Aedes spp. and Their Microbiota: A Review

1Dengue is the most rapidly spreading vector-borne disease in the world with 2.5 billion people at risk and approximately 500,000 people developing severe dengue cases annually. The increasing negative impact of dengue viruses on humans is partly associated with the range expansions of their primary vectors, Aedes aegypti and Aedes albopictus. Besides dengue viruses, Ae. aegypti and Ae. albopictus are also efficient vectors of Chikungunya, Zika, and other arboviruses, as well as dog heartworm and filarial nematodes.

2Currently, control of mosquito populations is the only available strategy to prevent arboviral diseases because there are no therapeutic treatments for arboviruses and vaccines are limited.

3Mosquitoes are holometabolous organisms with a life cycle involving two different types of habitats: larvae and pupae live in aquatic habitats, hereafter called “breeding sites,” and adults are subaerial. Only adult females transmit arboviruses, but controlling the juvenile stages is effective because significant reduction of larvae results in a decreased number of adults, thus reducing not only chances of disease transmission, but also nuisance. Consequently, a number of strategies have been developed to control larvae, including environmental sanitation, the use of insecticides or biological agents. These conventional vector control methods are facing challenges because of their sustainability and organizational complexity. For instance, the Region Plan that was established in the Italian region of Emilia Romagna after the 2007 Chikungunya outbreak involved 280 municipalities and had a cost of 5.3 million euros over 3 years. Additionally, resistance to insecticides is emerging in natural Ae. albopictus populations and is widespread in Ae. aegypti, challenging the sustainability of this control measure. Thus, the development of novel, eco-friendly and easy to manage products or systems for vector control is urgently needed to complement traditional mosquito control methods.

4Manipulation of mosquito microbiota is emerging as a promising field to develop novel vector control strategies. Examples that are already being implemented in the field include the use of entomopathogenic fungi such as Beauveria bassiana, which can be found on the water surface of breeding sites and kills larvae and adults of a number of mosquito species, and some strains of the alpha-proteobacteria Wolbachia, which induces cytoplasmic incompatibility and, when introduced into its not-natural host Ae. aegypti, it negatively impacts mosquito vector competence to dengue viruses. Additional strategies aim at identifying natural symbionts of mosquitoes and either alter them genetically to express anti-pathogen effectors or disrupt their natural symbiosis with the insect host.

5Here we provide an overview of the current knowledge on the composition, structure and function of Aedes spp. symbionts, with a focus on gut microbiota. We also highlight the technological progresses that are shaping our knowledge of mosquito microbiota and the exploitation of microbiota for vector control. The literature describing mosquito microbiota is ample and, in certain cases, controversial. The present review provides a summary of the available knowledge and may inadvertently omit some information. For these omissions, the authors apologize. For those interested in expanding on the topic, a number of helpful reviews have been published also in this Research Topic (see for example;;;;; ).

6The workflow for the study of Aedes spp. microbiota is organized in three main phases, i.e., data generation, analysis and exploitation, as summarized in Figure 1.

7The first paper that described the microbiota of Ae. aegypti was published in 2001. In this study, the authors used a culture-dependent approach including isolation of Ae. aegypti guts of larvae collected from natural breeding sites in Thailand, plating of the homogenates onto Luria-Bertani (LB) agar plates and analysis of resulting colonies. Colonies were differentiated by morphology, as well as Gram-staining and standard biochemical assays. In this first study, two strains of Bacillus cereus were identified as particularly abundant and, given the identified stable association with Ae. aegypti, authors suggested their potential exploitation for the development of mosquito larvicidal systems. A similar approach was adopted in a subsequent work focused on the characterization of the bacterial symbionts from Ae. aegypti crop. Besides identification of bacteria by morphology and biochemical approaches, authors also extracted the DNA and Sanger-sequenced their 16S rRNA gene to characterize them. Serratia sp. were the predominant bacteria in this tissue, and also Bacillus sp. and Bacillus subtilis were identified.

8Culture-dependent methods were increasingly adopted in the following years based on different media and isolation techniques to investigate the composition and diversity of the microbiota in both Ae. aegypti and Ae. albopictus. Culture-independent methods were also developed to overcome difficulties in recreating the physiological conditions necessary to cultivate bacteria. Culture-independent approaches include low-throughput techniques such as Denaturating Gradient Gel Electrophoresis (DGGE), taxonomic microarray hybridizations, as well as more recent molecular strategies based on High Throughput DNA Sequencing technologies (HTS), such as DNA metabarcoding (or 16S rDNA amplicon sequencing) and metagenomics. These approaches allow researchers to achieve a more comprehensive and informative culture-independent picture of the bacterial communities that reside in mosquitoes.

