# The mosquito holobiont: fresh insight into mosquito-microbiota interactions

Morgane Guégan, Karima Zouache, Colin Démichel, Guillaume Minard, Van Tran Van, Patrick Potier, Patrick Mavingui, Claire Valiente Moro

► To cite this version:

Morgane Guégan, Karima Zouache, Colin Démichel, Guillaume Minard, Van Tran Van, et al. The mosquito holobiont: fresh insight into mosquito-microbiota interactions. Microbiome, 2018, 6, pp.49. 10.1186/s40168-018-0435-2. hal-02479073

HAL Id: hal-02479073

https://univ-lyon1.hal.science/hal-02479073

Submitted on 26 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

# The mosquito holobiont: fresh insight into mosquito-microbiota interactions

Morgane Guégan<fnref n="1" />,<fnref n="2" />,³,⁴, Karima Zouache<fnref n="1" />,<fnref n="2" />,³,⁴, Colin Démichel<fnref n="1" />,<fnref n="2" />,³,⁴, Guillaume Minard<fnref n="1" />,<fnref n="2" />,³,⁴, Van Tran Van<fnref n="1" />,<fnref n="2" />,³,⁴, Patrick Potier<fnref n="1" />,<fnref n="2" />,³,⁴, Patrick Mavingui<fnref n="1" />,<fnref n="2" />,³,⁴,⁵ and Claire Valiente Moro<fnref n="1" />,<fnref n="2" />,³,⁴*

## Abstract

The holobiont concept was first developed for coral ecosystems but has been extended to multiple organisms, including plants and other animals. Studies on insect-associated microbial communities have produced strong evidence that symbiotic bacteria play a major role in host biology. However, the understanding of these symbiotic relationships has mainly been limited to phytophagous insects, while the role of host-associated microbiota in haematophagous insect vectors remains largely unexplored. Mosquitoes are a major global public health concern, with a concomitant increase in people at risk of infection. The global emergence and re-emergence of mosquito-borne diseases has led many researchers to study both the mosquito host and its associated microbiota. Although most of these studies have been descriptive, they have led to a broad description of the bacterial communities hosted by mosquito populations. This review describes key advances and progress in the field of the mosquito microbiota research while also encompassing other microbes and the environmental factors driving their composition and diversity. The discussion includes recent findings on the microbiota functional roles and underlines their interactions with the host biology and pathogen transmission. Insight into the ecology of multipartite interactions, we consider that conferring the term holobiont to the mosquito and its microbiota is useful to get a comprehensive understanding of the vector pathosystem functioning so as to be able to develop innovative and efficient novel vector control strategies.

**Keywords:** Mosquito, Pan-microbiota, Multiple interactions, Symbiosis, Adaptation, Pathogen transmission

## Background

A holistic view of organisms is necessary to understand the biology of metazoa. The host can no longer be considered as an isolated entity and instead should be considered as a chimera with close interactions with microbial communities. The extent interplay between both partners and its consequences on their evolutionary trajectory has given rise to the holobiont concept (i.e. the host and its community of associated microorganisms as well as their interactions) [1]. Under changing environmental conditions, associated microbiota, i.e. all microorganisms (bacteria, fungi, protists, viruses, etc.), can evolve rapidly and influence the acclimation, adaptation and evolution of host organisms. Changes in microorganism diversity and abundance may have a direct

impact on the holobiont's evolution by modifying the multipartite interaction dynamics.

The holobiont concept—originally developed for coral ecosystems—can also be transposed to other organisms. Most recent advances have shown that human gut microbiota plays a key role in regulating the host's metabolic functions, immunity, nutrition, physiology and even behaviour [2]. In arthropods, first studies focusing on the molecular mechanisms underlying the role of microbiota such as the host's immune function, nutrition, physiology and even behaviour concerned phytophagous insects [3]. However, there is still little evidence on the role of microbiota in haematophagous insects of medical importance (pathogen transmission to humans and/or animals) as well as the molecular mechanisms underlying their interactions with the host. The best-known examples concern the mutualistic symbiosis between tsetse flies (Diptera: Glossinidae) and its obligate bacterial symbionts *Wigglesworthia* spp. [4], even

<footnote>* Correspondence: claire.valiente-moro@univ-lyon1.fr</footnote>

<footnote n="1">¹Université de Lyon, Lyon, France</footnote>

<footnote n="2">²Université Lyon 1, Villeurbanne, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
though available data on specific *Wolbachia*-mosquitoes associations have considerably increased in the past decades [5]. These bacteria participate in nutrient provisioning, insect fitness, host immunity maturation or pathogen transmission [6]. According to the holobiont concept, an arthropod vector should no longer be considered as an isolated organism but rather as a complex system in which the different partners (host and microbiota) interact.

According to the World Health Organization (WHO), mosquito (Diptera: Culicidae) vectors of arthropod-borne pathogens such as *Anopheles* sp., *Aedes* sp. and *Culex* sp. mosquitoes are the greatest threat to public health [7]. *Anopheles* mosquitoes are able to transmit to humans the causal agent of malaria, which is the deadliest vector-borne disease, with about 212 million cases and an estimated 429,000 deaths reported in 2015 [8]. *Culex* sp. mosquitoes are able to transmit both arboviruses and parasites [9] and *Aedes* sp. (mainly *Aedes aegypti* and *Aedes albopictus*) can transmit arboviruses of medical importance to animals and humans, including West Nile (WNV) (Flaviviridae, *Flavivirus*), dengue (DENV) (Flaviviridae, *Flavivirus*), Zika (ZIKV) (Flaviviridae, *Flavivirus*) and chikungunya (CHIKV) (Togaviridae, *Togavirus*) viruses [10, 11]. Dengue is the most serious mosquito-borne viral disease and major chikungunya outbreaks have occurred worldwide over the last decade. Zika virus infection has also recently become a major public health concern, with the global spread of the disease and foetal microcephaly cases arising in women during pregnancy [12]. In addition, some of these mosquito species are invasive and their geographical distribution has been expanding as they take advantage of human activities such as international trade, emphasized by global warming [13, 14]. The lack of effective and preventive treatments against most mosquito-borne diseases restricts control strategies to individual protection and mosquito population control using insecticides. However, intensive and repeated of bio-cides use leads to the development of mosquito resistance and unwanted effects on non-target species.

There is recent increased interest in studies on mosquito-associated microbiota, which encompass bacteria, fungi, protists and viruses, i.e. both mosquito-specific viruses (MSVs) and the transmitted pathogens. Research on the role of microbial communities in the host biology and pathogen interference has led to the development of new vector control approaches based on the use of “symbiotically” modified mosquitoes [15]. Knowledge on the roles of microbes in the development, physiology or immunity of their hosts, as well as interference with transmitted pathogens, is henceforth essential to be able to develop these alternative strategies. This review summarizes current knowledge on mosquito-associated microbiota. We discuss recent advances and current prospects on the ecology and

functions of the mosquito microbiome as well as interactions with the host and transmitted pathogens.

## Influence of the breeding site ecology on mosquito bacterial microbiota

The origin of microbes colonizing mosquitoes and the role of the environment in microbial acquisition are issues that have long been debated [16]. This aspect is essential to define the dynamics of microbial communities in the mosquito holobiont. Most studies carried out to date on microbial acquisition in mosquitoes have been focused mainly on the bacterial component.

Recent studies confirmed that a substantial fraction of bacteria colonizing mosquitoes is acquired during the aquatic life stage, through the aquatic larval habitats. The microbial composition and environmental characteristics of breeding sites could partially explain the different colonization patterns of bacteria in immature and adult mosquito stages (Fig. 1). These effects likely accentuate interspecific variations in the microbiota due to the habitat tropism of the different mosquito species. Duguma et al. [17] showed some association patterns between nutrient contents and microbial composition in larval habitats and bacterial communities associated with *Culex nigripalpus* adults. While mosquitoes originating from high-nutrient habitats were associated with members of the Clostridiales order, those from low-nutrient habitats were instead associated with Burkholderiales order members. A specific community profile depending on environmental factors has also been associated with the bacterial composition in *Anopheles gambiae* [18]. Interestingly, the difference in bacterial diversity of larvae from different mosquito species sharing the same breeding site was shown to be lower than that of larvae of similar species living in different collection sites [19]. In *Anopheles coluzzii* and *An. gambiae*, some bacterial communities are shared among fourth instar larvae, water of the larval habitat and adults [20]. Another interspecific comparison indicated that the bacterial communities present in aquatic larval habitats and in larva guts were similar to each other and differed from the bacterial communities of the adult guts [21]. Similarly, only Firmicutes and Actinobacteria phyla were commonly found in both *Ae. aegypti* larvae and aquatic ecosystems, with higher bacterial diversity found in water than in larvae [22]. This suggests that although the bacterial community is acquired from water, the insect's gut is a more selective habitat for bacteria. This selectivity could be explained by the physicochemical conditions present in the gut (e.g. alkaline pH, redox potential, oxygen level below 5%, etc.) as well as other factors such as the immune response, peristalsis or presence of lytic enzymes or microbial interactions. Moreover, the findings of other
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Venn diagrams illustrating overlapping of bacterial composition between mosquito species, development stages and habitats. **a** Number of bacterial taxa specific and common between mosquito larvae, habitats and adults of *Aedes japonicus*, *Aedes triseriatus* and *Anopheles gambiae* [20, 21]. **b** Number of bacterial taxa specific and common to larvae of *Anopheles gambiae*, *Culex pipiens*, *Culex nigripalpus*, *Aedes aegypti* and *Aedes japonicus* [17, 18, 20, 22, 23, 27]. **c** Number of bacterial taxa specific and common to adults of *Anopheles gambiae*, *Anopheles stephensi*, *Culex nigripalpus*, *Aedes albopictus* and *Aedes aegypti* [17, 18, 20, 21, 23, 24, 27, 29–33, 35–37, 39]. An additional table shows in more detail the identification of bacterial species/genera in mosquito species [see Additional file 1]</figcaption></figure>

studies indicate that some larvae-borne bacteria persist in adults (Fig. 1). Interestingly, the *Thorsellia* genus was detected in both immature (early and late larval instars and pupae) and adult stages in *Culex tarsalis* [23]. In *Ae. albopictus*, some bacteria belonging to Micrococcaceae, Pseudomonadaceae and Staphylococcaceae families are common to larvae, adult males, as well as sugar-fed and blood-fed females [24].

