According to the World Health Organization (WHO), vector diseases represent 17% of the estimated emerging infectious diseases in the world (WHO, 2020). This results from vector’s geographical expansion, world transport, unplanned urbanization, and climate change (Jupatanakul et al., 2014). Viruses like dengue, zika, and chikungunya are the most important arboviruses, which are the leading cause of the emerging infectious diseases worldwide (Patterson et al., 2016). The main vectors of these diseases are mosquitoes belonging to the genus Aedes (Diptera: Culicidae) (Mayer et al., 2017). In Mexico, the control of these mosquitoes depends on integrated management with insecticides (CENAPRECE, 2018); however, as in many other countries, the effectiveness of vector control programs is compromised by the development of resistance to insecticides (Musso and Gubler, 2015). Moreover, there is concern about the environmental impact of this approach (Nkya et al., 2013; Smith et al., 2016). Recent studies have shown that insecticide resistance is a diverse and global process related to metabolic mechanisms (increased enzymatic activity) and resistance to the target site (Smith et al., 2016; Dada et al., 2019). These problems have created the need to develop alternative methods for controlling mosquito populations (Ramirez et al., 2012).

Previous research indicates that mosquitoes can host different bacterial communities that vary depending on sex, stage of development, and environmental factors (Cirimotich et al., 2010; Ramirez et al., 2012; Terenius et al., 2012; Coon et al., 2016). The functions of these microorganisms in the insects are correlated with nutrition, immune response, protection against pathogens, digestion, and development, among others (Gaio et al., 2011; Douglas, 2014). Multiple studies have addressed the bacterial community in A. aegypti, where species of the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas have been found (Terenius et al., 2012). Other studies have demonstrated the role of microbiota as a modulator of vector competence in Anopheles during infection with Plasmodium (Abdul-Ghani et al., 2012; Dennison et al., 2014). In A. atropalpus, the intestinal microbiota has been found to be associated with variations in survival, size, and egg production (Coon et al., 2016). In addition, new control strategies and environmentally friendly measures have focused on endosymbiotic bacteria such as Wolbachia to reduce or block the transmission of pathogens such as dengue, chikungunya, and Zika viruses (Bian et al., 2010; Bennett et al., 2019; Scolari et al., 2019). Recently, the microbiota of insects has been found to be involved in the detoxification processes (Aislabie and Lloyd-Jones, 1995; Itoh et al., 2018). Studies have shown that bacteria such as Bacillus cereus, Enterobacter asburiae, and Pantoea agglomerans can degrade acephate, an organophosphate in the diamondback moth Plutella xylostella (Kikuchi et al., 2012; van den Bosch and Welte, 2017). Knowing that the detoxification mechanism is a complex process that is regulated by metabolic (increase in enzyme activity) or genetic (mutations) systems, our study aimed to characterize the resident microbiota in A. aegypti mosquitoes and their potential role in their susceptibility to the insecticides: permethrin and deltamethrin.

Insects are a broad group of organisms with a large variety of lifestyles, which depends directly on microorganism associations (Douglas, 2014; Guégan et al., 2018). Adaptations of mosquitoes to selective pressure have made it necessary to determine new ways to approach their control. There are only a few studies regarding the effect of insecticides on mosquito microbiota, and thus in this study, we determined the microbiota of a field population of A. aegypti in southern Mexico with sequencing techniques and the characterization of key bacteria in response to two pyrethroid insecticides. Previous studies have been conducted to determine the microbiota of mosquitoes in different geographical regions, which have allowed the determination of the microbial diversity of these species of mosquitoes (Ramirez et al., 2012). In our study, the natural microbiota of the A. aegypti population (control) was mainly dominated by the phyla Proteobacteria and Bacteriodetes; these results are like those previously reported by Wang et al. (2011). Bacterial populations differ because of the habitat in which they interact as it depends on food, through plants for sugar sources, or through blood in female mosquitoes (Guégan et al., 2018). There are several studies about the microbiota in A. aegypti populations of endemic areas of Panama where the predominant phyla are Proteobacteria and Firmicutes (Ramirez et al., 2012).