9The protocol for the amplification of 16S rDNA became a universal tool for determining the phylogenetic relationships among bacteria since its development in the early nineties. Nowadays, DNA metabarcoding is the most common sequencing approach to characterize the microbial community in a sample. This method is based on the amplification and sequencing of hypervariable region(s) of the 16S rDNA, nowadays most-frequently using Illumina technology, primarily the MiSeq system, to achieve the most accurate longest reads with high throughput. Variability of the 16S rDNA is usually high enough to allow accurate taxa characterization but may not always allow unambiguous identification at a lower classification level such as genus or species. DNA metabarcoding has been applied to both Ae. aegypti and Ae. albopictus (Table 1), allowing to analyze samples in a very cost-efficient manner.

10More recently, Shotgun Metagenomic Sequencing (SMS) was implemented through HTS. This approach does not rely on an initial PCR step with universal primers (for instance targeting bacterial 16S rDNAs), thus allowing to extend the analyses of insect microbiota beyond bacteria to fungi and viruses and allowing bacteria identification beyond the 16S rRNA genes. SMS was initially applied to identify viruses infecting wild mosquitoes, including those of the Culex, Anopheles, Ochlerotatus, and Aedes genera. SMS was used to analyze Ae. aegypti and Ae. albopictus strains artificially infected with dengue virus type 1 and 3 (DENV-1 and DENV-3), chikungunya (CHIKV), or yellow fever (YFV) viruses. The results of these studies showed that SMS has the potential to be integrated in the framework of arbovirus surveillance programs, with the advantages of obviating the need for culture-based approaches and prior knowledge of etiologic agents. This is possible since DNA and RNA viruses can be detected in mosquito blood meal for up to 24 h after initial ingestion. Despite the power of SMS, this technique has not been extensively applied to Aedes spp.

11Culturomics recently emerged as a novel tool to discover still unknown microbes. This method consists in the combination of multiple culture conditions using a high-throughput approach (i.e., use of different selective and/or enrichment culture conditions) followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) or 16S rDNA amplification and sequencing to identify the growing colonies. This method was applied to Anopheles gambiae, Culex quinquefasciatus, and Ae. albopictus to characterize the bacterial diversity of mosquito midguts. With this approach, 17 previously unknown bacterial species were identified in An. gambiae, suggesting the potential of culturomics for expanding our knowledge of the microbiota composition. The advantages provided by culturomics include the ability to detect minor community members, the capacity to provide information about the viability of the detected symbionts, and the potential for further improvements due to innovations in automation and miniaturization.

12Protocols to produce axenic individuals (i.e., bacteria-free) or gnotobiotic larvae (i.e., larvae colonized by a single bacterial species or a simplified bacterial community) were generated to study the physiological impact of the microbiota. Early studies on the functions of microbiota in Ae. aegypti were based on the use of sterile conditions and diet supplementation with vitamins and nutrients. More recent studies used a combination of ethanol and bleach to sterilize the egg surface and standard larval food, previously sterilized by irradiation. Gnotobiotic larvae are generated by inoculation of a given bacterial isolate in flasks containing sterile water, sterilized standard diet and the axenic first instar larvae. Interestingly, despite the use of antibiotic treatments to manipulate insect bacterial communities, a recent study clearly indicated that several antibiotics failed in achieving the full elimination of bacteria in Ae. aegypti (UGAL strain) and showed adverse effects on the fitness of first instars larvae. An alternative approach to rear axenic adult mosquitoes was recently described based on maintaining larvae hatched from surface-sterilized eggs on agar plugs containing yeast and liver extract. This method was also used for the production of adult mosquitoes with simplified microbiota (i.e., from one to three symbiont species).

13Concerns about the microbial quality of drinking water together with the increasingly recognized importance of free-living and host-associated microbes to the function of both the ecosystems and living organisms greatly stimulated the development of protocols for the analysis of the microbiota in aquatic environments. The complexity of aquatic environments requires the adoption of integrated analytic systems in which stringent water filtration methods, HTS technologies and bioinformatics are combined to cope with the low concentrations of organisms in aquatic environments and with the ultrasmall cell size of some aquatic bacteria. Such integrated approaches began to be applied to isolate and characterize the bacteria present in Aedes spp. larval breeding sites, showing that a substantial fraction of the microbiota in mosquitoes is acquired through larval feeding in breeding sites. Most analyses involve water filtration allowing to retain microorganisms >0.2 μm in size. Recently, novel and more stringent protocols were developed, which, through serial water filtration with membrane filters of decreasing pore sizes, allow to collect and concentrate the bacterial samples in the water. Such methods make use of tangential flow filtration (TFF) systems combined with filtration modules able to retain particles <0.1 μm in size, thus allowing to physically separate macro-organisms from micro-organisms and viruses. DNA extracted from these water samples is then sequenced using HTS approaches. Studies in Anopheles spp. mosquitoes showed that the depth in which breeding site water is sampled may influence the composition of bacteria. Aedes spp. mosquitoes tend to breed in small, often human-made and not stable breeding sites, for which there should be no depth differences.