Overall, these results imply that there is a continuum of bacteria from the aquatic environment to immature stages and adult mosquitoes, as shown by the overlap in bacterial composition between water, larvae and adults [20, 25] (Fig. 1). Contrary to previous assumptions [26], bacterial clearance during mosquito metamorphosis from pupae to adults would not be complete, clearly suggesting that a subset of such environmental-acquired
bacterial microbiota will intrinsically be part of the holobiont cell components.

## Mosquito core- and pan-microbiota

### From mosquito core-microbiota...

The concept of a core microbiota in mosquitoes (i.e. shared by mosquito populations belonging to the same species) has been addressed in recent years [20]. Generally defined as being a microbial community associated with hosts from a given group (e.g. individual, population, genus, species, etc.), a more flexible definition considering the microbial species most prevalent in the hosts (≥ 90%) has been proposed [27, 28]. Many studies have described core microbiota shared by different adult mosquito populations and/or species collected in distinct geographic areas [see Additional file 1]. For instance, *Pseudomonas*, *Acinetobacter* and *Aeromonas* spp. bacteria were detected in different *Ae. aegypti* populations from Brazil [29]. Likewise, *An. gambiae* populations collected at different sites in Burkina Faso shared *Thorsellia*, *Wolbachia*, *Massilia* and *Acinetobacter* spp. bacteria, which correspond to the most abundant taxa associated with those populations [18]. In Vietnam, *Acinetobacter* was found to be the sole core microbiota constituent of 11 *Anopheles* species [30, 31]. Indigenous and invasive populations of *Ae. albopictus* from Vietnam and France, respectively, also shared core bacterial microbiota, with *Dysgonomonas* being the most prevalent and abundant genus [32]. The tissue tropism of core microbiota was also examined. *Staphylococcus*, *Corynebacterium*, *Geobacillus*, *Micrococcus*, *Acinetobacter* and *Pseudomonas* spp. bacteria are present in male and female *An. gambiae* and *An. coluzzii* reproductive tissues [27]. Tchioffo et al. [33] detected core microbiota composed of *Pseudomonas*, *Comamonas*, *Acinetobacter*, *Rhizobium*, *Burkholderia* and members of the Enterobacteriaceae family in different *Anopheles* mosquito tissues, i.e. ovaries, salivary glands and midgut. Further studies are needed to investigate whether this tissue tropism is correlated with potential core microbiota functions.

### ...to mosquito pan-microbiota

As suggested for other organisms, the ecology modulates host-associated microbiota, thus prompting us to apply the pan-microbiota concept to mosquito, e.g. microbiota shared by different mosquito species regardless of their geographic origin [34]. Indeed, environmental factors influence the microbial composition of breeding sites and food resources (plants, sugar, blood). However, as mentioned above, the fact that a common bacterial fraction is shared by different mosquito species at various developmental stages is in favour of the environmental acquisition hypothesis [see Additional file 1]. This repeated-bacterial colonization leads to a stable association between

mosquitoes and their environmentally acquired microbiota. For instance, cultivable core microbiota of *Ae. aegypti* and *Ae. albopictus* females in north-eastern India was found to be composed of the same bacterial species, i.e. *Enterobacter cloacae*, *Klebsiella michiganensis*, *Pseudomonas monteilii*, *Bacillus aryabhattai*, *Lysinibacillus fusiformis* and *Staphylococcus hominis* [35]. In *Culex pipiens* and *Culex restuans*, 44% of operational taxonomic units (OTUs) were common in both species [36]. However, bacterial diversity was reportedly lower in distant mosquito species. Only *Bacillus* and *Escherichia/Shigella* spp. were common in *Cx. pipiens*, *Culiseta incidens* and *Ochlerotatus sierrensis* [37]. Similarly, *Pseudomonas* and *Wolbachia* were the two genera shared by *Culiseta melanura* and *Coquillettidia perturbans* [38]. A broader study targeting 12 mosquito species in the USA, including *Aedes*, *Anopheles* and *Culex*, revealed similarities among their bacterial communities [see Additional file 1]. These communities were dominated by *Gluconobacter*, *Propionibacterium* and *Staphylococcus* bacterial taxa [39]. Part of the microbiota in adult mosquitoes would thus be acquired from the aquatic larval habitat, while the other fraction would be dependent on food resources, through natural plant-based sugar sources or through blood meals for females. Bacterial composition and diversity are modified subsequently to a sugar- and/or blood-meal which increase inter-individual differences [20]. Notably, blood meal leads to a progressive shift in oxidative conditions in the gut through the modification of microbial communities' composition and structure [40, 41]. Diversity and composition of the bacterial populations are influenced by both infection status and time after the blood meal in *Ae. albopictus* [42]. The bacterial composition but not its structure is influenced by the blood meal whereas only few taxa varied significantly due to chikungunya virus infection [42]. The bacterial abundance of La Crosse virus (LACV)-infected *Aedes japonicus* and *Aedes triseriatus* increased while richness and evenness of resident fungi decreased [43]. Otherwise, the bacterial abundance is reduced in *Ae. aegypti* infected by DENV [44]. ZIKV infection also modulates the dynamics of the bacterial families *Rhodobacteraceae* and *Desulfuromonadaceae* in *Ae. aegypti*, suggested as potential markers for ZIKV [45]. The associated effects of blood meal and infection tend to accentuate microbiota-mosquito immune interactions, such as immune response as well as redox and detoxifying enzyme metabolisms mentioned below [41]. Similarly, a study conducted by Short et al. [46] showed that part of the host amino acid metabolic pathway, which involves branched chain amino acid degradation, did affect midgut microbial communities in *Ae. aegypti*. This could partly explain variations in the midgut microbiota of mosquitoes in the field.

To summarize, recent reports have confirmed that the ecology of breeding sites drives environmental bacterial
acquisition in mosquitoes. Above all, core and pan-
microbiota might represent an assemblage fraction of
the mosquito that belongs to the extended genome of
the mosquito hologenome and contributes to key fea-
tures of the holobiont. Further studies are necessary to
effectively link these core microbes and their genomes
with the functions displayed and to determine which
host genetic factors govern host-microbe interactions.

"Neglected" microbes in the mosquito holobiont
Mosquito virobiota

A novel group of insect-specific viruses has recently been described [47]. In contrast to arthropod-borne viruses which have a dual host tropism (i.e. can replicate in mosquitoes and vertebrates), these insect-specific viruses (ISVs) are host restricted and do not replicate in vertebrate cells. Although most have been discovered in mosquitoes, ISV sequences have also been detected in other haematophagous insects such as sandflies [47]. The cell fusing agent virus (CFAV), Kamiti River virus (KRV) and *Culex* flavivirus (CxFV) were the first endogenous mosquito-specific viruses (MSVs) identified in *Aedes* and *Culex* sp. mosquitoes [see Additional file 1] [48–50].

In the past decade, next-generation sequencing ana-
lysis and increasing interest in both microbiome and
arbovirus transmission have led to the description of
many MSVs [see Additional file 1]. Despite the host
restriction, these RNA viruses are related to mosquito-
borne viruses (MBVs) and they essentially belong to the
Bunyaviridae, Birnaviridae, Flaviviridae, Mesonoviridae,
Negoviridae, Reoviridae, Rhabdoviridae and Togaviridae
families [51–53]. Most ISVs described to date belong to
the Flaviviridae family [54]. Mosquito-specific flavi-
viruses are divided into two distinct phylogenetic and
serologic clades. The first group is closely related to
mosquito-borne flaviviruses, including dengue (DENV),
yellow fever (YFV) and West Nile (WNV) viruses, while
also comprising a dozen virus species [51, 55]. The
second one forms a clade distinct from mosquito-borne
flaviviruses and includes the previously mentioned
viruses CFAV, KRV, CxFV and *Aedes* flavivirus (AeFV),
and others [55]. These mosquito-specific flaviviruses are
distributed worldwide and have been isolated or detected
in diverse mosquito species [see Additional file 1]. For
instance, CFAV, KRV, CxFV or AeFV have been isolated
or detected in laboratory reared and field-caught *Aedes*,
*Anopheles* or *Culex* populations from Africa, Australia,
South America or Japan [55]. Mosquito-specific alpha-
viruses are less diversified as only two viral species have
been described [56, 57]. The first one, i.e. the Eilat virus
(EILV), was isolated from collected *Anopheles coustani*
in Israel [56] and the second one, i.e. the Taï Forest
virus, was recently detected in *Culex decens* from Côte
d'Ivoire [57].

