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Mosquito breeding sites are complex aquatic environments with wide microbial diversity and physicochemical parameters that can change over time during the development of immature insect stages. Changes in biotic and abiotic conditions in water can alter life-history traits of adult mosquitos but this area remains understudied. Here, using microbial genomic and metabolomics analyses, we explored the metabolites associated with Aedes aegypti breeding sites as well as the potential contribution of Klebsiella sp., symbiotic bacteria highly associated with mosquitoes. We sought to address whether breeding sites have a signature metabolic profile and understand the metabolite contribution of the bacteria in the aquatic niches where Ae. aegypti larvae develop. An analysis of 32 mosquito-associated bacterial genomes, including Klebsiella, allowed us to identify gene clusters involved in primary metabolic pathways. From them, we inferred metabolites that could impact larval development (e.g., spermidine), as well as influence the quality assessment of a breeding site by a gravid female (e.g., putrescine), if produced by bacteria in the water. We also detected significant variance in metabolite presence profiles between water samples representing a decoupled oviposition event (oviposition by single females and manually deposited eggs) versus a control where no mosquito interactions occurred (PERMANOVA: p < 0.05; R2 = 24.64% and R2 = 30.07%). Five Klebsiella metabolites were exclusively linked to water samples where oviposition and development occurred. These data suggest metabolomics can be applied to identify compounds potentially used by female Ae. aegypti to evaluate the quality of a breeding site. Elucidating the physiological mechanisms by which the females could integrate these sensory cues while ovipositing constitutes a growing field of interest, which could benefit from a more depurated list of candidate molecules.

Pathogens may manipulate their human and mosquito hosts to enhance disease transmission. Dengue, caused by four viral serotypes, is the fastest-growing transmissible disease globally resulting in 50–100 million infections annually. Transmission of the disease relies on the interaction between humans and the vector Aedes aegypti and is largely dependent on the odor-mediated host seeking of female mosquitoes. In this study, we use activity monitors to demonstrate that dengue virus-1 affects the locomotion and odor-mediated behavior of Ae. aegypti, reflecting the progression of infection within the mosquito. Mosquitoes 4–6 days post-infection increase locomotion, but do not alter their odor-driven host-seeking response. In contrast, females 14–16 days post-infection are less active, yet more sensitive to human odors as assessed by behavioral and electrophysiological assays. Such an increase in physiological and behavioral sensitivity is reflected by the antennal-specific increase in abundance of neural signaling transcripts in 14 days post-infection females, as determined by transcriptome analysis. This suggests that the sensitivity of the mosquito peripheral olfactory system is altered by the dengue virus by enhancing the overall neural responsiveness of the antenna, rather than the selective regulation of chemosensory-related genes. Our study reveals that dengue virus-1 enhances vector-related behaviors in the early stages post-infection that aid in avoiding predation and increasing spatial exploration. On the other hand, at the later stages of infection, the virus enhances the host-seeking capacity of the vector, thereby increasing the risk of virus transmission. A potential mechanism is discussed. Author summary The mosquito Aedes aegypti is a major urban vector for human pathogens, including dengue, the most rapidly-spreading infectious disease globally. We show that one of the four strains of the dengue virus differentially, and inversely, affects the locomotion and odour-mediated behaviour of female Aedes aegypti. During the early stages after infection, female mosquitoes show an increased locomotion activity suggesting that the virus likely promotes mosquito dispersion and increases its spatial exploration. In contrast, in the later infective stages, dengue infection modulates the mosquito’s host-seeking capacity, by enhancing the physiological and behavioural sensitivity to human odors, thus increasing the risk of virus transmission. At this stage of infection, neural signalling gene expression is enhanced, providing a mechanism for the increased sensitivity to human odor, while genes involved in chemosensation and neuromodulation are likely playing a secondary role. A better understanding of alterations of mosquito host-seeking behavior upon pathogen infection will contribute to the development of novel control strategies against vector borne diseases.

Microbiota studies of Aedes aegypti and other mosquitoes generally focus on the bacterial communities found in adult female midguts. However, other compartments of the digestive tract maintain communities of bacteria which remain almost entirely unstudied. For example, the Dipteran crop is a food storage organ, but few studies have looked at the microbiome of crops in mosquitoes, and only a single previous study has investigated the crop in Ae. aegypti. In this study, we used both culture-dependent and culture-independent methods to compare the bacterial communities in midguts and crops of laboratory reared Ae. aegypti. Both methods revealed a trend towards higher abundance, but also higher variability, of bacteria in the midgut than the crop. When present, bacteria from the genus Elizabethkingia (family Weeksellaceae) dominated midgut bacterial communities. In crops, we found a higher diversity of bacteria, and these communities were generally dominated by acetic acid bacteria (family Acetobacteriaceae) from the genera Tanticharoenia and Asaia. These three taxa drove significant community structure differences between the tissues. We used FAPROTAX to predict the metabolic functions of these communities and found that crop bacterial communities were significantly more likely to contain bacteria capable of methanol oxidation and methylotrophy. Both the presence of acetic acid bacteria (which commonly catabolize sugar to produce acetic acid) and the functional profile that includes methanol oxidation (which is correlated with bacteria found with natural sources like nectar) may relate to the presence of sugar, which is stored in the mosquito crop. A better understanding of what bacteria are present in the digestive tract of mosquitoes and how these communities assemble will inform how the microbiota impacts mosquito physiology and the full spectrum of functions provided by the microbiota. It may also facilitate better methods of engineering the mosquito microbiome for vector control or prevention of disease transmission.