14Depending on their life-stage, mosquitoes interact with microbiota differently. At the larval stage, Aedes spp. microbiota is acquired primarily through feeding in breeding site water. In both Ae. aegypti and Ae. albopictus, the composition of larval microbiota represents a subset of the Operational Taxonomic Units (OTUs) found in the breeding site water. The lower abundance of bacterial taxa in the larvae as compared to what found in breeding site water suggests that bacteria that establish symbiosis early during larval development may inhibit the colonization by additional taxa. The composition of larval microbiota varies greatly among sites, but strong similarities are found among larvae of different species that breed in the same site. These data support the relevance of larval habitat in shaping Aedes spp. microbiota. Distinct bacterial communities were identified between domestic and sylvatic Ae. aegypti habitats further supporting the important effect of the ecological context of the breeding sites in defining the composition of mosquito microbiota.

15Adults can acquire bacteria from their breeding water while they emerge from their pupal cases, as shown for An. gambiae. In addition, mosquitoes have been proposed to be able to transmit symbionts back to the breeding sites while laying eggs, thus affecting the microbial community which larvae are exposed to, and supporting a certain level of vertical transmission. This is particularly important for Ae. aegypti and Ae. albopictus, which are able to exploit small and temporary water containers as larval breeding sites. In these conditions, water biogeochemical properties such as pH and concentration of ions, temperature, food sources and microorganism abundance may undergo sharp and rapid variations, which affect microbial composition. For instance, in microcosm-based experiments performed with Ae. triseriatus, the presence of larvae in the water was shown to contribute to create enriched and anoxic conditions which favored the growth of Enterobacteriaceae. Besides influencing the composition of larval gut microbiota, the microbiota of the breeding site also plays a role in mediating attraction and oviposition responses of mated Aedes spp. females. For example, Bacillus cereus and Pseudomonas aeruginosa elicit oviposition responses in Ae. aegypti. Similarly, Ae. aegypti females were shown to be significantly induced to oviposit in water containing a suspended solution of Acinetobacter calcoaceticus. In Ae. albopictus, Psychrobacter immobilis isolated from the water of larval breeding sites elicited higher oviposition responses from gravid females than did water deprived of that bacterial species.

16The variable nature of aquatic environments, including fluctuations in temperature, pH and oxygen content that impact microbial growth, prevents mosquitoes from reliably encountering particular and standard bacterial species and support the hypothesis of a dynamic host-symbiont interaction.

17Despite habitat-related differences in the composition of larval microbiota were observed, a number of bacterial taxa have been consistently found in all tested Aedes spp. and have been proposed to constitute the ‘core microbiota’ of mosquitoes. Aedes spp. microbiota is composed primarily by Gram-negative aerobic and facultative-anaerobic bacteria, as generally occurring in other mosquito species. Only two obligate anaerobe taxa have been detected in Aedes spp. so far. These anaerobe taxa are Clostridium, found in Ae. aegypti, and Blautia, detected in Ae. albopictus. Members of the Blautia genus were previously isolated from mammalian gut and were suggested to play a role in nutrient assimilation.

18Microbiota composition changes during the development from larvae to adults. Adults eliminate larval midgut bacteria during metamorphosis, for instance Ae. aegypti expel more than 90% of the bacterial species during molting and metamorphosis, similarly to what is observed in other insects. Nevertheless, several bacteria are trans-stadially transmitted and bacteria may be acquired also during blood feeding, as reported for the arboviral vector Culicoides imicola.

19Actinobacteria and Bacteroidetes, members of Proteobacteria, were found to be consistently present in 4th instar larvae of both Ae. aegypti and Ae. albopictus. Other genera that are frequently found in the gut of larvae include Chryseobacterium, Elizabethkingia, Pseudomonas, Nisseria, and Enterobacter. The Actinobacteria Leucobacter and Microbacterium, both belonging to Microbacteriaceae family, are abundant in Ae. aegypti larvae, but nearly absent in adults. In contrast, Chryseobacterium (Flavobacteriaceae) was a common component of mosquito microbiota at all life-stages.

20In the case of adult mosquitoes, Proteobacteria, Bacteroides, Firmicutes, and Actinobacteria are the phyla grouping more than 99% of the total microbiota community components. More specifically, members of Enterobacteriaceae (e.g., Enterobacter, Klebsiella, Kluyvera), Erwiniaceae (e.g., Pantoea), Yersiniaceae (e.g., Serratia), Acetobacteraceae (e.g., Asaia), Enterococcaceae (e.g., Enterococcus), and of Bacillaceae (e.g., Bacillus) are the most-frequently described bacteria from the gut of adult Aedes spp.. Characterization of microbiota is biased by the technique used and the level of variability within the 16S rRNA genes. Thus, while higher taxa assignment is certain, lower classification may be problematic and lead to contrasting results.