Despite the high prevalence of MSVs in nature, their
acquisition, transmission cycle and maintenance in mos-
quitoes are poorly known. Like some bacteria, vertical
transmission from mother to progeny has been reported.
*Culex* and *Aedes* flaviviruses are vertically transmitted in
*Cx. pipiens* and *Ae. albopictus*, respectively [58, 59].
However, horizontal transmission could occur as the
same MSVs can be detected in different mosquito spe-
cies from the same geographical area. Both venereal and
ectoparasite transmission have been reported. As for
phytophagous insects, MSVs could be acquired from the
environment and transmitted through infected plants
during nectar feeding [51, 53, 60]. MSVs thus have to in-
fect mosquito salivary glands and/or saliva "injected"
during feeding. Mosquito flaviviruses CFAV are absent
from saliva and salivary glands of *Ae. aegypti* and *Culex
annulirostris*, while AeFV was detected in *Ae. albopictus*
saliva [61]. Finally, CxFV was only found in *Culex quin-
quefasciatus* saliva bi-infected with WNV [61]. The Eilat
alphavirus was detected in salivary glands of *Ae. aegypti*,
*Ae. albopictus* and *Cx. quinquefasciatus* [61]. As no hori-
zontal transmission was detected between *Cx. pipiens*
mosquitoes infected or not by CxFv and reared in the
same cage with common food resources [62], further in-
vestigations are required to corroborate these horizontal
modes of transmission. Temperature could modulate
their prevalence and/or maintenance in field populations
since mosquitoes are ectotherms and some MSVs are
temperature sensitive [63].

Reverse genetic tools have revealed that restrictions
occurred during cell entry and replication steps and dur-
ing viral assembly for Eilat and Niénokoué viruses,
respectively [56, 64, 65]. Mammal innate immunity
could also explain host restriction, as demonstrated for
KRV, which can complete its viral replication cycle in
cells deficient in some interferon regulatory factors [66].

Mosquito-associated eukaryotes

In addition to viruses and bacteria, the mosquito micro-
biota is also composed of fungal (mycobiota) and protist
communities [see Additional file 1]. However, few stud-
ies have focused on these communities in mosquitoes.
Recently, Belda et al. [67] developed an efficient method
that enabled the identification of eukaryotic microbiota
associated with *Anopheles* mosquito larvae. Ichthyospor-
eans of the Pseudoperkinsus group were the most abun-
dant protist members. Steyn et al. [68] also provided the
first description of yeast microbiota of *Cx. pipiens* and
*Culex theileri* larvae. The authors identified Candida,
Cryptococcus, Galactomyces, Hannaella, Meyerozyma,
Pichia, Rhodosporidium, Rhodotorula, Trichosporon and
Wickerhamomyces genera. Apart from non-pathogenic
fungi, mosquitoes also harbor yeasts of clinical import-
ance. A recent study reported the isolation of the
opportunistic pathogen *Candida parapsilosis* from dif-
ferent developmental stages and organs of several
laboratory-reared mosquito species, including *An. gam-
biae*, *An. stephensi*, *Cx. quinquefasciatus*, *Ae. albopictus*
and *Ae. aegypti* [69]. Moreover, culturable yeast micro-
biota analysis from wild *Cx. pipiens* and *Cx. theileri*
mosquito larvae revealed the presence of clinically rele-
vant species, including the well-known opportunistic
human pathogen *Candida albicans*.

Overall, there has been much consideration of bacter-
ial microbiota in the holobiont concept as it represents
the abundant fraction of the host microbiota and conse-
quently has been shown to impact the extended pheno-
type. The advent of high-throughput sequencing
methods has made it possible to describe other commu-
nities of microbes including viruses and protists. Some
of them, even at lower abundances, established notable
interactions with their host. In mammals, retroviral
genes are thought to be involved in the placenta forma-
tion [70]. We provided here some published data on
virobiota and eukaryotic microbiota associated with
mosquitoes. We encourage experimental evolutionary
and functional researches to be performed on this
neglected microbiota and we anticipate, in light of what
was demonstrated with bacterial microbiota, that it will
build a foundation to extend the list of microbial taxa in-
volved in the mosquito holobiont.

Microbiome-mosquito interactions and mosquito holobiont success

As previously mentioned, most studies on mosquito
microbiota have been descriptive and focused on bacter-
teria with the aim of drawing up an inventory of the
microbial communities [see Additional file 1] and their
variation factors. While recent studies have demon-
strated interactions between bacteria and mosquitoes as
well as between fungi and mosquitoes, the role of MSVs
in the host biology remains to be determined. Cytophatic
effects have been observed in cell cultures, but it is still
unknown whether and how MSVs influence host life
history traits (lifespan, fecundity, oviposition).

Growth and larval development

In recent years, major studies have focused on the role
of microbes in mosquito development (Fig. 2). One of
the most relevant examples is about the *Asaia* genus,
which is closely associated with *An. stephensi* and is one
of the principal members of its microbiota. Up- and
downregulation of genes involved in cuticle synthesis in
*Asaia*-infected larvae promotes the insect's growth (size
and development stages) [71]. Similarly, both bacteria
(*Klebsiella* and *Aeromonas*) and yeasts (*Saccharomyces
cerevisiae*) promote *Cx. pipiens* development [72]. An-
other study showed that the survival and pupation of

*Cx. pipiens* larvae were negatively impacted when they
were fed with yeast isolates compared to fish food [68].

Besides experimental infections, antibiotic treatments
are the main approaches used to manipulate mosquito
bacterial communities for the purpose of studying the
functional roles of insect microbiota. However, these
methods fail to eliminate all the bacteria. An alternative
approach was therefore developed to generate axenic
(i.e. without microorganisms) mosquitoes using a com-
bination of ethanol and bleach to sterilize the egg
surface. Gnotobiotic (i.e. with known microbiota) indi-
viduals can thus be obtained via inoculation of a given
bacterium [25] or other microbes. Recent studies using
this experimental approach, together with functional as-
says, clearly demonstrated that larvae rely on bacteria
for their development. All axenic *Ae. aegypti*, *An. gam-
biae* and *Aedes atropalpus* larvae fed with a standard
sterile diet died during the first larval instar [25]. How-
ever, mono-associated inoculation of *Acinetobacter*,
*Aeromonas*, *Aquitalea*, *Chryseobacterium* or *Paenibacil-
lus* restored *Ae. aegypti* larval development. Likewise, *Ae.
albopictus* and *Cx. quinquefasciatus* require gut
colonization by living bacteria for their development [19].
Overall, these results suggest that mosquitoes do not rely
on specific bacterial taxa for their development but rather
on exchangeable symbiont combinations that could be ac-
quired through larval aquatic habitats.

Some mechanisms involved in larval development
have been described. Genes involved in nutrient acquisi-
tion, metabolism and stress responses are differentially
expressed in the first axenic *Ae. aegypti* larval stage
compared to conventional and gnotobiotic ones, sug-
gesting a putative role of gut bacteria in nutrient acquisi-
tion and/or assimilation after hatching [73]. Mechanisms
were detected with *Ae. aegypti* gnotobiotic larvae colo-
nized by different *E. coli* mutants [74]. The cytochrome
bd oxidase gene appeared to be a key component in this
interaction by reducing the gut oxygen level. Conse-
quently, hypoxia induces the stabilization of hypoxia-
induced transcription factors (HIFs) that enables larval
growth and ecdysone-induced molting [74, 75]. To
summarize, larvae acquire some of their bacterial micro-
biota from aquatic habitats and these bacteria contribute
to the host development, survival and pupation. Any
disturbance of the microbial community in the larval
aquatic habitat could therefore impact the mosquito
biology and ecology [76].

Egg production and oviposition

Coon et al. [77] demonstrated the contribution of gut
bacterial microbiota in egg production by *Ae. aegypti*
and *Ae. atropalpus* while comparing gnotobiotic larvae
to their axenic relatives (Fig. 2). *Ae. atropalpus* can pro-
duce their first clutch of eggs without blood-feeding and
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Putative functions of mosquito-associated microbiota (bacteria and fungi). Functions related to metabolism are indicated in orange and those related to life history traits are indicated in yellow: (1) Blood digestion (*Acinetobacter*, *Pantoea*, *Enterobacter*, *Dysgonomonas*), (2) Sugar digestion (*Acinetobacter*, *Elizabethkingia*, *Thorsellia*, *Sphingomonadaceae* family, *Meyerozyma*), (3) Supply of vitamins and amino acids (*Dysgonomonas*, *Klebsiella*, *Aeromonas*, *Saccharomyces cerevisiae*), (4) Survival (*Escherichia coli*, *Beauveria bassiana*), (5) Mediating oviposition site choice (*Klebsiella*, *Aeromonas*), (6) Egg production (*Comamonas*), (7) Larval development (*Acinetobacter*, *Asaia*, *Aeromonas*, *Chryseobacterium*, *Paenibacillus*, *Aquitalea*, *Escherichia coli*) [19, 25, 68, 71–75, 77, 80]. Pictures from JM Hosatte, with permission</figcaption></figure>

depends on specific members of their gut microbiota to produce eggs whereas a blood meal is mandatory for *Ae. aegypti*. *Ae. atropalpus* microbiota probably provides nutrient reserves during larval development which are necessary for the first clutch in the absence of a blood meal. Interestingly, *Cx. pipiens* females were shown to select media containing *Klebsiella* and *Aeromonas* bacteria for oviposition [72].

## Mosquito pathogens

Nowadays, *Bacillus thuringiensis subsp. israelensis* (Bti) producing Cry, Cyt, Vip and Sip insecticidal proteins is a safer alternative to chemical insecticides. However, its repeated and intensive use was found to generate strong selection pressure that could promote Bti resistance. A reduction in bacterial microbiota diversity in *An. stephensi* larvae has been shown to increase their susceptibility to Bti [78]. This finding differs from what was previously known in other insect models where

microbiota was a key component in Bti efficiency [79]. In addition, the microaerophilic conditions of the larval gut would also favour the use of insecticidal proteins as a source of nitrogen by bacteria [78]. Intestinal bacteria would increase larval resistance through Bti toxin degradation. There has been recent increased interest in the identification of mosquito-killing fungi to tackle insecticide resistance. It was recently shown that the pathogenic fungus *Beauveria bassiana* could interact with gut bacterial microbiota and accelerate *Anopheles* mosquito death [80]. The fungus induced an increase of the opportunistic pathogenic bacterium *Serratia marcescens* density that lead to its dissemination in the haemocoel and promote death of their host [80].

