que podem estar associados à resistência a piretroides, foram recentemente observados na barata Periplaneta americana (Moignot et al. 2009) e no mosquito Culex quinquefasciatus (Xu et al. 2011). Estas ocorrências corroboraram nossa hipótese de duplicação do NaV em populações naturais de Ae. aegypti do Brasil, apresentadas no capítulo 2 desta dissertação. 1.7 Custo evolutivo da resistência A característica da resistência a inseticidas é geralmente um fator rapidamente selecionado, uma vez que os compostos aplicados tem o poder de matar os susceptíveis ou, quando não, podem torna-los reprodutivamente pouco competitivos. Desta forma, sob aplicação maciça de inseticida, os genes de resistência tendem a se dispersar mais livremente na população, mesmo se carregarem algum efeito prejudicial. O custo evolutivo da resistência, também chamado de efeitos de perda de fitness do inseto, pode ser notado principalmente na ausência de seleção, ou seja, em ambientes livres de inseticida (Belinato et al 2012). Tempo de desenvolvimento larvar, capacidade e frequência de realização de repasto sanguíneo, comportamento, eficiência na fecundidade e fertilidade dos ovos são parâmetros diretamente relacionados à capacidade vetorial e à dispersão dos genes, que podem sofrer alterações devido à resistência. Entre outros, destaca-se o tempo de desenvolvimento larvar: quanto maior o período larva-adulto, maiores serão a possibilidade de predação, a necessidade de 31 manutenção do criadouro e o tempo de geração, além da desvantagem na competição pela inseminação, devido à demora no amadurecimento sexual (Kingsolver & Raymond 2008). Como acima apresentado, a resistência pode ser selecionada pela superexpressão de genes codificantes de enzimas envolvidas no metabolismo de xenobióticos (resistência metabólica). Nestes casos, o inseto resistente precisa desviar recursos energéticos de suas funções fisiológicas e reprodutivas para a síntese exacerbada daquelas enzimas. Este fenômeno, mais conhecido como trade-off energético, é bastante estudado em mosquitos do gênero Culex (Chevillon et al. 1997; Gazave et al. 2001). Quanto maior o desvio, maiores os efeitos colaterais no desenvolvimento, longevidade, competição por alimentação e por cópula. Em populações brasileiras de Ae. aegypti, já foi observado que quanto maior a razão de resistência ao organofosforado temephos (via resistência metabólica), maiores os prejuízos na tabela de vida do inseto (Belinato et al. 2012; Martins et al. 2012). Com relação aos efeitos colaterais de alterações nos genes dos sítios alvo de inseticidas, o exemplo mais conhecido é o da mutação no gene ace-1, acima descrito. Esta mutação aumentou rapidamente em frequência em populações de Culex pipens do sul da França, em localidades sob forte pressão de seleção com organofosforado. Contudo, não avançou para localidades sem tratamento. Isto pode ser explicado pelo fato de que a enzima mutante tinha atividade depreciada em 60% comparada à selvagem (Labbe et al. 2007b). Mutações em canais iônicos, como é o caso do NaV, podem estar diretamente relacionadas à alterações no comportamento. Por exemplo, a mutação kdr clássica Leu1014Phe na mosca de frutas Drosophila melanogaster causa nos mutantes a perda de preferência por temperaturas mais altas (Foster, 2003) e a perda da percepção de feromônios de alarme no pulgão Myzus persicae (Foster, 2011). Além da identificação dos mecanismos de resistência é também necessário saber se os que foram selecionados geram algum custo evolutivo no inseto. Se assim ocorrer, é provável que a resistência diminua ao longo do tempo em um ambiente livre de inseticida. A avaliação do custo evolutivo da mutação kdr em Ae. aegypti ajudará a entender a dinâmica de dispersão dos alelos mutantes em populações naturais, o que seria de grande importância para os programas de manejo do vetor. 32 2. OBJETIVOS Objetivo Geral Avaliar os efeitos de mutações kdr no gene do canal de sódio de Ae. aegyti na resistência à piretroide e no fitness do vetor, e investigar a ocorrência de duplicação neste gente. Objetivos Específicos Capítulo 1 – Distribuição das mutações kdr no Brasil - Descrever o padrão de distribuição dos alelos kdr em populações brasileiras através de metodologias de PCR alelo-específicas (AS-PCR) para os sítios 1016 e 1534. Capítulo 2 – Duplicação gênica no NaV de Ae. aegypti - Observar o padrão de distribuição da mutação no sítio 1011 do NaV em populações naturais. - Sequenciar da região IIS6 do NaV para identificação de haplótipos/indivíduo em algumas populações - Genotipar a prole de casais específicos para avaliação da segregação dos alelos do NaV, considerando o sítio 1011. - Estimar o número de cópias gênicas em uma linhagem previamente selecionada para resistência, homozigota para a duplicação. Capítulo 3 – Custo evolutivo da mutação kdr - Avaliar o perfil de resistência a piretroide em linhagem de laboratório homozigota para ambos os sítios. - Comparar diversos parâmetros de desenvolvimento e reprodução da linhagem estabelecida com a cepa Rock. - Observar se a mutação kdr diminui de frequência ao longo de gerações em ambiente livre de inseticida. 33 3. APRESENTAÇÃO DOS CAPÍTULOS Os resultados desta dissertação estão apresentados em rês capítulos, cada um contendo na íntegra um artigo publicado no escopo dos objetivos aqui propostos. O Capítulo 1 refere-se à distribuição dos alelos do gene do canal de sódio regulado por voltagem (NaV) em populações naturais de Ae. aegypti, considerando dois sítios (1016 e 1534) deste mesmo gene. O trabalho, publicado na revista Parasites & Vectors (Linss et al. 2014), apresenta o primeiro registro da mutação kdr no sítio 1534 no Brasil. Genotipagem dos sítios 1016 e 1534 do NaV, considerando ambos na formação de um locus único, revelou altas frequências de dois alelos kdr mutantes, com distribuição regionalizada pelo país. Além disso, acompanhamento de algumas localidades no período de uma década indicou que aquelas mutações vêm aumentando de frequência rapidamente. No Capítulo 2, o artigo apresenta a hipótese de duplicação no NaV de Ae. aegypti, sustentada por ensaios de genotipagem de populações naturais para o sítio 1011 do NaV, sequenciamento individual da região IIS6 do gene, experimentos de análise da prole de cruzamentos de parentais com genótipo conhecido e, finalmente, a quantificação do número de cópias gênicas de uma população selecionada em laboratório, em comparação com a cepa Rock. O artigo do Capítulo 3, aborda os experimentos de obtenção da linhagem homozigota para as mutações kdr, bem como a avaliação da resistência e efeitos no fitness do inseto. Apresentamos o artigo intitulado “Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost”, publicado em 2013 na revista PLos One. Neste trabalho, mostramos i) o estabelecimento da linhagem Rock-kdr, homozigota para mutação em ambos os sítios 1016 e 1534, com background genético da cepa controle de susceptibilidade Rockefeller (Rock); ii) avaliação da susceptibilidade desta linhagem ao piretroide deltametrina; iii) efeitos da mutação kdr em diversos parâmetros da tabela de vida do inseto (fitness) e iv) flutuação do alelo mutante ao longo de 15 gerações na ausência de inseticida. Observamos que a linhagem Rock-kdr (daqui por diante chamada de R2R2) é resistente, em caráter recessivo, não apresenta resistência metabólica, e possui alterações em alguns parâmetros do desenvolvimento e reprodução, em comparação à cepa Rock. Além disso, a frequência do alelo kdr diminuiu significativamente ao longo das gerações, em ambiente livre de inseticida, reforçando a hipótese de efeito daquelas mutações no fitness de Ae. aegypti. Por último, apresentamos uma discussão geral agrupando os três artigos desta dissertação e alguns resultados preliminaries em andamento. 34 4. CAPITULO 1 35 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 RESEARCH Open Access Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations Jutta Gerlinde Birggitt Linss1,2†, Luiz Paulo Brito1,2†, Gabriela Azambuja Garcia1,2, Alejandra Saori Araki3, Rafaela Vieira Bruno3,4, José Bento Pereira Lima1,2, Denise Valle4,5* and Ademir Jesus Martins1,2,4* Abstract Background: The chemical control of the mosquito Aedes aegypti, the major vector of dengue, is being seriously threatened due to the development of pyrethroid resistance. Substitutions in the 1016 and 1534 sites of the voltage gated sodium channel (AaNaV), commonly known as kdr mutations, confer the mosquito with knockdown resistance. Our aim was to evaluate the allelic composition of natural populations of Brazilian Ae. aegypti at both kdr sites. Methods: The AaNaV IIIS6 region was cloned and sequenced from three Brazilian populations. Additionally, individual mosquitoes from 30 populations throughout the country were genotyped for 1016 and 1534 sites, based in allele-specific PCR. For individual genotypes both sites were considered as a single locus. Results: The 350 bp sequence spanning the IIIS6 region of the AaNaV gene revealed the occurrence of the kdr mutation Phe1534Cys in Brazil. Concerning the individual genotyping, beyond the susceptible wild-type (NaVS), two kdr alleles were identified: substitutions restricted to the 1534 position (NaVR1) or simultaneous substitutions in both 1016 and 1534 sites (NaVR2). A clear regional distribution pattern of these alleles was observed. The NaVR1 kdr allele occurred in all localities, while NaVR2 was more frequent in the Central and Southeastern localities. Locations that were sampled multiple times in the course of a decade revealed an increase in frequency of the kdr mutations, mainly the double mutant allele NaVR2. Recent samples also indicate that NaVR2 is spreading towards the Northern region. Conclusions: We have found that in addition to the previously reported Val1016Ile kdr mutation, the Phe1534Cys mutation also occurs in Brazil. Allelic composition at both sites was important to elucidate the actual distribution of kdr mutations throughout the country. Studies to determine gene flow and the fitness costs of these kdr alleles are underway and will be important to better understand the dynamics of Ae. aegypti pyrethroid resistance. Keywords: kdr mutation, Pyrethroid resistance, Vector control, Aedes aegypti, Dengue in Brazil, Sodium channel Background Dengue is currently the most important arbovirus in the world. Dengue has spread widely in urban areas of tropical and subtropical regions during the last decades, including countries of Southeast Asia, Pacific and Latin America [1]. Between 2001–2011, almost 10 million dengue cases were reported in Latin America, almost 60% of these * Correspondence: [email protected]; [email protected] † Equal contributors 4 Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brazil 1 Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, RJ, Brazil Full list of author information is available at the end of the article were registered in Brazil [2]. Dengue mortality can reach up to 5% of the confirmed infection cases. In addition, in tropical dengue endemic countries a loss of 1,300 disabilityadjust life years (DALYs) per million people is estimated [1]. Aedes aegypti is the main dengue vector throughout the world. Control of this mosquito consists primarily of the elimination of artificial and disposable water flooded larvae breeding sites and application of insecticides. The WHO Pesticide Evaluate Scheme (WHOPES) recommends ten different compounds to eliminate larvae, including neurotoxicants (organophosphates, pyrethroids and neocotinoids), Insect Growth Regulators (chitin synthesis inhibitors and juvenile hormone analogs), and Bacillus © 2014 Linss et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 36 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 2 of 11 Ile allele previously found. The simultaneous occurrence of both kdr mutations at the 1016 and 1534 was found in several localities. Spatial and temporal analysis of these alleles point to a significant role of the kdr mutations in pyrethroid resistance in Brazil. (like B. thuringiensis var israelensis) as larvicides. However, fewer formulations are recommended for the control of adult mosquitoes, mostly five pyrethroids and one organophosphate [3]. Given their rapid mode of action and low hazardous effect to the environment, compared to organophosphate insecticides, the use of pyrethroids has increased significantly in the last two decades. Nowadays, pyrethroids are widely employed in and around households, even for pet protection and mosquito control [4]. Since Ae. aegypti is essentially an urban mosquito, it is constantly exposed to strong pyrethroid selection. As a consequence, many Ae. aegypti populations worldwide are becoming resistant to this class of insecticides [5]. Pyrethroids target the transmembrane voltage gated sodium channel (NaV ) from the insect nervous system, triggering rapid convulsions followed by death, a phenomenon known as knockdown effect [6]. The NaV is composed of four homologous domains (I-IV), each with six hydrophobic segments (S1-S6) [7]. Because the NaV is a very conserved protein among invertebrates, small changes are permissive without impairing its physiological role [8]. A series of mutations have been identified in different orders of insects and acarids that affect pyrethroid susceptibility, thus being referred to as ‘knockdown resistance’ or kdr mutations [9]. These kdr mutations may lead to conformational changes in the whole channel that maintain its physiological role but avoid insecticide action [10]. In insects, the most common kdr mutation is the substitution Leu/Phe in the 1014 site (numbered according to the Musca domestica NaV primary sequence), followed by the Leu/Ser substitution in the same position, in Anopheles and Culex mosquitoes [11]. In the Ae. aegypti NaV (AaNaV), the 1014 Leu codon is encoded by CTA, rather than TTA as in Anopheles and Culex mosquitoes. This means that two substitutions would have to be simultaneously selected in the same codon in order to change Leu to Phe (TTT) or Ser (TCA) [12]. Although several mutations have been identified in natural populations at AaNaV [13], only the Val1016Ile and Phe1534Cys substitutions were clearly related to the loss of pyrethroid susceptibility [12,14]. These sites are placed respectively in the IIS6 and IIIS6 regions of the channels that are known to be involved in the interaction with pyrethroids [10]. It has been previously observed that in Latin America the 1016 Ile kdr is highly disseminated [12,15,16] and its frequency is rapidly increasing in localities with intense pyrethroid use, such as Brazil and Mexico [15,16]. High frequencies of 1534 Cys kdr were also observed in Grand Cayman and Martinique [14,17]. In the current study, we demonstrate that the1534 Cys kdr mutation is present in Brazil together with the 1016 Methods Mosquito samples Ae. aegypti used for kdr genotyping originated from the same samples evaluated by the Brazilian Aedes agypti Insecticide Resistance Monitoring Network, collected with ovitraps according to recommendations of the Brazilian Dengue Control Program [18]. Adult mosquitoes resulting from the eggs collected in the field (F0 generation) were preferentially used. However, in some cases only the following generations reared in the laboratory were available. Details regarding sampling as well as individual data from mosquitoes used for kdr genotyping are found in Table 1. A total of 30 localities were analyzed at least once, with AJU, SGO, MSR and VIT analyzed for two-four time-points. Genotyping assays Thirty individual mosquitoes from each locality were genotyped at both 1016 and 1534 positions from genomic DNA by allele-specific PCR (AS-PCR) which contains a common primer and two specific primers targeting each polymorphic site. The specificity is attained in the 3′-end, strengthened by a transition three nucleotides before [19]. Additionally, a GC-tail of different sizes was added at the 5′-end of these primers so products can be distinguished by their melting temperature (Tm) in a melting curve analysis or by electrophoresis [12,20,21]. Primer sequences are shown in Table 2. DNA extraction and amplification of the 1016 (Val/Ile) site were conducted as previously described [15]. The reaction for the 1534 (Phe/Cys) site was optimized from previous work [16,22]. In both cases, PCR was carried out with the GoTaq Green Master Mix kit (Promega), 0.5 μL of genomic DNA, 0.24 μM of the common primer, 0.12 and 0.24 μM of the specific primers (1534 Cyskdr and 1534 Phe), in a total volume of 12.5 μL. Denaturing, annealing and extension conditions were, respectively, 95°C ⁄ 30″, 54°C ⁄ 40″ and 72°C ⁄ 45″, in 32 cycles. Alternatively, real-time PCR was conducted with the SYBR Green PCR Master Mix kit (LifeTechnologies/Applied Biosystems), 1 μL genomic DNA and 0.24 μM of each primer, in a total volume of 10 μL. The best conditions for denaturing, annealing and extension were respectively 95°C ⁄ 15″, 54°C ⁄15″ and 60°C ⁄ 30″, in 33 cycles, followed by a standard melting curve stage. The amplification reaction and melting curve analyses were performed in a StepOne Plus or in a 7500 Real-time PCR system (LifeTechnologies/Applied Biosystems). DNA pools of individuals from CGR, STR 37 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 3 of 11 Table 1 Aedes aegypti populations used in this study Code Municipality Locality state Coordinates Brazilian macroregion Year of sampling Generation used in the assays Gender AJU Aracajú Sergipe 10°54'AJU S, 37°04'O Northeast 2002 F1 Males 2006 F1 Females 2010 F1 Females 2012 F0 Males APG Aparecida de Goiânia Goiás 16°48'S, 49°14'O Central-west 2012 F0 Males BEL Belém Pará 1°27'S, 48°30'O North 2010 F1 Males BVT Boa Vista Roraima 2°49'N, 60°40'O North 2011 F1 Males CAC Caicó Rio Grande do Norte 6°27'S, 37°05'O Northeast 2010 F1 Females CAS Castanhal Pará 1°17'S, 47°55'O North 2011 F0 Males CBL Campos Belos Goiás 13°02'S, 46°45'O Central-west 2011 F0 Males CGR Campo Grande Mato Grosso do Sul 20°26'S, 54°38'O Central-west 2010 F0 Males CIT Cachoeiro do Itapemirim Espírito Santo 20°51'S, 41°06'O Southeast 2012 F0 Males CLT Colatina Espírito Santo 19°32'S, 40°37'O Southeast 2011 F0 Males DQC Duque de Caxias Rio de Janeiro 22°47'S, 43°18'O Southeast 2001 F3 Females 2010 F1 Males 2012 F0 Males FOZ Foz do Iguaçú Paraná 25°32'S, 54°35'O South 2009 F2 Females GVD Governador Valadares Minas Gerais 18°50'S, 41°56'O Southeast 2011 F1 Males ITP Itaperuna Rio de Janeiro 21°12'S, 41°53'O Southeast 2011 F2 Males LZN Luziânia Goiás 16°15'S, 47°55'O Central-west 2011 F2 Females MRB Marabá Pará 5°22'S, 49°07'O North 2011 F0 Males MSR Mossoró Rio Grande do Norte 5°11'S, 37°20'O Northeast 2009 F0 Males 2011 F0 Males PCR Pacaraima Roraima 4°25'N, 61°08'O North 2011 F0 Males PGT Porangatu Goiás 13°25'S, 49°08'O Central-west 2012 F0 Males PNM Parnamirim Rio Grande do Norte 5°54'S, 35°15'O Northeast 2010 F0 Males RVD Rio Verde Goiás 17°47'S, 50°55'O Central-west 2011 F0 Males SGO São Gonçalo Rio de Janeiro 22°49'S, 43°03'O Southeast 2002 F2 Males 2008 F2 Males Maless SIP Santana do Ipanema Alagoas 9°21'S, 37°14'O Northeast 2010 F2 SMA São Miguel do Araguaia Goiás 13°15'S, 50°09'O Central-west 2012 F0 Males SRO Santa Rosa Rio Grande do Sul 27°52'S, 54°28'O South 2011 F1 Males SSO São Simão Goiás 18°59'S, 50°32'O Central-west 2011 ? Males STR Santarém Pará 2°26'S, 54°41'O North 2010 F0 Males TCR Tucuruí Pará 3°46'S, 49°40'O North 2010 F0 Males URU Uruaçu Goiás 14°31'S, 49°09'O Central-west 2011 F0 Males VIT Vitória Espírito Santo 20°18'S, 40°18'O Southeast 2006 F1 Males 2010 F0 Males and cloned with CloneJet PCR Cloning Kit (Thermo Scientific). The DNA sequencing was carried out in an ABI377 Sequencer with the Big Dye 3.1 Kit (LifeTechnologies/Applied Biosystems). Sequence analysis was performed using the BioEdit software version 7.2. and PNM were used to amplify the region spanning the NaV IIIS6 segment with the primers AaEx31P and AaEx31Q (Table 2), as specified elsewhere [14]. The PCR products were purified in S-400 microcolumns (GE Healthcare) according to manufacturer instructions 38 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 4 of 11 Table 2 Primer sequences Primer name Sequence (5′ - 3′) References 1016 Val+ (for) ## [12,15] kdr 1016 Ile (for) # ACAAATTGTTTCCCACCCGCACCGG ACAAATTGTTTCCCACCCGCACTGA 1016 comom (rev) GGATGAACCGAAATTGGACAAAAGC 1534 Phe+ (for) # 1534 Cyskdr (for) ## TCTACTTTGTGTTCTTCATCATATT [22] TCTACTTTGTGTTCTTCATCATGTG 1534 comom (rev) TCTGCTCGTTGAAGTTGTCGAT AaEx31P (for) TCGCGGGAGGTAAGTTATTG AaEx31Q (rev) GTTGATGTGCGATGGAAATG long 5'-tail GCGGGCAGGGCGGCGGGGGCGGGGCC short 5'-tail GCGGGC + wild-type specific primer, 5′tail attached. [14] kdr specific primer, #short 5′tail attached, ##long kdr All individuals were genotyped for both 1016 and 1534 sites. Linkage disequilibrium was tested by the online Genepop version 4.2 [23], and since the 1016 and 1534 sites are linked (see Results section), genotypic and allelic frequencies were taken as a single locus. Hardy-Weinberg equilibrium was evaluated by the classical equation [24], being the null hypothesis of equilibrium checked by a chi-square test with three or one degrees of freedom, respectively, when six or three genotypes were evidenced. Results Allele-specific discrimination A 20 bp size difference, due to the 5′-GC tail of allele specific primers, enabled the easy discrimination of homozygous and heterozygous genotypes in either a polyacrylamide gel electrophoresis or in dissociation curves through real-time PCR (Figure 1). Electrophoresis revealed products of around 80 and 100 bp, respectively for Ilekdr and Val+ (1016 reaction), and 90 and 110 bp, respectively for Phe+ and Cyskdr (1534 reaction). The dissociation curve exhibited Tm of around 76 and 84°C, respectively for Ilekdr and Val (1016 reaction), and 77 and 82°C, respectively for Phe and Cyskdr (1534 reaction). The PCR conditions of annealing temperature, number of cycles and concentration of each primer were crucial to avoid unspecific amplification. All reactions were accompanied by positive controls, each one consisting of the three potential genotypes at the 1016 and 1534 positions, which were obtained by previously genotyped individuals: homozygous wild type, heterozygous, and homozygous kdr. As the Phe1534Cys mutation was detected for first time in Brazilian samples, we cloned and sequenced the IIIS6 region (exon 31) of the AaNaV gene of three genotyped populations (CGR, STR and PNM), confirming the primers’ specificity. The 350 bp fragments were submitted to GenBank (accession numbers: KF527414 and KF527415, for 1534 Cyskdr and 1534 Phe+, respectively). Excluding the site of the 1534 kdr mutation (TTC/TGC), no other polymorphic site was detected relative to the sequence deposited in VectorBase (Liverpool strain). Genotyping 1016 and 1534 AaNaV sites in natural populations Around 30 Ae. aegypti individuals from each one of 30 distinct Brazilian localities were genotyped for both 1016 and 1534 NaV sites, totalling 1,112 analyzed mosquitoes. Some localities were sampled two to four times within a ten-year interval. The genotypes of individual mosquitoes for both sites were first calculated independently: 1016 Val+/Val+, Val+/Ilekdr and Ilekdr/Ilekdr, and 1534 Phe+/Phe+, Phe+/Cyskdr and Cyskdr/Cyskdr. These data were used to perform a genotypic linkage disequilibrium analysis and total linkage between them was demonstrated (Fisher’s method, p < 0.001), as expected from two sites placed in the same gene. In this sense both sites were considered as constituents of a single locus, thus evidencing the occurrence of six genotypes in individual mosquitoes (Table 3). Based on the composition of these genotypes, we concluded that three alleles were present in the evaluated samples: ‘1016 Val+ + 1534 Phe+’ (wild-type), ‘1016 Val+ + 1534 Cyskdr’ (1534 kdr) and ‘1016 Ilekdr + 1534 Cyskdr’ (1016 kdr + 1534 kdr). Hereafter these alleles will be simply referred to as ‘NaVS’, ‘NaVR1’ and ‘NaVR2’, respectively (Figure 2). Double mutants and individuals with mutation only in the 1534 position were found (respectively, NaVR2 and ‘NaVR1); however, in no case was the 1016 kdr mutation observed alone, precluding the existence of a 1016 Ilekdr + 1534 Phe+ allele in the evaluated populations. Figure 3 shows the frequencies for NaVS, NaVR1 and NaVR2 alleles in the most recent samples obtained from each locality. The 95% CI of the allele frequencies is shown in the Additional file 1: Table S1. According to the alleles, the genotypes were named SS, SR1, SR2, R1R1, R1R2 and R2R2. Their frequencies and the Hardy-Weinberg Equilibrium deviation test are presented in Table 3. In only seven out of 38 samplings the Hardy-Weinberg Equilibrium assumption was rejected (p < 0.05). No specific genotype contributed to the deviation in these seven localities. Overall, the distribution of the three alleles differed according to the geographical region (Figure 3). In the North and Northeast Regions, the NaVR1 allele, mutant only at position 1534, was found in all localities, nevertheless the NaVS wild-type allele was the most representative in six of the localities (BEL, CTL, MRB, CAC, SIP and PNM). The highest frequency of NaVR1, was found in the North: 0.750 (STR), among all populations analyzed. On the other hand, with exception of the most recent AJU (AJU2012), the NaVR2 double mutant allele was either absent or < 5% in the North and Northeast of Brazil. In contrast, the wild-type allele, NaVS, was absent from 39 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 A Page 5 of 11 B (bp) 300 200 150 100 75 50 - Tm: 76,70C C D Tm2: 82,20C Tm: 82,20C Tm1: 76,70C Figure 1 Allele specific PCR (AS-PCR) for genotyping kdr mutations in the Aedes aegypti voltage gated sodium channel. All panels represent reactions for the 1534 site. (A) Visualization of the amplicons in a 10% polyacrylamide gel electrophoresis, run under 170 V/45' and stained with ethidium bromide (1 μg/mL). Amplicons of approximately 90 and 110 bp correspond to alleles 1534 Phe+ and 1534 Cyskdr, respectively. DNA ladder was used as size marker (O’GeneRuler DNA Ladder, Ultra Low Range/Fermentas, 150 ng). Dissociation curve analysis in real time PCR differentiating the Phe/Phe (B), Phe/Cys (C), and Cys/Cys (D) genotypes. The Tm for the respective alleles are indicated. 