21Most studies focus on the microbiota of the gut because of its direct implications with mosquito vector biology. However, microorganisms can colonize other organs, including reproductive tissues and salivary glands, both in Anopheles and Aedes spp. mosquitoes. An overview of the bacterial genera so far identified in Aedes spp. tissues is reported in Figure 2.

22In both Ae. aegypti and Ae. albopictus adults of different laboratory strains, the phylum Proteobacteria is dominant in gut, ovaries, salivary glands, testes and male accessory glands, with tissue-specific tropism being detected. For instance, the microbiota of reproductive organs of both sexes appears more diverse than that colonizing either the gut or salivary glands. Inter-specific differences in the tissue distribution of the microbiota were also detected. For instance, while Alphaproteobacteria represent the 97% of female and the 73% of male total microbiota in reproductive tissues of Ae. albopictus, they constitute about 30% of the total bacteria in reproductive tissues of Ae. aegypti. Wolbachia dominates in Ae. albopictus ovaries (94%), while it co-exists with bacteria of the genera Sphingomonas, Cupriavidus, and Serratia in testes. Gammaproteobacteria are the dominant taxon in salivary glands of female Ae. albopictus, while the microbiota of Ae. aegypti salivary glands is richer and includes representatives of the genera Serratia, Escherichia–Shigella, Pantoea, Acetobacter, Sphingomonas, Burkholderia, and Cupriavidus.

23In the gut of both Aedes species, Alpha-, Beta- and Gammaproteobacteria are equally represented, with Sphingomonas, Asaia, Cupriavidus, Escherichia-Shigella, Pseudomonas and Serratia being the most-frequent taxa. Sex differences in the composition of the microbiota are detected, with the dominance of Alphaproteobacteria in male guts. Whether the composition of Aedes spp. microbiota is richer than what is found in other species is still controversial. A number of factors, such as the number of generations in the insectary, the age and the genetic background of the species studied, along with the rearing conditions may influence microbiota composition.

24Microbiota composition was analyzed in both laboratory strains and wild populations. Laboratory strains include UGAL, Galveston, Rockefeller and MOYO for Ae. aegypti and CDC, Foshan and Galveston for Ae. albopictus (see Table 1). Field mosquitoes were sampled across the global distribution of both species. Most of the studies were performed on adult female midguts, followed by 4th instar larvae and their breeding site water.

25Upon laboratory colonization, the composition of adult midgut microbiota among different strains derived from distinct Ae. aegypti geographic populations was highly similar at the phylum level. The landscape of the overall microbiota across strains does not change even in presence of differences in the abundance of specific taxa within each phylum. These results pose the challenging question of whether preferential associations between Aedes spp. genotypes and specific bacterial taxa exist or are selected for during laboratory colonization.

26Aedes spp. sampled in different areas showed a limited diversity within bacterial communities at high taxonomic levels (i.e., phylum and family), similarly to data reported for field-collected Anopheles spp.. Micrococcus of the Actinobacteria phylum, Staphylococcus of the Firmicutes phylum, and primarily members of the Proteobacteria phylum, such as Comamonas, Acinetobacter, Enterobacter, and Pseudomonas genera, are consistently present in wild mosquitoes. Proteobacteria include Gram-negative bacteria that have been suggested to be abundant in insects due to a more effective capacity to invade and proliferate within new hosts and/or their active recruiting by insects. Similarly, Ae. albopictus samples from Northern Italy (Trentino region) display a lower degree of microbial diversity when compared to French and Vietnamese populations. Whether this result is dependent on differences in breeding sites water or the fact that populations from Italy and France are new populations is an open question. The absence of a relation between population genetic origin and midgut microbiota was recently shown in two Ae. albopictus populations from tropical (La Réunion) and temperate (Montpellier, Continental France) regions reared under controlled laboratory conditions. These contrasting data highlight the need to continue investigating the impact of Aedes spp. population dynamics on microbiota composition.

27One common trait revealed by several studies is the high inter-individual variability in the composition of the microbiota, especially at a lower classification level. Specific OTUs may be found exclusively in one specimen, and individual OTUs may represent over 90% of the microbiota of one mosquito. This aspect is particularly relevant since it may be related to variations in vector competence as well as mate choice, as occurring in the fruit fly Drosophila melanogaster, where flies prefer mates sharing a similar bacterial gut community.

28Habitat-related parameters affect bacterial communities, thus impacting the composition of mosquito microbiota. For example, increase of water temperature in breeding sites results in higher abundance of Betaproteobacteria and this correlates with higher abundance of Anopheles vs. Aedes spp. larvae. Anthropogenic activities also impact the bio-geochemical properties of breeding sites, and, in turn, affect mosquito microbiota. Fertilizers containing ammonium and phosphorous contaminating aquatic habitats are known to affect the development and abundance of bacteria, which are a nutrient source for Culicidae larvae. The increasing abundance of residual antibiotics in the environment has been shown to impact the composition of the microbiota to which mosquitoes are exposed. Antibiotics in human blood disrupt gut microbiota of An. gambiae females and enhance susceptibility to Plasmodium spp. infection. In addition, larval ingestion of antibiotics alters bacterial composition in Ae. albopictus adults, with reduction or even elimination of specific taxa and concomitant proliferation of Wolbachia and Dysgonomonas spp.. Dysgonomonas spp. is abundant in Ae. albopictus populations and is known to produce vitamin B12 in termites. Whether this bacterium plays a similar role in Ae. albopictus remains to be determined.

