J. Basic Microbiol. 43 (2003) 5, 393 – 398
DOI: 10.1002/jobm.200310263
(Departamento de Parasitologia Animal, Instituto de Veterinária, Universidade Federal Rural do Rio
de Janeiro, Seropédica, Brazil; 1Departamento de Micologia, Instituto Oswaldo Cruz/ FIOCRUZ,
Rio de Janeiro, RJ, Brazil)
Beauveria bassiana isolated from engorged females
and tested against eggs and larvae of Boophilus microplus
(Acari: Ixodidae)
ÉVERTON KORT KAMP FERNANDES, GISELA LARA DA COSTA1*, EDSON JESUS DE SOUZA,
AUREA MARIA LAGE DE MORAES1 and VANIA RITA ELIAS PINHEIRO BITTENCOURT
(Received 02 January 2003/Accepted 17 March 2003)
The purpose of this work was to evaluate the in vitro virulence of three isolates of Beauveria bassiana
to eggs and larvae of the tick Boophilus microplus. The fungus tested were isolated from engorged
females of B. microplus collected on the field, and identified as Bb28, Bb29 and Bb30. These isolates
were evaluated by immersion of eggs and larvae in suspensions with different conidial concentrations:
108, 107, 106 and 105 conidia/ml and compared to the control groups. In the treated eggs, there was a
percentage much smaller of hatching than that observed in the controls. The egg hatch was inversely
proportional to the conidial concentration. In larval bioassays, all isolates resulted in a higher mortality
of larvae compared to the control according to the conidial concentrations/ml, 10 days after treatment
inoculation.
The tick Boophilus microplus (CANESTRINI) is the most common arthropod attacking Brazilian cattle, causing significant losses to the country’s ranchers. Infestantion of B. microplus on cattle reduces value of the hides and increase the transmission of pathogens such as
anaplasmosis and babesiosis, reflected in lower production or even death of the animals
(HORN and ARTECHE 1985).
Various methods have been tried to control this tick, during both the non-parasitic and
parasitic phases. The use of acaricides to control the parasitic phase of this arthropod is
common among ranchers. However, indiscriminate use of acaricides has caused serious
problems of environmental pollution and development of resistance. Hence, the exclusive
use of chemical agents is becoming less and less advisable from both a practical and economic standpoint. Alternative methods of control are needed (BARROS and EVANS 1989).
The use of entomopathogenic fungi in the biological control of vectors has been growing
steadily. Various fungi are being evaluated as promising agents to use against arthropod
pests that spread pathogens to both humans and animals.
The fungus Beauveria bassiana (BALSAMO) VUILLEMIN has already been found to cause
natural infections in ticks of the species Hyalomma anatolicum and Ixodes ricinus (SAMSINAKOVA 1957). It has also been used in artificial infections, with promising results in the
latter species (BOYCEV and RIZVANOV 1960), as well as in Hyalomma sp. (TIAN 1984),
which demonstrates its pathogenic potential against ticks.
BITTENCOURT et al. (1992, 1994, 1996 and 1997) evaluated the action of different lineages of Beauveria bassiana (BALSAMO) VUILLEMIN and Metarhizium anisopliae (METSCHNIKOFF) SOROKIN against different growth stages of the tick B. microplus. The pathogenicity of these fungi has already been proven against some tick species, however, a few fungi
* Corresponding author: Dr. DA COSTA; e-mail: [email protected]
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/03/0509-0393
394
É. K. K. FERNANDES et al.
species has been isolated from ticks. It is believed that isolates associated with ticks, if
obtained in their natural state, could be more effective in the integrated control of this arthropod.
Based on this belief, strains of B. bassiana isolated naturally from engorged females of
B. microplus collected from the soil of a ranch in the municipality of Paracambi, Rio de
Janeiro State, were evaluated for their entomopathogenic potential against eggs and larvae
of B. microplus.
Materials and methods
Three strains of Beauveria bassiana – Bb28, Bb29 and Bb30 – were isolated from engorged females
of B. microplus collected from the soil in the municipality of Paracambi, Rio de Janeiro, Brazil
(COSTA 2001).
Engorged females of B. microplus were collected from artificially infested cattle at the W. O. NEITZ
Parasitology Research Station of the Department of Animal Parasitology, Veterinary Institute, of the
Universidade Federal Rural do Rio de Janeiro (UFRRJ). They were subsequently washed in distilled
water, dried and placed in Petri dishes kept in a controlled environment chamber at 27 ± 1 °C and
relative humidity ≥ 80%. After the end of egg laying, the egg masses were weighed and separated into
samples of 50 mg and placed in test tubes sealed with cotton. Then the tubes were maintained in the
same temperature and humidity.