40 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 6 of 11 Table 3 Genotype frequencies of Brazilian Aedes aegypti populations at the 1016 and 1534 sites of the NaV locus Macro-region North Northeast Central-west Southeast South Population Genpotype frequencies Total (n) HWE test χ2 p 30 0.0 0.879 0.071 28 0.0 0.993 0.000 30 2.3 0.512 0.000 0.000 28 0.4 0.932 0.700 0.000 0.000 30 16.1 0.000 0.000 0.500 0.000 0.000 30 3.5 0.062 0.000 0.241 0.000 0.000 29 13.3 0.000 0.367 0.000 0.033 0.000 0.000 30 0.2 0.660 0.000 0.767 0.000 0.200 0.000 0.033 30 14.9 0.002 0.704 0.111 0.037 0.111 0.037 0.000 27 9.3 0.025 CAC10 0.833 0.133 0.033 0.000 0.000 0.000 30 0.0 0.998 SIP10 0.433 0.500 0.067 0.000 0.000 0.000 30 4.7 0.199 AJU02 1.000 0.000 0.000 0.000 0.000 0.000 30 0.0 1.000 AJU06 0.767 0.033 0.167 0.000 0.033 0.000 30 0.3 0.955 AJU10 0.269 0.038 0.308 0.000 0.000 0.385 26 3.6 0.306 AJU12 0.200 0.033 0.333 0.033 0.100 0.300 30 3.4 0.338 CBL11 0.069 0.069 0.414 0.000 0.103 0.345 29 0.5 0.918 SMA12 0.207 0.172 0.241 0.103 0.207 0.069 29 1.2 0.750 PGT12 0.000 0.069 0.241 0.241 0.241 0.207 29 5.9 0.115 URU11 0.233 0.133 0.300 0.000 0.100 0.233 30 1.3 0.723 LZN11 0.200 0.333 0.200 0.033 0.167 0.067 30 1.7 0.639 APG12 0.000 0.207 0.207 0.138 0.241 0.207 29 2.5 0.466 RVD11 0.103 0.034 0.241 0.069 0.241 0.310 29 2.8 0.421 SSO11 0.000 0.133 0.033 0.200 0.233 0.400 30 7.6 0.056 CGR10 0.000 0.033 0.100 0.000 0.267 0.600 30 1.2 0.749 SS SR1 SR2 R1R1 R1R2 R2R2 0.000 0.000 0.000 0.367 0.467 0.167 BVT11 0.000 0.000 0.000 0.536 0.393 CAS11 0.400 0.500 0.033 0.033 0.033 BEL10 0.536 0.357 0.000 0.107 STR10 0.200 0.100 0.000 TCR10 0.200 0.300 MRB11 0.621 0.138 MSR09 0.600 MSR11 PNM10 PCR11 GVD11 0.000 0.033 0.200 0.267 0.067 0.433 30 18.3 0.000 CLT11 0.067 0.333 0.300 0.000 0.100 0.200 30 9.0 0.029 VIT06 0.267 0.100 0.333 0.000 0.033 0.267 30 2.4 0.492 VIT10 0.000 0.067 0.100 0.000 0.000 0.833 30 2.3 0.507 CIT12 0.000 0.069 0.138 0.103 0.172 0.517 29 3.8 0.281 ITP11 0.148 0.111 0.259 0.074 0.074 0.333 27 5.0 0.172 SGO02 1.000 0.000 0.000 0.000 0.000 0.000 30 0.0 1.000 SGO08 0.192 0.231 0.308 0.115 0.115 0.038 26 1.6 0.669 DQC01 1.000 0.000 0.000 0.000 0.000 0.000 30 0.0 1.000 DQC10 0.000 0.033 0.067 0.100 0.067 0.733 30 13.0 0.005 DQC12 0.000 0.033 0.000 0.000 0.433 0.533 30 5.6 0.136 FOZ09 0.133 0.100 0.400 0.033 0.000 0.333 30 3.6 0.311 SRO11 0.296 0.259 0.222 0.037 0.000 0.185 27 7.4 0.059 the two northernmost localities evaluated (PCR and BVT, both in the State of Roraima), where both mutant alleles were at high frequencies. In all localities from Central-West, Southeast and South regions, all three alleles were present. The most frequent allele was the NaVR2 double mutant. Exceptions were LZN, SMA, URU, SGO and SRO, where the NaVS wild-type allele was the most representative (Figure 3). The dynamics of the genotype frequencies was analyzed in AJU, MSR, VIT and DQC. Samples from AJU were 41 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 7 of 11 Figure 2 Voltage gated sodium channel and the 1016 and 1534 alleles found in Brazilian Aedes aegypti populations. The NaV is represented with its four domains (I-IV), each with the six transmembrane segments (S1-S6). The voltage sensitive S4 and the pore forming S6 segments are colored in blue and green, respectively (scheme adapted from [9]). The 1016 and 1534 kdr sites in Aedes aegypti are indicated. Mutant amino acids are underlined. Figure 3 Distribution of the kdr alleles in Brazilian Aedes aegypti populations. For each locality, only the most recent samples evaluated are shown. Details of the localities are shown in Table 1. Alleles are represented according to the colors used in Figure 2. 42 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 8 of 11 collected four times in the course of a decade, between 2002 and 2012. In 2002, only the NaVS wild-type allele was detected. The kdr alleles appeared first in 2006 and the double mutant NaVR2 was the most frequent allele by 2012 (Figure 4). Accordingly, the ‘SS’ wild-genotype progressively decayed from 100% in 2002 to 20% in 2012, when the double mutant ‘R2R2’ represented 30% of the individuals, and was the most frequent genotype (AJU2012, Table 3). The frequency of the NaVS wild-type allele also decreased in all other localities evaluated where the kdr alleles increased in frequency (Figure 4). Except for MSR, the NaVR2 double mutant is likely to be the most favorably selected allele. It is noteworthy that in AJU, the NaVR1 allele showed the larger frequency increase, probably because NaVR2 must have arrived to the Northeast more recently. Discussion The genotyping of mutations directly related to insecticide resistance is an important surveillance tool for agricultural and sanitary purposes. Among selected mechanisms of pyrethroid resistance, kdr mutations in the voltage gated sodium channel (NaV ) are those that better correlate particular genotypes with insecticide resistance [25]. The increased efficiency of insecticide detoxification, known as metabolic resistance – involving super families of enzymes such as GST, esterases and especially the multi function oxidases P450 – may also confer resistance to pyrethroids. However, identification of these mechanisms is mainly based on enzymatic assays of low specificity [26] or on bioassays with synergist compounds [27], and are not clearly linked to particular genes. More recently, many successful transcriptome tools for metabolic resistance genes have emerged, pointing to a very complex and diverse scenario regarding insecticide selected genes and their pattern of expression among insect populations [28,29]. Because the metabolic resistance based selection seems to have a high fitness cost, due to reallocation of energetic resources, this mechanism is expected to induce lower resistance levels, if compared to mutations in the target site molecules [30]. This was corroborated by laboratory selection with pyrethroids in an Ae. aegypti lineage: increase of the 1016 Ilekdr frequency was inversely proportional to the number of ‘metabolic’ genes differentially transcribed [29]. It was hypothesized that, in the presence of pyrethroid, kdr mutations are preferentially selected among other mechanisms, contributing to higher resistance levels and/or resulting in less deleterious effects. In addition to the classical Leu1014Phe kdr mutation, several others have been associated with pyrethroid resistance [6]. Interactions of multiple NaV mutations may modulate pyrethroid resistance levels. For instance, certain NaV haplotypes, including synonymous substitutions, were found in two distinct field populations of Culex quinquefasciatus selected for pyrethroid resistance during 6–8 generations in the laboratory. It was suggested that some of these haplotypes were selected at an early stage of permethrin resistance and later evolved to other mutation combinations in the course of selection pressure [31]. In Ae. aegypti, a synonymous substitution at exon 20, Figure 4 Time-course of kdr alleles frequencies in four Brazilian Aedes aegypti populations. Localities: A - Aracaju (AJU), B - Mossoró (MSR), C - Vitória (VIT) and D - Duque de Caxias (DCQ). Bars indicate the 95% CI of allele frequencies. 43 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 together with an extensive polymorphism in the following intron, were linked to both Ile1011Met and Val1016Ile mutations [15,32]. Additionally, a gene duplication event was recently described in the AaNaV of natural populations and in a laboratory strain selected for pyrethroid resistance [33]. Although there are at least seven different mutations described in the AaNaV, only those corresponding to the 1016 and 1534 positions are clearly related to resistance; both are placed in a domain of the sodium channel that interacts directly with the pyrethroid molecule [34]. There are two mutations described in the AaNaV 1016 site, Val to Ile or Gly, respectively in Latin America [12,14,15] and in Southeast Asia [35]. In Brazil, we found no evidence of a haplotype that contains exclusively the 1016 Ilekdr mutation, since it was always found together with 1534 Cyskdr (NaVR2 allele, herein). Nevertheless, we are aware that it is possible for a haplotype carrying the 1016 kdr mutation to occur in the populations examined, however, it would be present at very low frequencies. Actually, this putative allele must have occurred in two out of three Ae. aegypti populations from Grand Cayman, given that the 1016 Ilekdr presented a higher frequency than the 1534 Cyskdr substitution [14]. Differently from the 1016 position, only one substitution, Phe/Cys, was found in the 1534 site by far [14,36]. This 1534 substitution can be linked with another one. In Thailand, the 1534 Cyskdr co-occurred with 1016 Glykdr and 989 Prokdr in the same molecule [22]. In that region an allele 1534 Cyskdr without mutation in 1016 site (NaVR1 allele, herein) seemed to be very common, since its frequency was higher than the 1016 Glykdr [35]. Here we presented the distribution of the kdr variants for the AaNaV, considering both 1016 and 1534 sites screen from several natural Brazilian populations. We considered that once these sites are very close in the genome, reporting the allele/genotypic frequencies of each site separately would not be fully informative. However, because there are still some gaps concerning the actual role of these mutations in pyrethroid resistance, regarding whether they are acting alone or synergistically, and present in cis or trans mutations, we are reporting the allele frequencies of each site rather than as an haplotype. The implication of the 1016 Ilekdr allele in resistance to pyrethroids was corroborated by laboratory selection, which highly increased the allelic frequency up to fixation in only five generations [29]. Accordingly, in the last decade this mutation has been rapidly spreading in natural populations from Brazil and Mexico, concomitantly with the intensification of pyrethroid usage due to the emergence of severe dengue outbreaks [15,16]. In these cases however, the co-occurrence of the 1534 Cyskdr mutation has been overlooked. A recent study reported high frequencies of 1534 Cyskdr in Grand Cayman [14], suggesting it is not a novel mutation in Latin America. In a recent report, Page 9 of 11 nine single and two double AaNaV mutants were constructed and inserted in a Xenopus oocyte system in order to perform functional evaluations of these substitutions in the presence of type I or II pyrethroids [34]. The 1016 Ilekdr construct did not result in sensitivity reduction, to either pyrethroid types. On the other hand, the 1534 Cyskdr significantly diminished the AaNaV sensibility to type I but not to type II pyrethroids. This same substitution in the homologous kdr site of the cockroach NaV exhibited similar results [37]. An Ae. aegypti lineage, selected for permethrin resistance in the laboratory, exhibited high frequencies of 1016 Glykdr + 1794 Tyrkdr substitutions in the same molecule, which suggested a synergistic effect towards pyrethroid resistance [38]. We hypothesize that mutation in the 1016 site should be important when in synergism with other specific mutations. In Brazil, the 1534 Cyskdr mutation is widespread throughout the territory. The NaVR1 allele is more frequent in North/Northeast regions whereas NaVR2 is more commonly present in Central/ Southeast regions, generally where the highest resistance levels to pyrethroids are observed [18]. Both mutant haplotypes appear to be rapid and favorably selected in all evaluated populations. However, in the most recent samplings the NaVR2 double mutant was the more frequent kdr allele. The exception was MSR, in the Northeast Region, where NaVR2 was only recently introduced. Together these data suggest that NaVR2 allele would be more advantageous for pyrethroid resistance, or impose a lower fitness cost when compared to NaVR1. We recently demonstrated that an NaVR2 homozygous Ae. aegypti lineage, highly resistant to pyrethroids, exhibited a fitness cost in a series of life-trait parameters [39]. Further comparisons between NaVR1 and NaVR2 lineages will be of importance to better clarify those assumptions. It is of note that since 2001 and up to 2009 the Brazilian Dengue Control Program employed pyrethroids in ultralow volume applications in several municipalities as part of the effort to control the dengue vector [18]. With very few exceptions, the basis for pyrethroid selection pressure derived from national campaigns is essentially the same in the whole country. Therefore, differential selection pressures would not explain the aforementioned regionalization of the kdr alleles. It is likely that the current distribution of the kdr alleles reflects distinct Ae. aegypti populations that colonized the continent. Population genetics analysis of neutral loci will help us to unravel the evolutionary routes of these resistance genes. Conclusions In conclusion, pyrethroids are the most employed insecticides worldwide and the only chemical class presently allowed in long lasting treated materials, such as nets 44 Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 and curtains [40]. Although novel control strategies are being tested in the field, such as those based on transgenic and on Wolbachia-infected mosquitoes [2,41,42], insecticides will certainly play an important role for yet a long time. Knowledge of the sodium channel diversity in natural populations together with the role of each allele regarding pyrethroid resistance as well as their fitness effects are crucial for preserving the effectiveness of this class of compounds as a viable tool against Ae. aegypti. Page 10 of 11 8. 9. 10. 11. 12. Additional file Additional file 1: Table S1. Kdr allele frequencies of Aedes aegypti natural populations from Brazil. The CI95%* is under parentheses. Competing interests The authors declare that they have no competing interests. Authors’ contributions Conceived and designed the experiments: JGBL, LPB, AJM. Performed the experiments: JGBL, LPB, GAG. Analyzed the data: JGBL, LPB, ASA, RVB, AJM. Contributed reagents/materials/analysis tools: RVB, JBPL, DV. Wrote the paper: AJM, DV. All authors read and approved the final version of the manuscript. Acknowledgements We thank Dr Alexandre Afranio Peixoto for his friendship and orientation throughout this study. This work is dedicated to his memory. We also thank the DNA sequencing facility of FIOCRUZ (Plataforma de Sequenciamento/ PDTIS/Fiocruz) and the Brazilian Dengue Control Program that allowed utilization of samples collected in the scope of the Brazilian Aedes aegypti Insecticide Resistance Monitoring Network (MoReNAa). We are grateful to Dr Andrea Gloria-Soria for critical reading the manuscript. Author details 1 Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, RJ, Brazil. 2Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ, Brazil. 3Laboratório de Biologia Molecular de Insetos, Instituto Oswaldo Cruz – FIOCRUZ, Rio de Janeiro, RJ, Brazil. 4 Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brazil. 5Laboratório de Biologia Molecular de Flavivirus, Instituto Oswaldo Cruz - FIOCRUZ, Rio de Janeiro, RJ, Brazil. Received: 12 August 2013 Accepted: 18 December 2013 Published: 15 January 2014 References 1. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, et al: (2010) Dengue: a continuing global threat. Nat Rev Microbiol 2010, 8:S7–S16. 2. Maciel-de-Freitas R, Aguiar R, Bruno RV, Guimaraes MC, Lourenco-de-Oliveira R, et al: Why do we need alternative tools to control mosquito-borne diseases in Latin America? Mem Inst Oswaldo Cruz 2012, 107:828–829. 3. WHOPES: Pesticides and their application for the control of vectors and pests of public health importance (WHO/CDS/NTD/WHOPES/GCDPP/2006.1). Geneva: World Health Organization; 2006. 4. Agency USEP: Pesticides: Regulating Pesticides. U.S. Environmental Protection Agency; 2012. http://www.epa.gov/oppsrrd1/reevaluation/pyrethroidspyrethrins.html. 5. Ranson H, Burhani J, Lumjuan N, Black WC IV: Insecticide resistance in dengue vectors. TropIKAnet 2010, 1:1. cited 2013-12-02], pp. 0–0. Available from: http://journal.tropika.net/scielo.php?script=sci_arttext&pid=S207886062010000100003&lng=en&nrm=iso. ISSN 2078–8606. 6. Dong K: Insect sodium channels and insecticide resistance. Invert Neurosci 2007, 7:17–30. 7. Catterall WA: From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000, 26:13–25. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 45 ffrench-Constant RH, Pittendrigh B, Vaughan A, Anthony N: Why are there so few resistance-associated mutations in insecticide target genes? Philos Trans R Soc Lond B Biol Sci 1998, 353:1685–1693. Martins AJ, Valle D: The pyrethroid knockdown resistance. In Insecticides - Basic and Other Applications. Edited by Soloneski S, Larramendy M. Rijeka: InTech; 2012:17–38. O’Reilly AO, Khambay BP, Williamson MS, Field LM, Wallace BA, et al: Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochem J 2006, 396:255–263. Davies TG, Field LM, Usherwood PN, Williamson MS: A comparative study of voltage-gated sodium channels in the Insecta: implications for pyrethroid resistance in Anopheline and other Neopteran species. Insect Mol Biol 2007, 16:361–375. Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M, Flores AE, et al: A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol Biol 2007, 16:785–798. Brengues C, Hawkes NJ, Chandre F, McCarroll L, Duchon S, et al: Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med Vet Entomol 2003, 17:87–94. Harris AF, Rajatileka S, Ranson H: Pyrethroid resistance in Aedes aegypti from Grand Cayman. Am J Trop Med Hyg 2010, 83:277–284. Martins AJ, Lima JB, Peixoto AA, Valle D: Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations. Trop Med Int Health 2009, 14:1351–1355. Garcia GP, Flores AE, Fernandez-Salas I, Saavedra-Rodriguez K, Reyes-Solis G, et al: Recent rapid rise of a permethrin knock down resistance allele in Aedes aegypti in Mexico. PLoS Negl Trop Dis 2009, 3:e531. Marcombe S, Mathieu RB, Pocquet N, Riaz MA, Poupardin R, et al: Insecticide resistance in the dengue vector Aedes aegypti from Martinique: distribution, mechanisms and relations with environmental factors. PLoS One 2012, 7:e30989. Montella IR, Martins AJ, Viana-Medeiros PF, Lima JB, Braga IA, et al: Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. Am J Trop Med Hyg 2007, 77:467–477. Okimoto R, Dodgson JB: Improved PCR amplification of multiple specific alleles (PAMSA) using internally mismatched primers. Biotechniques 1996, 21:20–22. 24, 26. Germer S, Higuchi R: Single-tube genotyping without oligonucleotide probes. Genome Res 1996, 9:72–78. Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, et al: High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. Biotechniques 2005, 39:885–893. Yanola J, Somboon P, Walton C, Nachaiwieng W, Somwang P, et al: High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand. Trop Med Int Health 2011, 16:501–509. Raymond M, Rousset F: Genepop (Version-1.2) - population-genetics software for exact tests and ecumenicism. J Hered 1995, 86:248–249. Shorrocks B: The Genesis of Diversity. London: Hodder and Stoughton; 1978. Donnelly MJ, Corbel V, Weetman D, Wilding CS, Williamson MS, et al: Does kdr genotype predict insecticide-resistance phenotype in mosquitoes? Trends Parasitol 2009, 25:213–219. Valle D, Montella IR, Medeiros PFV, Ribeiro RA, Martins AJ, et al: Quantification methodology for enzyme activity related to insecticide resistance in Aedes aegypti. Ministério da Saúde/Brasil: Brasília; 2006. Brogdon WG, McAllister JC: Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles. J Am Mosq Control Assoc 1998, 14:159–164. Bariami V, Jones CM, Poupardin R, Vontas J, Ranson H: Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti. PLoS Negl Trop Dis 2012, 6:e1692. Saavedra-Rodriguez K, Suarez AF, Salas IF, Strode C, Ranson H, et al: Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti. Insect Mol Biol 2012, 21:61–77. Martins AJ, Ribeiro CD, Bellinato DF, Peixoto AA, Valle D, et al: Effect of insecticide resistance on development, longevity and reproduction of field or laboratory selected Aedes aegypti populations. PLoS One 2012, 7:e31889. Linss et al. Parasites & Vectors 2014, 7:25 http://www.parasitesandvectors.com/content/7/1/25 Page 11 of 11 31. Xu Q, Zhang L, Li T, Zhang L, He L, et al: Evolutionary adaptation of the amino acid and codon usage of the mosquito sodium channel following insecticide selection in the field mosquitoes. PLoS One 2012, 7:e47609. 32. Martins AJ, Lins RM, Linss JG, Peixoto AA, Valle D: Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil. Am J Trop Med Hyg 2009, 81:108–115. 33. Martins AJ, Brito LP, Linss JGB, Rivas GB, Machado R, et al: Evidence for gene duplication in the voltage gated sodium channel gene of Aedes aegypti. EMPH 2013:eoto12v1–eot012. 34. Du Y, Nomura Y, Satar G, Hu Z, Nauen R, et al: Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel. Proc Natl Acad Sci USA 2013:1305118110v1–1305118110v201305118. 35. Kawada H, Higa Y, Komagata O, Kasai S, Tomita T, et al: Widespread distribution of a newly found point mutation in voltage-gated sodium channel in pyrethroid-resistant Aedes aegypti populations in Vietnam. PLoS Negl Trop Dis 2009, 3:e527. 36. Yanola J, Somboon P, Walton C, Nachaiwieng W, Prapanthadara LA: A novel F1552/C1552 point mutation in the Aedes aegypti voltage-gated sodium channel gene associated with permethrin resistance. Pestic Biochem Physiol 2010, 96:127–131. 37. Hu Z, Du Y, Nomura Y, Dong K: A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids. Insect Biochem Mol Biol 2011, 41:9–13. 38. Chang C, Shen WK, Wang TT, Lin YH, Hsu EL, et al: A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti. Insect Biochem Mol Biol 2009, 39:272–278. 39. Brito LP, Linss JG, Lima-Camara TN, Belinato TA, Peixoto AA, et al: Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost. PLoS One 2013, 8:e60878. 40. Zaim M, Aitio A, Nakashima N: Safety of pyrethroid-treated mosquito nets. Med Vet Entomol 2000, 14:1–5. 41. Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, et al: The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature 2011, 476:450–453. 42. Harris AF, Nimmo D, McKemey AR, Kelly N, Scaife S, et al: Field performance of engineered male mosquitoes. Nat Biotechnol 2011, 29:1034–1037. doi:10.1186/1756-3305-7-25 Cite this article as: Linss et al.: Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations. Parasites & Vectors 2014 7:25. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit 46 4. CAPITULO 2 47 148 o r i gi na l research article Evolution, Medicine, and Public Health [2013] pp. 148–160 doi:10.1093/emph/eot012 Ademir Jesus Martins*1,2, Luiz Paulo Brito1, Jutta Gerlinde Birggitt Linss1, Gustavo Bueno da Silva Rivas3, Ricardo Machado3, Rafaela Vieira Bruno2,3, José Bento Pereira Lima1, Denise Valle2,4 and Alexandre Afranio Peixoto2,3,y 1 Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz—FIOCRUZ and Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ, 21040-360, Brazil, 2Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil, 3Laboratório de Biologia Molecular de Insetos, Instituto Oswaldo Cruz— FIOCRUZ, Rio de Janeiro, RJ, 21040-360, Brazil and 4Laboratório de Biologia Molecular de Flavivirus, Instituto Oswaldo Cruz—FIOCRUZ, Rio de Janeiro, RJ, 21040-360, Brazil *Correspondence address. Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz— FIOCRUZ and Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ, 21040-360, Brazil. Tel:+55 21 25621398; Fax:+55 21 25621308; E-mail: [email protected] y In memoriam. Received 9 March 2013; revised version accepted 9 June 2013 ABSTRACT Background and objectives: Mutations in the voltage-gated sodium channel gene (NaV), known as kdr mutations, are associated with pyrethroid and DDT insecticide resistance in a number of species. In the mosquito dengue vector Aedes aegypti, besides kdr, other polymorphisms allowed grouping AaNaV sequences as type ‘A’ or ‘B’. Here, we point a series of evidences that these polymorphisms are actually involved in a gene duplication event. Methodology: Four series of methods were employed: (i) genotypying, with allele-specific PCR (AS-PCR), of two AaNaV sites that can harbor kdr mutations (Ile1011Met and Val1016Ile), (ii) cloning and sequencing of part of the AaNaV gene, (iii) crosses with specific lineages and analysis of the offspring genotypes and (iv) copy number variation assays, with TaqMan quantitative real-time PCR. Results: kdr mutations in 1011 and 1016 sites were present only in type ‘A’ sequences, but never in the same haplotype. In addition, although the 1011Met-mutant allele is widely disseminated, no homozygous (1011Met/Met) was detected. Sequencing revealed three distinct haplotypes in some individuals, raising the hypothesis of gene duplication, which was supported by the genotype frequencies in the offspring of specific crosses. Furthermore, it was estimated that a laboratory strain selected for ! The Author(s) 2013. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 48 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti Martins et al. | Sodium channel gene duplication in Aedes aegypti 149 insecticide resistance had 5-fold more copies of the sodium channel gene compared with a susceptible reference strain. Conclusions and implications: The AaNaV duplication here found might be a recent adaptive response to the intense use of insecticides, maintaining together wild-type and mutant alleles in the same organism, conferring resistance and reducing some of its deleterious effects. K E Y W O R D S : gene duplication; kdr mutation; sodium channel; pyrethroid resistance; Aedes aegypti BACKGROUND AND OBJECTIVES Several mutations have been identified in the Ae. aegypti NaV gene (AaNaV) comprising the IIS5–S6 region: Gly923Val, Leu982Trp, Ile1011Met, Ile1011Val, Val1016Ile and Val1016Gly [12–16]. The Ile1011Met substitution was associated with low sensitivity to pyrethroids evidenced by electrophysiological assays [12] and was the most frequent in a resistant Brazilian natural Ae. aegypti population [14]. However, substitutions in another position, 1016 (Val/Ile in South and Central America and Val/Gly in Thailand), are presently attributed with a more important role in pyrethroid resistance, the 1016 substitutions appearing as a recessive trait [13, 16–18]. Outside domain II, a Phe1534Cys substitution in the IIIS6 region was also related to pyrethroid resistance [19]. Besides amino acid changes, nucleotide and insertion/deletion polymorphisms have been detected in intron 20 in the AaNaV IIS6 genomic region that enable grouping the sequences in two categories, type ‘A’ or type ‘B’. The Ile1011Met and Val1016Ile mutations are found only in type ‘A’ sequences [14]. Herein, we further investigated the nature of this polymorphism. Sequencing of the AaNaV IIS6 genomic region and alelle specific-PCR (AS-PCR) typing of the 1011 and 1016 sites revealed, in several cases, three haplotypes in the same mosquito. Besides, in no case were homozygous specimens for the 1011Met mutation in natural populations detected. Crosses between laboratory-selected genotypes and copy number variation assays strongly suggested the occurrence of duplication events in the sodium channel gene, at least for the studied genomic region. MATERIALS AND METHODS Mosquitoes Rockefeller strain, continuously reared in the laboratory as a standard for insecticide susceptibility and life-history trait parameters, was used as reference for wild-type alleles for the voltage-gated sodium 49 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 The use of DDT as public health insecticide was one of the factors responsible for the yellow fever mosquito eradication in many Latin American countries in the 1950s [1]. Since the reintroduction of Aedes aegypti to South America, organophosphates and, subsequently, pyrethroid insecticides have been extensively used in governmental campaigns as well as in residential or private services. Pyrethroids have similar effects as DDT but with a lower residual effect in the environment, and they represent nowadays the main class of insecticide against arthropods, not only those of medical and veterinary importance but also in relation to agriculture and livestock [2]. In Brazil, despite the recent introduction of pyrethroids in campaigns for dengue control throughout the whole country, resistance to these compounds has already been detected in many Ae. aegypti populations [3, 4]. Pyrethroids and DDT have a rapid effect on the insect central nervous system, leading to repetitive and involuntary muscular contractions, followed by paralysis and death, commonly reported as knockdown effect [5, 6]. Accordingly, resistance to this is referred to as knockdown resistance (kdr), the principal cause being a mutation in the pyrethroid/DDT target site, the voltage-gated sodium channel (NaV). The NaV is an axonic transmembrane protein composed of four homologous domains (I–IV), each one with six hydrophobic segments (S1–S6) [7]. To date, most of the kdr mutations described lie in the NaV IIS6 region, and the Leu/ Phe substitution in the 1014 site (numbered according to the Musca domestica amino acid primary sequence) is by far the most common among all studied insects. Relatively recent analyses of kdr mutations in a series of arthropod species contributed to the knowledge concerning evolution and dynamics of pyrethroid resistance in natural populations. This effort is essential to formulate strategies able to prolong the effectiveness of pyrethroids in the field and to develop new compounds targeting the sodium channel [8, 9]. Some extensive reviews of kdr mutations are available [2, 10, 11]. 150 | Martins et al. Evolution, Medicine, and Public Health channel gene. The EE lineage was originated from laboratory selection pressure for nine consecutive generations with the pyrethroid deltamethrin using a sample of a natural population from Natal (a locality from the Northeast of Brazil) that did not harbor the mutation in the 1016 site [20]. Rearing and maintenance of the colonies were conducted according to standard laboratory conditions [21]. Field populations were obtained by sampling as described elsewhere [13]. Molecular assays 50 Crossing experiments Crosses were performed between mosquitoes from Rockefeller and EE strains, respectively, homozygous (Ile/Ile) and apparently ‘heterozygous’ (Ile/Met) for the 1011 site. Each couple of one male and one virgin female was maintained for at least 3 days in conical 50 ml tubes covered with a mesh tulle under a cotton wool soaked in sugar solution. Females were then blood-fed on anesthetized mice, 24 h after sugar removal. Individual females were induced to lay eggs in small Petri dishes lined with wet filter paper [22]. Resulting F1 larvae were reared Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 Genotyping by allele-specific PCR (AS-PCR) for the AaNaV 1011 site and sequencing of the IIS6 genomic region were performed with the DNA from the same specimens genotyped for the 1016 alleles, described in a previous report [13]. PCR discriminating type ‘A’ or ‘B’ sequences (see [14]) was carried out in 12.5 ml reactions containing 1 mM of each primer ‘forward’ (50 -AGGCTGACTGAAAGTAAATTGG-30 ) and ‘reverse’ (50 -CAAAAGCAAGGCTAAGAAAAGG-30 ), 6.25 ml of GoTaq Green Master Mix 2X (Promega) and 0.5 ml of genomic DNA, submitted for 30 denaturation, annealing and extension cycles under, respectively, 94! C/30", 60! C/1’ and 72! C/45". The amplified region includes the intron 20, polymorphic in size, in the AaNaV IIS6 region. For the 1011 site genotyping, PCR with 0.24 mM of common and 0.12 mM of each of the two specific primers [17] was performed as above, with 30 cycles of denaturation, annealing and extension under, respectively, 94! C/30", 57! C/1’ and 72! C/45’’ conditions. The PCR products were analyzed in 10% polyacrylamide gel electrophoresis stained in 1 mg/ml ethidium bromide solution. The AaNaV IIS6 region was amplified, cloned and sequenced as previously reported [14] in individual specimens from Uberaba, Cuiabá, Aparecida de Goiânia, Maceió and Fortaleza. Sequences of at least eight clones of each insect were analyzed. The numbers of copies of the AaNaV IIS6 genomic region were compared among the Rockefeller strain, the EE lineage and their F1 offspring (Hyb). DNA was extracted from pools of 10 L3 larvae ("20 mg) with the kit Insect DNA Extraction (Zymo Research) according to the manufacturer’s instructions, brought to 5 ng/ml in H2O and aliquoted. Real-time PCR reactions were carried out based on instructions of customized TaqMan Copy Number Assay (Applied Biosystems) in 15 ml, containing 7.5 ml of 2# TaqMan Genotyping Master Mix (Applied Biosystems), 0.75 ml of 20# mix composed of primers and probes for both target and reference genes, 20 ng of DNA and H2O. The chosen single copy reference was the ribosomal gene RP49 (GenBank accession number AY539746), with primers AaRP49_F: 50 -ACATCGGTTACGGATCGAACA AG-30 , AaRP49_R: 50 -TGTGGACCAGGAACTTCTTG AAG-30 and probe AaRP49_M: 50 -VIC-CACCCGCCA TATGCT-MGB-NFQ-30 . The target was determined based on the AaNaV IIS6 region (GenBank accession number FJ479613) with primers AaNaVex20_F: 50 ACCGACTTCATGCACTCATTCAT-30 , AaNaVex20_R: 50 -ACAAGCATACAATCCCACATGGA-30 and probe AaNaVex20_M: 50 -FAM-CCACTCGCCGCATAAT0 MGB-NFQ-3 . Three assays were performed with DNA from three distinct pools of each lineage, in triplicate/assay. Reactions were conducted in an ABI StepOne Thermocycler (Applied Biosystems), following standard cycling conditions for TaqMan Genotyping assays. The CTs for the target (AaNaV) and reference (RP49) genes were determined based on automatic threshold indicated by the StepOne Software v2.0. Given the CT of each sample, their !CTs were established, intended to normalize the amount of amplified products from AaNaV by RP49, and then the average of the replicates from each pool !CT (![!CT]) was calculated. The !!CT of the test lineages (EE and Hyb) were obtained by the difference between their ![!CT] and that of Rockefeller. Finally, the average of !!CTs from the three assays (![!!CT]) was calculated in order to estimate the number of AaNaV copies, normalized by RP49, related to Rockefeller. The diploid number of the target sequence of the tested sample was determined by the formula: cnc2!!CT, where cnc is the copy number of the target sequence in the reference sample and !!CT is the difference between the !CT for the tested sample and the reference sample. Martins et al. | Sodium channel gene duplication in Aedes aegypti until adults for genotyping by AS-PCR or for subsequent crossings to obtain F2, performed as above. Ethics statement Mosquito blood feeding Aedes aegypti females were fed on anesthetized mice (ketamine:xylazine 80–120:10–16 mg/kg), according to institutional procedures, oriented by the national guideline ‘the Brazilian legal framework on the scientific use of animals’ [23]. This study was reviewed and approved by the Fiocruz Ethics Committee on Animal Use (CEUA/FIOCRUZ), license number: L-011/09. RESULTS Typing of 1011 and 1016 sodium channel sites in Ae. aegypti natural populations by AS-PCR The allele frequencies of the AaNaV 1011 site were evaluated in the same mosquitoes which had the 1016 site analyzed previously, belonging to samples from 15 Brazilian localities [13]. The 1011Met-mutant allele was found in all localities, except in Boa Vista. In seven localities, specimens were divided into pyrethroid susceptible (S) or resistant (R) [13]. Table 1 shows allele frequencies considering both 1011 and 1016 sites together, combined in six molecular phenotypes, derived from three potential haplotypes (1011Ile+1016Val, 1011Ile+1016Ile and 1011Met+1016Val). We assumed that the recombinant haplotype containing both mutant alleles (1011Met+1016Ile) was not expected, because these sites are very close in the genome and both mutations are likely to be very recent. We observed that the 1011Ile/Ile+1016Ile/Ile combination, i.e. homozygous for the wild-type and for the mutant allele, respectively, in the 1011 and 1016 sites, was far more frequent among resistant than susceptible insects. This suggests that the 1016 site is probably more important for pyrethroid resistance than the 1011 site. Two other striking results can also be observed. First, we did not detect any specimen ‘homozygous’ Sequencing of the IIS6 region of the Ae. aegypti sodium channel gene We obtained sequences of the AaNaV IIS6 region from a number of mosquitoes from five Brazilian populations (see ‘Materials and Methods’ section for details) and confirmed the polymorphism in this genomic region. Figure 1 shows the haplotypes and their respective submission numbers in GenBank. Sequences were classified as ‘A’ or ‘B’, according to two synonymous substitutions in exon 20 and differences in the intron (see [14] for details). The Ile1011Met substitution was seen in all studied populations, whereas Val1016Ile was not detected in the Northeastern localities (Maceió and Fortaleza). Both substitutions were present only in sequences type ‘A’, and among sequences from 40 individuals, no haplotype shared substitutions in both the 1011 and 1016 sites, indicating no recombinants between the two mutations. As mentioned above, this was expected considering that these sites are very close, and the mutations are likely to be very recent. Hence, only four haplotypes were observed 51 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 Entomological survey All field egg collections were conducted by agents from each respective State Health Secretariat, following procedures designed by the National Program of Dengue Control/Brazilian Ministry of Health. All ovitraps were installed and collected in the houses with residents’ permission. for the 1011Met (1011Met/Met+1016Val/Val) mutation. Second, there is a higher than expected frequency of the 1011Ile/Met+1016Val/Val molecular phenotype in all samples, except the near monomorphic Boa Vista population (Table 1). Although the individual tests of the Hardy–Weinberg expectations for each sample were significant only in four cases, likely due to the small sample sizes, the lack of the 1011Met/Met+1016Val/Val molecular phenotype and the excess of 1011Ile/Met+1016Val/Val were observed in almost all populations. Two simple hypotheses were considered to explain this pattern. One possibility is that the 1011Met mutation is involved in a gene duplication, carrying both the mutant (1011Met+1016Val) and the wild-type allele (1011Ile+1016Val). In this case, the 1011Met/Met genotype would never be detected by the AS-PCR, because that duplication would generate a molecular phenotype mimicking a heterozygous 1011Ile/ Met. Alternatively, one might argue that the 1011Met mutation is lethal when in homozygosis. However, this is not the case ([16], see ‘Discussion’ section herein), and it does not explain the increased frequency of 1011Ile/Met+1016Val/Val, unless one also assumes this particular combination has a higher fitness. In order to better understand these data, we cloned and sequenced the IIS6 region from a number of mosquitoes. 151 R S R S R S R S R S R S R S * * * * * * * * Aparecida de Goiânia 0.056 (0.094) 0.105 (0.305) 0.045 (0.052) 0.118 (0.221) 0.231 (0.148) 0.571 (0.617) 0 (0.035) 0.250 (0.303) 0.250 (0.391) 0.313 (0.431) 0.467 (0.538) 0.333 (0.444) 0.043 (0.030) 0.300 (0.276) 0.950 (0.930) 0.200 (0.090) 0 (0.191) 0 (0.003) 0.900 (0.903) 0.300 (0.423) 0.938 (0.938) 0.650 (0.681) 1016Val/Val 0 (0.204) 0.053 (0.029) 0.273 (0.299) 0.118 (0.138) 0 (0.325) 0.143 (0.112) 0.063 (0.223) 0.250 (0.275) – – – – 0.087 (0.204) 0.050 (0.184) 0 (0.095) 0 (0.255) 0.250 (0.191) 0.053 (0.078) – – – – 1016Val/Ile 1011Ile/Ile 0.222 (0.111) 0 (0.001) 0.455 (0.435) 0 (0.022) 0.385 (0.179) 0 (0.005) 0.500 (0.353) 0.100 (0.063) – – – – 0.391 (0.345) 0.050 (0.031) 0.050 (0.003) 0.250 (0.181) 0.063 (0.048) 0.526 (0.543) – – – – 1016Ile/Ile 0.500 0.842 0.091 0.588 0.308 0.286 0.313 0.350 0.750 0.688 0.533 0.667 0.174 0.400 – 0.200 0.625 0.053 0.100 0.700 0.063 0.350 (0.165) (0.301) (0.022) (0.095) (0.455) (0.061) (0.289) (0.221) (0.465) (0.052) (0.360) (0.148) (0.020) (0.260) (0.220) (0.469) (0.451) (0.391) (0.444) (0.083) (0.315) 1016Val/Val 0.222 (0.240) 0 (0.022) 0.136 (0.150) 0.176 (0.112) 0.077 (0.163) 0 (0.020) 0.125 (0.048) 0.050 (0.100) – – – – 0.304 (0.281) 0.200 (0.240) – 0.350 (0.234) 0.063 (0.150) 0.368 (0.310) – – – – 1016Val/Ile 1011Ile/Met 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (0.130) (0.177) (0.013) (0.146) (0.037) (0.037) (0.037) (0.040) (0.141) (0.118) (0.071) (0.111) (0.057) (0.090) – (0.076) (0.118) (0.044) (0.003) (0.123) (0.001) (0.031) 1016Val/Val 1011Met/Met Frequency of genotypes: observed (and expected assuming Hardy–Weinberg equilibrium) 14.7, 5, 0.0119 2.9, 5, 0.7204 1.1, 5, 0.9571 6.8, 5, 0.2347 11.2, 5, 0.0473 1.0, 5, 0.9589 15.6, 5, 0.0080 3.5, 5, 0.6213 5.8, 2, 0.0561 4.4, 2, 0.1114 2.0, 2, 0.3709 3.8, 2, 0.1534 5.5, 5, 0.3619 6.2, 5, 0.2860 1.9, 2, 0.3772 11.1, 5, 0.0487 11.7, 5, 0.0388 2.1, 5, 0.8408 0.06, 5, 0.9727 5.8, 2, 0.0551 0.02, 2, 0.9917 0.9, 2, 0.6377 !2, df, P HWE Frequencies observed and expected (for Hardy–Weinberg equilibrium) of the molecular phenotypes derived by AS-PCR for the sites 1011 and 1016 in the same insects. In the header, the mutant alleles are underlined. Some populations are divided regarding their resistant (R) or susceptible (S) status to pyrethroid resistance. Populations whose individuals were not divided in R or S are marked with an asterisk (*) in status. The absence of the mutations 1011Ile/Met and 1016Val/Ile in a population is represented as endash (–). The last column gives the result of !2 analyses for testing Hardy–Weinberg equilibrium (HWE). The 1016 genotyping data were already presented elsewhere [13]. Boa Vista Cachoeiro do Itapemirim Colatina Foz do Iguaçu Ijuı́ Macapá Santa Bárbara Santa Rosa Uberaba Maceió 18 19 22 17 13 14 16 20 16 16 15 15 23 20 20 20 16 19 20 20 16 20 n | Martins et al. Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 52 Fortaleza Dourados Cuiabá Campo Grande Status Locality Table 1. Phenotypic frequency, considering AaNaV 1011 and 1016 sites, of Ae. aegypti natural populations from Brazil 152 Evolution, Medicine, and Public Health Martins et al. | Sodium channel gene duplication in Aedes aegypti Figure 1. Diversity of a voltage-gated sodium channel gene region observed in Ae. aegypti Brazilian populations. Part of the region corresponding to the AaNaV exons 20 and 21, and the intron between them, are represented. A and B indicate the type of intron, as previously stated [14]. In red, the presumed amino acids for the sites 1011 and 1016. Genomic sequences representative for each haplotype were submitted to GenBank: 1011Ile+B+1016Val (GenBank accession number: FJ479613), 1011Ile+A+1016Val (FJ479611), 1011Met+A+ 1016Val (FJ479612) and 1011Ile+A+1016Ile (JX275501). from Ae. aegypti genome project (Vectorbase) (1011Ile+A+1016Val, 1011Ile+A+1016Ile, 1011Ile+B+1016Val and 1011Met+A+1016Val) out of six possibilities, considering the type of sequence (‘A’ or ‘B’) and the sites 1011 (Ile or Met) and 1016 (Val or Ile) (Table 2). Moreover, the 1011Met+A+1016Val haplotype was only present in specimens which also harbored the 1011Ile+ B+1016Val haplotype, therefore, classified as ‘heterozygous’. Accordingly, typing of various natural populations had revealed the absence of ‘homozygous’ for the 1011Met mutation (Table 1). Curiously, some specimens presented three haplotypes, which were in all cases: 1011Met+A+1016Val, 1011Ile+ A+1016Ile and 1011Ile+B+1016Val (Table 2). It is important to mention that females had their abdomen removed prior to DNA extraction in order to avoid eventual amplification of DNA from spermatozoids stored in the spermatechae, and there was no evidence of contamination in PCR negative controls. The last column of Table 2 presents the expected ‘genotypes’ through sequence typing (A or B) and the 1011 and 1016 sites. Sequencing confirmed the results for all insects genotyped by AS-PCR (data not shown). The presence of three alleles in one specimen suggests the gene duplication, at least in the genomic region analyzed. However, search in the Ae. aegypti genome project database (http://aaegypti. vectorbase.org/) did not indicate any evidence that the original Liverpool strain has more than one copy of any part, let alone the whole voltage-gated sodium channel gene. Based on the available sequences, this strain would be classified as homozygous for the 1011Ile+B+1016Val allele, just like the Rockefeller strain used here. Hence, the putative duplication Crossing experiments In order to test the duplication hypothesis, we performed crosses between specimens with known molecular phenotypes (based on AS-PCR) and determined the frequency of the variants in the AaNaV 1011 site in their offspring. Initially, we evaluated the F1 of seven couples, each composed of a homozygous wild-type (1011Ile/Ile) and a putative heterozygous or duplicated (1011Ile/Met) progenitor, belonging, respectively, to the Rockefeller and the EE lineages. The latter originated from a laboratory population selection for pyrethroid resistance using a sample from a natural population that did not harbor the mutation Val1016Ile [20]. The results are shown in Table 3, with expected values and the Fisher tests for the three different hypotheses in Fig. 3, assuming either a duplication or no duplication. If the 1011Ile/Met parent did not harbor the duplicated haplotype, the offspring would present the Ile/Ile and Ile/Met genotypes in equal frequencies (Hypothesis 1). Assuming the occurrence of a duplication, one would expect the offspring genotyped as either 100% Ile/Met or alternatively Ile/Ile and Ile/Met in equal frequencies, respectively, if the parent was homozygous (Hypothesis 2a) or heterozygous (Hypothesis 2b) for the duplicated haplotype (Fig. 3). Two out of seven crosses (#3 and #4) had the 1011Ile/Ile genotype in around half of their offspring, which was thus not informative. In these two cases, this could be explained if the progenitor harboring the 1011Met mutation was heterozygous for the duplication (1011Ile/Ile_Met) as well as if it was heterozygous for non-duplicated haplotypes. 53 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 TIGR = sequence does not occur in all individuals, being therefore a polymorphic trait. In the samples analyzed, we detected mosquitoes ‘homozygous’ for the 1011Ile+ B+1016Val, 1011Ile+A+1016Val and 1011Ile+ A+1016Ile haplotypes, all having the wild-type allele for the 1011 site. However, the ‘1011Met+ A+1016Val’ (mutant in the 1011 site) haplotype was never detected in ‘homozygosis’, but always in association with ‘1011Ile+B+1016Val’, suggesting that the duplication involves these two variants (Table 2). Figure 2 presents a schematic representation of AaNaV haplotypes proposed for the populations analyzed based on our duplication hypothesis. The offspring of crosses between some combinations of parental genotypes was further analyzed in order to test this hypothesis. 