29Host-related factors, including nutrition, development and sex, also influence microbiota composition. For instance, blood-meal induces an overall decrease of OTU numbers with an increase in relative abundance of bacteria of the genera Chryseobacterium and Delftia in Ae. aegypti, and blood- and sugar-fed females harbor distinct bacterial communities. Changes in the composition of the gut microbiota following a blood meal may be due to the oxidative stress associated with the catabolism of the blood meal, as proposed for An. gambiae. The two above-mentioned dominant genera were also detected on the surface of eggs, supporting the idea of vertical transmission. The source of blood meal may also influence the composition of the microbiota in mosquito midguts, similar to what occurs in Ixodes pacificus ticks. In particular, members of the genera Leucobacter, Chryseobacterium, Elizabethkingia, and Serratia were characteristic of either newly emerged Ae. aegypti or adults fed on chicken, rabbit, and human blood, respectively. Sugar-fed mosquitoes displayed higher abundance of Pseudomonas spp. and unclassified Acetobacteraceae, which were previously found in associations with insects relying on sugar-based diets. While blood directly goes to the midgut, sugar meals are stored in the crop as food reserves. The crop of Ae. aegypti harbor bacteria including Serratia (Yersiniaceae) and the yeast Pichia sp., which can be transferred to the midgut along with food. Elizabethkingia spp. (Flavobacteriaceae) was found only in sugar-fed females, in agreement with findings in An. gambiae, where Elizabethkingia spp. abundance was reduced after blood feeding. The different nutritional behavior of male and female mosquitoes may contribute in the observed sex-related differences in the composition of microbiota. Such differences may also relate to the different tropism of endosymbionts for female and male reproductive organs, as observed in Anopheles spp..

30The composition of microbiota also changes with mosquito age, probably as the result of interspecific competition among symbionts for sugar sources, or, as occurring in other insects, nutrient availability in the gut or mosquito immunity.

31The importance of bacteria interactions, as well as potential interaction with viruses, is becoming more and more evident as a regulator of the composition and abundance of the microbiota and has practical implications that we describe below. Microbiota composition of Ae. aegypti adults changes following ZIKV infection, with Rhodobacteraceae and Desulfuromonadaceae emerging as biomarkers of ZIKV infection. When stable symbiosis is artificially established in Ae. aegypti, Wolbachia dynamically interacts with other members of the microbiota community but has minimal effects on microbiota composition.

32Studies aiming at clarifying patterns of co-occurrence and co-exclusion among the components of the microbiota are being perused to decipher the physiological impact of the microbiota and shed light on complex phenotypes. For example, Serratia and Cedecea spp. displayed several co-exclusionary relationships with dominant taxa such as members of the genera Asaia, Pseudomonas, and Enterobacter in the microbiota of Ae. aegypti, Ae. albopictus, and C. quinquefasciatus from both the field and the laboratory. Additionally, the first whole genome metagenomic analysis of An. albimanus revealed links between microbiota and phenotypic resistance to the insecticide fenitrothion, suggesting a role of microbiota in insecticide resistance.

33Aedes aegypti mosquitoes were initially thought to require living bacteria for development, as axenic larvae die as first instars differently than for Anopheles spp.. This interaction did not appear to depend on a particular bacterial species or community assemblage, as several different bacterial species rescued development of gnotobiotic larvae. In such gnotobiotic mosquitoes, each of the individual bacteria tested proliferated in absence of other community members, with the exception of Microbacterium and Leucobacter spp. These last taxa require other bacteria to survive in Ae. aegypti. Taken together, these results suggest that several members of the larval gut microbiota support development and egg production comparably to conventionally reared individuals with a mixed bacterial community.

34As described above, survival of axenic larvae may be achieved under specific conditions. In contrast to previous experiments, managed to rear axenic larvae to adulthood by providing high concentrations of liver and yeast extract in a semi-solid form. Axenic larval developmental time was longer than that of larvae with an unaltered microbiota. These data support the idea that the primary symbiotic association between gut bacteria and Ae. aegypti is essentially nutritional, as live bacteria and fungi do not appear to be essential to mosquito development.

35Adult gut microbiota affects blood meal digestion. Treatment with antibiotics reduced the abundance of culturable gut bacteria, resulting in slower digestion of the blood bolus and statistically significant reductions in the number of laid eggs. Enterobacter and Serratia, in particular, are involved in hemolytic activity.

