Abstract
BackgroundInsect microbiota is a dynamic microbial community that can actively participate in defense against pathogens. Bacillus thuringiensis (Bt) is a natural entomopathogen widely used as a bioinsecticide for pest control. Although Bt’s mode of action has been extensively studied, whether the presence of microbiota is mandatory for Bt to effectively kill the insect is still under debate. An association between a higher tolerance and a modified microbiota was already evidenced but a critical point remained to be solved: is the modified microbiota a cause or a consequence of a higher tolerance to Bt?MethodsIn this study we focused on the mosquito species Aedes aegypti, as no work has been performed on Diptera on this topic to date, and on B. thuringiensis israelensis (Bti), which is used worldwide for mosquito control. To avoid using antibiotics to cure bacterial microbiota, mosquito larvae were exposed to an hourly increasing dose of Bti during 25 hours to separate the most susceptible larvae dying quickly from more tolerant individuals, with longer survival.ResultsDenaturing gradient gel electrophoresis (DGGE) fingerprinting revealed that mosquito larval bacterial microbiota was strongly affected by Bti infection after only a few hours of exposure. Bacterial microbiota from the most tolerant larvae showed the lowest diversity but the highest inter-individual differences. The proportion of Bti in the host tissue was reduced in the most tolerant larvae as compared to the most susceptible ones, suggesting an active control of Bti infection by the host.ConclusionsHere we show that a modified microbiota is associated with a higher tolerance of mosquitoes to Bti, but that it is rather a consequence of Bti infection than the cause of the higher tolerance. This study paves the way to future investigations aiming at unraveling the role of host immunity, inter-species bacterial competition and kinetics of host colonization by Bti that could be at the basis of the phenotype observed in this study.
Highlights
Insect microbiota is a dynamic microbial community that can actively participate in defense against pathogens
To avoid the bias associated with microbiota curation procedures, we studied the dynamics of the microbiota upon exposure to increasing Bacillus thuringiensis (Bt) dose, which allowed us to conclude that microbiota modification is a rapid process occurring as a consequence of Bacillus thuringiensis israelensis (Bti) infection
Larvae from the Bora-Bora Ae. aegypti mosquito strain were individually exposed to an hourly increasing dose of Bti (1 μg suspension of Bti spores/crystals applied every hour) in order to separate the most susceptible individuals from the most tolerant ones (Fig. 1). While this mosquito strain has been reported as highly susceptible to Bti [52], our phenotyping experiment revealed that individual survival upon Bti exposure was highly variable, with some larvae dying after 2.5 h of exposure while others survived 25 h exposure to a final dose of Bti 15 times greater
Summary
Insect microbiota is a dynamic microbial community that can actively participate in defense against pathogens. Each Bt subspecies exhibits a high level of specificity toward an insect phylogenetic group [5, 6] This adaptation to their host with the selection and conservation of key virulence factors makes Bt a pathogen rather than an opportunistic bacterium [7, 8]. This view has been challenged during the last decade with contrasting reports about the role of the insect gut microbiota on the toxicity and infection capacity of Bt [7]; some authors suggested that host-associated microbiota might be mandatory for Bt to kill insects [9]
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