153 154 | Martins et al. Evolution, Medicine, and Public Health Table 2. Sequencing of the AaNaV IIS6 genomic region of specimens from Ae. aegypti Brazilian natural populations Locality Sample Haplotype (1011+intron+1016) Ile + A + Val Uberaba Ap Goiânia Maceió Fortaleza Ile + A + Ile X X X X X X X X X X X X X X X X X X X X X X X X Ile + B + Val X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Met + B + Val Ile + B + Ile Ile/Ile+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Ile Ile/Ile+AA+Val/Ile Ile/Ile+AA+Val/Ile Ile/Ile+AA+Ile/Ile Ile/Ile+AA+Ile/Ile Ile/Ile+BB+Val/Val Ile/Ile+AB+Val/Ile Ile/Met+AB+Val/Ile Ile/Ile+AA+Ile/Ile Ile/Ile+AA+Ile/Ile Ile/Ile+AA+Ile/Ile Ile/Ile+AB+Val/Ile Ile/Ile+AA+Val/Val Ile/Ile+AB+Val/Val Ile/Ile+AB+Val/Val Ile/Ile+AB+Val/Val Ile/Ile+AB+Val/Val Ile/Ile+AB+Val/Val Ile/Ile+AB+Val/Val Ile/Met+AB+Val/Val Ile/Ile+AA+Val/Ile Ile/Met+AB+Val/Ile Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Ile Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Met+AB+Val/Val Ile/Ile+BB+Val/Val Ile/Ile+BB+Val/Val Ile/Met+AB+Val/Val Ile/Ile+AA+Val/Val Ile/Ile+BB+Val/Val Ile/Ile+AB+Val/Val Identification of each sample corresponds to the sampling locality: UBR, Uberaba; CUI, Cuiabá; APG, Aparecida de Goiânia; COM, Maceió and hrjg, Henrique Jorge (a district of Fortaleza). ‘Haplotypes’ indicate the combination among site 1011 (Ile or Met)+type of intron (A or B)+site 1016 (Val or Ile). The haplotype observed for each insect is marked by an ‘X’. In the header, the mutations are indicated in bold letters. The last column shows the phenotypic classification, confirmed by AS-PCR. 54 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 Cuiabá UBR-04 UBR-08 UBR-10 UBR-S25 UBR-S26 UBR-R1 UBR-R3 UBR-R10 UBR-R11 UBR-R13 UBR-R20 UBR-R22 UBR-R26 CUI-01 CUI-02 CUI-03 CUI-04 CUI-07 CUI-08 CUI-12 CUI-R16 CUI-S15 APG-01 APG-02 APG-04 APG-05 APG-06 APG-07 APG-08 APG-09 APG-10 APG-11 APG-12 COM-02 COM-07 COM-09 hrjg-21 hrjg-22 hrjg-23 hrjg-28 Met + A + Val Molecular phenotype (1011+intron+1016) Martins et al. | Sodium channel gene duplication in Aedes aegypti 155 Figure 2. Schematic representation of AaNaV haplotypes. Blue boxes indicate exons 20 and 21 with the intron between them, the latter used to classify the haplotypes as A (orange) or B (green). Sites 1011 and 1016 are represented by the variant wild-type (blue box) or mutant (red box). According to our hypothesis, there is a duplication in some populations, comprised of haplotypes 1011Ile+B+1016Val and 1011Met+A+1016Val. Dashed line suggests linkage of the haplotypes, but which one is upstream was not determined Crossings Hypothesesa F1 observed (n) Without duplication With duplication Hypothesis 1 #1 #2 #3 #4 #5 #6 #7 (, Ile/Met x < Ile/Ile) (, Ile/Met x < Ile/Ile) (, Ile/Met x < Ile/Ile) (,Ile/Ile x < Ile/Met) (, Ile/Ile x < Ile/Met) (, Ile/Met x < Ile/Ile) (, Ile/Met x < Ile/Ile) Hypothesis 2a Hypothesis 2b Ile/Ile Ile/Met Ile/Ile Ile/Met P Ile/Ile Ile/Met P Ile/Ile Ile/Met P 0 0 8 9 0 0 0 20 20 12 9 30 30 22 10 10 10 9 15 15 11 10 10 10 9 15 15 11 *** *** NS NS *** *** *** 0 0 0 0 0 0 0 20 20 20 18 30 30 22 NS NS ** *** NS NS *** 10 10 10 9 15 15 11 10 10 10 9 15 15 11 *** *** NS NS *** NS NS Molecular phenotype frequencies were determined by AS-PCR for the AaNaV 1011 site (see ‘Materials and Methods’ section). aExpected numbers of F1 individuals of each molecular phenotype based on the three hypotheses of parental haplotype constitution (Fig. 3). Significance of the deviations of the tested hypotheses obtained through Fisher’s exact test: NS = non-significant, **P < 0.01, ***P < 0.001. However, as all the offspring from the other five crosses were 1011Ile/Met, the progenitor who harbored the mutation was necessarily homozygous for the duplication (Ile_Met/Ile_Met) (Fig. 3). In addition, the F2 offspring from crosses #1 (#1.1) and #2 (#2.1) revealed segregation in the approximated proportion of 3Ile/Met:1Ile/Ile (Table 4), corroborating the duplication hypothesis. Figure 3. Three hypotheses with the expected genotypes and molecular phenotypes in the AaNaV 1011 site for the parental Copy number assay We analyzed the AaNaV copy number variation through molecular assays using DNA from pools of larvae from the Rockefeller reference strain, homozygous for the wild-type alleles, and a strain (EE) selected in the laboratory for pyrethroid resistance [20] and harboring the putative duplication in and their respective expected frequency in the F1 offspring. The 1011Met mutation is shown in red. See text for further details the AaNaV, as suggested by the assays described above. In this sense, we assessed the relative amount of DNA molecules containing the genomic region spanning the AaNaV 1011 site normalized by 55 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 Table 3. Testing the gene duplication hypothesis: molecular phenotype frequencies for the AaNav 1011 site in F1 offspring from crossings between Ae. aegypti Ile/Ile X Ile/Met 156 | Martins et al. Evolution, Medicine, and Public Health a reference gene (RP49). Assuming that the Rockefeller strain has only two copies of AaNaV as expected for a diploid with a single copy gene, the EE lineage selected for resistance and ‘homozygous’ for the duplication, revealed to have in fact 10 copies (Table 5 and Supplementary Table S1). Accordingly, the F1 resulting from Rockefeller and EE had six copies. The results therefore indicate further duplication events and amplification in this locus. DISCUSSION Table 4. Testing the gene duplication hypothesis: molecular phenotype frequencies for the AaNav 1011 site in F2 offspring from crosses #1 and #2 (Table 3) Crossings (F1) F2 (n) Observed #1.1 (, Ile/Met x < Ile/Met) #2.1 (, Ile/Met x < Ile/Met) Expected Ile/Ile Ile/Met Ile/Ile Ile/Met P 5 7 25 23 8 8 22 22 NS NS Observed and expected numbers for each molecular phenotype in the F2 of crosses #1 and #2 (Table 3) assuming parents carry the following haplotypes Ile/Ile_Met " Ile/Ile_Met, in agreement with the duplication hypothesis (Fig. 3). The expected frequencies are 0.25 Ile/Ile and 0.75 Ile/Met (0.50Ile/Ile_Met+0.25Ile_Met/Ile_Met). Deviations from the proposed hypotheses are non-significant (Fisher’s exact test; P > 0.05). Table 5. Copy number variation assay for AaNaV Assay 1 2 3 Rock EE Hib ![!CT] (SD) !!Cq ![!CT] (SD) !!Cq ![!CT] (SD) !!Cq !0.4 0 -0.7 (0.09) (0.11) (0.07) 0 0 0 !2.7 !2.4 !3 (0.03) (0.04) (0.04) !2.3 !2.4 !2.4 !2 !1.7 -2.4 (0.07) (0.05) (0.06) !1.6 !1.6 !1.7 ![!!CT] (SD) 0 Cn 2 !2.3 (0.03) 10 !1.6 (0.07) 6 Average and standard deviation !CT (target ! reference) followed by the !!Cq (lineage test ! Rock) values from each lineage in each assay. Bottom: mean and standard deviation of !!CT from the three assays and the resulting number of copies (cn) of AaNaV relative to rp49. 56 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 DDT and pyrethroids target the voltage-gated sodium channel (NaV) of insects, a key component of axon membranes exhibiting a fundamental physiological function in neural current propagation, with a complex but highly conserved structure among animals [24]. Vertebrate genomes present 6–10 NaVcoding genes, whereas invertebrate classes, such as Cnidaria and Annelida, have only 2–4 NaV genes [25]. In insects, there is only one NaV, also commonly referred to as ‘paralytic’ (para), due to its relationship with the phenotype of reversible paralysis under high temperatures in Drosophila melanogaster-mutant lineages [26, 27]. An important source of NaV protein variability in different tissues relies on alternative splicing and RNA editing [28]. However, to date no association between pyrethroid resistance and variation derived from post-transcriptional modifications in the Ae. aegypti NaV gene has been uncovered [18]. Another possible source of molecular diversity might be polymorphism generated by recent gene duplications. Putative additional NaV in insects (the orthologous channels DSC1 in D. melanogaster and BSC1 in Blattella germanica) were later grouped close to calcium channels, both functionally and evolutionarily [29, 30]. Recently, two NaV distantly related proteins were characterized in the Periplaneta americana cockroach, coded by the PaNaV and PaFPC para-like genes, a finding that Martins et al. | Sodium channel gene duplication in Aedes aegypti metabolic resistance was demonstrated in Caribbean Ae. aegypti populations. Compared with the susceptible strain, two genes (CYP9J26 and the ABC transporter ABCB4) were amplified up to eight and seven copies, respectively [44]. Besides insecticide resistance, duplication of metabolic-resistance genes may also be selectively advantageous to the organism by increasing its general ability of detoxify xenobiotics. Moreover, new functions might be generated due to accumulation of substitutions in duplicated genes [45]. Such events would be more ‘free’ to occur, since the detoxifying enzyme system is redundant, reliant upon different enzymes with a similar function. Hence, the accumulation of potential loss of function alterations might not significantly compromise the metabolism [46]. By contrast, gene duplication events in molecules which are targets of neurotoxic insecticides are thought to be less likely, since they carry out very specific and essential activities, highly conserved throughout evolution. The increase in number might compromise the neurological functioning of the organism, an event described as dosage-balance hypothesis [47]. For instance, a Culex pipiens lineage with an acetilcolinesterase gene (ace-1) duplication presents 60% increase in enzyme activity. However, the acquired organophosphate resistance status is accompanied by an elevated cost of several life-history trait parameters [48]. Indeed, in a number of Cx. pipiens populations, the frequency of the ace-1R-mutant allele decays quickly in the absence of insecticide [49, 50], the same tendency observed for ace-1R in An. gambiae [51]. However, Cx. pipiens’ natural populations with a putative recent ace-1 gene duplication (<40 years) have also been described. In these cases, both copies, with and without the mutation selected for organophosphate resistance, lie in the same chromosome. These mosquitoes, with a ‘heterozygous’ molecular phenotype, are resistant to organophosphates but have a lower fitness loss [52], suggesting a mechanism which favors the occurrence of duplications in neurotoxic insecticide target-coding genes. Herein, we initially hypothesized a duplication in a region of the NaV gene of Ae. aegypti (AaNaV) as a polymorphic trait in natural populations of this important vector, which would include one-mutant haplotype for the 1011 site together with one wildtype for both sites, 1011Met+1016Val and 1011Ile+1016Val, respectively, supported by a fund 57 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 suggested a possible early duplication event and subsequent loss of the NaV gene in some lineages [31]. The role of gene duplication and/or amplification in insecticide resistance has been described in at least 10 arthropod species, including mosquitoes [32]. The most classic case involves overexpression of Culex Esterase genes, leading to organophosphate resistance. This is the consequence of duplication of two genes (named esterase A and esterase B) or at least the esterase B [33– 35]. Amplification of esterase B1 in Californian Culex mosquitoes was the first event described in this context [36]. Variation in the number of copies among insects was also observed, being directly proportional to organophosphate resistance levels [37]. In agreement, laboratory insecticide selection pressure resulted in an increase in the gene copy numbers. However, it is likely that this process has a limit, since gene amplification is associated with a high fitness cost [38]. In fact, unequal crossingover in the duplicated locus [37] may cause a reduction in copy number over time in the absence of insecticide pressure. Gene duplication was also associated with another class of enzymes related to metabolic resistance, the multi-function oxidases (MFOs) or P450 [39]. Two genes of this class (CYP6P9 and CYP6P4) were overexpressed in pyrethroid-resistant lineages of the malaria vector, Anopheles funestus. This overexpression is associated within tandem gene duplications, mapped in a quantitative trait locus (QTL locus rp1) and responsible for 87% of the genetic variation for pyrethroid resistance in this lineage. Besides, single nucleotide polimorphisms (SNPs) observed in these genes were described as insecticide-resistance markers [39]. Another gene duplication event was associated with overexpression of a P450 gene (CYP9M10) in a pyrethroid-resistant strain of Culex quinquefasciatus [40]. Duplications in genes coding for enzymes involved in metabolic resistance are somewhat expected, since they are components of supergene families bearing many paralogous genes, generally organized in genome clusters [41]. These are rapidly evolving families and few orthologs are identified among insect species [42]. In the Ae. aegypti genome, at least 26, 49 and 160 genes of the main detoxifying enzymes were identified corresponding, respectively, to GST, Esterases and MFO. These numbers represent an increase of 36% compared with Anopheles gambiae [43]. Recently, the importance of gene amplification for pyrethroid 157 158 | Martins et al. Evolution, Medicine, and Public Health 58 when pools of 10 larvae were employed. The variation in the number of copies in natural populations remains to be investigated as an important clue for this evolutionary process. Amplification of the NaV gene was also recently demonstrated in a pyrethroid-resistant C. quinquefasciatus lineage. The classical kdr mutation (Leu1014Phe), strongly associated to pyrethroid resistance, was present in one type of sequence. The other type of sequence lacked the intron close to the 1014 site and was not related to resistance. This haplotype was suggested to be a pseudogene [55]. To the best of our knowledge, we present here the first evidence of a duplication event in the sodium channel gene of the dengue vector, Ae. aegypti. Although the available data point to a more important role of the mutations in the 1016 site for pyrethroid resistance, there is clear evidence that the 1011Met mutation, which is associated with the duplication/amplification event(s), is also associated with some resistance [12, 14]. Therefore, the gene duplication and amplification in the Ae. aegypti NaV gene might be a recent adaptive response to the intense use of insecticides, maintaining together wild-type and mutant alleles in the same organism conferring some resistance at the same time as reducing some of its deleterious effects on other aspects of fitness. It will be very interesting to investigate how much diversity in copy number variation there is in natural populations, besides its possible association with pyrethroid resistance and fitness cost. It is also intriguing whether the mosquito sodium channel gene is more prone to duplications than that of other pyrethroid-selected insects as well as what the potential evolutionary interpretation and implications of this process are. supplementary data Supplementary data is available at EMPH online. acknowledgments The authors thank Dr Alexandre Afranio Peixoto for his friendship and orientation throughout this study. This work is dedicated to his memory. They also thank Andre Torres and Heloisa Diniz for their assistance with the figures, the DNA sequencing facility of FIOCRUZ (Plataforma de Sequenciamento/PDTIS/Fiocruz) and to the Brazilian Dengue Control Program that allowed utilization of samples collected in the scope of the Brazilian A. aegypti Insecticide Resistance Monitoring Network (MoReNAa). Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 of evidence. AS-PCR genotyping confirmed that all individuals carrying the 1011Met mutation were (phenotypically) ‘heterozygous’. In addition, sequencing of the AaNaV IIS6 genomic region revealed some individuals with three haplotypes, suggesting the existence of a duplication with the proposed aforementioned composition. Similar results of mosquitoes harboring three alleles were recently reported for the An. gambiae acetilcolinesterase ace-1 gene and interpreted as evidence of a gene duplication event [53]. Saavedra-Rodriguez et al. [16] evaluated the role of AaNaV mutations in pyrethroid resistance by analyzing the susceptibility of the F3 offspring from the parental crossing ,1011Ile/Met+1016Ile/Ile (from Isla Mujeres, Mexico) ! <1011Ile/Ile+ 1016Val/Val (from New Orleans, lineage control of susceptibility). Interestingly, if the presence of a duplicated sodium channel had been considered, interpretation of some results would have been made easier since they would have better explained the different genotypes in the crosses. In addition, it is remarkable that the Ile1011Met substitution seems to appear in ‘homozygosis’ (1011Met/Met) in high frequency in other localities in Latin America [16, 54], indicating that this mutation is not recessive-lethal and that different types of duplicated haplotypes probably coexist in Ae. aegypti populations. This might also suggest that the gene duplication in the Ae. aegypti NaV gene we observed in Brazilian populations is a relatively recent event. Our initial hypothesis was that, at least for the Ae. aegypti populations studied herein, the 1011Met mutation occurs only in a duplicated haplotype containing a type ‘A’ sequence and the 1016Val wild-type allele, together and in linkage disequilibrium with a type ‘B’ sequence, containing the wild-type allele for both the 1011 and 1016 positions (Fig. 2). The high frequency of ‘heterozygous’ A/B, the lack of 1011Met/ Met specimens, 1011Ile/Met+1016Ile/Ile genotypes and the molecular phenotype of the offspring analyzed here support this hypothesis. However, the results obtained by the copy number variation assay show a ratio of five copies of the AaNaV gene in the EEselected lineage when compared with the Rockefeller strain, indicating that further duplication events might have taken place, possibly as a result of unequal crossing-over. Moreover, it is presumed that the number of copies is a polymorphic trait, given the large variation observed when using single mosquito DNA (data not shown), which was diminished Martins et al. | Sodium channel gene duplication in Aedes aegypti funding 159 13. Martins AJ, Lima JB, Peixoto AA et al. Frequency of Val1016Ile mutation in the voltage-gated sodium channel This work was supported by the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnolóico (CNPq - Pronex gene of Aedes aegypti Brazilian populations. Trop Med Int Health 2009;14:1351–5. Dengue), Fundação Carlos Chagas Filho de Amparo à 14. Martins AJ, Lins RM, Linss JG et al. Voltage-gated sodium Pesquisa do Estado do Rio de Janeiro (FAPERJ - Cientistas channel polymorphism and metabolic resistance in pyr- do nosso estado), the Howard Hughes Medical Institute ethroid-resistant Aedes aegypti from Brazil. Am J Trop Med (HHMI) and the Instituto Nacional de Ciêmcia e Tecnologia Hyg 2009;81:108–15. - Entomologia Molecular (INCT-EM). The funders had no role 15. Rajatileka S, Black WC 4th, Saavedra-Rodriguez K et al. in study design, data collection and analysis, decision to Development and application of a simple colorimetric publish, or preparation of the manuscript. Funding to pay assay reveals widespread distribution of sodium channel the Open Access publication charges for this article was mutations in Thai populations of Aedes aegypti. Acta Trop provided by Fundação Oswaldo Cruz (FIOCRUZ). 2008;108:54–7. Conflict of interest: None declared. 16. Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S et al. A mutation in the voltage-gated sodium channel gene Aedes aegypti. Insect Mol Biol 2007;16:785–98. references 17. Garcia GP, Flores AE, Fernandez-Salas I et al. Recent rapid rise of a permethrin knock down resistance allele in Aedes 1. Braga IA, Valle D. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde 2007;16:295–302. 2. Soderlund DM. Pyrethroids, knockdown resistance and sodium channels. Pest Manag Sci 2008;64:610–6. aegypti in Mexico. PLoS Negl Trop Dis 2009;3:e531. 18. Chang C, Shen WK, Wang TT et al. A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti. Insect Biochem Mol Biol 2009;39:272–8. 3. da-Cunha MP, Lima JB, Brogdon WG et al. Monitoring of 19. Harris AF, Rajatileka S, Ranson H. Pyrethroid resistance in resistance to the pyrethroid cypermethrin in Brazilian Aedes Aedes aegypti from Grand Cayman. Am J Trop Med Hyg aegypti (Diptera: Culicidae) populations collected between 2001 and 2003. Mem Inst Oswaldo Cruz 2005;100:441–4. 2010;83:277–84. 20. Martins AJ, Ribeiro CD, Bellinato DF et al. Effect of insecti- 4. Montella IR, Martins AJ, Viana-Medeiros PF et al. cide resistance on development, longevity and reproduc- Insecticide resistance mechanisms of Brazilian Aedes tion of field or laboratory selected Aedes aegypti aegypti populations from 2001 to 2004. Am J Trop Med populations. PLoS One 2012;7:e31889. Hyg 2007;77:467–77. 5. Busvine JR. Mechanism of resistance to insecticide in 21. Lima JB, Da-Cunha MP, Da Silva RC et al. Resistance of houseflies. Nature 1951;168:193–5. 6. Harrison CM. Inheritance of resistance of DDT in the municipalities in the State of Rio de Janeiro and Espirito housefly, Musca domestica L. Nature 1951;167:855–6. 7. Catterall WA. From ionic currents to molecular mechan- 22. Belinato TA, Martins AJ, Lima JB et al. Effect of the chitin isms: the structure and function of voltage-gated sodium bility and reproduction of Aedes aegypti. Mem Inst Oswaldo channels. Neuron 2000;26:13–25. 8. Du Y, Nomura Y, Luo N et al. Molecular determinants on 23. Filipecki AT, Machado CJ, Valle S et al. The Brazilian legal the insect sodium channel for the specific action of type II framework on the scientific use of animals. ILAR J 2011;52: pyrethroid insecticides. Toxicol Appl Pharmacol 2009;234: 266–72. 9. O’Reilly AO, Khambay BP, Williamson MS et al. Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochem J 2006;396:255–63. 10. Davies TE, O’Reilly AO, Field LM et al. Knockdown resist- Aedes aegypti to organophosphates in several Santo, Brazil. Am J Trop Med Hyg 2003;68:329–33. synthesis inhibitor triflumuron on the development, viaCruz 2009;104:43–7. E8–15. 24. Martins AJ, Valle D. The pyrethroid knockdown resistance. In: Soloneski,S, Larramendy,MS (eds), Insecticides—Basic and Other Applications. Rijeka: InTech, 2012, 17–38. 25. Goldin AL. Evolution of voltage-gated Na+ channels. J Exp Biol 2002;205(Pt 5): 575–84. ance to DDT and pyrethroids: from target-site mutations 26. Suzuki DT, Grigliatti T, Williamson R. Temperature-sensi- to molecular modelling. Pest Manag Sci 2008;64:1126–30. tive mutations in Drosophila melanogaster. VII. A mutation 11. Davies TG, Field LM, Usherwood PN et al. DDT, pyreth- (para-ts) causing reversible adult paralysis. Proc Natl Acad rins, pyrethroids and insect sodium channels. IUBMB Life 2007;59:151–62. 12. Brengues C, Hawkes NJ, Chandre F et al. Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med Veterinary Entomol 2003;17:87–94. Sci USA 1971;68:890–3. 27. Loughney K, Kreber R, Ganetzky B. Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 1989;58:1143–54. 28. Davies TG, Field LM, Usherwood PN et al. A comparative study of voltage-gated sodium channels in the Insecta: 59 Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 associated with pyrethroid resistance in Latin American 160 | Martins et al. Evolution, Medicine, and Public Health implications for pyrethroid resistance in Anopheline and other Neopteran species. Insect Mol Biol 2007;16:361–75. environmental response in the honeybee. Insect Mol Biol 2006;15:615–36. 29. Zhou W, Chung I, Liu Z et al. A voltage-gated calcium- 43. Strode C, Wondji CS, David JP et al. Genomic analysis of selective channel encoded by a sodium channel-like gene. detoxification genes in the mosquito Aedes aegypti. Insect Neuron 2004;42:101–12. Biochem Mol Biol 2008;38:113–23. 30. Cui YJ, Yu LL, Xu HJ et al. Molecular characterization of DSC1 orthologs in invertebrate species. Insect Biochem Mol Biol 2012;42:353–9. 44. Bariami V, Jones CM, Poupardin R et al. Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the 31. Moignot B, Lemaire C, Quinchard S et al. The discovery of a novel sodium channel in the cockroach Periplaneta americana: evidence for an early duplication of the paralike gene. Insect Biochem Mol Biol 2009;39:814–23. 32. Bass C, Field LM. Gene ampliEcation and insecticide resistance. Pest Manag Sci 2011;67:886–9. 33. Raymond M, Chevillon C, Guillemaud T et al. An overview quito Culex pipiens. Philos Trans R Soc Lond B Biol Sci 1998;353:1707–11. 45. Kimura M, King JL. Fixation of a deleterious allele at one of two ‘‘duplicate’’ loci by mutation pressure and random drift. Proc Natl Acad Sci USA 1979;76:2858–61. 46. Conant GC, Wolfe KH. Turning a hobby into a job: how duplicated genes find new functions. Nat Rev Genet 2008; 9:938–50. 