36Exposure to bacteria during larval development affects adult traits related to pathogen transmission, suggesting that a better understanding of larval ecology has the potential to reveal determinants of pathogen transmission by Aedes spp.. Earlier studies in Ae. aegypti showed that removal of the gut microbiota with antibiotics increases mosquito susceptibility to DENV-2 infection, and that Serratia odorifera is able to enhance DENV-2 susceptibility. The increase in DENV loads and prevalence correlate with the presence of Serratia because Serratia secretes SmEnhancin, a protein that cleaves off membrane-bound mucins and weakens the peritrophic matrix favoring viral dissemination out of the midgut. This effect on viral dissemination was not observed when other 20 commensal bacteria were tested, supporting the idea of a species-specific effect of the microbiota on Ae. aegypti vector competence. Serratia-positive mosquitoes were obtained from DENV endemic regions, while Serratia-negative mosquitoes were caught in non-DENV-endemic regions supporting the hypothesis that microbiota composition may contribute to the observed differences in vector competence across Ae. aegypti populations.

37The bacterial component of mosquito microbiota is by far the most widely investigated. However, Aedes spp. microbiota comprise also other entities such as non-pathogenic fungi, pathogenic yeasts and viruses.

38Less than five fungal species were identified in Ae. aegypti and Ae. albopictus. Whether this limited number is indicative of a streamlined fungal community remains to be determined. Penicillium was found in wild adults and larvae based on the morphological analysis of fungal colonies. Subsequently, a combination of culture-dependent methods and PCR amplification of the 28S rRNA gene (i.e., 28rRNA and 16rRNA amplification are analogous as fungi do not have 16 rRNA) allowed the identification of Pichia in the crop of newly emerged unfed females of the Rockefeller strain. Members from the genus Pichia and Candida were also found in the midgut and in midgut and ovaries of Ae. aegypti mosquitoes, respectively. Pichia was isolated only from sugar-fed females. By using culture-dependent method, Candida parapsilosis and Meyerozyma guilliermondii were identified in larvae, pupae and adults (in both gut and gonads) of Ae. aegypti and Ae. albopictus laboratory strains and wild-collected adults from Brazil, Bangladesh and Italy. Candida parapsilosis and Meyerozyma guilliermondii can become opportunistic human pathogens under specific physiological conditions. The identification of these fungi in Aedes spp., which complete their life cycle in anthropized environments, suggests these mosquitoes could contribute to the dissemination of pathogenic yeasts, thus increasing their public health relevance. Meyerozyma guilliermondii colonizes the guts of insects from several taxa; for instance, it is the dominant species in the mycobiota of the leishmaniasis vector Phlebotomus perniciosus where it was proposed to contribute in uric acid degradation. Metabolic interactions between members of the mycobiota and the mosquito host are being discovered. As an example, a fungus from the Talaromyces genus was identified to be naturally present in the midgut of field-caught Ae. aegypti females from Puerto Rico using a combination of microscopy and sequencing of the rRNA internal transcribed spacer (ITS). Talaromyces was found to enhance DENV2 infection by transcriptional and enzymatic inhibition of trypsins in the midgut, thus increasing mosquito vector competence.

39Mosquito virome includes arthropod-borne viruses (i.e., arboviruses) able to replicate in mosquitoes and vertebrates, and recently identified insect-specific viruses (ISVs), which are restricted to insects and do not replicate in vertebrates.

40Metagenomic approaches were initially used for discovery and surveillance of specific viruses, such as DENV-1 and Phasi Charoen-like virus (PCLV) in Ae. aegypti and CHIKV, DENV-3 and YFV in Ae. albopictus. The first study using mosquito virus metagenomic sequencing to describe the diversity of DNA viruses was performed on wild mosquitoes from California. This study analyzed pools of female mosquitoes from different species collected in three geographical sites, comprising Culex erythrothorax as well as other undetermined species. This study revealed that the viral community was highly diverse across samples and most of its members were uncharacterized. The identified viral sequences showed similarity to members of the Anelloviridae, Circoviridae, Herpesviridae, Poxviridae, and Papillomaviridae families, which infect mammals and birds. This study also showed for the first time that mosquito virome includes plant viruses, such as Geminiviruses and Nanoviruses.

41As described above, the SMS method significantly improves the detection of viruses in mosquitoes, allowing the identification of previously unknown entities, and the characterization of the virome of individual mosquitoes. Recent work supports the conclusion that mosquito virome is frequently dominated by specific ISVs. For instance, ISVs of the Flaviridae family account for 88.5% of the virome of Culex spp. mosquitoes from Mozambique. Similarly, ISVs are 88% of the virome of Culex tritaeniorhynchus from China. The virome of Culex spp. mosquitoes collected in different sites in Kenya and China was shown to differ both in terms of number and in relative abundance of arboviruses vs. ISVs. Three known ISVs dominated the virome of wild-caught Ae. aegypti mosquitoes from Thailand and Australia: the phlebovirus PCLV (family Bunyaviridae), which represents >75% of the viral community in both sites; the unclassified Humaita-Tubiacanga virus (HTV), and the flavivirus Cell fusing agent virus (CFAV), which was previously found to be common in wild Ae. aegypti samples. The similarity of the virome in mosquitoes from Thailand and Australia contrasted with substantial differences in the composition and abundance of their bacterial community and mycobiota.