Published data reported here illustrate that hypothesis- and experimentally-driven researches are key elements to demonstrate the contribution of bacterial microbiota in extended phenotypes of the mosquito holobiont. Notably, the manipulation of microbial assemblages allows to deduce their important impact on the mosquito life
history traits and somehow which host-genes were
modulated.

## Mosquito-microbiota co-evolution

Recent studies have highlighted host-microbiota phylo-
symbiosis, i.e. a congruency between the host phylogeny
and the divergence in its associated microbial commu-
nity composition [81, 82]. A field study conducted by
Novakova et al. [82] in 11 mosquito species from
Canada revealed congruency between the phylogeny of
hosts and differences in their associated bacterial com-
munities. However, this co-evolutionary pattern was not
observed for all related host species. Indeed, in standard
laboratory conditions, *Ae. aegypti* showed more diver-
gent microbiota from the closely related species *Ae.
atropalpus* than from the distantly related species *An.
gambiae* [25]. Conversely, a congruence pattern in
microbial communities was also observed in species
belonging to the same subgroup. Analysis of gut bacter-
ial microbiota revealed a strong similarity between *Ae.
albopictus* and a cryptic species living in sympatry in
Vietnam [81]. Those findings could possibly be ex-
plained by recent host-microbe co-adaptation. Add-
itional studies are necessary to clarify the influence of
local environmental parameters and host genotypes on
the mosquito microbiota composition. Evolutionary
prospects of *Wolbachia*-mosquito associations have also
recently been highlighted. Besides, horizontal gene trans-
fers (HGTs) were described between *Aedes* mosquitoes
and *Wolbachia* [83, 84]. The evolution of *Wolbachia*-
mosquito associations raises the question of the out-
come of bacterial-to-mosquito HGTs on adaptive capaci-
ties of the holobiont or/and mosquito vector
competence, as *Wolbachia* can modulate the transmis-
sion of some pathogens depending on the mosquito spe-
cies [5]. The adaptive capacities of the holobiont could
also be impacted by functional bacterial HGTs. This
event was demonstrated for the coffee-crop insect that
acquired a gene encoding a polysaccharide enzyme from
bacteria belonging to Bacilli class through HGT, that
could extend its host-plant range and so promote its
adaptation to new ecological niches [85].

Likewise, phylogenetic studies have highlighted that some
MSVs belonging to Bunyaviridae and Flaviviridae families
have co-evolved and diversified with their mosquito host
[63, 86]. Transovarial transmission as well as the detection
of integrated viral sequences in mosquito genomes (i.e.
endogenous viral elements [EVEs]) [87] supports the
hypothesis of an “ancient” association and possible co-
evolution. A description of new lineages of mosquito-
specific bunyaviruses associated with phylogenetic ancestral
reconstruction indicated that pathogenic bunyaviruses have
evolved from an arthropod-specific ancestor [63]. Those
data suggest possible adaptation of MSVs to vertebrates,

following a spillover phenomenon (i.e. crossing of species
barrier), and the emergence of other pathogenic viruses.

In this evolutionary context, in addition to bacterial
microbiota, attention should be paid on MSVs as they
may represent drivers of biological traits linked to the
ability of the mosquito holobiont to transmit or not
pathogens (see below).

## Microbial interactions

### Intra-microbial community interactions

Microbial interactions shape mosquito bacterial diversity
and structure. To date, few data on interactions of gut
microbes are available. Most of studies on microbe-
microbe interactions focused on the influence of *Wolba-
chia* on the microbiome, or vice versa [88]. *Wolbachia*
are intracellular, vertically transmitted bacterial symbi-
onts that naturally infect many mosquito species and are
known to manipulate their reproduction through cyto-
plasmic incompatibility (e.g. the offspring of infected
males and uninfected females are not viable) [89]. In
*Anopheles* mosquitoes, the identification of new factors
modulating *Wolbachia* transmission in artificially trans-
fected adults highlighted the importance of native
mosquito microbiota and interaction with *Wolbachia*
[90]. The disturbance of bacterial microbiota by anti-
biotic treatment reduced the ability of *Anopheles* to
transmit the *Wolbachia w*AlbB strain (from *Ae. albopic-
tus*) to offspring. Additional experiments combining
high-throughput sequencing and oral infection of bact-
teria revealed that their native microbiota, especially
*Asaia*, impeded vertical transmission of *Wolbachia*. This
co-exclusion pattern between *Wolbachia* and *Asaia* is
also found in *Ae. albopictus* and *Cx. quinquefasciatus*
naturally bi-infected by both bacteria for which *Asaia*
tissue tropism is restrained to the gut. Conversely, *Asaia*
is also able to colonize reproductive organs and salivary
glands in species uninfected by *Wolbachia* such as *An.
gambiae*, *An. stephensi* and *Ae. aegypti* [91]. These
observations suggest co-exclusion or competition be-
tween the two bacterial genera for reproductive organ
colonization.

Bacteria could also interact with mosquito-specific
viruses but so far only *Wolbachia*-ISV interactions have
been considered [92]. An *Ae. aegypti* derived-cell line
(Aag2) transfected with a *Drosophila melanogaster*-de-
rived *Wolbachia* strain (*w*Melpop) was infected with the
mosquito-specific CFAV flavivirus or with the Phasi
Charoen-like bunyavirus. Molecular analysis revealed in-
hibition only for CFAV in Aag2-*w*Melpop cells, possibly
related to the production of CFAV-specific small RNAs
[92]. Indeed, MSVs could induce the RNA interference
(RNAi) pathway by producing small interfering
RNAs (siRNAs), as demonstrated for MBV [93]. The
extent to which these interactions are genotype-by-
genotype dependant is unknown. Moreover, it is also un-
known whether *Wolbachia* can inhibit other single nega-
tive- or positive-strand RNA viruses using in vitro and/
or in vivo systems. *Wolbachia* and MSV interactions in
mosquitoes are nevertheless conceivable, as both are ver-
tically transmitted intracellular organisms, suggesting
co-localisation in reproductive organs. Their co-
localisation at the cellular level still needs to be deter-
mined as it could impact the maintenance and transmis-
sion of both microbes in field bi-infected populations.

Microbial interference with transmitted pathogens

Mosquito infections with pathogens, including transmit-
ted pathogens, trigger a complex crosstalk between
different metabolic and immune pathways. Innate im-
mune systems such as immune deficiency (Imd), the
Toll, Janus kinases and signal transducers and activators
of transcription (JAK-STAT) are activated in response to
diverse microbes (viruses, bacteria, fungi or parasites)
whilst RNAi modulates virus replication [41, 94, 95].
This general immune homeostatic response can be asso-
ciated with the induction of autophagy, apoptosis as well
as oxidative stress [40]. Transmitted pathogens can alter
metabolisms through the modulation of stress-inducible
genes involved for instance in redox and detoxifying
enzyme metabolisms. However, molecular responses to
transmitted pathogens are more complex if we consider
the holobiont. The interplay between microbiota and
mosquito immune system may result in an enhanced
synergistic effect on the expression of effector mole-
cules of the mosquito immune system, as previously
reviewed [96].

Bacteria-mosquito-borne pathogen interference

As shown in Table 1, recent reports have stressed the
importance of microbiota in the modulation of vector
competence (ability of a susceptible mosquito to get
infected by a pathogen, to support the pathogen’s repli-
cation and/or development and to transmit the pathogen
to a susceptible vertebrate host)—this phenomenon is
called microbial interference [97]. Multipartite interac-
tions between the pathogen, the mosquito and its bacter-
ial microbiota have become a major target for
developing new control strategies in order to stop patho-
gen transmission and related epidemics.

In *An. gambiae* mosquitoes, the presence of *Escheri-
chia coli*, *Serratia marcescens* or *Pseudomonas stutzeri*
resulted in a significant reduction in the prevalence and
intensity of *Plasmodium falciparum* infection [98]. The
abundance of *Serratia* was positively correlated with *P.
falciparum* infection in both the midgut and salivary
glands, suggesting a potential interaction between
bacteria and the malaria parasite. The bacterium *Chro-
mobacterium* was shown to increase *An. gambiae*

resistance against *P. falciparum* [99]. It was suggested
that the underlying mechanism behind interference was
the production of cyanide by the bacterium. *An. gambiae*
mosquitoes are also naturally colonized by the *Entero-
bacter Esp_Z* bacterial strain which inhibits the develop-
ment of *Plasmodium* parasites prior to midgut
colonization [100]. Specific genes associated with react-
ive oxygen species (ROS) production were found to be
involved in mosquito midgut colonization by Esp_Z bac-
teria [101]. Recent studies have demonstrated the ability
of gut bacteria to produce antiparasitic effectors that in-
hibit parasite growth. For instance, *Chromobacterium*
was suggested to be a powerful immune elicitor since it
increases mosquito immune gene expression. This fea-
ture, combined with its ability to rapidly invade the mos-
quito gut and reduce the lifespan of immature stages
and adult mosquitoes, makes this bacterium a promising
candidate for vector control applications [99]. Another
study showed that the *E. coli* clone 444ST95 previously
isolated from *Anopheles* mosquito midgut is able to
markedly decrease the survival of these mosquitoes as
well as the development of their *Plasmodium* parasites
[102]. Hemolysin F or other toxins released by the bac-
terium are virulence factors associated with this effect
[102]. Finally, a positive correlation between intensive
antibiotic therapy in humans and increased risk of mal-
aria transmission by *An. gambiae* mosquitoes has been
suggested [103]. Indeed, antibiotics ingested by humans
and circulating in their blood would enhance the suscep-
tibility of blood-sucking *An. gambiae* females to malaria
infection by disturbing their gut microbiota [103]. Des-
pite the overall decrease in the microbial load in the
mosquito, some specific changes have occurred in the
microbial community, including a reduction in *Serratia*
density, associated with an increase in *Asaia* abundance.
Gendrin et al. [104] subsequently showed that critical
parameters for the mosquito vector capacity, such as
lifespan, permissiveness to *P. falciparum*, the mosquito
microbiota composition and gut homeostasis were spe-
cific to the antibiotic treatment used. Moreover, micro-
biota disruption is closely related to gut homeostasis
regulation [105]. For instance, the peritrophic matrix
(PM) is a membrane that physically separates the blood
meal from epithelium cells and plays a key role in regu-
lation of mosquito gut homeostasis. Rodgers et al. [105]
demonstrated that PM synthesis and integrity are related
to gut microbiota. Importantly, microbial metabolites
trigger the host oxidative response in mosquitoes, while
maintaining redox homeostasis in the midgut [40].

