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
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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
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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
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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.
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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
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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.
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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.
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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
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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.
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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
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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
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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
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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.
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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
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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
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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.
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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
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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
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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
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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
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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.
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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
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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.
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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).
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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.
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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)
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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.
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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.
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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).
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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
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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
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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
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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
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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)
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FIGURE S2
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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
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