47. Papp B, Pal C, Hurst LD. Dosage sensitivity and the evolution of gene families in yeast. Nature 2003;424: 34. Rooker S, Guillemaud T, Berge J et al. Coamplification of esterase A and B genes as a single unit in Culex pipiens mosquitoes. Heredity (Edinb) 1996;77(Pt 5): 555–61. 194–7. 48. Bourguet D, Raymond M, Fournier D et al. Existence of two acetylcholinesterases in the mosquito Culex pipiens 35. Montella IR, Schama R, Valle D. The classification of esterases: an important gene family involved in insecticide (Diptera:Culicidae). J Neurochem 1996;67:2115–23. 49. Berticat C, Boquien G, Raymond M et al. Insecticide resist- resistance—a review. Mem Inst Oswaldo Cruz 2012;107: ance genes induce a mating competition cost in Culex 437–49. 36. Mouches C, Pasteur N, Berge JB et al. Amplification of an pipiens mosquitoes. Genet Res 2002;79:41–7. 50. Berticat C, Duron O, Heyse D et al. Insecticide resistance esterase gene is responsible for insecticide resistance in a genes confer a predation cost on mosquitoes, Culex California Culex mosquito. Science 1986;233:778–80. pipiens. Genet Res 2004;83:189–96. 37. Guillemaud T, Lenormand T, Bourguet D et al. Evolution of 51. Alout H, Djogbenou L, Berticat C et al. Comparison of resistance in Culex pipiens: Allele replacement and changing environment. Evolution 1998;52:443–53. Anopheles gambiae and Culex pipiens Acetycholinesterase 1 biochemical properties. Comp Biochem Physiol B 38. Raymond M, Poulin E, Boiroux V et al. Stability of insecti- Biochem Mol Biol 2008;150:271–7. cide resistance due to amplification of esterase genes in 52. Labbe P, Berthomieu A, Berticat C et al. Independent Culex pipiens. Heredity 1993;70:301–7. 39. Wondji CS, Irving H, Morgan J et al. Two duplicated P450 duplications of the acetylcholinesterase gene conferring genes are associated with pyrethroid resistance in insecticide resistance in the mosquito Culex pipiens. Mol Biol Evol 2007;24:1056–67. Anopheles funestus, a major malaria vector. Genome Res 53. Djogbenou L, Chandre F, Berthomieu A et al. Evidence of 2009;19:452–9. 40. Itokawa K, Komagata O, Kasai S et al. Genomic structures introgression of the ace-1R mutation and of the ace-1 duplication in West African Anopheles gambiae s. s. PLoS One of Cyp9m10 in pyrethroid resistant and susceptible strains of Culex quinquefasciatus. Insect Biochem Mol Biol 2010;40: 2008;3:e2172. 54. Lima EP, Paiva MH, de Araujo AP et al. Insecticide resistance in Aedes aegypti populations from Ceara, Brazil. 631–40. 41. Ranson H, Claudianos C, Ortelli F et al. Evolution of supergene families associated with insecticide resistance. Parasit Vectors 2011;4:5. 55. Xu Q, Tian L, Zhang L et al. Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the Science 2002;298:179–81. 42. Claudianos C, Ranson H, Johnson RM et al. A deficit of detoxification enzymes: pesticide sensitivity and 60 mosquito Culex quinquefasciatus. Biochem Biophys Res Commun 2011;407:645–9. Downloaded from http://emph.oxfordjournals.org/ at Fundação Oswaldo Cruz on July 15, 2013 of the evolution of overproduced esterases in the mos- dengue vector, Aedes aegypti. PLoS Negl Trop Dis 2012;6: e1692. 61 6. CAPITULO 3 62 Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid Resistance and Its Fitness Cost Luiz Paulo Brito1, Jutta G. B. Linss1, Tamara N. Lima-Camara2, Thiago A. Belinato1,3, Alexandre A. Peixoto2,3, José Bento P. Lima1, Denise Valle1,3, Ademir J. Martins1,3* 1 Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil, 2 Laboratório de Biologia Molecular de Insetos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil, 3 Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brasil Abstract Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects. Citation: Brito LP, Linss JGB, Lima-Camara TN, Belinato TA, Peixoto AA, et al. (2013) Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid Resistance and Its Fitness Cost. PLoS ONE 8(4): e60878. doi:10.1371/journal.pone.0060878 Editor: Nirbhay Kumar, Tulane University, United States of America Received December 10, 2012; Accepted March 4, 2013; Published April 8, 2013 Copyright: ! 2013 Brito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Programa Nacional de Controle da Dengue/Secretaria de Vigilância em Saúde/Ministério da Saúde (PNCD/SVS/MS), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnolóico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ, the Howard Hughes Medical Institute (HHMI) and the Instituto Nacional de Ciência e Tecnologia - Entomologia Molecular (INCT-EM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] participation campaigns that strengthen the importance of mechanical control through the elimination of potential breading sites. Thousands of health agents are responsible for visiting residences and orienting dwellers. Ideally, treatment with larvicides takes place, in 4–6 annual cycles as a complementary measure, only on water reservoirs that cannot be eliminated. Presently two different larvicides are employed, temephos or, in localities with confirmed resistance to this organophosphate, a chitin synthesis inhibitor. Control of adult mosquitoes, performed by ultra-low volume (ULV) space spraying of pyrethroids, is theoretically restricted to epidemic seasons. However, the domestic use of this insecticide class is massive, both through aerosol cans or even space spraying services hired privately by residential complexes. At a global scale, pyrethroids are by far the most popular insecticide class, in terms of surface area [5], against adult mosquitoes since they act very rapidly (the knockdown effect), are easily applied and less harmful to both the environment and man. Up to now, they are also the only class of products recommended by the World Health Organization (WHO) for use in insecticide treated materials Introduction Diseases like dengue, malaria, lymphatic filariasis, leishmaniasis and Chagas’ disease are caused by pathogens transmitted by insect vectors and represent a significant part of all morbidity and mortality records in tropical countries. In the last decades, urban areas in most of these countries have faced an accelerated and disorganized growth, with deficient sanitation and general infrastructure, a scenario that favors the expansion of insect vector populations [1]. Since there are still no effective vaccines against these etiologic agents, disease control strongly relies on actions against their insect vectors. In this sense, insecticides are expected to remain a key component in the control of insect populations for a long time [2]. Aedes aegypti is the main dengue vector, currently the most important arthropod-borne viral infection of man [3]. Brazil is hyperendemic for the dengue virus, with confirmed cocirculation of the four serotypes since 2010 when more than one million cases of the disease were registered [4]. In this country, Ae. aegypti control is increasingly based on community PLOS ONE | www.plosone.org 1 63 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations Results (ITMs), which are currently largely distributed for personal protection against malaria and dengue vectors [6,7]. However, insecticide resistant populations are spreading all over the world, representing a growing obstacle to vector control programs [8]. Aedes aegypti pyrethroid resistance may be the consequence of the selection of detoxifying enzymes with altered expression, mainly from the multi function oxidases (MFO) [9] and glutathione-S transferases (GST) super-families [10], but also esterases (EST) [11]. Nevertheless, point mutations in the molecular pyrethroid target site in the mosquito central nervous system, the voltage gated sodium channel (NaV), generally referred to as kdr mutations, are the major reported causes of resistance to this insecticide class [12]. NaV is a transmembrane protein present in the neuronal axons and composed of four homologous domains (I-IV) each with six hydrophobic segments (S1–S6) [13]. In many insect species, mutations related to both pyrethroid and DDT resistance are placed mainly in the IIS6 region. The replacement of a Leu for a Phe in the 1014 site (Leu1014Phe), first described in a DDT resistant Musca domestica strain [14], is the most common. Besides, other substitutions in the same homologous site have been described in a series of species, such as Leu1014Ser in the mosquitoes Anopheles gambiae [15] and Culex pipiens [16] and Leu1014His in the tobacco budworm Heliothis virescens [17]. In Ae. aegypti, these substitutions at the 1014 site are unlikely to occur since two independent changes in the same codon would be necessary [18]. Instead, mutations in different positions have been observed in Ae. aegypti populations from Latin America and Southeast Asia. At least two sites are indeed related to pyrethroid resistance, 1016 (Val to Ile or Gly) and 1534 (Phe to Cys) in the IIS6 and IIIS6 segments, respectively [18–22]. There is also another polymorphic site in the IIS6 region, 1011 (Ile to Met or Val), but its relative role in pyrethroid resistance remains to be elucidated [19,21]. Recent reports from Brazil and Mexico reveal a fast dissemination of resistance to pyrethroids together with a drastic increase in the rates of the Val1016Ile mutation in Ae. aegypti populations [7,20,23]. Selection pressure under laboratory conditions confirmed this tendency [18,24]. In an evolutionary perspective, selection for insecticide resistance may lead to a series of side effects in the life history traits of an insect population. This may be achieved as a result of pleiotropic effects in the resistance genes themselves or as a consequence of a hitchhiking effect, where certain alleles of nonrelated loci increase in frequency in consequence of a strong linkage to a resistance gene under directional selection [25]. In some cases these effects may be associated with a reduced fitness in the absence of insecticide [26]. Consequently, there is a decrease in the vector population resistance levels after the insecticide is withdrawn. Ultimately, this could contribute to a rational utilization of a given insecticide, here represented by the possibility of its re-introduction once susceptible levels are recovered by the vector population. In this sense, knowledge of the resistance status and its underlying mechanisms are the first steps toward a more effective resistance management. In addition, the fitness cost of insecticide resistance should be more thoroughly explored in a comparative perspective, both in the presence and in the absence of the insecticide. Herein, we present a study of the effect of the Aedes aegypti kdr mutations on several life-history parameters, in order to evaluate the fitness cost. We also examine the changes in mutant allele frequencies along 15 generations in population cages without insecticide selection pressure. PLOS ONE | www.plosone.org 1. Establishment and Characterization of a kdr Homozygous Strain We initially assembled 68 crosses recruiting insects from a natural population of Ae. aegypti from Cachoeiro do Itapemirim, a Brazilian municipality. Allele specific PCR (see Methods) revealed that both parents of strain CIT-32 had genotype 1011 Ile/Ile (wild) +1016 Ile/Ile (mutant) for the AaNaV. However, besides homozygous for alteration in the pyrethroid target site, this strain displayed highly altered activity of enzymes related to metabolic resistance, particularly a-EST, pNPA-EST and GST, when compared to the susceptible reference Rockefeller strain, Rock (Figure 1, CIT-32). Since we aimed to determine the actual role of the kdr mutation on resistance and assess its fitness cost, it was necessary to reduce the contribution of metabolic resistance. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (Rock), a laboratory reference strain, for eight generations in order to bring the kdr mutation into the Rock susceptible genetic background, identifying the heterozygotes from each cross by allele specific PCR (see Methods). Afterwards, 1016 Ile/Ile homozygous individuals were produced by crosses between heterozygous parents, originating the Rock-kdr strain. This procedure resulted in a considerable decrease of the insecticide metabolic resistance of Rock-kdr as compared to CIT-32 (Figure 1). One exception was observed for MFO, an enzyme class exhibiting a slightly higher rate of altered individuals in Rockkdr than in CIT-32. It is of note that in the course of this study after establishing the CIT-32 and Rock-kdr strains we further evaluated the NaV 1534 site, carrying an additional substitution, Phe/Cys, recently related to pyrethroid resistance in Ae. aegypti natural populations from Grand Cayman and Thailand [22,27]. We confirmed that both strains were also homozygous for the mutation in that site indicating that they were probably in linkage disequilibrium in the original population. Bioassay. Dose-response bioassays with deltamethrin-impregnated papers showed that both Rock and the heterozygous Figure 1. Activity of enzymes related to insecticide metabolic resistance in Aedes aegypti strains. The cut-offs are (dashed lines) determined by the Rockefeller 99 percentile value of each enzyme (see [11]). Rockefeller is a reference strain of insecticide susceptibility and vigor. Distributions with less than 15% of individuals beyond the cut-off are considered unaltered. Between 15 and 50% are altered and above 50% are highly altered. CIT-32 is the original kdr strain, derived from a pyrethroid resistant Brazilian Aedes aegypti population. Rock-kdr is the kdr strain, backcrossed for eight generations with Rockefeller in order to reduce the contribution of detoxification enzymes to pyrethroid resistance. doi:10.1371/journal.pone.0060878.g001 2 64 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations Hib-F1 mosquitoes, derived from CIT-32 and Rock crosses, share a susceptible profile while CIT-32 and Rock-kdr strains exhibit high resistant levels (Figure 2). This result corroborates the recessive nature of the kdr mutations for pyrethroid resistance. Besides, since in Rock-kdr the insecticide metabolic resistance has been greatly weakened, the similarity of Rock-kdr and CIT-32 pyrethroid resistant profiles indicates that most of the resistance can be attributed to the kdr allele. 2. Fitness Cost – Developmental Parameters Larval development time and pupae formation rate. When Rock-kdr and Rock larvae were reared under controlled conditions, the latter developed faster. Regarding pupae which managed to develop up to the seventh day after larvae eclosion, there were around 40% more Rock than Rock-kdr (Figure 3). Although resistant larvae took more time to develop, there was no significant difference in the amount of pupation, i.e. viability from egg to pupae, between strains (t = 0.3055; df = 16, p = 0.764), reached 93.9% (61.3) and 94.6% (62.1) for Rock-kdr and Rock, respectively. Adult longevity. This parameter was evaluated under two distinct food regimens, sugar offered ad libitum (to males and females) or supplemented with blood feeding (females). The results are shown in Figure S1. As expected, male longevity (Figure S1-A) was lower than female (Figure S1-B). The analysis of the survival curves indicated no significant difference between Rock and Rockkdr for both males (x2 = 0.4153, df = 1, p = 0.5195) and females fed only with sugar (x2 = 3.809, df = 1, p = 0.0510). Mortality of bloodfed females, for both Rock and Rock-kdr, was far lower than the females fed only with sugar. Mortality of blood-fed females reached only 12.0% (62.8) and 6.5% (60.7) for Rock and Rockkdr, respectively, after 60 days of accompaniment (Figure S1-C). Their survival curves difference was also non-significant (x2 = 0.8671, df = 1, p = 0.3518). Therefore, we found no evidences that the kdr mutations interfere with adults’ longevity. Locomotor activity and circadian rhythm. Both Rock-kdr and Rock females presented diurnal habits, with peaks of activity at the beginning and end of the photophase. Although the pattern of activity was not altered in the pyrethroid resistant strain, the level of diurnal activity (during photophase) was significantly increased (t = 22.059; df = 206; p = 0.041) in pyrethroid resistant Figure 3. Comparison of larval development time between Rockefeller and Rock-kdr Ae. aegypti strains. Numbers represent the cumulative daily proportion of Rock and Rock-kdr pupae formation after larvae eclosion under standard laboratory conditions. SEM is indicated. Gray dotted line indicates equal proportion (rate = 1) between strains. doi:10.1371/journal.pone.0060878.g003 females (Figure 4). The same result was verified when the activity during the first 30 minutes after lights-on was not considered (t = 22.11; df = 170.87; p = 0.036), meaning that this significant increase observed in the pyrethroid resistant strain during the photophase was not due to the lights-on startle response. Blood feeding. The difference in average weight between Rock and Rock-kdr females before blood feeding was not significant (t = 0.3796, df = 4, p = 0.7235), indicating that there should be no differences in their total body size. Rock and Rockkdr females respectively, engorged approximately 100 and 87% of their weight (Figure S2), this difference neither being significant (t = 0.4418, df = 4, p = 0.6815). 3. Fitness Cost – Reproductive Parameters Female fecundity. In general, compared to Rock, fewer Rock-kdr females laid eggs and in smaller amounts (Table 1), females with few eggs or without eggs at all considered as noninseminated [28]. In this sense, fewer Rock-kdr females must have been inseminated (x2 = 13.83, df = 1, p,0.0002). Rock laid more eggs than Rock-kdr both considering exclusively females with more than 50 eggs (t = 2.580, df = 45, p,0.05) or including those with less than 50 eggs (t = 4.095, df = 74, p,0.001). The Figure 2. Linear regression curves of Aedes aegypti mortality after exposure to deltamethrin impregnated papers. Strains evaluated correspond to the susceptibility control (Rock), the 1016 Ile/ Ile selected strains with genetic background from a natural population (CIT-32) or from Rock (Rock-kdr), and the F1 offspring between Rock-kdr and Rock (Hib-F1). doi:10.1371/journal.pone.0060878.g002 PLOS ONE | www.plosone.org Figure 4. Locomotor activity Rock and Rock-kdr Ae. aegypti strains. Locomotor activity of susceptible (Rockefeller strain – blue line) and pyrethroid resistant (Rock-kdr – red line) Aedes aegypti females exposed two days under LD 12:12, at 25uC. Dotted lines represent standard errors. doi:10.1371/journal.pone.0060878.g004 3 65 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations df = 1, p = 0.8887). This indicates that there was no apparent insemination advantage or female preference for Rock males over Rock-kdr males. Population cage experiments. The fitness cost of the kdr mutations was also investigated in population cages examining the changes in the frequency of the 1016 Ile mutation, which is in complete linkage disequilibrium with the 1534 Cys mutation, over 15 non-overlapping generations under an insecticide free environment. This was performed for two initial 1016 Ile mutant allele frequencies, 50 and 75%. If the disadvantages herein noted for the Rock-kdr resulted in a real impact on fitness, one should expect that the wild allele would increase in frequency, which was indeed confirmed. In all cages, the mutant allele frequency tended to decay over the course of the 15 evaluated generations (Figure 7). When the initial 1016 Ile frequency was 50%, in the last generation the mutant allele decreased to an average frequency of 21.7% (32, 13 and 20%, respectively in cages 1, 2 and 3) (Figure 7-A). The same trend occurred in the cages where the initial frequency of the mutant allele was 75%, dropping to 30, 26 and 3%, respectively in cages 4, 5 and 6 (Figure 7-B). Detailed numbers for each cage throughout generations are presented in Table S3. Table 1. Oviposition performance of Rockefeller and Rockkdr Ae. aegypti females. Females with Rock (n) Rock-kdr (n) No eggs 11.1% (7) 18.8% (9) 1- 50 eggs 9.5% (6) 27.1% (13) .50 eggs 79.4% (50) 54.2% (26) Total (n) 63 48 doi:10.1371/journal.pone.0060878.t001 distribution of females of both Rock and Rock-kdr laying at least one egg is presented in Figure 5. Egg viability. The average rates (6 standard deviation) of hatched larvae were 90.8 (65.04) and 86.9 (63.80)% for Rock and Rock-kdr, respectively. This difference was not significant (t = 1.051, df = 4, p = 0.3527). 4. Competition Analysis Development time until adult. As observed under standard laboratory conditions (see Figure 3), Rock-kdr took more time to develop than Rock, also with higher larval density and lower food supply (Figure 6-A). When Rock and Rock-kdr were reared together, i.e. under inter-strain competition, most of the newly emerged adult males by the fourth day were Rock (Figure 6-B). Daily numbers of emerging adults, as well as data for determination of their strain in the inter-strain condition, are presented in Tables S1 and S2. Competition for insemination. A potential difference in insemination success between Rock and Rock-kdr males was tested in a pair of cages, both containing a similar number of males from each strain but only females Rock (in cage A) or Rock-kdr (in cage B). Considering that Ae. aegypti females mate only once, becoming refractory to further inseminations [29], if the offspring of a female was genotyped as homozygous (see Methods) it would mean that a male of the same strain was the father. In contrast, heterozygous offspring indicated insemination by a male from the other strain. For each cage, the offspring of 11 females was genotyped, and the number of homo or heterozygous offspring did not differ neither in cage A (x2 = 0.1640, df = 1, p = 0.6855) nor in cage B (x2 = 0.0196, Discussion The diagnostic and quantification of the kdr mutations in Ae. aegypti natural populations is nowadays an important tool to predict resistance to pyrethroids in the field [18,20,22,23,30]. In many localities the mutant allele Val1016Ile was found in high frequencies with tendency of rapid increase towards fixation [20,23]. If the mutation harbors a fitness cost in an insecticide-free environment, it is expected that once pyrethroid pressure ceases, the frequency of the mutant allele, and consequently pyrethroid resistance, will decrease. To date, evidences of physiological commitment in Ae. aegypti kdr mosquitoes were mainly observed in laboratory selected strains for pyrethroid resistance. These once established strains are generally difficult to maintain due to high mortality in early larval stages [23]. This might be by pleiotropy of the selected alleles or hitchhiking of deleterious alleles at other linked loci not specifically related to resistance. In this study we analyzed the fitness cost of the kdr mutation in Ae. aegypti, containing both Val1016Ile and Phe1534Cys substitutions. Differently from other reports, and aiming to avoid interference of other resistance mechanisms eventually co-selected with the kdr mutation, we selected neither a kdr strain from a field population nor a laboratory strain under insecticide pressure. Instead, we assembled crosses of randomly chosen mosquitoes belonging to a field population already exhibiting a high incidence of the 1016Ile mutant allele (42,5%) [20]. The resulting CIT-32 strain was homozygous for the kdr allele but also presented altered GST and esterase profiles, enzymes possibly enrolled in pyrethroid metabolic resistance [31]. In this sense and in order to guarantee unbiased comparisons of life-history trait parameters between the two strains, the CIT-32 strain genetic background was enriched with that of Rockefeller (Rock), a reference susceptibility and vigor strain which has been kept under laboratory conditions for many decades [32]. This procedure originated the Rock-kdr strain. The biochemical assay revealed that major esterase and GST activities were greatly reduced in Rock-kdr, although some pNPA-esterase and MFO remained. Contrastingly, pyrethroid resistance prevailed, with a resistance profile similar to CIT-32, suggesting the kdr mutation is indeed the major factor contributing to resistance, although we cannot exclude possible effects associated with closely linked genes. These results indicated that the developed Rock-kdr Figure 5. Number of eggs laid by females from Rock and Rockkdr Ae. aegypti strains. Each dot represents a single female. Only females that laid at least one egg were included. Median value with interquartile range is shown for each distribution. Dotted line points 50 eggs/female, which was herein empirically considered as discriminative of successful insemination. ***Difference between strains was highly