The predominant species in our control group were P. agglomerans-eucrina, S. marcescens-nematodiphila, P. azotoformans-fluorescens-synxantha, and E. meningoseptica. Previously, these bacterial genera have been isolated from other species of mosquitoes such as Anopheles gambiae and A. funestus from Kenia and Mali (Straif et al., 1998). Similarly, in A. aegypti (Rockefeller), the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas (Gusmão et al., 2010; Terenius et al., 2012) have been isolated. In Brazil, the most abundant genera detected in A. aegypti populations have been Pseudomonas, Acinetobacter, and Aeromonas (David et al., 2016). In addition, in Anopheles species from Vietnam, Acinetobacter spp. has been found to be the main component of the gut microbiota (Ngo et al., 2016). In the same way, microbiota in Anopheles has included Pseudomonas, Comamonas, Acinetobacter, Rhizobium, Burkholderia, and members of the family Enterobacteriaceae (Tchioffo et al., 2016). The midgut microbiota of Culex quinquefasciatus has been shown to harbor bacterial species such as Acinetobacter junii, Ac. calcoaceticus, Aeromonas culicicola, Bacillus thuringiensis, Microbacterium oxydans, P. agglomerans, P. aeruginosa, Staphylococcus epidermidis, and Stenotrophomonas maltophila (Pidiyar et al., 2004).

Adult mosquitoes may contain a bacterial diversity concentrated mainly in aerobic and anaerobic gram-negative bacteria (Coon et al., 2014, 2016). Many of the related associations between bacterial communities of an insect are known to be mutualists where the host provides bacteria with nutrients and habitat, having selectivity due to the physicochemical conditions present in the host intestine (e.g., alkaline pH, redox potential, oxygen level below 5%, etc.) (Dada et al., 2019). It has been reported that many gram-negative bacteria are frequently found in the middle intestine of vector insects, influencing differential growth by contributing to the modulation of vector competition (Azambuja et al., 2005). In mosquitoes, the bacteria of the phylum Proteobacteria, especially the family Enterobacteriaceae, which are the main components of the middle gut microbiome, can tolerate the redox stress of blood digestion (Jupatanakul et al., 2014). In our study, the most representative species in A. aegypti population (control group) was P. agglomerans. It is common to find this bacterium as a symbiont in mosquitoes since it is a natural inhabitant of the environment (Segado Arenas et al., 2012; Mayer et al., 2017). In mosquitoes, this bacterium participates in nitrogen fixation, creating a nitrogen-rich environment ideal for the development of eggs and larvae (MacCollom et al., 2009).

The microbiota of our population under study was modified by reducing important enterobacteria species to observe their impact on insecticide response. The populations of A. aegypti treated with penicillin/streptomycin and then exposed to permethrin showed a decrease in survival of 16% compared to the mosquitoes not treated. The populations modified with gentamicin decreased by 28% compared to those mosquitoes not treated but exposed to permethrin. The results showed that >90% of bacteria in these treatments were represented by the species P. azotoformans-fluorescens-synxantha with penicillin/streptomycin treatment, while in treatments with gentamicin, A. dhakensis-hydrophila-taiwanensis and S. marcescens-nematodiphila accounted for 29.10%. The strains of Pseudomonas are gram-negative, ubiquitous bacteria, characterized by primary nutritional needs and presence in various environments (soil, decaying organic material, atmospheric dust, vegetation, and water), with a wide range of plants and animals (Andreani et al., 2015). This genus has been found as part of the bacterial community in insects. Some research points to the toxic effectiveness of P. aeruginosa in the larvae of different mosquito species of the genera Aedes, Anopheles, Culex, and Culiseta. In vivo experiments with P. fluoresences cultures have found effectiveness against pupae of three mosquito species: An. stephensi, Cx. quinquefasciatus and Ae. aegypti (Prabakaran et al., 2009). Similarly, Nabar and Lokegaonkar (2015) reported the larvicidal power of metabolites extracted from Pseudomonas spp. The species P. fluorescens has active metabolites against the larvae and pupae of Cx quinquefasciatus mosquitoes (Sadanandane et al., 2003; Brammacharry and Paily, 2012).

On the other hand, with the modification of the microbiota with gentamicin, we found the complex A. dhakensis-hydrophila-taiwanensis. This genus of gram-negative bacteria inhabits many environments (aquatic, fish, food, domesticated pets, invertebrate species, birds, ticks and insects, and natural soils) (Figueras et al., 2005; Janda and Abbott, 2010), and has been isolated from the intestines of Cx. quinquefasciatus (Pidiyar et al., 2002, 2004). Similarly, the species A. hydrophila has been reported to possess chitinolytic enzymes with activity against Cx. quinquefasciatus under laboratory conditions (Halder et al., 2012). As we observed in our results, when modifying the microbiota, enterobacteria were eliminated, indicating that these bacteria may play an important role in maintaining the response to insecticides. Certain bacterial communities such as Pseudomonas spp. and Aeromonas spp. were increased; it has been reported that some of these bacteria may produce metabolites that can compromise the physiological functioning of the mosquitoes when they are exposed to insecticides. Therefore, we could associate this type of response to this event resulting in decreased survival in our results.