The three strains of B. bassiana were grown in rice corn contained in polypropylene sacks. The
conidia produced in rice corn were suspended in 0.1% (v/v) sterile aqueous Tween 80 and the
suspension were quantified in a Neubauer chamber, obtaining concentrations of 108, 107, 106 and 105
conidia/ml. The control group suspension were prepared as described above but without conidia.
The methodology used with eggs of B. microplus was similar to that used in the bioassays with
larvae, based on the work of TORRADO and GUTIERREZ (1969). Each treatment group was made up of
ten repetitions, each one constituted in a test tube with 50 mg of eggs, receiving 1ml of the conidia
suspension to be tested, with the eggs immersed for 5 min. After this period, the excess suspension
was eliminated. The tubes, duly identified, were kept in the controlled environment chamber
(27 ± 1 °C and RH ≥ 80%) for observation of the following parameters: periods of incubation (number
of days understood between the date of the beginning of the posture and the date of beginning eclosion
of eggs), periods of eclosion ( number of days understood between the date of the beginning and the
finishing of eclosion) and percentage of eclosion of larvae (BELLATO 1995).
To identify the mortality of larvae vis-à-vis the different isolates, the test tubes containing 50 mg of
eggs were kept in the controlled environment chamber as already specified and after hatching each
tube-received 1 ml of the suspension to be tested. After 5 min, the excess suspension was removed and
the larvae were returned to the controlled environment chamber. Ten days after inoculation of the
suspensions, the mortality percentage was estimated visually through a stereoscopic microscope
(DAVEY et al. 1984).
The unhatched eggs and dead larvae were placed in a moist chamber to allow the development of
the fungus and evaluation of the infection following the Koch postulates (ALVES et al. 1998). After
observation for fungal growth, the structures were transferred to PETRI dishes with Potato dextrose
agar (PDA) and again kept in the controlled environment chamber under controlled temperature and
relative humidity. The microscopic characteristics were evaluated in mycelial preparations in a drop of
Lactophenol Amann with cotton blue between a slide and a coverslip (HAWKSWORTH 1977) for
confirmation and authentication of the entomopathogen.
Statistical testing was through analysis of the variance (ANOVA), followed by the TUKEY test
(p ≤ 0.05) for comparison among the media, calculating the coefficient of variance to check the
precision of the data.
Results and discussion
The incubation period observed in this study, both for the control groups and the groups
treated with isolates of B. bassiana, was 25 days, with no variation among the averages.
Beauveria bassiana isolated from engorged females and tested stages from Boophilus microplus 395
These data differ from those found by BITTENCOURT et al. (1996), where on testing the
effect of isolates 986 and 747 of the same fungus on eggs of B. microplus, they observed an
incubation period in the control groups between 21.3 and 22.0 days, increasing in proportion
to the concentration of the isolates. Being thus, it is observed that the isolated ones of
Beauveria bassiana tested had exactly presented different behavior of the excessively
isolated ones of fungus, being indifferent in the infection of eggs of Boophilus microplus in
that it says respect to the incubation period.
Regarding the period for hatching of larvae, the average number of days for the groups
treated with the different isolates tested ranged from 2.8 to 6.4 days, while for the control
groups this variation was between 6.3 and 7.2 days. Statistical analysis showed that the
isolates Bb28 and Bb30 caused a significant reduction in the larval hatching period, while
the reduction for the Bb29 isolate was not significant at p ≤ 0.05 (Bb28: P < 0.0001,
F = 8.294, dF = 49; Bb29: P = 0.0779, F = 2.256, dF = 49 and Bb30: P = 0.0043,
F = 4.415, dF = 49). BITTENCOURT et al. (1996), working with different isolates of the same
fungus on B. microplus eggs, found that the eclosion period for larvae of both groups treated
with different suspensions was longer than that observed in the control groups, and that
the increase was directly proportional to the concentration of conidia/ml. These data are not
the same as ours – once again we observed different behavior among the isolates regarding
this parameter, contrary to that of the isolates 986 and 747 tested by BITTENCOURT et al.
(1996).
The eclosion percentages from B. microplus eggs treated with the different isolates of
B. bassiana varied from 30% to 80%, while in the control groups the variation was between
80% and 96%. The different isolates differed significantly in larval hatching percentage,
with the treated groups havinglower hatching percentages than the control groups (Bb28:
P < 0.0001, F = 8.307, dF = 49; Bb29: P < 0.0001, F = 12.220, dF = 49 and Bb30:
P < 0.0001, F = 8.882, dF = 49). Bittencourt et al. (1996) observed eclosion percentages
among the groups treated with different isolates of B. bassiana ranging from 20.0% to
86.6%, uniformly smaller than the percentage for the control groups, where this figure was
93.3%. This result is similar ours in the present work. In this respect, we point out that the
isolates tested in the two works, although differing regarding the previous two parameters,
are similar for this last one, which is of greater importance because it means a lesser number
of larvae hatching. Hence, we can conclude that the isolates of B. bassiana tested are
pathogenic to eggs of B. microplus (Table 1).