As previously mentioned, the immune system is
enhanced during microbial infections in mosquitoes.
Stathopoulos et al. [106] characterized the molecular
processes driving the mosquito immune response
following infection by the enterobacterium *S. marcescens*
<table><caption>**Table 1** Examples of microbial interference between microbiota and vector-borne pathogens</caption><thead><tr><th>Pathogen</th><th>Mosquito</th><th>Microorganism</th><th>Interference</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Arboviruses</td><td rowspan="2">Dengue virus</td><td><i>Serratia odorifera</i></td><td>Enhances susceptibility to the virus</td><td>[115]</td></tr><tr><td><i>Chromobacterium</i></td><td>Increases infection resistance<br>Antiviral activity<br>Immune elicitor</td><td>[99]</td></tr><tr><td rowspan="2">West Nile virus</td><td><i>Enterobacteriaceae, Esp_ivi isolate, alternatively Salmonella, Escherichia or Shigella</i></td><td>Decreases antibacterial activity<br>Reduces virus dissemination titer</td><td>[113]</td></tr><tr><td><i>Wolbachia</i></td><td>Reduces susceptibility to the virus</td><td>[153]</td></tr><tr><td rowspan="2">Culex pipiens Colorado strain</td><td><i>Culex Flavivirus (CxFV)</i></td><td>Reduces virus dissemination</td><td>[62]</td></tr><tr><td><i>Culex quinquefasciatus<br>Honduras strain</i></td><td><i>Culex Flavivirus (CxFV)</i></td><td>Enhances virus dissemination</td><td>[132]</td></tr><tr><td rowspan="2">Chikungunya virus</td><td><i>Culex quinquefasciatus</i></td><td><i>Nhumirim virus (NHUV)</i></td><td>Reduces virus infection</td><td>[135]</td></tr><tr><td><i>Aedes aegypti</i></td><td><i>Serratia odorifera</i></td><td>Enhances susceptibility to the virus</td><td>[115]</td></tr><tr><td rowspan="4">La Crosse virus</td><td><i>Eilat virus</i></td><td>Reduces virus replication and dissemination</td><td>[64]</td></tr><tr><td><i>Enterobacter ludwigii</i></td><td>Antiviral activity</td><td>[116]</td></tr><tr><td><i>Pseudomonas rhodesiae</i></td><td>Antiviral activity</td><td>[116]</td></tr><tr><td><i>Vagococcus salmoninarium</i></td><td>Antiviral activity</td><td>[116]</td></tr><tr><td rowspan="12">Parasites</td><td><i>Plasmodium yoelii</i></td><td><i>Anopheles dirus</i></td><td>Protects against infection by regulating <i>tep1</i> expression</td><td>[109]</td></tr><tr><td rowspan="5">Plasmodium falciparum</td><td><i>Anopheles gambiae</i></td><td><i>Chromobacterium</i></td><td>Increases infection resistance by forming a protective biofilm against parasite</td><td>[99]</td></tr><tr><td><i>Enterobacter</i></td><td>Inhibits parasite development by stimulating oxydative stress</td><td>[100, 101]</td></tr><tr><td><i>Escherichia coli</i></td><td>Reduces infection prevalence and intensity<br>Reduces parasite development</td><td>[98]</td></tr><tr><td><i>Pseudomonas stutzeri</i></td><td>Reduces infection prevalence and intensity</td><td>[98]</td></tr><tr><td><i>Serratia</i></td><td>Protects against infection</td><td>[103]</td></tr><tr><td rowspan="4">Plasmodium berghei</td><td><i>Serratia marcescens</i></td><td>Reduces infection prevalence and intensity<br>Actives IMD/REL2 immune pathway</td><td>[98, 106]</td></tr><tr><td><i>Penicillium chrysogenum</i></td><td>Enhances susceptibility to the parasite</td><td>[130]</td></tr><tr><td><i>Asaia bogorensis</i></td><td>Inhibits parasite development by secreting a siderophore receptor protein and a YVTN beta-propeller repeat protein</td><td>[140]</td></tr><tr><td><i>Wickerhamomyces anomalus</i></td><td>Anti-<i>Plasmodium</i> activity</td><td>[129]</td></tr></tbody></table>

as well as its consequences on transmission of the parasite. Peptidoglycan recognition proteins (PGRPs) are key regulators of the innate immune response [107]. These proteins specifically recognize microbe-associated molecular patterns (MAMPs) and therefore are influenced by microbiota variations. *S. marcescens* infections were found to result in the activation of some host genes, including PGRP-LC which activates the IMD/REL2 immune pathway involved in a reduction in

*Plasmodium* infection. The authors also showed that some effectors of *Anopheles* immunity could impact the microbiota composition and load [107]. Similarly, Gendrin et al. [108] demonstrated that PGRP are important regulators of mosquito epithelial immunity and vector competence. PGRP-LA and PGRP-S2/ PGRP-S3 would be involved in the antiparasitic defense system, while PGRP-LB would promote mosquito permissiveness to *P. falciparum*. Immune system modulation by gut microbiota has
also been demonstrated in *Anopheles dirus* mosquitoes naturally resistant to *Plasmodium yoelii* infection [109]. Its microbiota regulates the expression of a thioester-containing protein 1 (TEP1) following parasite infection. *tep1* inactivation prevents microbiota from protecting the mosquito against parasitic infections [109]. This result suggests an important role of TEP1 related to microbiota in the refractoriness to *P. yoelii* infection. The immune regulation through microRNAs (miRNAs) can also modulate anti-*Plasmodium* defense and midgut microbiota [110]. The use of transgenic mosquitoes engineering to express miRNAs targeting endogenous-miRNAs offers new perspectives for the development of alternative malaria control.

There is abundant literature on *Wolbachia*-mediated interference of arbovirus transmission, as previously reviewed [111]. Here we will mainly focus on mosquito-borne pathogen interference with other members of bacterial microbiota. Indeed, few studies have examined the role of the microbiota in the modulation of arbovirus replication and transmission in mosquitoes. Interference mechanisms evidenced include production of bacterial metabolites with an anti-viral activity or nutrient competition between arbovirus and resident microbiota. As for *Plasmodium*, the bacterium *Chromobacterium* produces a metabolite with an anti-DENV activity in *Ae. aegypti* [99]. By comparing untreated and antibiotic-treated mosquitoes, Audsley et al. [112] demonstrated that the microbiota composition was not essential for blocking DENV in laboratory-reared *Ae. aegypti*. However, it seems that this assessment cannot be generalized since larval exposure to an Enterobacterium isolate *Esp_ivi* (genus-level classification undetermined, alternatively *Salmonella*, *Escherichia* or *Shigella*) resulted in decreased antibacterial activity in the hemolymph of *Ae. aegypti* females and reduced DENV dissemination titers [113]. Conversely, *Serratia odorifera* enhanced the susceptibility of *Aedes* mosquitoes to dengue and chikungunya viruses [114, 115]. A recent study also showed that *Enterobacter ludwigii*, *Pseudomonas rhodesiae* and *Vagococcus salmoninarium* isolated from *Ae. albopictus* could have an anti-viral effect on the La Crosse virus in vitro [116]. Interestingly, Novakova et al. [82] demonstrated that bacterial microbiota could be an important factor in the variability of vector competence in mosquitoes for WNV. The findings of other studies have also confirmed this observation. *Ae. albopictus* populations from France were shown to be more efficient in chikungunya virus dissemination compared to Vietnamese autochthonous relatives [117, 118]. In parallel, these invasive populations exhibited a reduction in their gut bacterial diversity compared to the Vietnamese populations [32].

