The use of entomopathogenic fungi against Anopheles funestus giles (Diptera: Culicidae)
Date
2011-04-01
Authors
Mouatcho, Joel Claude
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Abstract
Malaria vector control relies primarily on the application of chemical insecticides.
The increasing incidence of insecticide resistance in target vector populations coupled
with the threat of environmental contamination are of major concern in terms of this
approach. The use of biological agents to complement existing insecticide based
control strategies has been proposed, e.g. Metarhizium anisopliae and Beauveria
bassiana. The efficacy of M. anisopliae (Metschnikoff) Sorokin strain ICIPE-30 and
Beauveria bassiana (Balsamo) Vuillemin isolate I93-825 alone or in combination was
assessed against laboratory strains of the major malaria vector Anopheles funestus.
Samples of adult females from three laboratory strains of An. funestus were exposed
to dry conidia of M. anisopliae and B. bassiana for 3, 6 or 24 hours. Of these An.
funestus strains, Fang was fully susceptible to all insecticides, Fumoz was partly
resistant to pyrethroid insecticides and Fumoz-R has been intensively selected for
pyrethroid resistance. Following inoculations, the rate of mortality in all strains was
approximately 6-fold higher in fungus infected cohorts compared to their
corresponding uninfected control cohorts. Susceptibility to fungal infection in the
colonies appeared to follow their pattern of susceptibility to insecticide whereby Fang
showed higher rates of mortality following fungus infection than Fumoz and Fumoz-
R. Further, Mosquitoes placed in forced proximity of fungal spores for only three
hours showed significantly lower rates of mortality than those placed in similar
circumstances for 24 hours, showing that the probability of acquiring an infection is a
function of time and that a longer potential exposure time leads to the acquisition of
greater numbers of infective spores. Approximately 99% of all fungus infected
mosquitoes (infection confirmed by followup sporulation tests on cadavers) died
within 14 days of acquiring their infection. Fourteen days is the maximum time
required by malarial parasites to reach the infective sporozoite stage.
Experiments were designed to quantify a possible interaction between susceptibility to
the pyrethroid insecticide permethrin and susceptibility to B. bassiana or M.
anisopliae infection, based on the hypothesis that intoxication or infection with one of
these agents exerts a synergistic effect on susceptibility to the other. Further, the
effect of a non insecticidal substance that inhibits the activity of monooxygenases -
piperonyl butoxide (PBO) – on subsequent susceptibility to fungus infection in
pyrethroid resistant An. funestus was tested. Fumoz-R infected with fungus proved
significantly more susceptible to pyrethroid intoxication post fungus infection than
uninfected samples from the same cohort. Pre-exposure to PBO did not affect
subsequent susceptibility to fungus infection, suggesting that monooxygenases play
a negligible role in protection against fungus infection.
Experiments were designed to test for variation in fungus induced mortality rates
between blood fed and unfed cohorts of female An. funestus as well as to test for
differences in fecundity in response to fungal infection. Females blood fed post
exposure to fungus showed a slightly higher rate of mortality compared to unfed
fungus infected females. Females blood fed prior to fungus infection showed
comparable rates of mortality with those of unfed fungus infected cohorts. These
results suggest that blood-feeding may affect susceptibility to fungus infection,
although the effect is slight at best and of no concern in terms of fungal pathogenicity. Fungus infected females produced significantly fewer eggs than
uninfected females. The proportion of progeny from fungus infected females
surviving to adulthood was also significantly reduced by comparison to the progeny
of uninfected females.
In order to test the effectiveness of entomopathogenic fungi in semi-field conditions it
is important to first survey the local malaria vector species composition including
their insecticide susceptibility status. Baseline mosquito surveillance was conducted
in the Mamfene region of northern KwaZulu-Natal, South Africa, in order to assess
the feasibility of using clay pots treated with dry conidia of entomopathogenic fungi
as a delivery/infection system in a field environment. Anopheles arabiensis, An.
parensis, An. funestus, An. merus and An. quadriannulatus were collected inside
houses between February and September, 2005. A sample of An. parensis, a nonvector,
was shown to present false positives for the presence of P. falciparum
circumsporozoites using the standard ELISA method. This result highlighted the
importance of accurate species identification and vector incrimination, and showed
how over-reliance on standard methodologies without suitable quality assurance can
lead to inaccurate information about malaria transmission dynamics in a given area.
Resistance to permethrin (pyrethroid) was detected in An. arabiensis and An.
parensis. Biochemical analysis and insecticide-synergist assays showed
monooxygenase elevation leading to monooxygenase based permethrin detoxification
in An. arabiensis. Preliminary laboratory tests revealed that clay pots treated with dry
conidia of B. bassiana or M. anisopliae are suitable for spore delivery to anopheline
mosquitoes resting inside them. However, the efficacy of treated pots, measured in
terms of relative infectivity, decreased with increasing time lapse since treatment, so
that by 3 months post treatment their efficacy was negligible under standard
laboratory conditions.
I conclude that dry conidia of B. bassiana and M. anisopliae are effective pathogens
against An. funestus. Infective spores can be delivered using either an attractant such
as sucrose or by treating the surfaces of preferred resting sites such as clay pots. Their
pathogenicity is not significantly affected by monooxygenase based insecticide
resistance, and in almost all cases fungus infected females die within 14 days of
acquiring an infection, regardless of bloodfeeding status. Further, fungal infection
significantly attenuates the expression of insecticide resistance, and also significantly
reduces fecundity and fertility in infected An. funestus females. These factors enhance
the potential of entomopathogenic fungi as biocontrol agents in areas where resistance
to insecticide occurs in target vector populations.