significant by t test. doi:10.1371/journal.pone.0060878.g005 PLOS ONE | www.plosone.org 4 66 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations Figure 6. Developmental timing of Ae. aegypti Rock and Rock-kdr male adult emergence competing under a stringent condition. A – Cumulative rate of male emergence up to the 8th day after the beginning of adult emergence when the controls Rock and Rock-kdr were reared separately (‘intra-strain’ conditions). B – Cumulative proportion of Rock or Rock-kdr male emergence from the inter-strain competition. Male strain was daily determined by randomly genotyping 30% of emerging individuals. doi:10.1371/journal.pone.0060878.g006 organophosphate resistant Culex mosquitoes, such as decrease in the overwintering survival, reduced adult size, increased predation, longer developmental time and decreased male reproductive success, as reviewed elsewhere [35]. These examples are likely to derive from a trade-off between energetic resources and insecticide resistance since, for instance, an extreme over production of esterases was involved in most of the cases [12]. The affected parameters noted herein, however, are probably not related to deviation of resources originally destined to development and reproduction since we dealt with a single nucleotide polymorphism in the coding region of the sodium channel molecule, the pyrethroid target site. It cannot be ruled out that some gene variant involved with an unexplored characteristic hitchhiked together with the mutant sodium channel allele in the process of selection. However, in the lack of such evidence, it seems more parsimonious to assume that the kdr mutation itself was directly strain, pyrethroid resistant by target site alteration, was ready to be compared to Rock in order to specifically evaluate the kdr mutation fitness in an environment free of insecticide. Although our aim was to develop a kdr homozygous mutant lineage in the 1016 site, we later realized that the 1534 position also harbored a mutation in the original couple (1016 Ile +1534 Cys) that gave rise to the CIT-32 and Rock-kdr strains, suggesting that the two mutations are in linkage disequilibrium in the wild. Therefore, all the analyses we carried out accessed the combined fitness cost of both kdr mutations in this double mutant allele. Two main mechanisms, extensively studied in Culex mosquitoes, are commonly associated with fitness costs, resource based tradeoffs and oxidative stress [26,33,34]. This is easy to understand in the case of metabolic resistance, since an increased production of detoxifying enzymes likely implies commitment of resources that would be important for aspects of the fitness such as longevity and fecundity. Impairment of some life history traits has been noted in Figure 7. Population cage assays with pyrethroid resistant Aedes aegypti (Rock-kdr) and Rockefeller strains. The frequency of AaNaV alleles in the 1016 site was followed in independent cages kept under the same conditions, without insecticide exposure for 15 generations. The initial frequency of the 1016Ile kdr allele in cages 1–3 (A) was 0.50 and in cages 3–6 (B) was 0.75. Lines represent the linear regression analysis taken by the means of the mutant allele frequencies of the respective three cages in A (r2 = 0.5273, p = 0,0006) and B (r2 = 0,5690, p = 0,0003). doi:10.1371/journal.pone.0060878.g007 PLOS ONE | www.plosone.org 5 67 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations aspects of the vectorial capacity [42,43]. Under laboratory conditions, there was no significant mortality during the immature phase, and the resulting adults presented the same body weight and equivalent adult longevity when compared to the reference strain. Although the load of ingested blood did not differ between Rock and Rock-kdr females, the latter displayed reduction in the rate of insemination and number of laid eggs. This was surprising, since the number of eggs is generally directly related to the amount of ingested blood [28,44]. Notwithstanding, Rock-kdr larvae succeeded to hatch normally from inseminated eggs. Taken together, our results suggest that the kdr mutation does not interfere with embryonic development itself but with fecundity. Since rearing conditions are usually optimized in the laboratory, the fitness costs of some parameters here evaluated could be underestimated. Moreover, it is known that Ae. aegypti shows strong phenotypic responses to larval competition [45,46]. For instance, when reared under high larval density, the immature development time was longer, and the adults, besides being smaller and lighter, had reduced longevity [46]. In this sense, we also evaluated some life-history trait parameters under more stringent conditions and under inter-strain competition between susceptible and resistant kdr genotypes. Larvae from Rock-kdr and Rock genotypes were reared together under high larval density and limited food supply conditions, in parallel with controls consisting of only one genotype under the same conditions. The Rock-kdr larvae took more time to develop when competing with Rock, compared to the controls reared alone. We suggest that, despite larval density being the same in both situations, Rock-kdr larvae are less skilled to compete with Rock for food and space and the latter end up developing faster. In other words, the susceptible genotype was more competitive in terms of development timing. In this sense, simple food resource sharing implies additional and indirect costs. Competition derived fitness costs might be related to: i) higher accumulation of nitrogenous wastes at higher densities, ii) increased physical contact which might induce stress and iii) faster resource depletion due to a higher feeding rate and a reduced energetic efficiency [46]. In the present work, whichever were the effects impairing the normal fitness, we showed that individuals homozygous for the kdr mutations (and possibly other closely linked loci) were more susceptible to stringent conditions than their wild-type counterparts. Mating, copulation and finally, insemination efficiency are key factors in species whose females are inseminated only once during their lifespan, even though they have the potential to mate several times, such as Ae. aegypti [47]. In these cases, males must be able to compete for copula, since the first to inseminate the female will increase the chance of propagating its genes. In Culex pipiens mosquitoes, males from a susceptible strain exhibited an advantage over males bearing three distinct organophosphate resistant genotypes, when competing for mating with both insecticide susceptible or resistant females. Apart from specific resistance traits, all these strains shared the same genetic background, corroborating the relevant role of each evaluated resistance mechanism in this reproductive disadvantage [48]. Evolution of the kdr genotype and its effects on reproductive aspects have been better studied on the peach-potato aphid Myzus persicae, with at least one record of reproductive potential impairment of a kdr strain [49]. Here, we compared the ability of Rock-kdr and Rock males to inseminate females from both strains. Since Rock-kdr exhibited altered locomotor activity and reduction in the rate of inseminated females and in the number of eggs, one might expect differences in the competition for insemination. However, no differences between susceptible and resistant males were noted. linked to the fitness costs here presented in the absence of pyrethroid. In a very recent study [24], a laboratory selection for pyrethroid resistance starting from field Ae. aegypti populations with different resistance profiles showed consistent frequency increase of the 1016 Ile kdr allele. Its frequency was negatively correlated with the number of detoxifying genes differentially expressed (regardless of the down or up-regulation). The authors suggested that selection pressure for pyrethroid resistance favored the kdr mutation rather than metabolic alterations. Besides increase in the expression of two sigma class GST and at least 10 CYP genes related to metabolic resistance, the authors argued that there was weak selection for additional metabolic resistance genes in the insects previously ‘‘protected’’ with the kdr mutation [24]. It is not possible to generalize the resistance pleiotropic effects as negative, mainly when vector-parasite relationship is involved. For instance in Culex quinquefasciatus populations from Sri Lanka, a strong negative correlation was found between esterase activity accounting for organophosphate resistance and levels of the filariasis worm, Wuchereria bancrofti [36]. It was proposed that upregulation of carboxylesterases in response to insecticide resistance might also improve the insect immune system against pathogens [25]. Concerning kdr mutations, a study covering spatial and seasonal variation of malaria in Uganda showed the Leu1014Ser mutation frequency was significantly higher in An. gambiae infected with Plasmodium falciparum. The authors correlated the mutation with an increased adult longevity, resulting in higher chances of infection [37]. Target site resistance, as is the case herein, can directly influence behavioral aspects of the insect. For example, when exposed to a temperature gradient, houseflies with susceptible genotypes prefer warmer temperatures whilst individuals with the classical kdr mutation, Leu1014Phe, have no preferences [38]. The most striking results correlating kdr mutation with fitness cost were developed with the peach-potato aphids, Myzus persicae. Kdr insects presented reduced answer to the alarm pheromone, affecting the response to external stimuli as the presence of parasitoids [39]. Our Ae. aegypti Rock-kdr strain maintained the normal circadian activity, but the females’ locomotor activity was significantly increased. Although it is difficult to say whether this might cause an increase or decrease in fitness, as it probably depends on the specific environmental conditions, it is likely to have potential epidemiological consequences. Recently, it has been shown that infection with dengue virus also increases the locomotor activity of Ae. aegypti females [40]. It was assumed that this altered behavior might be translated into an increased biting rate displayed by infected mosquitoes which, based on a mathematical model, could result in dengue outbreaks with higher incidence of primary and secondary infections with severe biennial epidemics [41]. Hence, this behavioral change may directly influence traits directly related to vectorial capacity, e.g. longevity, blood feeding, intraspecific competition for resources and reproduction, here explored. An extensive review concerning pleiotropic effects of insecticide resistance mechanisms influencing insect vector capacity, either positively or negatively, was recently presented [26]. Nevertheless, kdr mutations were not cited in any context. We demonstrated that the kdr mutations (1016 Ile +1534 Cys) have a fitness cost in Ae. aegypti. Among several evaluated life-history trait parameters, the Rock-kdr strain presented some negative effects on larval development timing and reproductive aspects. In the field, a delay of larval development up to the adult stage is crucial. A slower development increases the chances of larvae predation, parasitism or even breeding site destruction. Moreover, larval developmental kinetics can be related to vector density, one of the determinant PLOS ONE | www.plosone.org 6 68 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations Despite no apparent cost on mating/insemination potential having been noted for the Rock-kdr strain, population cage assays definitively confirmed the fitness cost of the kdr mutation. In 15 consecutive non-overlapping generations, the 1016 Ile allele frequency dropped from 75 or 50 to less than 30% in most cages. The longer developmental time and the reproductive disadvantages of the Rock-kdr strain certainly contributed to this profile. Here we presented evidences of a consistent fitness cost of the Ae. aegypti kdr mutations (1016 Ile +1534 Cys) in homozygosis when exposed to an insecticide free environment, although as mentioned before we cannot exclude a possible effect of closely linked loci. The assays included the dynamics under competition with a susceptible genotype, when the mutant allele tendency to decrease in frequency became evident. These results are important for insecticide management activities, mainly those based on pyrethroids. Although this is presently the preferred class of insecticides, pyrethroid use is growingly precluded due to fast resistance dissemination in Ae. aegypti natural populations [21–23]. It is expected that wild-type NaV alleles could overlap kdr mutations in the absence of selection pressure with pyrethroids. However, the frequency of kdr mutations tends to increase very rapidly under positive selection [7]. Additionally, under continued selection pressure, it is likely that the fitness cost attributed to kdr mutations tends to decrease due to co-selection of modifier genes [35,50]. In this sense, enhanced surveillance for resistance should be a priority in areas where the kdr mutations are found. We are presently interested in determining the contribution of the isolated kdr alleles at positions 1016 and 1534 to both pyrethroid resistance and their related fitness cost. field population, backcrosses of CIT-32 and Rockefeller (Rock), a reference strain for vigor and insecticide susceptibility [32] were performed. The heterozygous offspring of Rock and CIT-32 was allowed to copulate and lay eggs. New isolated couples were then assembled as above and only eggs derived from heterozygous parents were induced to hatch. At the ninth generation, in order to restore the homozygous genotype for the kdr mutation, couples were assembled with 1016 Ile/Ile specimens, originating the strain herein named Rock-kdr. The Rock-kdr strain is then homozygous for the 1016 mutation but is expected to carry a general background genotype susceptible to insecticides, similar to Rockefeller mosquitoes. The F1 offspring between Rock-kdr females and Rock males (Hib-F1), heterozygous for the 1016 site (1016 Ile/Val), was also employed in some assays. Materials and Methods 3. Bioassays 2. Metabolic Resistance Assays The activity of the main enzymes related to metabolic resistance: glutathione-S-transferases (GST), mixed function oxidases (MFO) and esterases (EST), were evaluated in one-day-old adult females. With respect to esterases, substrates a-naphthyl, bnaphthyl and p-nitrophenyl acetate (herein referred to as a-EST, b-EST and pNPA-EST) were employed. Assays for each specimen and enzyme were performed in duplicate samples, in 96 microtiter plates, totaling 35–45 Rockefeller, 41–45 CIT-32, 39–45 Hib-H1 and 58–60 Rock-kdr individuals, according to the enzyme activity evaluated. Standard susceptible profiles were taken from Rockefeller values. Mosquitoes from this reference strain were also included in all plates as internal controls. Details of reaction and analysis are extensively described elsewhere [11,53]. The established strains (CIT-32, Rock-kdr and Rock) and HibF1 mosquitoes were submitted to dose-response bioassays in order to evaluate their profile of susceptibility to the pyrethroid, deltamethrin. The test was adapted from World Health Organization (WHO) and consists in confining mosquitoes in acrylic chamber tubes internally lined with Whatman grade nu1 papers [54] that had been previously impregnated in the lab with deltamethrin concentrations ranging from 4.2 to 1,050.0 mg/ paper. Approximately 20 females, around three-days-old, were exposed to the insecticide for 1 hour and then transferred to insecticide free rescue tubes, mortality scored 24 hours later. Three replicates/dosage were used, and each assay was executed three times. Control conditions, consisting of acrylic chambers lined only with paper impregnated with the solvent (silicone oil), were run in parallel. 1. Strains Establishment Isolation of 1016 Ile/Ile kdr homozygous strains. Larvae and adult mosquitoes were reared according to standard conditions previously described [51]. Mosquitoes from Cachoeiro do Itapemirim (CIT), Espı́rito Santo State, at the Southeastern Brazil, were chosen due to previous detection of the resistant allele at a high frequency in that locality [20]. The approach consisted of obtaining eggs from isolated couples. After genotyping all the adults, the offspring of those 1016Ile/Ile homozygous parents were selected to proceed up to the next generation. To assure virgin females would be used in the initial crosses, F1 specimens from Cachoeiro do Itapemirim, collected as described elsewhere [51], were reared until pupae, which were transferred to individual chambers. A total of 68 crosses were assembled with one male and three virgin females each inside 50 mL conical mesh covered tubes. The tubes were kept for at least three days, the insects fed ad libitum with a 10% sugar solution soaked cotton. One day after the sugar solution removal, females were allowed to feed on blood from anesthetized mice. After additional three days, females were individually induced to lay eggs in small Petri dishes lined with wet filter papers [52], as detailed further in the section 2.5. Meanwhile, males were genotyped for the 1011 and 1016 sites of the AaNaV, as described below. After egglaying, those females inseminated by 1016 Ile/Ile (mutant) and 1011 Ile/Ile (wild) homozygous males were also genotyped. Only eggs from crosses of the desired genotype, both parents homozygous 1011 Ile/Ile +1016 Ile/Ile, were induced to hatch. The progeny of one of the females used in cross number 32 originated strain CIT-32, which was further used in bioassays and biochemical tests. The CIT-32 strain was also adopted to establish the purified Rock-kdr strain, as stated below. In order to isolate the 1016 Ile/Ile mutation from other potential insecticide resistance mechanisms present in the original PLOS ONE | www.plosone.org 4. Evaluation of Developmental Parameters All parameters were evaluated by simultaneously comparing Rock and Rock-kdr, reared under identical conditions, such as initial larval density and feeding, temperature and illumination regimens. Eggs were induced to hatch for approximately 24 hours. Three replicates of 500 newly emerged larvae were then randomly transferred to plastic trays (3062165 cm) with 1 L dechlorinated water and a 0.5 g of cat food (FriskiesH, Purina, São Paulo/SP). New food supplement was offered every two days. Larval development time and pupae formation. The kinetics of pupae formation under the above conditions was accompanied daily as indicative of larval development time. This assay was performed three times. Adult longevity. Adults resulting from the item above, three to seven days after emergence, were randomly pooled in cylindrical cardboard cages (18630 cm) and submitted to two alternative food regimens: 1) groups of 50 couples received sugar 7 69 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations solution ad libitum as the only food source and 2) groups of 50 females, without males, received blood meals in addition to sugar solution. In this case, anesthetized mice were offered during 30 minutes on the second and 11th days after cage assembling. Mortality was scored every two or three days for approximately two months. This assay was conducted twice. Comparisons of survival curves were based on the Gehan-Breslow-Wilcoxon test using GraphPad Prism version 5.00. Locomotor activity and circadian rhythm. These parameters were evaluated with a Locomotor Activity Monitor (TriKinetics) as described in previous studies [28,55]. Four to five-day old females were individually placed in glass tubes with a cotton plug soaked in 10% sucrose solution and the tubes were placed in the Monitor inside a Precision Scientific Incubator Mod. 818 under constant temperature (25uC) and a 12 h light, 12 h dark photoperiod (LD 12:12). The locomotor activity was individually registered every time a mosquito crossed the middle of the tube, interrupting an incident infrared light. For every mosquito, 48 data points (representing the total locomotor activity of 30 min intervals) were obtained for every day of monitoring. Mosquitoes were allowed to acclimatize to the conditions inside the monitor tubes for two days and data from the third up to the sixth day of locomotor activity monitoring were used in the statistical analysis. Only data from mosquitoes that were alive up to the seventh day of monitoring were considered for the analysis, which was performed through calculation of the Williams average of their activity. In each assay a total of 32 individual females of each strain were evaluated. This assay was carried out twice. For statistical analysis, pyrethroid resistant and control Ae. aegypti groups were compared by t test with the SPSS software. Blood feeding. The amount of blood ingested by females was inferred as the weight ratio after and before the blood meal, as performed elsewhere [56]. To accomplish this, 100 three-day old females were deprived of sugar solution during 24 hours before the assay. Six to seven pools of five females each were killed (by rapid freezing) and weighed in an analytical balance (APX –200, Denver Instrument). In parallel, anesthetized mice were offered during 30 minutes to the remaining alive females. Additional six to seven groups of five engorged females were killed and weighed as above. The relative amount of ingested blood was obtained by comparing the average value of both groups. This assay was performed twice. and humid brush. Seven days after the eggs had dried, each replicate was individually submerged in 300 mL of dechlorinated water with 0.25 g of cat food (FriskiesH, Purina, São Paulo/SP) during 24h, and hatching larvae were counted. This assay was performed three times. 6. Competition Analysis Larval development time. Competition between Rock and Rock-kdr larvae was evaluated under space and food stringent conditions. Triplicates of 250 one-day old larvae of each strain were placed together in a small tray (3061865 cm) containing 800 mL dechlorinated water. Two pellets of cat food were added every four days. Controls consisted of trays with 500 Rock or Rock-kdr larvae under the same conditions and also in triplicate. Pupae were counted daily and transferred to cages. A total of 30% of emerged males, daily discriminated, were genotyped for the 1016 site (see [21]) in order to determine their strain. Competition for mating. Virgin adults reared under laboratory standard conditions were grouped in two cages, both containing 15 Rock and 15 Rock-kdr males. Cage A was completed with 30 Rock females and cage B, with 30 Rock-kdr females. After three days, females were blood fed and individually induced to oviposit. Larvae were stimulated to hatch as aforementioned. Aedes aegypti females are monogamous, meaning that they are inseminated only once [29]. Nevertheless, we analyzed the genotype of several larvae of an offspring, by pooling them in three groups of seven F1 L3 larvae of each female, by allelic-specific PCR [21]. Population cage experiments. The dynamics of kdr frequency in the absence of insecticide pressure was achieved by cage trial assays. Each cage had an initial 1016 Ile (the mutant allele) frequency of 50 or 75%. For the first case, cages were mounted with 30 Rock-kdr females and 30 Rock males and the other with 30 Rock-kdr females and 15 Rock-kdr +15 Rock males, rendering the initial allele proportion required. Three cages with each start frequency were rigorously kept under the same lab standard conditions during 15 generations without any gene flow among them. At each generation, females fed on blood twice, the first blood meal being offered at least seven days after the end of pupation. The first oviposition was used to rear the next generation, and the second one was kept as a backup. Eggs were induced to hatch in 24 hours, and two days later, 500 larvae were randomly transferred to a new tray. Nearly 30 males in each cage were genotyped at each generation. 