Evidences underscore the importance of RNAi pathways in antiviral defense by the modulation of the expression of host or virus RNA-derived small RNAs,

including siRNAs, PIWI interacting RNAs (piRNAs) and miRNAs. The siRNAs are the predominant virus-derived RNAs detected in infected mosquitoes, but piRNAs can also be detected in late/persistent infection [119, 120]. Zika virus modulates expression of both virus-derived siRNAs and piRNAs and host-derived miRNAs in *Ae. aegypti* [121]. Silencing of RNAi enzyme effectors such as Argonaute-2 (Ago-2) or Dicer 2/ R2D2 complex can promote flaviviruses (DENV) or alphaviruses (CHIKV, O'nyong'nyong virus [ONNV], Sindbis virus [SINV]) replication and/or transmission by *Aedes* and *Anopheles* mosquitoes [119, 120]. In a same way, RNAi-mediated knockdown of Imd and JAK-STAT pathways increased DENV replication in some *Ae. aegypti* lines [119]. Arboviruses can also induce protein synthesis involved in ROS production, carbohydrate or lipid metabolisms. In particular, midgut infection by DENV-2 and CHIKV triggered an antioxidant response through the production of proteins involved in detoxification. Other antiviral responses such as apoptosis and autophagy can also contribute to innate antiviral immunity [119, 120], but detailed mechanisms involved remains poorly understood. Overall, the relative implications and crosstalk of these metabolic and immune pathways remain to be clarified and seem to be dependent on multiple factors, notably the transmitted virus-mosquito combination of the holobiont.

These crosstalk pathways become more complex as mosquito immune responses to transmitted pathogens can influence resident microbiota and vice versa. Only few data on anti-arbovirus responses in the context of holobiont are available. One of the most documented "system" is the *Wolbachia*-transfected mosquito. *Wolbachia* transfection in *Wolbachia*-free mosquitoes induces oxidative stress that activates the Toll pathway through the production of ROS [122]. The subsequent production of the antimicrobial peptides (AMPs) (cecropin and defensin) modulates DENV replication [122]. *Wolbachia* can also modify host-derived miRNA expression in *wMelPop-CLA*-transfected mosquitoes resulting in DENV interference [123]. In *An. gambiae*, Carissimo et al. [124] demonstrated that the siRNA pathway is not involved in midgut antiviral defense, but instead protects the post-midgut systemic compartment, which is the site of subsequent disseminated viral infection. While *Anopheles* microbiota hampers ONNV multiplication, viral infection is positively related to the microbiota. These data indicate distinct protective mechanisms that would allow an adapted response specific to each body compartment, infection stages and pathogens.

Overall, these results highlight the need to decipher genetic and molecular mechanisms of interactions in vector pathosystems and their impacts on pathogen transmission. Complex and potentially conflicting interactions in the
gut mentioned above might have some direct implications in the design of new vector control strategies based on host microbiota manipulation. Caution is necessary to avoid using mosquito colonies that could potentially host new or circulating pathogenic agents in nature.

In accordance with the recent "pathobiome" concept, i.e. the pathogenic agent integrated within its biotic environment, disease transmission is modulated by interactions between host-transmitted pathogens and commensal and mutualistic microbes [125]. The gut microbiota would not be simply a passive commensal population with limited functions but an active sensor that would contribute to a local or systemic immune response, as previously demonstrated in *Drosophila* and *Anopheles* [126–128]. The mechanisms underlying these complex multipartite interactions [host-microbial community-environment] that modulate persistence, transmission and evolution of infectious pathogens remain to be deciphered. Understanding these interactions can open new avenues for controlling transmitted pathogen infection in vector insects.

**Mosquito-associated eukaryotes and pathogen interactions**

Some yeasts can directly, or via the host, interfere with parasites. For instance, *Wickerhamomyces* produces an anti-plasmodial toxin in vitro, while *Penicillium chrysogenum* promotes *Plasmodium* infection by suppressing the host innate immune response [129, 130]. Muturi et al. [43] recently showed that the *Meyerozyma* yeast dominated fungal communities in response to LACV infections in field-collected *Ae. triseriatus* and *Ae. japonicus* females. Secreted factors by the fungus *Talaromyces* downregulate digestive enzymes of its natural host *Ae. aegypti* that modulate DENV infection [131].

**Mosquito-borne and mosquito-specific virus interference**

Evidences of interference between MSVs and MBVs are inconsistent between studies. For instance, WNV replication was reported to decrease in the *Ae. albopictus* C6/36 cell line when co-infected with a CxFV strain isolated from Colorado [62] but not with one from Guatemala [132]. The CxFV Japan strain promotes dengue and Japanese encephalitis (JEEV) virus infection in *Culex tritaeniorhynchus* cells [133]. However, replication of JEEV and WNV decreased when co-infected with the Nhumirim flavivirus (NHUV) in C6/36 cells [134, 135]. Superinfection exclusion, which corresponds to an infected cell that is refractory to be secondarily infected with another closely related (or not) virus, could explain the observed interference effects. Different combinations of mosquito cell lines, MBV and MSV strains could also explain the disparities in the reported results.

In vivo experiments are essential to corroborate the interference between MSVs and MBVs, especially since

in vitro studies have recurrently used the C6/36 cell line that is deficient in the RNAi immune pathway. Nasar et al. [64] demonstrated negative effects of EILV on CHIKV replication or dissemination in both in C7/10 *Ae. albopictus* cells and *Ae. aegypti* mosquitoes, respectively (Table 1) [64].

Most other in vivo studies have focused on CFxV and WNV interactions in *Culex* sp. but with contrasting results (Table 1). West Nile virus dissemination decreased when *Cx. pipiens* were intrathoracically infected with CxFV but not with NHUV [62, 135]. The same phenotype was observed in a CxFV-positive colony from Colorado in comparison to a CxFV-negative colony from Iowa [62]. Co-inoculation of WNV and CxFV led to a reduction of WNV transmission in *Cx. quinquefasciatus* populations from Honduras while no effect was observed when the experiment was reproduced with Floridians populations [132]. Moreover, a *Cx. quinquefasciatus* NHUV-positive colony was found to transmit WNV less efficiently at days 7 and 9 post-infection (pi) (Table 1) [135]. Viral interference in *Culex* sp. thus depends on the mosquito species, MSV and MBV strain combination and/or the mosquito infection status with ISVs. Overall, genotype-by-genotype-by-genotype interactions (mosquito-MSV-MBV), the status, time and mode (natural, oral or intrathoracic) of infection, as well as intra-microbial interactions in the mosquito holobiont, could explain the differences observed in these studies.

The evolutionary and phylogenetic aspects mentioned above indicate that the function and evolution of the holobiont-associated microbiota can shape keystone phenotypes such as the vectorial capacity, a key component of the dynamic of vector-borne disease transmission. This also underscores the need to decipher factors and mechanisms involved in host restriction or permis-siveness and virus interference in the development of new bio-control strategies or vaccines [65].

**Microbiota-based control strategies: promising perspectives?**

To date, paratransgenesis approach using symbionts to produce molecules that inhibit pathogen development and/or transmission has been mainly restricted to *Anopheles* mosquitoes to prevent *Plasmodium* development [136, 137]. The most promising candidates are the bacteria *Pantoea agglomerans* and *Asaia* spp. [138, 139]. Paratransgenic strains of *Asaia bogorensis* were recently engineered using induced-fusion system of antiplasmodial effectors and bacterial secreted putative genes (encoding a siderophore receptor protein and YVTN beta-propeller repeat proteins) [140]. These effectors expressed in *An. stephensi* significantly inhibit *P. berghei* development [140]. Mancini et al. [139] evaluated paratransgenesis efficiency in semi-field conditions with
genetically modified strains of *Asaia* and demonstrated their ability to colonize both *An. stephensi* and *An. gambiae* mosquito populations. Paratransgenesis still needs to be assessed in *Aedes* and *Culex* mosquitoes to impede arbovirus replication and/or transmission. The use and persistence in the environment of microbes that induce mosquito immune response to transmitted viruses (RNA interference) or produce toxins are currently unknown. Complementary data and semi-field studies are needed to evaluate the feasibility of these strategies to control or suppress mosquito populations. The development and implementation of paratransgenesis needs to be carefully evaluated to ensure safety issues for both humans and the environment, as reviewed in Touré et al. [141] and Gabrieli et al. [136].

Currently, phenotypes such as viral and parasite inter-
ferences induced by several *Wolbachia* strains (as *wMel*
and *wMelpop* of *Drosophila* transfected in mosquitoes)
and cytoplasmic incompatibility are used to control
mosquito populations [142, 143]. Field applications
highlighted the efficiency of using *Wolbachia* transfected
in *Ae. aegypti* to block DENV transmission [144]. Devel-
opment of future *Wolbachia*-based strategies to prevent
malaria transmission are promising, as the first known
*Wolbachia-Anopheles* associations were recently identi-
fied [145, 146] and that *Plasmodium* development seems
to be related to the presence of *Wolbachia* in *Anopheles*
*coluzzii* field populations [146].

The incompatible insect technique (IIT) can also be
combined with the sterile insect technique (SIT) to im-
prove vector control [147]. For instance, Zhang et al.
[148] studied the effect of *wPip* (*Wolbachia* strain from
*Cx. pipiens*) on *Ae. albopictus* that is originally naturally
bi-infected by *wAlbA* and *wAlbB*. They showed that
*wPip* has a low effect on mosquito fitness. Thereby, the
authors conclude that the competitiveness with natural
populations combined with the strong cytoplasmic
incompatibility of this triple *Wolbachia*-infected *Ae.
albopictus* line supports its use in SIT/IIT strategies to
control *Ae. albopictus* populations.

To conclude, field applications of these different strat-
egies, combined or not, need properly ethical, ecological
and social issues, especially if the strategies are related to
the release of symbiotically modified mosquitoes [136].
Particular attention should be given to ecological and
evolutionary aspects. More data will improve our under-
standing on the implications, the outcome and the environ-
mental sustainability of these engineering systems, notably
for potential gene flows (HGT), the emergence of resistance
in mosquito populations and/or the accidental spread to
non-target species. In this context, future innovative
control strategies should favour the reduction of mosquito
density below threshold levels of disease transmission
rather than the population-replacement strategy.

