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, 11 (2)

Aedes aegypti (Diptera: Culicidae) Immune Responses With Different Feeding Regimes Following Infection by the Entomopathogenic Fungus Metarhizium anisopliae

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Aedes aegypti (Diptera: Culicidae) Immune Responses With Different Feeding Regimes Following Infection by the Entomopathogenic Fungus Metarhizium anisopliae

Sara Cabral et al. Insects.

Abstract

The mosquito Aedes aegypti is the most notorious vector of illness-causing viruses. The use of entomopathogenic fungi as bioinsecticides is a promising alternative for the development of novel mosquito control strategies. We investigate whether differences in immune responses could be responsible for modifications in survival rates of insects following different feeding regimes. Sucrose and blood-fed adult A. aegypti females were sprayed with M. anisopliae 1 × 106 conidia mL-1, and after 48 h, the midgut and fat body were dissected. We used RT-qPCR to monitor the expression of Cactus and REL1 (Toll pathway), IMD, REL2, and Caspar (IMD pathway), STAT and PIAS (JAK-STAT pathway), as well as the expression of antimicrobial peptides (Defensin A, Attacin and Cecropin G). REL1 and REL2 expression in both the midgut and fat body were higher in blood-fed fungus-challenged A. aegypti than in sucrose-fed counterparts. Interestingly, infection of sucrose-fed insects induced Cactus expression in the fat body, a negative regulator of the Toll pathway. The IMD gene was upregulated in the fat body in response to fungal infection after a blood meal. Additionally, we observed the induction of antimicrobial peptides in the blood-fed fungus-challenged insects. This study suggests that blood-fed A. aegypti are less susceptible to fungal infection due to the rapid induction of Toll and IMD immune pathways.

Keywords: Zika; dengue; gene expression; immune response; vector.

Conflict of interest statement

The authors declare no conflict of interest. Futhermore, the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Susceptibility of Aedes aegypti adult female mosquitoes to fungal infection. SUC, sucrose-fed mosquitoes treated with Tween 80 (control); SUC + I, sucrose-fed mosquitoes infected with M. anisopliae; BLD, blood-fed mosquitoes treated with Tween 80 (control); BLD + I, blood-fed mosquitoes infected with M. anisopliae. Survival rates of A. aegypti (n = 45 per treatment) exposed to conidia of M. anisopliae ESALQ (ESALQ818) at a final dose of 106 spores mL−1. The graph represents three independent experiments and data were analyzed with Kaplan–Meier survival analysis (GraphPad Prism 6 software). Error bars indicate the SEM. The fungus significantly increased SUC + I mortality compared to all other treatments (*** p < 0.001).
Figure 2
Figure 2
Expression analysis of TOLL pathway marker genes after exposure of A. aegypti to M. anisopliae. Gene expression analysis of Cactus and transcription factor REL1 in the midgut and fat body of sugar-fed or blood-fed insects at 48 h post-infection. The insects were fed with sucrose (10%) or mouse blood. After feeding, the insects were sprayed with M. anisopliae. Females at 48 h post fungal infection were dissected for total RNA extraction followed by synthesis of cDNA. cDNA was subsequently used to perform the RT-qPCR analysis. Each bar represents the results from a pool of 15 female mosquitoes. Results are the means of four independent experiments. The p-values from pair-wise comparisons (comparing Tween–treated to sugar-fed/blood-fed) were calculated using Student’s t-test and level of significance indicated for each bar (*: p < 0.05; b: ** p < 0.01; ***: p < 0.001; ****: p < 0.0001). Negative controls were treated with 0.05% aqueous Tween 80.
Figure 3
Figure 3
Expression analysis of IMD pathway marker genes after exposure of A. aegypti to M. anisopliae. Gene expression analysis of transcription factors REL2, IMD, and Caspar in the midgut and fat body of both sugar-fed and blood-fed insects at 48 h post-infection. The insects were fed with sucrose (10%) or mouse blood. Immediately after feeding, mosquitoes were sprayed with 0.05% Tween 80 solution, with or without M. anisopliae. Forty-eight hours post-infection, total RNA was extracted from mosquitoes, cDNA obtained, and RT-qPCR analyses subsequently performed. Each bar represents the results from a pool of 15 female mosquitoes. Results are the means of four independent experiments. The p-values from pair-wise comparisons (comparing Tween-treated to sugar-fed/blood-fed) were calculated using Student’s t-test and levels of significance were indicated for each bar (* p < 0.05; b: ** p < 0.01; *** p < 0.001; **** p < 0.0001). Negative controls were treated with 0.05% aqueous Tween 80.
Figure 4
Figure 4
Expression analysis of the JAK-STAT pathway marker genes after exposure of A. aegypti to M. anisopliae. Gene expression analysis of PIAS, and signal transducer and activator of transcription STAT both in the midgut and fat body of sugar-fed and blood-fed mosquitoes at 48 h post infection. The insects were fed with either sucrose (10%) or mouse blood. Immediately after feeding, mosquitoes were sprayed with 0.05% Tween 80 with or without M. anisopliae. Forty-eight hours post-infection, total RNA was extracted from mosquitoes, cDNA obtained, and RT-qPCR analyses were subsequently performed. Each bar represents the results from a pool of 15 female mosquitoes. Results are the means of four independent experiments. The p-values from pair-wise comparisons (comparing Tween treated to sugar-fed/blood-fed) were calculated using Student’s t-test and levels of significance were indicated for each bar (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Negative controls were treated with 0.05% aqueous Tween 80.
Figure 5
Figure 5
Expression analysis of antimicrobial peptides genes following infection with M. anisopliae. Gene expression analyses of defensin A, cecropin G and attacin in the midgut and fat body of sugar-fed or blood-fed at 48 h post-infection. The insects were fed with sucrose (10%) or mouse blood. Immediately after feeding, the mosquitoes were sprayed with 0.05% Tween 80 with or without M. anisopliae. Forty-eight hours post-infection, total RNA was extracted from mosquitoes, cDNA obtained, and RT-qPCR analyses were subsequently performed. Each bar represents the results from a pool of 15 female mosquitoes. The p-values from pair-wise comparisons (comparing Tween treated to sugar-fed/blood-fed) were calculated using Student’s t-test and levels of significance were indicated for each bar (* p < 0.05; b: ** p < 0.01; *** p < 0.001; **** p < 0.0001). Negative controls were treated with 0.05% aqueous Tween 80.

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