5. Reproductive Parameters Female fecundity. Roughly equivalent numbers of males and females were confined in cages for at least three days before a blood meal was offered to females, as detailed above. According to our rearing conditions, this period is sufficient for insemination of all healthy females [28]. Three days after blood feeding, oviposition was induced [52]. Briefly, around 30 females from each strain were individualized in small Petri dishes (6 cm in diameter) lined with filter paper in the lids. After moistening the filter paper with 700 mL dechlorinated water, the Petri dishes remained in a humid chamber inside the insectary for two days, when the number of egglaying females and the amount of eggs/ female were recorded. Virgin females generally lay a smaller number of eggs or do not lay eggs at all [28]. Females were then classified in three groups: i) those that lay no eggs, ii) females laying less than 50 eggs and iii) females that oviposit 50 or more eggs. This assay was performed twice. Egg viability. Three days after blood feeding, around 100 females were transferred to a new cage, containing a black cup internally lined with filter paper and filed with dechlorinated water to receive ovipositing eggs. For each strain, three to four groups of 100 eggs, 24–48 hours old, were randomly gathered with a smooth PLOS ONE | www.plosone.org 7. Statistical Analysis The hypothesis tests for comparing the parameters between Rock and Rock-kdr were indicated in each assay methods and together with the results. Otherwise stated, graphs and analysis were performed with the software GraphPad Prism version 5.04 for Windows, GraphPad Software, La Jolla California USA. 8. Ethics Statement Mosquito blood feeding. Ae. aegypti females were fed on anesthetized mice (Ketamine:Xylazine 80–120 mg/kg:10–16 mg/ kg), accordingly to the institutional proceedings, [57] which is oriented by the national guideline ‘‘The Brazilian legal framework on the scientific use of animals’’ [58]. This study was reviewed and approved by the Fiocruz institutional committee ‘‘Comissão de Ética no Estudo de Animais’’ (CEUA/FIOCRUZ), license number: L-011/09. Entomological survey. The egg collections at Cachoeiro do Itapemirim were conducted by agents from the Health Secretariat of Espı́rito Santo State, following procedures designed by the 8 70 April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations National Program of Dengue Control/Ministry of Health-Brazil. Ovitraps were installed and collected in the dwellings with the residents’ permission. Table S2 Competition analysis, development time until adult. Number of individuals collected from each tray and observed genotypes. (PDF) Supporting Information Table S3 Population cage experiments. Numbers of individuals genotyped from each cage throughout generations. (PDF) Figure S1 Adult longevity of Rock-kdr and Rock Ae. aegypti strains. Survival curves of males (A) and females (B, C) fed exclusively with sugar solution (A, B) or with sugar and two blood meals (offered at days 2 and 11). (TIF) Acknowledgments We dedicate this paper to the memory of Alexandre A Peixoto. We thank the Brazilian Dengue Control Program that allowed utilization of samples collected in the scope of the Brazilian A. aegypti Insecticide Resistance Monitoring Network (MoReNAa) Blood feeding. Each dot represents a pool of females weight before and after blood feeding. Median and SE were evidenced. (TIF) Figure S2 Author Contributions Table S1 Competition analysis, development time until adult. Conceived and designed the experiments: AJM JBPL AAP DV. Performed the experiments: LPB JGBL AJM TAB TNLC. Analyzed the data: LPB AJM AAP DV. Contributed reagents/materials/analysis tools: AAP DV. Wrote the paper: AJM DV AAP. Number of daily emerged males. (PDF) References 1. WHO (2009) Dengue: guidelines for diagnosis, treatment, prevention and control. Geneva: World Healt Organization. 147 p. 2. Townson H, Nathan MB, Zaim M, Guillet P, Manga L, et al. (2005) Exploiting the potential of vector control for disease prevention. Bulletin of the World Health Organization 83: 942–947. 3. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, et al. (2010) Dengue: a continuing global threat. Nat Rev Microbiol 8: S7–16. 4. Nogueira RM, Eppinghaus AL (2011) Dengue virus type 4 arrives in the state of Rio de Janeiro: a challenge for epidemiological surveillance and control. Mem Inst Oswaldo Cruz 106: 255–256. 5. van den Berg H, Zaim M, Yadav RS, Soares A, Ameneshewa B, et al. (2012) Global Trends in the Use of Insecticides to Control Vector-Borne Diseases. Environmental Health Perspectives 120: 577–582. 6. Okumu FO, Moore SJ (2011) Combining indoor residual spraying and insecticide-treated nets for malaria control in Africa: a review of possible outcomes and an outline of suggestions for the future. Malar J 10: 208. 7. Barbosa S, Black WC 4th, Hastings I (2011) Challenges in estimating insecticide selection pressures from mosquito field data. PLoS Negl Trop Dis 5: e1387. 8. Marcombe S, Darriet F, Agnew P, Etienne M, Yp-Tcha MM, et al. (2011) Field efficacy of new larvicide products for control of multi-resistant Aedes aegypti populations in Martinique (French West Indies). American Journal of Tropical Medicine and Hygiene 84: 118–126. 9. Rodpradit P, Boonsuepsakul S, Chareonviriyaphap T, Bangs MJ, Rongnoparut P (2005) Cytochrome P450 genes: molecular cloning and overexpression in a pyrethroid-resistant strain of Anopheles minimus mosquito. J Am Mosq Control Assoc 21: 71–79. 10. Lumjuan N, Rajatileka S, Changsom D, Wicheer J, Leelapat P, et al. (2011) The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides. Insect Biochem Mol Biol 41: 203–209. 11. Montella IR, Martins AJ, Viana-Medeiros PF, Lima JB, Braga IA, et al. (2007) Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. American Journal of Tropical Medicine and Hygiene 77: 467– 477. 12. Montella IR, Schama R, Valle D (2012) The classification of esterases: an important gene family involved in insecticide resistance - A review. Mem Inst Oswaldo Cruz 107: 437–449. 13. Catterall WA, Chandy KG, Clapham DE, Gutman GA, Hofmann F, et al. (2003) International Union of Pharmacology: Approaches to the nomenclature of voltage-gated ion channels. Pharmacological Reviews 55: 573–574. 14. Ingles PJ, Adams PM, Knipple DC, Soderlund DM (1996) Characterization of voltage-sensitive sodium channel gene coding sequences from insecticidesusceptible and knockdown-resistant house fly strains. Insect Biochem Mol Biol 26: 319–326. 15. Pinto J, Lynd A, Elissa N, Donnelly MJ, Costa C, et al. (2006) Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon. Medical and Veterinary Entomology 20: 27–32. 16. Chen L, Zhong D, Zhang D, Shi L, Zhou G, et al. (2010) Molecular ecology of pyrethroid knockdown resistance in Culex pipiens pallens mosquitoes. PLoS One 5: e11681. 17. Park Y, Taylor MFJ (1997) A novel mutation L1029H in sodium channel gene hscp associated with pyrethroid resistance for Heliothis virescens (Lepidoptera: Noctuidae). Insect Biochemistry and Molecular Biology 27: 9–13. 18. Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M, Flores AE, et al. (2007) A mutation in the voltage-gated sodium channel gene associated PLOS ONE | www.plosone.org 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 9 71 with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol Biol 16: 785–798. Brengues C, Hawkes NJ, Chandre F, McCarroll L, Duchon S, et al. (2003) Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Medical and Veterinary Entomology 17: 87–94. Martins AJ, Lima JB, Peixoto AA, Valle D (2009) Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations. Tropical Medicine & International Health 14: 1351–1355. Martins AJ, Lins RM, Linss JG, Peixoto AA, Valle D (2009) Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil. American Journal of Tropical Medicine and Hygiene 81: 108–115. Harris AF, Rajatileka S, Ranson H (2010) Pyrethroid resistance in Aedes aegypti from Grand Cayman. American Journal of Tropical Medicine and Hygiene 83: 277–284. Garcia GP, Flores AE, Fernandez-Salas I, Saavedra-Rodriguez K, Reyes-Solis G, et al. (2009) Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in Mexico. Plos Neglected Tropical Diseases 3. Saavedra-Rodriguez K, Suarez AF, Salas IF, Strode C, Ranson H, et al. (2012) Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti. Insect Mol Biol 21: 61–77. Martins AJ, Ribeiro CD, Bellinato DF, Peixoto AA, Valle D, et al. (2012) Effect of insecticide resistance on development, longevity and reproduction of field or laboratory selected Aedes aegypti populations. PLoS One 7: e31889. Rivero A, Vezilier J, Weill M, Read AF, Gandon S (2010) Insecticide control of vector-borne diseases: when is insecticide resistance a problem? PLoS Pathog 6: e1001000. Yanola J, Somboon P, Walton C, Nachaiwieng W, Somwang P, et al. (2011) High-throughput assays for detection of the F1534C mutation in the voltagegated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand. Tropical Medicine & International Health 16: 501–509. Belinato TA, Martins AJ, Lima JB, Lima-Camara TN, Peixoto AA, et al. (2009) Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti. Mem Inst Oswaldo Cruz 104: 43–47. Bargielowski I, Alphey L, Koella JC (2011) Cost of mating and insemination capacity of a genetically modified mosquito Aedes aegypti OX513A compared to its wild type counterpart. PLoS One 6: e26086. Marcombe S, Poupardin R, Darriet F, Reynaud S, Bonnet J, et al. (2009) Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies). BMC Genomics 10: 494. Ranson H, Claudianos C, Ortelli F, Abgrall C, Hemingway J, et al. (2002) Evolution of supergene families associated with insecticide resistance. Science 298: 179–181. Kuno G (2010) Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains. Journal of Medical Entomology 47: 957–971. Chevillon C, Bourguet D, Rousset F, Pasteur N, Raymond M (1997) Pleiotropy of adaptive changes in populations: comparisons among insecticide resistance genes in Culex pipiens. Genetical Research 70: 195–203. Gazave E, Chevillon C, Lenormand T, Marquine M, Raymond M (2001) Dissecting the cost of insecticide resistance genes during the overwintering period of the mosquito Culex pipiens. Heredity (Edinb) 87: 441–448. April 2013 | Volume 8 | Issue 4 | e60878 Fitness Cost of Ae. aegypti kdr Mutations 35. Raymond M, Berticat C, Weill M, Pasteur N, Chevillon C (2001) Insecticide resistance in the mosquito Culex pipiens: what have we learned about adaptation? Genetica 112–113: 287–296. 36. McCarroll L, Paton MG, Karunaratne SH, Jayasuryia HT, Kalpage KS, et al. (2000) Insecticides and mosquito-borne disease. Nature 407: 961–962. 37. Verhaeghen K, Van Bortel W, Roelants P, Okello PE, Talisuna A, et al. (2010) Spatio-Temporal Patterns in kdr Frequency in Permethrin and DDT Resistant Anopheles gambiae s.s. from Uganda. American Journal of Tropical Medicine and Hygiene 82: 566–573. 38. Foster SP, Young S, Williamson MS, Duce I, Denholm I, et al. (2003) Analogous pleiotropic effects of insecticide resistance genotypes in peach-potato aphids and houseflies. Heredity (Edinb) 91: 98–106. 39. Foster SP, Denholm I, Poppy GM, Thompson R, Powell W (2011) Fitness tradeoff in peach-potato aphids (Myzus persicae) between insecticide resistance and vulnerability to parasitoid attack at several spatial scales. Bulletin of Entomological Research 101: 659–666. 40. Lima-Camara TN, Bruno RV, Luz PM, Castro MG, Lourenco-de-Oliveira R, et al. (2011) Dengue infection increases the locomotor activity of Aedes aegypti females. PLoS One 6: e17690. 41. Luz PM, Lima-Camara TN, Bruno RV, Castro MG, Sorgine MH, et al. (2011) Potential impact of a presumed increase in the biting activity of dengue-virusinfected Aedes aegypti (Diptera: Culicidae) females on virus transmission dynamics. Mem Inst Oswaldo Cruz 106: 755–758. 42. van Uitregt VO, Hurst TP, Wilson RS (2012) Reduced size and starvation resistance in adult mosquitoes, Aedes notoscriptus, exposed to predation cues as larvae. J Anim Ecol 81: 108–115. 43. Bourguet D, Guillemaud T, Chevillon C, Raymond M (2004) Fitness costs of insecticide resistance in natural breeding sites of the mosquito Culex pipiens. Evolution 58: 128–135. 44. Grech K, Maung LA, Read AF (2007) The effect of parental rearing conditions on offspring life history in Anopheles stephensi. Malar J 6: 130. 45. Agnew P, Hide M, Sidobre C, Michalakis Y (2002) A minimalist approach to the effects of density-dependent competition on insect life-history traits. Ecological Entomology 27: 396–402. 46. Bedhomme S, Agnew P, Sidobre C, Michalakis Y (2003) Sex-specific reaction norms to intraspecific larval competition in the mosquito Aedes aegypti. Journal of Evolutionary Biology 16: 721–730. PLOS ONE | www.plosone.org 47. Roth LM (1948) A study of mosqutio behavior. An experimental laboratory study of the sexual behaviour of Aedes aegypti Linnaeus. Am Midl Nat 40: 265– 352. 48. Berticat C, Boquien G, Raymond M, Chevillon C (2002) Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes. Genetical Research 79: 41–47. 49. Fenton B, Kasprowicz L, Malloch G, Pickup J (2010) Reproductive performance of asexual clones of the peach-potato aphid, (Myzus persicae, Homoptera: Aphididae), colonising Scotland in relation to host plant and field ecology. Bulletin of Entomological Research 100: 451–460. 50. Labbe P, Berticat C, Berthomieu A, Unal S, Bernard C, et al. (2007) Forty years of erratic insecticide resistance evolution in the mosquito Culex pipiens. PLoS Genet 3: e205. 51. Lima JB, Da-Cunha MP, Da Silva RC, Galardo AK, da Silva Soares S, et al. (2003) Resistance of Aedes aegypti to organophosphates in several municipalities in the State of Rio de Janeiro and Espirito Santo, Brazil. American Journal of Tropical Medicine and Hygiene 68: 329–333. 52. Farnesi LC, Martins AJ, Valle D, Rezende GL (2009) Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures. Memorias Do Instituto Oswaldo Cruz 104: 124–126. 53. Valle D, Montella IR, Medeiros PFV, Ribeiro RA, Martins AJ, et al. (2006) Quantification metodology for enzyme activity related to insecticide resistance in Aedes aegypti. Brası́lia: Ministério da Saúde/Brasil. 54. WHO (2006) Guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mosquito nets. Geneva: World Health Organization. 60 p. 55. Gentile C, Meireles-Filho AC, Britto C, Lima JB, Valle D, et al. (2006) Cloning and daily expression of the timeless gene in Aedes aegypti (Diptera:Culicidae). Insect Biochem Mol Biol 36: 878–884. 56. Belinato TA, Martins A, Valle D (2012) Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos. Memorias Do Instituto Oswaldo Cruz 107(7): 916–922. 57. CEUA (2008) [Manual de Utilização de Animais/Fiocruz]. Comissão de Ética no Uso de Animais de Experimentação. Rio de Janeiro: Fiocruz. 54. 58. Filipecki AT, Machado CJ, Valle S, Teixeira Mde O (2011) The Brazilian legal framework on the scientific use of animals. ILAR J 52: E8–15. 10 72 April 2013 | Volume 8 | Issue 4 | e60878 FIGURE S1 73 FIGURE S2 74 75 76 77 7. DISCUSSÃO Piretroides compõem a classe de inseticida mais comumente utilizada tanto na agricultura, quanto em campanhas de saúde pública, principalmente no uso doméstico. Isso se deve em grande parte a sua rápida ação contra o inseto, alta fotoestabilidade e baixa toxicidade para aves, répteis e mamíferos. No entanto, seu uso excessivo vem selecionando rapidamente populações de insetos resistentes de diferentes espécies e em diferentes localidades em todo o mundo. No Brasil, os piretroides tão logo foram introduzidos em escala nacional pelo PNCD para o controle de Ae. aegypti, poucos anos depois a resistência foi detectada (da-Cunha et al. 2005). Esta rápida seleção para resistência pode estar acontecendo devido a alguns fatores, tais como: i) prévia exposição ao inseticida, que já vinha selecionando as populações de Ae. aegypti antes das campanhas do PNCD; ii) resistência cruzada, selecionada pelos organofosforados via resistência metabólica; e iii) existência prévia de alelos de resistência, originárias das populações que possivelmente re-infestaram o país. No estado de São Paulo, os piretroides já vinham sendo utilizados há mais tempo em campanhas públicas contra o Ae. aegypti. Além disso, estes compostos vêm sendo aplicados contra os vetores de Chagas, malária e de leishmanioses, em várias regiões urbanas do país com ocorrência de Ae. aegypti. É possível, portanto, que a seleção à favor dos alelos de resistência a piretroides neste mosquito tenha se iniciado antes das campanhas do PNCD. Com relação à resistência cruzada, uma pré-disposição à detoxificação de piretroides selecionada pelo uso maciço de organofosforado pode ter selecionado incremento ou modificação na atividade detoxificante nos insetos. Apesar de avanços recentes na identificação de genes relacionados à resistência metabólica via detoxchip (Bariami et al. 2012; Poupardin et al. 2012; Strode et al. 2008), ainda é difícil sugerir alguma dinâmica de seleção pelos organofosforados, que também leve à metabolização eficiente de piretroides. Finalmente, há evidências de que as populações de Ae. aegypti presentes no país resultam de múltiplas introduções vindas, principalmente, de países latino americanos (onde não ocorreu erradicação do vetor nas campanhas das décadas de 1960-70) e asiáticos a partir da década de 1980 (Bracco et al. 2007). É possível, portanto, que novas infestações tenham ocorrido, trazendo populações já resistentes a piretroides. Com relação aos alelos kdr de Ae. aegypti, dois sítios do NaV vêm sendo consideradas importantes: 1016 e 1534. Embora estejam no mesmo gene, as referências que consideraram a genotipagem de ambos os sítios trataram-lhes como independentes (Harris et al. 2010a; Kawada et al. 2009; Seixas et al. 2013; Stenhouse et al. 2013), dificultando, a nosso ver, a compreensão da distribuição dos reais alelos de resistência, que podem ter um ou os dois sítios mutados. No artigo apresentado no capítulo 1 desta dissertação, a genotipagem de cada indivíduo considerou ambos os sítios, revelando a ocorrência de três alelos em populações brasileiras. O alelo NaVR1, mutante no sítio 78 1534, mostrou-se presente em todas as localidades avaliadas mais recentemente. No entanto, estava ausente nas amostras mais antigas de 2000, 2001 e 2002, respectivamente, em Duque de Caxias/RJ, Aracajú/SE e São Gonçalo/RJ. O alelo NaVR2, mutante nos dois sítios, também não estava presente nestas amostras, e nas mais recentes mostrou frequências altas nas regiões centro-sul do país, porém esteve ausente ou em baixas frequências nas localidades do estado do Pará e no nordeste, exceto Aracajú/SE. Resultados preliminares da genotipagem de populações de localidades do Grande Rio de Janeiro/ RJ, coletadas em 2012, mostraram alta frequência de ambos os alelos kdr. Estas frequências estavam bastante similares entre as localidades, sendo aquelas mais diferentes, com menores frequências de NaVR1 e NaVR2, observadas em Paquetá, que é uma ilha na Baía de Guanabara, com acesso restrito por via marítima (anexo I). O fato do alelo NaVR1 estar mais disseminado sugere que o mesmo tenha surgido no Brasil há mais tempo que o NaVR2, ou ainda que tenha um menor custo evolutivo. É possível que este segundo tenha derivado do primeiro, já que não observamos um alelo contendo mutação apenas no sítio 1016.Outra hipótese, não excludente, é que tenham ocorrido eventos independentes de seleção de distintos haplótipos contendo a mutação Phe1534Cys. Vale ainda ressaltar que substituição homóloga ao Phe1534Cys foi também observada em Ae. albopictus de Singapura (Kasai et al. 2011). Em An. gambiae, a clássica mutação kdr Leu1014Phe teve grande dispersão pelo continente africano, mas também surgiu de novo, pelo menos quatro vezes independentemente (Pinto et al. 2007). No caso do Ae. aegypti, é notável que duas mutações distintas ocorrem no sítio 1016: Val101Ile na América Latina e Val1016Gly na Ásia. Já a mesma mutação Phe1534Cys é observada em ambos os locais. Stenhouse e colaboradores (2013) mostraram que em populações tailandesas de Ae. aegypti, indivíduos homozigotos para a mutação Phe1534Cys não apresentavam a Val1016Gly, ou seja, a ocorrência asiática do alelo NaVR1 ou similar. Experimentos que definam a origem e dispersão dos haplótipos kdr, a partir do sequenciamento dos íntrons próximos às mutações, por exemplo, de populações de vários países e das diferentes regiões do Brasil, certamente ajudarão a traçar a rota evolutiva das mutações kdr no país. Com relação à contribuição dos alelos kdr para a resistência, há relato de que a mutação Phe1534Cys não seja importante para resistência a piretroides do tipo II, como a deltametrina (Hu et al. 2011), produto utilizado nas campanhas pelo PNCD. No entanto, observamos aumento da frequência de NaVR1 e NaVR2, ambos com a mutação Phe1534Cys. Além disso, resultados nossos, ainda que preliminares, mostraram que uma linhagem homozigota para o alelo NaVR1, sem evidência de alterações metabólicas, é resistente à deltametrina (anexo II). Estes mesmos ensaios mostram ainda que o alelo NaVR2 confere aproximadamente 1,7 vezes mais resistência que o alelo NaVR1, o que pode explicar a maior frequência daquele alelo nas localidades centro-sul do Brasil. 79 Além dos alelos NaVR1 e NaVR2, apresentamos evidências da ocorrência de duplicação gênica no NaV (capítulo 3). A investigação da mutação Ile1011Met nos sugeriu duplicação no NaV de Ae. aegypti, uma vez que todos os indivíduos portadores daquela mutação eram heterozigotos. Em seguida, o sequencimaneto da região IIS6 do NaV de indivíduos de populações naturais e as análise de cruzamentos de uma linhagem selecionada no laboratório fortaleceram nossa hipótese. Duplicações gênicas envolvendo estruturas alvo de inseticidas neurotóxicos já era conhecidas para o ace-1 de Culex e An. gambiae e, mais recentemente, para rdl, codificante do receptor de GABA, alvo do organoclorado dieldrien, de D. melanogaster (Remnant et al. 2013b). O primeiro trabalho sugerindo duplicação no NaV de inseto foi descrito para a barata Periplaneta amaericana (Moignot et al. 2009) e, em seguida, para o mosquito C. quinquefasciatus (Xu et al. 2011). Aqui, usamos abordagens diferentes daqueles trabalhos. No primeiro caso, os autores clonaram e sequenciaram a versão expressa do possível gene duplicado. Já em Culex, concluiu-se que havia múltiplas cópias do NaV, via análises de hibridização contra o genoma (Southern blot). A nossa linhagem ‘EE’ homozigota para a duplicação foi originária de pressão de seleção em laboratório com deltametrina, a qual elevou a resistência até a quinta geração e depois diminuiu (Martins et al. 2012). De fato, ensaios preliminares, mostraram que curiosamente esta linhagem não é resistente (anexo II). A frequência da mutação 1011 Met, que usamos como marcador da duplicação, vem progressivamente diminuindo no país à medida que os alelos kdr NaVR1 e NaVR2 estão aumentando (dados não mostrados). Desta forma, parece que esta duplicação não deve estar relacionada à resistência. Contudo, é provável que existam outros alelos duplicados para o NaV de Ae. aegypti. Realizamos alguns ensaios para investigar polimorfismo no número de cópias deste gene em populações naturais e linhagens de laboratório. Resultados preliminares apontam amplificação gênica, onde o número de cópias tende a ser maior e mais homogêneo na região centro-sul, com média de duas a três vezes o número de cópias da linhagem Rock, do que quando comparado à região nordeste do Brasil, com média de uma a duas vezes o número de cópias (anexo III). Na região centro-sul é onde há menor frequência da mutação 1011 Met, de forma que a amplificação observada deve ser por conta de outros alelos. Ou seja, é possível que hajam alelos NaVR1 e/ou NaVR2 duplicados. Ensaios de quantificação de cópias gênicas e de genotipagem dos sítios polimórficos individuais, em conjunto com bioensaios serão importantes para ajudar a definir melhor o caráter e o papel das duplicações no NaV de Ae. aegypti. A comparação de principais parâmetros da tabela de vida dos insetos entre linhagens susceptíveis e resistentes via mutação kdr são essenciais para estimativa do custo evolutivo dos alelos mutantes. Este conhecimento pode nos ajudar a compreender o efeito de dispersão dos alelos de resistência tanto na presença quanto na sua ausência do inseticida, contribuindo com estratégias no manejo correto de inseticidas. 