Future challenges in mosquito holobiont
investigations

Some key components and processes should be deter-
mined to gain further insight into the mosquito holobiont.
In particular, little is known on the (co)evolutionary
aspects of mosquito holobiont functioning, including the
involvement of associated-microbiota in adaptation and
speciation [149, 150]. Mosquito invasive capacities and
global spread could be emphasized through its microbial
compartment. Mutualistic symbionts are notably a key
factor for the ecological success and adaptation to novel
environments of some insect species. Most striking exam-
ples are the symbiont-mediated insecticide resistance in
the bean bug *Riptortus pedestris* [151] or thermal toler-
ance of insect aphids due to a mutation in their bacterial
symbiont *Buchnera aphidicola* [152]. Besides, microbe
genomes evolve relatively rapidly through mutations,
recombinations, rearrangements, horizontal transfers and
hybridizations. Consequently, the microbial hologenome
responds differentially and more quickly to selective envir-
onmental pressure than the host’s genome. These rapidly
evolving characteristics in the symbiotic community could
markedly contribute to extending the host and/or their
microbiota-associated phenotypes. Moreover, it is also es-
sential to enhance knowledge on holobiont (microbiota
and host) genome architecture and expression via high
throughput OMICS strategies (genomics, transcriptomics,
proteomics, metabolomics, etc.) in order to shape func-
tional networks and obtain a deeper understanding on the
multipartite interactions involved. Greater effort will thus
be needed in developing *ad hoc* protocols and tools.

Conclusions

The holobiont concept has raised considerable debate
on the functioning and evolution of organisms with
the aim of gaining greater insight into important bio-
logical issues. Host-associated microbiota involves a
complex network of cooperation and competition,
which makes it difficult to understand the role of
each microorganism. In mosquitoes, recent findings
have given rise to new hypotheses on holobiont func-
tioning and dynamics, with symbiotic interactions be-
ing the keystone of the vector pathosystem. Intra-
community microbial interaction dynamics within the
mosquito holobiont as well as the molecular mecha-
nisms involved in these multipartite interactions have
yet to be investigated in detail. A technical issue is
that deciphering the intricate interactions between
microbes, host and environment is challenging. Such
investigations could provide new targets and tools for
vector-borne pathogen control. Moreover, we believe
that studying the mosquito holobiont in the evolu-
tionary context (experimentally, modeling, etc.) could
help to predict, for instance, areas suitable for mosquito adaptation or even outbreaks, and thus lead to the development of strategies to avoid outbreaks, epidemics and epizootic diseases.

## Additional file

**Additional file 1:** List of bacteria, fungi and viruses found in different larva and adult mosquito species [15, 17, 18, 20–22, 24, 25, 27, 29, 32, 35–38, 52, 54, 61, 68, 69, 81, 103, 154–157]. List of articles mentioned are published since 2013. Previous publications on this research topic are available in our previous review [16]. (XLSX 56 kb)

### Abbreviations

AeFV: *Aedes flavivirus*; AMPs: Antimicrobial peptides; Bti: *Bacillus thuringiensis* subspecies *israelensis*; CFAV: Cell fusion agent virus; CHIKV: Chikungunya virus; CxFV: *Culex flavivirus*; DENV: Dengue virus; EILV: Eilat virus; EVEs: Endogenous viral elements; HGTs: Horizontal gene transfers; HIFs: Hypoxia-induced transcription factors; IIT: Incompatible insect technique; Imd: Immune deficiency; ISVs: Insect-specific viruses; JAK-STAT: Janus kinases and signal transducers and activators of transcription; JEEV: Japanese encephalitis; KRV: Kamiti River virus; LACV: La Crosse virus; MAMPs: Microbe-associated molecular patterns; MBVs: Mosquito-borne viruses; miRNAs: MicroRNAs; MSVs: Mosquito-specific viruses; NHUV: Nhumirim virus; ONNV: O'nyong'nyong virus; OTUs: Operational taxonomical units; PGRPs: Peptidoglycan recognition proteins; pi: Post-infection; piRNAs: PIWI interacting RNAs; PM: Peritrophic matrix; RNAi: RNA interference; SINV: Sindbis virus; SIT: Sterile insect technique; siRNA: Small interfering RNAs; TEP: Thioester-containing protein; WHO: World Health Organization; WNV: West Nile virus; YFV: Yellow fever virus; ZIKV: Zika virus

### Acknowledgements

We thank Jean-Marie Hosatte for pictures of *Aedes albopictus*.

### Funding

Morgane Guégan and Colin Démichel are supported by PhD fellowships from the French Ministère de l'Éducation Nationale, de la Recherche et des Nouvelles Technologies. Karima Zouache is funded by grant ANR-16-CE35-0004-02. This review was funded in the framework of the ERA-NET Biodiversité program with the national funders ANR-13-EBID-007-01, FWF I-1437, and DFG KL 2087/6–1.

### Availability of data and materials

Not applicable.

### Authors' contributions

MG, KZ and CVM performed the systematic review and wrote the manuscript. GM, CD and VTV assisted in reviewing literature and writing the manuscript. PM and PP provided critical and intellectual input to the manuscript. All authors read and approved the final manuscript.

### Ethics approval and consent to participate

Not applicable.

### Competing interests

The authors declare that they have no competing interests.

## Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

### Author details

Received: 7 November 2017 Accepted: 5 March 2018

Published online: 20 March 2018

## References

1. Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I. The role of microorganisms in coral health, disease and evolution. Nat Rev Microbiol. 2007;5:355–62.

2. Cresci GA, Bawden E. Gut microbiome: what we do and don't know. Nutr. Clin. Pract. Off. Publ. Am. Soc. Parenter. Enter. Nutr. 2015;30:734–46.

3. Douglas AE. Multiorganismal insects: diversity and function of resident microorganisms. Annu Rev Entomol. 2015;60:17–34.

4. Snyder AK, Rio RVM. Interwoven biology of the tsetse holobiont. J Bacteriol. 2013;195:4322–30.

5. Caragata EP, Dutra HLC, Moreira LA. Exploiting intimate relationships: controlling mosquito-transmitted disease with *Wolbachia*. Trends Parasitol. 2016;32:207–18.

6. Erkosar B, Storelli G, Defaye A, Leulier F. Host-intestinal microbiota mutualism: "learning on the fly.". Cell Host Microbe. 2013;13:8–14.

7. WHO | Executive summary [Internet]. WHO. [cited 2017 Aug 7]. Available from: http://www.who.int/whr/1996/media_centre/executive_summary1/en/index9.html.

8. WHO | Malaria [Internet]. WHO. [cited 2017 Aug 7]. Available from: http://www.who.int/mediacentre/factsheets/fs094/en/.

9. Organization WH. Progress report 2000–2009 and strategic plan 2010–2020 of the global programme to eliminate lymphatic filariasis: halfway towards eliminating lymphatic filariasis. Rapport de situation 2000–2009 et plan stratégique 2010–2020 du programme mondial pour l'élimination de la filariose lymphatique: à mi-parcours vers l'élimination de cette maladie [internet]. 2010 [cited 2017 Aug 14]; Available from: http://www.who.int/iris/handle/10665/44473

10. Patterson J, Sammon M, Garg M. Dengue, Zika and chikungunya: emerging arboviruses in the new world. West J Emerg Med. 2016;17:671–9.

11. Weaver SC, Charlier C, Vasilakis N, Lecuit M. Zika, chikungunya, and other emerging vectorborne viral diseases. Annu Rev Med. 2018;69:1–395–408.

12. Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet. 2017;390(10107):2099–2109.

13. Schaffner F, Medlock JM, Van Bortel W. Public health significance of invasive mosquitoes in Europe. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 2013;19:685–92.

14. Sutherst RW. Global change and human vulnerability to vector-borne diseases. Clin Microbiol Rev. 2004;17:136–73.

15. Ricci I, Damiani C, Capone A, DeFreece C, Rossi P, Favia G. Mosquito/ microbiota interactions: from complex relationships to biotechnological perspectives. Curr Opin Microbiol. 2012;15:278–84.

16. Minard G, Mavingui P, Moro CV. Diversity and function of bacterial microbiota in the mosquito holobiont. Parasit Vectors. 2013;6:146.

17. Duguma D, Hall MW, Smartt CT, Neufeld JD. Effects of Organic Amendments on Microbiota Associated with the Culex nigripalpus Mosquito Vector of the Saint Louis Encephalitis and West Nile Viruses. mSphere. 2017;2:e00387–16.

18. Buck M, Nilsson LKJ, Brunius C, Dabiré RK, Hopkins R, Terenius O. Bacterial associations reveal spatial population dynamics in *Anopheles gambiae* mosquitoes. Sci Rep. 2016;6:22806.

19. Coon KL, Brown MR, Strand MR. Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats. Mol Ecol. 2016;25:5806–26.

20. Gimonneau G, Tchioffo MT, Abate L, Boissière A, Awono-Ambéné PH, Nsango SE, et al. Composition of *Anopheles coluzzii* and *Anopheles gambiae* microbiota from larval to adult stages. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2014;28:715–24.

21. Kim C-H, Lampman RL, Muturi EJ. Bacterial communities and midgut microbiota associated with mosquito populations from waste tires in East- Central Illinois. J Med Entomol. 2015;52:63–75.

22. Dada N, Jumas-Bilak E, Manguin S, Seidu R, Stenström T-A, Overgaard HJ. Comparative assessment of the bacterial communities associated with *Aedes aegypti* larvae and water from domestic water storage containers. Parasit Vectors. 2014;7:391.

23. Duguma D, Hall MW, Rugman-Jones P, Stouthamer R, Terenius O, Neufeld JD, et al. Developmental succession of the microbiome of *Culex* mosquitoes. BMC Microbiol. 2015;15:140.