The response with deltamethrin was different from what we observed in the permethrin group. There was a non-significant 2% decrease in mosquito survival with exposure to deltamethrin and penicillin/streptomycin treatment and a reduction of 4% with gentamicin compared to those whose microbiota was not modified. However, we could observe a change in the microbiota populations exposed to deltamethrin. The mosquitoes treated with penicillin/streptomycin exposed to this insecticide showed as predominant species E. meningoseptica and P. azotoformans-fluorescens-synxantha, while B. hinzii-petrii and S. arletta in those exposed to gentamicin. E. meningoseptica is a non-motile gram-negative bacillus, ubiquitous in nature (Kim et al., 2005). The members of the genus Elizabethkingia are found in wet habitats, particularly in water supplies (Kämpfer et al., 2011). In mosquitoes, it has been reported that this bacterium can modulate anti-Plasmodium effects, thus prolonging the lifespan of the infected mosquito vector (Akhouayri et al., 2013). In addition, it has been consistently found in laboratory conditions to be an important part of the functionality of mosquitoes (Wang et al., 2011). E. anophelisis is known to be a dominant bacterium in the gut microbiota in the mosquito A. gambiae (Rani et al., 2009; Gusmão et al., 2010; Kämpfer et al., 2011; Boissière et al., 2012; Akhouayri et al., 2013), and E. meningoseptica has been found in Cx. quinquefasciatus (Terenius et al., 2012). Similarly, the genus Elizabethkingia was detected in 68% of mosquito populations collected in Cameroon (Chen et al., 2015). The species Bordetella sp., which was the most abundant bacterium in the modification with gentamicin, is found in various aquatic environments and terrestrial environmental sources, also associated with plants (Shamim et al., 2019). The genus Bordetella has been isolated from adult mosquitoes of An. stephensi (Chavshin et al., 2012). Although these species are not common among microbes in mosquitoes, they can be found in small amounts in the intestinal ecosystem or tissues of mosquitoes, having an ecological connection to behavior and protection against adverse conditions (Kukutla et al., 2013).

Recent studies have demonstrated the role of symbiotic bacteria in detoxification processes (Itoh et al., 2018). This was observed in arthropods with phenothion applied in agricultural fields which were shown to be degraded by Pseudomonas, Flavobacterium, and Burkholderia (Kikuchi et al., 2012). Bacillus cereus, isolated from the guts of the P. xylostella moth, has been found to have a high degradation capability for the pesticide indoxacarb (Ramya et al., 2016a). Similarly, Enterobacter asburiae and P. agglomerans were found to degrade acephalate (Ramya et al., 2016b). Also, Citrobacter sp. was isolated from the intestine of B. dorsalis, capable of hydrolyzing trichlorphon (McFall-Ngai et al., 2013). Recently, a study determined the effects of pyrethroid insecticides in An. albimanus microbiota, finding differences with respect to mosquitoes not exposed to pyrethroids, determining Klebsiella, Pantoea, and Asia as key species in resistance to pyrethroids (Dada et al., 2019). In our study, we did not find species with evidence of metabolism of deltamethrin; however, we observed that with exposure to deltamethrin, there were different patterns of bacterial profiles compared to those seen with exposure to permethrin, which could influence the detoxification process. Our results clearly showed a different response to two insecticides from the same family, previously noted in Dada et al. (2019)’s study, where they obtained different microbiota patterns against pyrethroid insecticides. It is very important in future investigations to study the participation of key bacteria in the mechanisms of detoxification in mosquitoes since there is limited information.

The results of this study describe the microbiota of A. aegypti, which were dominated by species of Proteobacteria and Bacteroidetes. In nature, insects frequently face unfavorable environmental conditions that can alter symbiotic bacteria. In this study, we observed that changes in the microbiota affect the mosquito’s response to exposure to pyrethroid insecticides. Symbiont microorganisms associated with mosquitoes have been shown to play a key role in response to insecticide exposure. The search for key symbionts related to response to insecticides will allow us to understand mosquito physiology and then create tools and new targets for controlling mosquito populations. The development of this research will generate basic knowledge of key bacteria of each population analyzed.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: National Center for Biotechnology Information (NCBI) BioProject database under accession numbers OK646404-OK646460 entitled “Uncultured Prokaryotic 16S rRNA/Microbiome Aedes aegypti Group 1” and OK648490-OK648570 entitled “Uncultured Prokaryotic 16SrRNA/Group 2”.

Ethical review and approval were not required for the animal study because the manuscript presents research results on invertebrate animals (Aedes aegypti).

All authors contributed to the Replication of experiments, data analysis, and manuscript review.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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