Sexual behavior of Aedes aegypti males during the auto-dissemination of the entomopathogenic fungi Metarhizium anisopliae

Background. Aedes aegypti transmits several important arboviral diseases such as yellow fever and dengue fever. Vectorial control programs aimed to reduce mosquito populations and halting transmission of dengue viruses, have been based on the application of chemical insecticides. The increment of this mosquito-borne disease over the last decades has led to the development of novel vector control methods, including the auto-dissemination of Metarhizium anisopliae conidia by mating behavior from male to female in A. aegypti. Nevertheless, the implementation of a biocontrol involving the contamination of male mosquitoes with conidia, requires a better understanding of Ae. aegypti ethology, particularly, the sexual behavior. The present laboratory study reports the behavior of the male of Ae. aegypti during the auto-dissemination of a highly virulent M. anisopliaestrain.

 

Methodology. Five bioassays (B) were established. In B1, the median lethal time (LT₅₀) of Ae. aegypti males exposed by indirect exposure for 24 hours to M. anisopliae was estimated using two treatments (T) with 3 replicates of 25 males each : T1 = conidia-contaminated  males (CM) and T2 = uncontaminated males (control). In B2, the mean (+/- standard error) of conidia attached on the whole body (n = 60 pools of 3 mosquitoes per pool) and frontal tarsus (n = 10) of Ae. aegypti males was determined  by using hemocytometer and a scanning electron microscope, respectively. In B3, the mean (+/- standard error) of conidia attached to the whole body of Ae. aegypti females after the  first and fifth mating with one conidia-contaminated  males was computed by using a hemocytometer  (n = 60 pools of 3 mosquitoes each ). In B4, the mean (+ / - standard error) of contaminated females (cf), uncontaminated (uf), mated (inseminated) (if) and attempted copulation (not inseminated) (nf) during exposure to the following treatments: T1 = 1 male contaminated with conidia and yellow fluorescent dust (YM), T2 = 1 male contaminated with conidia and red fluorescent dust (RM) and T3 = 1 male contaminated with conidia without fluorescent dust (control) were compared using a one-way ANOVA test for unbalanced experiments and followed by a Ryan post hoctest (P <.05). Here, 3 replications of 20 females per treatment were established. In B5, the mean (+/- standard error) of sexual encounters was evaluated in two treatments: T1 = fungus-contaminated males (CM) and uncontaminated males as control. Here, 3 replicates of 180 males per treatment were used. Data were analyzed by a polynomial regression and analyzed against the time (in minutes) after mosquito confinement. All data were analyzed using SAS 9.0.

 

Results. In B1, the survival rate in CM was 84.37 % (LT₅₀=3.69 +/- 0.16) and significantly (P < <0.0001) lower than the control (LT₅₀= 23.62 +/- 0.58). Herein, sporulation in CM cadavers was 98 %. In B2, the males showed a mean of 3.5 x 10⁴ conidia mL^-1 (SE +/- 1.93 x 10⁴) attached on whole body and a mean of 3.4 (SE +/- 1.2) “conidia groups” on their frontal tarsus, respectively. In B3, the mean of conidia loads in females after the first and fifth mating was of 1.08 x 10⁴ (SE +/- 6.82 x 10³) and 5.4 x 10³ (SE +/- 3.74 x 10³), respectively (conidia loads were 50 % less in the fifth mating compared with the first mating). In B4, the means in T1 were 16.66, 3.33, 10.0, and 6.66 for cf, if, nf and uf, respectively. For T2, the means were 15.32, 4.66, 9.0, and 6.33 for cf, if, nf and uf, respectively. In T3, the means were 14.0, 6.0, 8.0, and 6.0 hc, hs, hi y hn, respectively. In B5, the highest number of sexual encounters in Ae. aegyptiwas registered at 36 and 25 minutes post confinement in the CM and control group, respectively.

 

Conclusion: The fluorescent dust tagging of Ae. aegypti males and their contamination with M. anisopliae conidia did not affect their mating behavior and, hence the passage of conidia from males to females. Their sexual activity peaked at 25 minutes post-confinement and it was 30% lower for contaminated than for uncontaminated males. There was, a relatively, low level of conidia attached to the whole body male and his frontal tarsus, however, this amount of conidia is potentially available for transmission through 16 of 20 females in 24 h. Given that it appears to be a potent method of biological control for dengue vectors, therefore, field trials are desperately needed to evaluate the impact of the auto-dissemination of M. anisopliae on A. aegypti populations.