We further note that the different strains of the same fungus B. bassiana behave differently in infecting eggs of this arthropod – perhaps the strains have variations. CASTRILLO
Table 1
Parameters observed in the test with Boophilus microplus eggs treated with varying concentrations of
different isolates of Beauveria bassiana (Bb), maintained at 27 °C ± 1 and RH ≥ 80%
ISOLATES
Treatments
(conidia/ml)
Periods of eclosion (days)
Percentage of eclosion (%)
Bb28*
Bb29*
Bb30*
Bb28*
Bb29*
Bb30*
Control
105
106
107
108
6.5 a
6.4 a
4.8 ab
4.9 ab
2.8 b
7.2 a
5.6 a
5.4 a
5.2 a
3.9 a
6.3 a
4.2 ab
5.3 ab
4.9 ab
3.2 b
80 a
80 a
64 a
62 a
30 b
96 a
61 bc
68 b
43 c
45 bc
87 a
59 a
67 a
53 b
39 c
* Values followed by the same letter within the same column were not statistically different using
Tukey’s test at the 5% probability level
396
É. K. K. FERNANDES et al.
and BROOKS (1998) detected variations among strains of B. bassiana isolated from the darkling beetle, Alphitobius diaperinus (Coleoptera: Tenebrionidae) when they used RAPD
analysis, and showed that polymorphism even within a determined population was inconstant. It is possible that the presence of polymorphism among the B. bassiana strains tested
for biocontrol of B. microplus is responsible for the different infection rates among strains
of this fungus.
In the larval bioassay, the average mortality rate in the control groups was 0%, while for
the groups treated with the different isolates in varying suspensions this percentage ranged
from 10% to 96%. Analysis of the variance and TUKEY test (p ≤ 0.05) showed that the
eclosion percentages differed significantly among the treatments and/or when compared
with the control group (Bb28: P < 0.0001, F = 61.318, dF = 49; Bb29: P < 0.0001,
F = 55.929, dF = 49 and Bb30: P < 0.0001, F = 170.220, dF = 49). The action of these isolates on larvae of B. microplus is demonstrated by the fact that the treated groups have a
larva mortality percentage directly proportional to the concentration of conidia in the suspensions (Fig. 1).
The results obtained in this bioassay are similar to those of BITTENCOURT et al. (1996),
who found that the percentage of mortality in the treated groups varied from 18.8% to
88.0% depending on the concentration of conidia used.
The fungus B. bassiana has also been used to promote artificial infections in Ixodes
ricinus (LATREILLE 1804) and the results showed a reduction in the eclosion percentage and
a larva mortality rate varying between 86% and 100% (GORSKOVA 1966).
Re-isolation of the fungus was confirmed with the development of fungal species in the
same culture medium and controlled environment chamber , with classification and identification performed according to DE HOOG (1972), TULLOCH (1976) and ROMBACH
et al. (1986). The fungal species were found to be the same as those used in the bioassays,
thus confirming the infection of the eggs and larvae of B. microplus by the tested isolates.
Therefore, we can state that the isolate of B. bassiana tested in vitro were virulent
for eggs and larvae of B. microplus. The isolate Bb28 was the best isolate tested, presenting
a high larvae mortality percentage and low hatching of larvae in the treated eggs, and could
be promising for use as biocontrol agents in these two developmental stages of this arthropod.
Bb28
Bb29
Bb30
percentage of mortality (%)
100
80
60
40
20
0
control
conc.10E5
conc.10E6
conc.10E7
conc.10E8
concentration (conidia/ml)
Fig. 1
Percentage of larval mortality for Boophilus microplus treated with varying concentrations of different
isolates of Beauveria bassiana (Bb), kept at 27 °C ± 1 and RH ≥ 80%
Beauveria bassiana isolated from engorged females and tested stages from Boophilus microplus 397
Acknowledgements
We thank to the Consellho Nacional de Pesquisa e Tecnologia (CNPq) for financial support for the
work, ROSANA COLATINO REIS for statistical assistence and Dra CINTIA DE MORAES BORBA for helpful
comments in the manuscript.
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Mailing address: GISELA LARA DA COSTA, Departamento de Micologia, Instituto Oswaldo Cruz –
FIOCRUZ, Pavilhão Rocha Lima, sala 525, Av. Brasil, 4365 – Manguinhos – Rio de Janeiro, Rio de
Janeiro, Brasil. 21045-900
Phone: +55 21 2598-4423; Fax: +55 21 2270-6397;
E-mail: [email protected]