42Similar to findings on the bacterial component of the mosquito microbiota, viral diversity is likely shaped by host- and environmental-related factor, including sex, diet, environmental temperature and ecological features of the resting sites. For instance, arboviruses replicate at higher temperatures (i.e., 36.5–42°C in mammals and birds) than ISVs (i.e., around 28°C in tropical regions), supporting the idea that temperature is an important factor modulating viral prevalence and maintenance in mosquito field populations.

43The landscape of ISVs in the field as well as their prevalence in both laboratory and natural mosquito populations are still poorly described and require further investigation because ISVs may influence mosquito immunity, with effects on viral replication and gut microbiota diversity.

44The increasingly emerging interactions among mosquito host, viral infection and microbiota are stimulating the development of strategies to exploit Aedes spp. microbiota for vector control. The application of microbiota in vector control include strategies that aim at altering or using microbiota taxa that were shown to have physiological impacts on the host or displaying mosquitocidal and antipathogen effects. Alternative strategies, collectively regarded as paratransgenesis, aim at interfering with pathogens via the genetic modification of endosymbionts to express antipathogen effector molecules in the mosquito host. Figure 3 provides a summary of the currently available strategies.

45Several strains of Wolbachia pipientis (Alphaproteobacteria, Rickettsiales) are able to manipulate host reproduction through Cytoplasmic Incompatibility (CI). Establishment of a stable mosquito infection with a Wolbachia strain inducing CI can be exploited for mosquito control to induce sterility as a consequence of male release in the field (Incompatible Insect Technique, IIT). This strategy has been implemented also in Ae. albopictus, which naturally hosts two Wolbachia strains (wAlbA and wAlbB), generating a triple-infected strain with wPip from Culex pipiens molestus. Field releases of wPip Wolbachia-infected Ae. albopictus males in Kentucky (United States) were shown to be effective in achieving a significant reduction in the number of females in the treated area and large-scale population suppression trials are on the way in Guanzhou (China). The possibility to stably introduce Wolbachia in non-natural host species via transinfection opened new possibilities when Wolbachia infection was found to increase protection against arbovirues. Specifically, when transfected in Ae. aegypti, the Wolbachia wMelPop-CLA strain reduces mosquito lifespan and its vector competence for DENV-2 and CHIKV. After initial cage experiments, which showed that both wMelPop- and wMel-infected Ae. aegypti could invade wild populations and reach high frequencies, field releases in Australia showed that Wolbachia could be established in mosquito populations and continue to reduce vector competence following establishment in the field. Since then, several small- and large-scale releases of transinfected Ae. aegypti are ongoing in several locations worldwide, including Indonesia, Vietnam, Australia, and Brazil (see; ).

46The application of Wolbachia for the control of Ae. aegypti mosquitoes in population replacement strategies stimulated investigation to assess the occurrence of Wolbachia in wild-caught mosquitoes, with contrasting results. Wolbachia was detected in wild Ae. aegypti larvae and adults from Florida and in adults from Thailand and the Philippines. In contrast, there was no evidence of Wolbachia in over 2,500 mosquitoes from the whole species range. The presence of a natural Wolbachia infection in Ae. aegypti would be of great significance because a natural endosymbiont may circumvent the fitness loads related to the artificial mosquito-Drosophila system currently in use.

47A pathogen enhancement effect with respect to DENV-2 was seen in Ae. aegypti re-infected with Serratia odorifera, opposite to the phenotype observed for wMelPop- and wMel-infected Ae. aegypti. These findings emphasize the complex interplay among the host, the microbiota and the pathogens. These multifaced effects should also be considered in an ecological framework. For instance, it has been proposed that DENV inhibition of wMelPop- and wMel-infected Ae. aegypti mosquitoes may be temperature-sensitive.

48Other bacterial endosymbionts, such as Spiroplasma and Arsenophonus are capable of manipulating host reproduction, suggesting that further exploration of microbiota in Aedes spp. mosquitoes may reveal additional candidates to be explored as tools for mosquito population control.

49A number of microbiota members were shown to have antipathogen activities. For example, some entomopathogenic fungi shorten mosquito life span or reduce blood feeding success. Analogously of Wolbachia, Beauveria bassiana influences mosquito vector competence by activating the Toll/Jak-Stat immune pathways in Ae. aegypti thus indirectly decreasing DENV-2 infection and reducing vectorial capacity for ZIKV in Ae. albopictus. The presence of the ascomycota Metarhizium anisopliae was shown to correlate with reduced DENV-2 loads in Ae. aegypti females.