80 Como foi mostrado no artigo do capítulo 2 desta dissertação, isolamos uma linhagem homozigota para o alelo NaVR2 com o background genético da cepa Rock. Esta linhagem, então chamada de Rock-Kdr, apresentou alterações, que lhes renderam diminuição consistente da frequência alélica ao longo de gerações em ambiente livre de inseticida. Um trabalho recente que também visava avaliar o fitness da mutação kdr em Ae. aegypti, comparou duas linhagens originárias de mesma localidade tailandesa e mantidas no laboratório por mais de 10 anos. Uma delas (PMD) era resistente a DDT e a outra tanto a DDT quanto à permetrina (PMD-R), pois vem sofrendo processo constante de seleção com este inseticida. Diferentemente de nosso estudo, foi observada uma distorção sexual à favor das fêmeas na linhagem resistente à piretroide, embora com menor tamanho. Além disso, as fêmeas PMD-R tiveram maior taxa de viabilidade dos ovos. Outros fatores como longevidade e taxa de insemninação não difereiu entre as linhagens, de forma que os autores concluíram que a mutação Phe1534Cys não acarreta um alto custo evolutivo à espécie (Stenhouse et al. 2013). Entretanto, há que se considerar que a comparação foi feita entre duas linhagens já resistentes: ambas possuiam, por exemplo, uma atividade de P450 10 vezes acima da cepa Rock. Além disso, em uma linhagem que vem sendo há uma década constantemente selecionada em laboratório, é possível que genes modificadores venham sendo selecionados para amenizar os efeitos colaterais da resistência. Selecionamos uma linhagem homozigota para o alelo NaVR1, a partir de retrocurzamentos de uma população de campo (Santarém), onde o outro alelo kdr estava ausente, com nossa linhagem Rock-kdr, a fim de podermos comparar as linhagens kdr com Rock, todas com o mesmo background genético. Como anteriormente apresentado, a nova linhagem, R1R1, é resistente, ainda que com menor razão de resistência do que a mutante em ambos os sítios (anexo II). Bioensaios entre os possíveis genótipos, avaliação dos parâmetros da tabela de vida e de caixas de população partindo-se de diferentes frequências genotípicas iniciais nos ajudarão a definer melhor o efeito das mutações kdr na resistência e no fitness do inseto. Aspectos moleculares e evolutivos da resistência precisam ser melhores estudados e compreendidos com a finalidade de melhorar a dinâmica no controle do vetor, junto às estratégias alternativas de controle que vêm sendo propostas, como o uso de mosquitos geneticamente modificados (Kidwell & Ribeiro 1992; Speranca & Capurro 2007) ou ainda de bactérias endossimbiontes que diminuem a capacidade vetorial do inseto (Maciel-de-Freitas et al. 2012; Moreira et al. 2009). No entanto, até que o uso destas novas ferramentas esteja de fato disponível para aplicações em campo, os inseticidas devem continuar a desempenhar um papel importante, principalmente nos períodos epidêmicos. Atualmente, em decorrência da disseminação de resistência a piretroides em todo o país, o PNCD está substituindo a classe de inseticidas usados em aplicações espaciais contra mosquitos adultos (“fumacê”). Apesar disto, a população faz intenso uso doméstico de inseticidas, que em sua 81 quase totalidade pertencem à classe dos piretroides, devido a sua ação rápida contra insetos susceptíveis (efeito knockdown) e baixa toxicidade aos mamíferos. Tudo isso leva a necessidade do monitoramento da resistência a inseticidas nas populações de campo e a necessidade do estudo dos mecanismos de seleção, bem como dos seus efeitos na capacidade vetorial do inseto em relação ao funcionamento efetivo das estratégias de controle da doença. Em conjunto, os resultados aqui esperados podem contribuir para os estudos de genética evolutiva da resistência aos inseticidas e para o monitoramento da resistência a piretroides em populações naturais de Ae. aegypti. 82 ANEXOS I - Distribuição das mutações kdr em populações de Aedes aegypti do Grande Rio ! ! Phe ! !Val ! ! Cys ! !Ile ! ! Cys ! ! 1534 1016 Val ‘VP’ ‘VC’ CER CAB MOQ ‘IC’ HEL TUB JGU PAQ PAV Baixada Fluminense OLA VAZ VAL FON Rio de Janeiro TAQ SFR Niterói JUR MEI ITA PIR CAJ RDP GRA SCR RCO HUM URC GAM Figura com o padrão de distribuição dos alelos NaVR1 (laranja); NaVR2 (vermelho) e NaVS (azul) em populações do Grande Rio e entorno. CAB (Cabuçu); CER (Cerâmica); MOQ (Moquetá); HEL (Heliópolis); OLA (Olaria); TUB (Tubiacanga); JGU (Jardim Guanabara); PAQ (Paquetá); FON (Fonseca); SFR (São Francisco); JUR (Jurujuba); ITA (Itacoatiara); PIR (Piratininga); GAM (Gamboa); URC (Urca); HUM (Humaitá); RCO (Rio Comprido); SCR (São Cristóvão); GRA (Grajaú); RDP (Rio das Pedras); CAJ (Cajú); MEI (Méier); TAQ (Taquara); VAL (Valqueire); VAZ (Vaz Lobo) e PAV (Pavuna). 83 II - Perfil de susceptibilidade/resistência à deltametrina de linhagens kdr homozigotas de Aedes aegypti Deltametrina (340mg/L) Mortalidade (%) 100 Rock (S) EE R1R1 R2R2 80 60 40 20 0 0 10 20 30 40 50 60 Tempo (minutos) Bioensaio do tipo tempo-resposta, em papéis impregnados com deltametrina (340 mg/L). Rock = cepa Rockefeller; Linhagens kdr: EE (homizgota para duplicação), R1R1 (homozigota para alelo NaVR1) e R2R2 (homozigota para alelo NaVR2) 84 R ef número de cópias (pop/rock) D erê up n x cia D Ro up c lic k ad R a oc k B Wh or i a- te B or a III - Variação do número de cópias gênicas do NaV em populações naturais de Aedes aegypti 6 5 4 3 2 1 Sa B nta el fo Ro r Sã d R sa o ox - R Si o S C am G mã - R po oiâ o - J s nia GO B Sa el - G n os O C tar - G as é t m O O an - P ia ha A po l qu - P e A -A P 0 Ensaio do tipo copy number variation em PCR em tempo real, por amplificação do fragmento IIS6 do NaV em comparação ao rp49 de Ae. aegypti. À esquerda, cepas de laporatório (Rock, white, Bora-Bora) e linhagem duplicada (Duplicada). À direita, populações do campo. 85 8. PERSPECTIVAS As publicações presentes nesta dissertação geraram perguntas e hipóteses que devem ser investigadas. Precisamos definir a genotipagem de amostras de Ae. aegypti das localidades do Grande Rio, coletadas um ano após daquelas apresentadas no Anexo I. Uma linhagem homozigota para o alelo NaVR1, com background genético semelhante à cepa Rock e à linhagem Rock-kdr, já foi estabelecida e bioensaios para avaliação da resistência entre os híbridos destas linhagens estão em andamento. Esperamos avaliar o fitness dos diferentes alelos kdr. Além disso, precisamos entender melhor a questão da duplicação, partindo para uma análise mais molecular, na intenção de clonar o gene inteiro, avaliar sua localização física no genoma e seus níveis de expressão. 86 9. CONCLUSÕES - Há ocorrência de pelo menos dois alelos kdr em populações naturais brasileiras de Ae. aegypti: um com a mutação Phe1534Cys (NaVR1) e outro com ambas as mutações Val1016Ile + Phe1534Cys (NaVR2). - Estes alelos estão distribuídos no país de forma regionalizada: NaVR1 por todo o território e NaVR2 principalmente na região centro-sul, com indícios de rápido aumento de frequência. - O alelo kdr NaVR2 tem um alto custo evolutivo em ambiente livre de inseticida: maior tempo de desenvolvimento larvar e redução tanto na quantidade de fêmeas que colocam ovos, como no número destes. Além disso, apresentam alteração na atividade locomotora. - A frequência de NaVR2 diminuiu significativamente ao longo de 15 gerações, sem pressão de seleção, no laboratório. - Além das mutações kdr, levantamos a hipótese e mostramos uma série de evidências à favor da duplicação do gene do canal de sódio de Ae. aegypti. 87 10. REFERÊNCIAS BIBLIOGRÁFICAS Adams B, Holmes EC, Zhang C, Mammem Jr MP, Nimmannaitya S, Kalayanarooj S, Boots M 2006. Cross-protective immunity can account for the alternating epidemic pattern of dengue vírus serotypes circulating in Bangkok. PNAS, 103, 38, 14234 –14239. Ang LH, Nazni WA, Kuah MK, Shu-Chien AC, Lee CY 2013. Detection of the A302S Rdl mutation in fipronil bait-selected strains of the German cockroach (Dictyoptera: Blattellidae). J Econ Entomol, 106, 2167-2176. Asih PB, Syahrani L, Rozi IE, Pratama NR, Marantina SS, Arsyad DS, Mangunwardoyo W, Hawley W, Laihad F, Shinta, Sukowati S, Lobo NF, Syafruddin D 2012. Existence of the rdl mutant alleles among the anopheles malaria vector in Indonesia. Malaria journal, 11, 57. Bariami V, Jones CM, Poupardin R, Vontas J, Ranson H 2012. Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti. PLoS Negl Trop Dis, 6, e1692. Bass C, Field LM 2011. Gene amplification and insecticide resistance. Pest Manag Sci, 67, 886-889. Beaty BJ, Marquardt WC 1996. The Biology of Disease Vectors. University Press of Colorado, Colorado. Belinato TA, Martins A, Valle D 2012. Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos. Mem I Oswaldo Cruz, in press. Bingham G, Strode C, Tran L, Khoa PT, Jamet HP 2011. Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid-resistant Aedes aegypti? Trop Med Int Health, 16, 492-500. Bracco JE, Capurro ML, Lourenco-de-Oliveira R, Sallum MA 2007. Genetic variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of multiple introductions. Mem Inst Oswaldo Cruz, 102, 573-580. Braga IA, Lima JB, Soares Sda S, Valle D 2004. Aedes aegypti resistance to temephos during 2001 in several municipalities in the states of Rio de Janeiro, Sergipe, and Alagoas, Brazil. Mem Inst Oswaldo Cruz, 99, 199-203. Braga IA, Valle D 2007a. Aedes aegypti: histórico do controle no Brasil. Epidemiol Serv Saúde, 16, 113-118. —— 2007b. Aedes aegypti: inseticidas, mecanismos de ação e resistência. Epidemiol Serv Saúde, 16, 277-291. —— 2007c. Aedes aegypti: vigilância, monitoramento da resistência e alternativas de controle no Brasil. Epidemiol Serv Saúde, 16, 295-302. Nova classificação de caso de dengue – OMS [homepage on the Internet]: Ministério da Saúde 2014. Available from: http://dtr2004.saude.gov.br/sinanweb/novo/Download/Nova_classificacao_de_caso_de_dengue_OMS.pdf. Brathwaite Dick O, San Martin JL, Montoya RH, del Diego J, Zambrano B, Dayan GH 2012. The history of dengue outbreaks in the Americas. Am J Trop Med Hyg, 87, 584-593. Brengues C, Hawkes NJ, Chandre F, McCarroll L, Duchon S, Guillet P, Manguin S, Morgan JC, Hemingway J 2003. Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med Vet Entomol, 17, 87-94. Brun-Barale A, Bouvier JC, Pauron D, Berge JB, Sauphanor B 2005. Involvement of a sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and development of a diagnostic test. Pest Manag Sci, 61, 549-554. Busvine JR 1951. Mechanism of resistance to insecticide in houseflies. Nature, 168, 193-195. Casida JE 1980. Pyrethrum flowers and pyrethroid insecticides. Environ Health Perspect, 34, 189-202. Catão RF 2011. Dengue no Brasil : abordagem geográfica na escala nacional. Dissertação (mestrado), Universidade Estadual Paulista, Presidente Prudente 169 pp. Chandre F, Darriet F, Darder M, Cuany A, Doannio JMC, Pasteur N, Guillet P 1998. Pyrethroid resistance in Culex quinquefasciatus from West Africa. Med Vet Entomol, 12, 359-366. Chevillon C, Bourguet D, Rousset F, Pasteur N, Raymond M 1997. Pleiotropy of adaptive changes in populations: comparisons among insecticide resistance genes in Culex pipiens. Genet Res, 70, 195203. 88 Consoli R, Lourenço-de-Oliveira R 1994. Principais mosquitos de importância sanitária no Brasil. Ed Fiocruz, Rio de Janeiro. da-Cunha MP, Lima JB, Brogdon WG, Moya GE, Valle D 2005. Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003. Mem Inst Oswaldo Cruz, 100, 441-444. Dick OB, San Martin JL, Montoya RH, del Diego J, Zambrano B, Dayan GH 2012. The History of Dengue Outbreaks in the Americas. Am J Trop Med Hyg, 87, 584-593. Domingues LN, Guerrero FD, Becker ME, Alison MW, Foil LD 2013. Discovery of the Rdl mutation in association with a cyclodiene resistant population of horn flies, Haematobia irritans (Diptera: Muscidae). Veterinary parasitology, 198, 172-179. Donnelly MJ, Corbel V, Weetman D, Wilding CS, Williamson MS, Black WCt 2009. Does kdr genotype predict insecticide-resistance phenotype in mosquitoes? Trends in parasitology, 25, 213-219. Egan H 1966. Pesticide residues in fat-containing foods and in human fat. The Proceedings of the Nutrition Society, 25, 44-51. ffrench-Constant RH, Pittendrigh B, Vaughan A, Anthony N 1998. Why are there so few resistance-associated mutations in insecticide target genes? Philos Trans R Soc Lond B Biol Sci, 353, 16851693. Forattini OP 2002. Culicidologia Médica: Identificação, Biologia, Epidemiologia. Vol. 2, EdUSP, São Paulo, 549 pp. Force A, Lynch M, Pickett FB, Amores A, Yan YL, Postlethwait J 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics, 151, 1531-1545. Garcia GP, Flores AE, Fernandez-Salas I, Saavedra-Rodriguez K, Reyes-Solis G, LozanoFuentes S, Guillermo Bond J, Casas-Martinez M, Ramsey JM, Garcia-Rejon J, Dominguez-Galera M, Ranson H, Hemingway J, Eisen L, Black IW 2009. Recent rapid rise of a permethrin knock down resistance allele in Aedes aegypti in Mexico. PLoS Negl Trop Dis, 3, e531. Gazave E, Chevillon C, Lenormand T, Marquine M, Raymond M 2001. Dissecting the cost of insecticide resistance genes during the overwintering period of the mosquito Culex pipiens. Heredity, 87, 441448. Georgopoulos GD 1954. Extension to chlordane of the resistance to DDT observed in Anopheles sacharovi. Bull World Health Organ, 11, 855-864. Hanley KA, Weaver SC 2008. Origin and Evolution of Viruses. . Elsevier, Oxford. Harris AF, Nimmo D, McKemey AR, Kelly N, Scaife S, Donnelly CA, Beech C, Petrie WD, Alphey L 2011. Field performance of engineered male mosquitoes. Nat Biotechnol, 29, 1034-1037. Harris AF, Rajatileka S, Ranson H 2010a. Pyrethroid resistance in Aedes aegypti from Grand Cayman. Am J Trop Med Hyg, 83, 277-284. —— 2010b. Pyrethroid resistance in Aedes aegypti from Grand Cayman. The American journal of tropical medicine and hygiene, 83, 277-284. Hemingway J, Hawkes NJ, McCarroll L, Ranson H 2004. The molecular basis of insecticide resistance in mosquitoes. Insect Biochem Mol Biol, 34, 653-665. Hemingway J, Ranson H 2000. Insecticide resistance in insect vectors of human disease. Annual Review of Entomology, 45, 371-391. Hougard JM, Duchon S, Darriet F, Zaim M, Rogier C, Guillet P 2003. Comparative performances, under laboratory conditions, of seven pyrethroid insecticides used for impregnation of mosquito nets. Bull World Health Organ, 81, 324-333. Hu Z, Du Y, Nomura Y, Dong K 2011. A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids. Insect Biochem Mol Biol, 41, 9-13. Kasai S, Ng LC, Lam-Phua SG, Tang CS, Itokawa K, Komagata O, Kobayashi M, Tomita T 2011. First detection of a putative knockdown resistance gene in major mosquito vector, Aedes albopictus. Japanese journal of infectious diseases, 64, 217-221. Kawada H, Higa Y, Komagata O, Kasai S, Tomita T, Thi Yen N, Loan LL, Sanchez RA, Takagi M 2009. Widespread distribution of a newly found point mutation in voltage-gated sodium channel in pyrethroidresistant Aedes aegypti populations in Vietnam. PLoS Negl Trop Dis, 3, e527. Kidwell MG, Ribeiro JM 1992. Can transposable elements be used to drive disease refractoriness genes into vector populations? Parasitology today, 8, 325-329. Kimura M, King JL 1979. Fixation of a deleterious allele at one of two "duplicate" loci by mutation pressure and random drift. Proc Natl Acad Sci U S A, 76, 2858-2861. 89 Kingsolver J, Raymond BH 2008. Size, temperature, and fitness: three rules. Evolutionary Ecology Research, 10, 251-268. Kumar S, Thomas A, Sahgal A, Verma A, Samuel T, Pillai MK 2002. Effect of the synergist, piperonyl butoxide, on the development of deltamethrin resistance in yellow fever mosquito, Aedes aegypti L. (Diptera: Culicidae). Arch Insect Biochem Physiol, 50, 1-8. Labbe P, Berthomieu A, Berticat C, Alout H, Raymond M, Lenormand T, Weill M 2007a. Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens. Mol Biol Evol, 24, 1056-1067. Labbe P, Berticat C, Berthomieu A, Unal S, Bernard C, Weill M, Lenormand T 2007b. Forty years of erratic insecticide resistance evolution in the mosquito Culex pipiens. PLoS Genet, 3, e205. Lima EP, Paiva MH, de Araujo AP, da Silva EV, da Silva UM, de Oliveira LN, Santana AE, Barbosa CN, de Paiva Neto CC, Goulart MO, Wilding CS, Ayres CF, de Melo Santos MA 2011. Insecticide resistance in Aedes aegypti populations from Ceara, Brazil. Parasit Vectors, 4, 5. Lima JB, Da-Cunha MP, Da Silva RC, Galardo AK, Soares Sda S, Braga IA, Ramos RP, Valle D 2003. Resistance of Aedes aegypti to organophosphates in several municipalities in the State of Rio de Janeiro and Espirito Santo, Brazil. Am J Trop Med Hyg, 68, 329-333. Linss JG, Brito LP, Garcia GA, Araki AS, Bruno RV, Lima JB, Valle D, Martins AJ 2014. Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations. Parasit Vectors, 7, 25. Maciel-de-Freitas R, Aguiar R, Bruno RV, Guimaraes MC, Lourenco-de-Oliveira R, Sorgine MH, Struchiner CJ, Valle D, O'Neill SL, Moreira LA 2012. Why do we need alternative tools to control mosquitoborne diseases in Latin America? Mem Inst Oswaldo Cruz, 107, 828-829. Marcombe S, Poupardin R, Darriet F, Reynaud S, Bonnet J, Strode C, Brengues C, Yebakima A, Ranson H, Corbel V, David JP 2009. Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies). Bmc Genomics, 10, 494. Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Berge JB, Devonshire AL, Guillet P, Pasteur N, Pauron D 1998. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae S.S. Insect Mol Biol, 7, 179-184. Martins AJ, Lima JB, Peixoto AA, Valle D 2009a. Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations. Trop Med Int Health, 14, 1351-1355. —— 2009b. Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations. Trop Med Int Health, 14, 1351-1355. Martins AJ, Lins RM, Linss JG, Peixoto AA, Valle D 2009c. Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil. Am J Trop Med Hyg, 81, 108-115. —— 2009d. Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil. Am J Trop Med Hyg, 81, 108-115. Martins AJ, Ribeiro CD, Bellinato DF, Peixoto AA, Valle D, Lima JB 2012. Effect of insecticide resistance on development, longevity and reproduction of field or laboratory selected Aedes aegypti populations. PloS one, 7, e31889. Martins AJ, Valle D 2012. The Pyrethroid Knockdown Resistance. In S Soloneski, M Larramendy, Insecticides - Basic and Other Applications InTech, Rijeka, p. 17-38. Moignot B, Lemaire C, Quinchard S, Lapied B, Legros C 2009. The discovery of a novel sodium channel in the cockroach Periplaneta americana: evidence for an early duplication of the para-like gene. Insect Biochem Mol Biol, 39, 814-823. Montella IR, Martins AJ, Viana-Medeiros PF, Lima JB, Braga IA, Valle D 2007. Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. Am J Trop Med Hyg, 77, 467477. Montella IR, Schama R, Valle D 2012. The classification of esterases: an important gene family involved in insecticide resistance--a review. Mem Inst Oswaldo Cruz, 107, 437-449. Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, Rocha BC, HallMendelin S, Day A, Riegler M, Hugo LE, Johnson KN, Kay BH, McGraw EA, van den Hurk AF, Ryan PA, O'Neill SL 2009. A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium. Cell, 139, 1268-1278. Normile D 2013. Tropical medicine. Surprising new dengue virus throws a spanner in disease control efforts. Science, 342, 415. 90 OMS 2012. Global Strategy for dengue prevention and control 2010-2012. In, World Health Organization, Geneva. Pampana EJ 1954. Changing strategy in malaria control. Bull World Health Organ, 11, 513520. Pinto J, Lynd A, Vicente JL, Santolamazza F, Randle NP, Gentile G, Moreno M, Simard F, Charlwood JD, do Rosario VE, Caccone A, Della Torre A, Donnelly MJ 2007. Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae. PloS one, 2, e1243. Poupardin R, Reynaud S, Strode C, Ranson H, Vontas J, David JP 2008. Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides. Insect Biochem Mol Biol, 38, 540-551. Poupardin R, Riaz MA, Jones CM, Chandor-Proust A, Reynaud S, David JP 2012. Do pollutants affect insecticide-driven gene selection in mosquitoes? Experimental evidence from transcriptomics. Aquat Toxicol, 114-115, 49-57. Powell JR, Tabachnick WJ 2013. History of domestication and spread of Aedes aegypti - A Review. Mem Inst Oswaldo Cruz, 108 Suppl 1, 11-17. Ranson H, Claudianos C, Ortelli F, Abgrall C, Hemingway J, Sharakhova MV, Unger MF, Collins FH, Feyereisen R 2002. Evolution of supergene families associated with insecticide resistance. Science, 298, 179-181. Remnant EJ, Good RT, Schmidt JM, Lumb C, Robin C, Daborn PJ, Batterham P 2013a. Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster. P Natl Acad Sci USA, 110, 14705-14710. —— 2013b. Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster. Proc Natl Acad Sci U S A, 110, 14705-14710. Rinkevich FD, Du YZ, Dong K 2013. Diversity and convergence of sodium channel mutations involved in resistance to pyrethroids. Pestic Biochem Phys, 106, 93-100. Saavedra-Rodriguez K, Strode C, Flores AE, Garcia-Luna S, Reyes-Solis G, Ranson H, Hemingway J, Black WCt 2013. Differential transcription profiles in Aedes aegypti detoxification genes after temephos selection. Insect Mol Biol. Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M, Flores AE, FernandezSalas I, Bisset J, Rodriguez M, McCall PJ, Donnelly MJ, Ranson H, Hemingway J, Black WCt 2007. A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol Biol, 16, 785-798. Mapa da dengue aponta 157 municípios em situação de risco e 525 em alerta [homepage on the Internet]: Ministério da Saúde; 2013 [updated 19/11/2013]. Available from: http://portalsaude.saude.gov.br/index.php/profissional-e-gestor/vigilancia/noticias-vigilancia/7716-. Seixas G, Salgueiro P, Silva AC, Campos M, Spenassatto C, Reyes-Lugo M, Novo MT, Ribolla PE, Pinto JP, Sousa CA 2013. Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector. Mem Inst Oswaldo Cruz, 108 Suppl 1, 3-10. Smith TJ, Lee SH, Ingles PJ, Knipple DC, Soderlund DM 1997. The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids. Insect Biochem Mol Biol, 27, 807-812. Soderlund DM 2008. Pyrethroids, knockdown resistance and sodium channels. Pest Manag Sci, 64, 610-616. —— 2012. Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances. Arch Toxicol, 86, 165-181. Soderlund DM, Knipple DC 2003. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochem Mol Biol, 33, 563-577. Speranca MA, Capurro ML 2007. Perspectives in the control of infectious diseases by transgenic mosquitoes in the post-genomic era--a review. Mem Inst Oswaldo Cruz, 102, 425-433. Stenhouse SA, Plernsub S, Yanola J, Lumjuan N, Dantrakool A, Choochote W, Somboon P 2013. Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand. Parasit Vectors, 6, 253. Strode C, de Melo-Santos M, Magalhaes T, Araujo A, Ayres C 2012. Expression profile of genes during resistance reversal in a temephos selected strain of the dengue vector, Aedes aegypti. PloS one, 7, e39439. 91 Strode C, Wondji CS, David JP, Hawkes NJ, Lumjuan N, Nelson DR, Drane DR, Karunaratne SH, Hemingway J, Black WCt, Ranson H 2008. Genomic analysis of detoxification genes in the mosquito Aedes aegypti. Insect Biochem Mol Biol, 38, 113-123. Tapia-Conyer R, Mendez-Galvan J, Burciaga-Zuniga P 2012. Community participation in the prevention and control of dengue: the patio limpio strategy in Mexico. Paediatrics and international child health, 32 Suppl 1, 10-13. Trapido H 1954. Recent experiments on possible resistance to DDT by Anopheles albimanus in Panama. Bull World Health Organ, 11, 885-889. Vasilakis N, Cardosa J, Hanley KA, Holmes EC, Weaver SC 2011. Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health. Nat Rev Microbiol, 9, 532-541. Walker T, Johnson PH, Moreira LA, Iturbe-Ormaetxe I, Frentiu FD, McMeniman CJ, Leong YS, Dong Y, Axford J, Kriesner P, Lloyd AL, Ritchie SA, O'Neill SL, Hoffmann AA 2011. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature, 476, 450-453. Weill M, Lutfalla G, Mogensen K, Chandre F, Berthomieu A, Berticat C, Pasteur N, Philips A, Fort P, Raymond M 2003. Comparative genomics: Insecticide resistance in mosquito vectors. Nature, 423, 136137. WHOPES 2006. Pesticides and their application for the control of vectors and pests of public health importance. In WHO/CDS/NTD/WHOPES/GCDPP/2006.1, World Health Organization. WHO recommended insecticides for space spraying against mosqitoes [homepage on the Internet]2014a. WHO Pesticide Evaluation Scheme: "WHOPES". Available from: http://www.who.int/whopes/Insecticides_for_space_spraying_Jul_2012.pdf?ua=1. WHOPES-recommended compounds and formulations fro control of mosquito larvae [homepage on the Internet]: WHOPES; 2014b. WHO Pesticide Evaluation Scheme: "WHOPES". Williamson MS, Martinez-Torres D, Hick CA, Devonshire AL 1996. Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides. Mol Gen Genet, 252, 51-60. Wondji CS, Dabire RK, Tukur Z, Irving H, Djouaka R, Morgan JC 2011. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa. Insect Biochem Molec, 41, 484-491. Xu Q, Tian L, Zhang L, Liu N 2011. Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the mosquito Culex quinquefasciatus. Biochem Biophys Res Commun, 407, 645-649. Yanola J, Somboon P, Walton C, Nachaiwieng W, Prapanthadara LA 2010. A novel F1552/C1552 point mutation in the Aedes aegypti voltage-gated sodium channel gene associated with permethrin resistance. Pestic Biochem Phys, 96, 127-131. Zakon HH 2012. Adaptive evolution of voltage-gated sodium channels: the first 800 million years. Proc Natl Acad Sci U S A, 109 Suppl 1, 10619-10625. 92