50Insect-specific viruses appear to suppress arboviruses in mosquitoes not only through replicative interference due to their genetic similarity with arboviruses, but also by superinfection exclusion, a process whereby primary viral infections can block a secondary infection of a similar virus. So far, most studies involved in vitro systems and focused on IS-flaviviruses, with the exception of the IS-alphavirus Eilat (EILV) that could alter Sindbis virus titers in vivo.

51Isolation of cultivable bacteria from the midgut of field-collected Ae. aegypti mosquitoes from Panama and subsequent reintroduction of single-isolate bacterial species such as Proteus sp. and Paenibacillus sp. resulted in a significant decrease in DENV-2 infection. This effect was related to the transcriptional changes induced in a number of antimicrobial peptide genes in the midgut, including cecropin, gambicin, and attacin. In the same study, the authors identified a Chromobacterium sp. isolate that was later shown to be able to recolonize the gut of both An. gambiae and Ae. aegypti and block Plasmodium and DENV-2 infection, respectively. The Gram-negative Chromobacterium inhibits growth of other bacteria in the midgut, displays entomopathogenic activity on larvae and adults, and was suggested to exert in vitro and in vivo anti-pathogen activity through stable secondary metabolites. While romidepsin appeared to be the most likely Chromobacterium-produced metabolite responsible for antiplasmodial activity, the anti-DENV activity is mediated by an aminopeptidase interfering with DENV-2 attachment by promoting the degradation of the Flavivirus E protein. These effects on mosquitoes, together with its culturability, make Chromobacterium an ideal candidate to be integrated in strategies for controlling both mosquito populations and pathogen transmission.

52One key aspect of these approaches is the feasibility of their use in field applications. Indeed, the capacity to spread efficiently in a wild population is essential. To do so, further research focused on the identification of selective pressures that could confer modified microbes an advantage over their wildtype counterparts is important and requires a better understanding of the physiological and genetic mechanisms favoring the presence of specific microbes among the community.

53Paratransgenesis requires the identification of symbionts that can be isolated from host tissues and used for in vitro genetic transformation. Moreover, symbionts should show specific tissue tropism as the cycle of several pathogens initiates in the gut and ends with salivary gland, and vertical transmission to the progeny, thus allowing self-sustenance of the modified symbionts in the field. Moreover, such symbionts have to be well established in the mosquito host in order to survive long enough to produce the effectors in the necessary amounts and display or excrete the effector molecule on their surface.

54Asaia emerged as a promising candidate for the paratransgenic-based control of malaria, as it was shown to be important for larval development in Anopheles spp., can be genetically manipulated, can be easily acquired by mosquitoes and it is vertically transmitted (see for a review). This bacterium has been detected in both laboratory and field mosquitoes, in both Ae. albopictus and Ae. aegypti. Asaia was already modified to secrete anti-malaria molecules and the results of semi-field trials suggested it can rapidly spread in wild populations of An. stephensi and An. gambiae. Wolbachia and Asaia appear to negatively compete, with Asaia occurrence in Wolbachia-infected mosquito species being low. Thus, a potential use of Asaia in paratransgenic approaches to control Aedes spp. mosquitoes will require a better understanding of the dynamic interactions between these two endosymbionts in the field.

55The possibility to rear mosquitoes in which a particular bacterial species is dominant among the gut microbiota supports the development of strategies based on symbionts that induce antiviral responses or antiviral molecules in Aedes spp.. Achieving a deeper understanding of the molecular mechanisms underlying the interaction between microbiota and pathogens may also lead to the selection of mosquito strains resistant to infection. On this basis, it is important to further expand our understanding of the physiological and metabolic interactions between Aedes spp. mosquitoes and their microbiota, in particular providing consistent answers to key questions, such as: (i) what is the composition of Aedes spp. microbiota in the field? (ii) what is the level of its variability and which are the parameters affecting such differences? (iii) how are community members of Aedes spp. microbiota transmitted cross-generationally? (iv) how do endosymbionts released into the environment compete with the natural microbiota members of mosquitoes?

56Besides having practical applications, these questions will also shed new light on the establishment and maintenance of symbiotic interactions. Interestingly, apart from Wolbachia, the bacterial species that have been identified so far to contribute to vector competence (i.e., Serratia and Chromobacterium) are sporadically detected in field mosquitoes supporting the hypothesis that while the core microbiota may contribute to mosquito physiology, rare and differentially distributed bacterial species should be more carefully studied in relation to vector competence.

57MB and FS conceived, designed, and wrote the manuscript. FS prepared the figures. MC critically reviewed the manuscript and contributed to figure improvement. All authors approved the final version of the manuscript.

58The 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.