<footnote n="1">¹Université de Lyon, Lyon, France. ²Université Lyon 1, Villeurbanne, France. ³CNRS, UMR 5557, Ecologie Microbienne, Villeurbanne, France. ⁴INRA, UMR1418, Villeurbanne, France. ⁵Université de La Réunion, CNRS 9192, INSERM U1187, IRD 249, Unité Mixte Processus Infectieux en Milieu Insulaire Tropical (PIMIT), Plateforme Technologique CYROI, Sainte-Clotilde, La Réunion, France.</footnote>
24. Yadav KK, Datta S, Naglot A, Bora A, Hmuaka V, Bhagyawant S, et al. Diversity of cultivable midgut microbiota at different stages of the Asian Tiger mosquito, *Aedes albopictus* from Tezpur. India PLoS One. 2016;11:e0167409.

25. Coon KL, Vogel KJ, Brown MR, Strand MR. Mosquitoes rely on their gut microbiota for development. Mol Ecol. 2014;23:2727-39.

26. Moll RM, Romoser WS, Modrzakowski MC, Moncayo AC, Lerdthusnee K. Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol. 2001;38:29-32.

27. Segata N, Baldini F, Pompon J, Garrett WS, Truong DT, Dabiré RK, et al. The reproductive tracts of two malaria vectors are populated by a core microbiome and by gender- and swarm-enriched microbial biomarkers. Sci Rep. 2016;6:24207.

28. Astudillo-García C, Bell JJ, Webster NS, Glasl B, Jompa J, Montoya JM, et al. Evaluating the core microbiota in complex communities: a systematic investigation. Environ Microbiol. 2017;19:1450-62.

29. David MR, Santos LMBD, Vicente ACP, Maciel-de-Freitas R. Effects of environment, dietary regime and ageing on the dengue vector microbiota: evidence of a core microbiota throughout *Aedes aegypti* lifespan. Mem Inst Oswaldo Cruz. 2016;111:577-87.

30. Ngo CT, Aujoulat F, Veas F, Jumas-Bilak E, Manguin S. Bacterial diversity associated with wild caught *Anopheles* mosquitoes from Dak Nong Province, Vietnam using culture and DNA fingerprint. PLoS One. 2015;10(3):e0118634.

31. Ngo CT, Romano-Bertrand S, Manguin S, Jumas-Bilak E. Diversity of the bacterial microbiota of *Anopheles* mosquitoes from Binh Phuoc Province, Vietnam. Front Microbiol. 2016;7:2095.

32. Minard G, Tran FH, Van VT, Goubert C, Bellet C, Lambert G, et al. French invasive Asian tiger mosquito populations harbor reduced bacterial microbiota and genetic diversity compared to Vietnamese autochthonous relatives. Front Microbiol. 2015;6:970.

33. Tchioffo MT, Boissière A, Abate L, Nsango SE, Bayibéki AN, Awono-Ambéné PH, et al. Dynamics of bacterial community composition in the malaria mosquito's epithelia. Front Microbiol. 2016;6:1500.

34. Leung MHY, Wilkins D, Lee PKH. Insights into the pan-microbiome: skin microbial communities of Chinese individuals differ from other racial groups. Sci Rep. 2015;5:srep11845.

35. Yadav KK, Bora A, Datta S, Chandel K, Gogoi HK, Prasad GBKS, et al. Molecular characterization of midgut microbiota of *Aedes albopictus* and *Aedes aegypti* from Arunachal Pradesh, India. Parasit Vectors. 2015;8:641.

36. Muturi EJ, Kim C-H, Bara J, Bach EM, Siddappaji MH. *Culex pipiens* and *Culex restuans* mosquitoes harbor distinct microbiota dominated by few bacterial taxa. Parasit Vectors. 2016;9:18.

37. Chandler JA, Liu RM, Bennett SN. RNA shotgun metagenomic sequencing of northern California (USA) mosquitoes uncovers viruses, bacteria, and fungi. Front Microbiol. 2015;6:185.

38. Andrews ES, Xu G, Rich SM. Microbial communities within field-collected *Culiseta melanura* and *Coquillettidia perturbans*. Med Vet Entomol. 2014;28:125-32.

39. Muturi EJ, Ramirez JL, Rooney AP, Kim C-H. Comparative analysis of gut microbiota of mosquito communities in Central Illinois. PLoS Negl Trop Dis. 2017;11(2):e0005377.

40. Champion CJ, Xu J. The impact of metagenomic interplay on the mosquito redox homeostasis. Free Radic Biol Med. 2017;105:79-85.

41. van Tol S, Dimopoulos G. Chapter nine—influences of the mosquito microbiota on vector competence. In: Raikhel AS, editor. Advances in insect physiology, vol. 51; 2016. p. 243-91.

42. Zouache K, Michelland RJ, Failloux A-B, Grundmann GL, Mavingui P. Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector. Mol Ecol. 2012;21:2297-309.

43. Muturi EJ, Bara JJ, Rooney AP, Hansen AK. Midgut fungal and bacterial microbiota of *Aedes triseriatus* and *Aedes japonicus* shift in response to La Crosse virus infection. Mol Ecol. 2016;25:4075-90.

44. Ramirez JL, Souza-Neto J, Torres Cosme R, Rovira J, Ortiz A, Pascale JM, et al. Reciprocal tripartite interactions between the *Aedes aegypti* midgut microbiota, innate immune system and dengue virus influences vector competence. PLoS Negl Trop Dis. 2012;6(3):e1561.

45. Villegas LEM, Campolina TB, Barnabe NR, Orfano AS, Chaves BA, Norris DE, et al. Zika virus infection modulates the bacterial diversity associated with *Aedes aegypti* as revealed by metagenomic analysis. PLoS One. 2018;13:e0190352.

46. Short SM, Mongodin EF, MacLeod HJ, Talyuli OAC, Dimopoulos G. Amino acid metabolic signaling influences *Aedes aegypti* midgut microbiome variability. PLoS Negl Trop Dis. 2017;11:e0005677.

47. Moureau G, Ninove L, Izri A, Cook S, De Lamballerie X, Charrel RN. Flavivirus RNA in phlebotomine sandflies. Vector Borne Zoonotic Dis Larchmt N. 2010; 10:195-7.

48. Stollar V, Thomas VL. An agent in the *Aedes aegypti* cell line (Peleg) which causes fusion of *Aedes albopictus* cells. Virology. 1975;64:367-77.

49. Sang RC, Gichogo A, Gachoya J, Dunster MD, Ofula V, Hunt AR, et al. Isolation of a new flavivirus related to cell fusing agent virus (CFAV) from field-collected flood-water *Aedes* mosquitoes sampled from a dambo in Central Kenya. Arch Virol. 2003;148:1085-93.

50. Hoshino K, Isawa H, Tsuda Y, Yano K, Sasaki T, Yuda M, et al. Genetic characterization of a new insect flavivirus isolated from *Culex pipiens* mosquito in Japan. Virology. 2007;359:405-14.

51. Vasilakis N, Tesh RB. Insect-specific viruses and their potential impact on arbovirus transmission. Curr Opin Virol. 2015;15:69-74.

52. Bolling BG, Weaver SC, Tesh RB, Vasilakis N. Insect-specific virus discovery: significance for the arbovirus community. Viruses. 2015;7:4911-28.

53. Roundy CM, Azar SR, Rossi SL, Weaver SC, Vasilakis N. Insect-specific viruses: a historical overview and recent developments. Adv Virus Res. 2017;98:119-46.

54. Calzolari M, Zé-Zé L, Vázquez A, Sánchez Seco MP, Amaro F, Dottori M. Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects. Infect Genet Evol. 2016;40:381-8.

55. Blitvich BJ, Firth AE. Insect-specific flaviviruses: a systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization. Viruses. 2015;7:1927-59.

56. Nasar F, Palacios G, Gorchakov RV, Guzman H, Da Rosa APT, Savji N, et al. Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication. Proc Natl Acad Sci U S A. 2012;109:14622-7.

57. Hermanns K, Zirkel F, Kopp A, Marklewitz M, Rwego IB, Estrada A, et al. Discovery of a novel alphavirus related to Eilat virus. J Gen Virol. 2017;98:43-9.

58. Saiyasombat R, Bolling BG, Brault AC, Bartholomay LC, Blitvich BJ. Evidence of efficient transovarial transmission of *Culex* flavivirus by *Culex pipiens* (Diptera: Culicidae). J Med Entomol. 2011;48:1031-8.

59. Haddow AD, Guzman H, Popov VL, Wood TG, Widen S, Haddow AD, et al. First isolation of *Aedes* flavivirus in the western hemisphere and evidence of vertical transmission in the mosquito *Aedes* (Stegomyia) *albopictus* (Diptera: Culicidae). Virology. 2013;440:134-9.

60. Roossinck MJ. Plants, viruses and the environment: ecology and mutualism. Virology. 2015;479-480:271-7.

61. Halbach R, Junglen S, van Rij RP. Mosquito-specific and mosquito-borne viruses: evolution, infection, and host defense. Curr Opin Insect Sci. 2017;22:16-27.

62. Bolling BG, Olea-Popelka FJ, Eisen L, Moore CG, Blair CD. Transmission dynamics of an insect-specific flavivirus in a naturally infected *Culex pipiens* laboratory colony and effects of co-infection on vector competence for West Nile virus. Virology. 2012;427:90-7.

63. Marklewitz M, Zirkel F, Kurth A, Drosten C, Junglen S. Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family. Proc Natl Acad Sci U S A. 2015;112:7536-41.

64. Nasar F, Gorchakov RV, Tesh RB, Weaver SC. Eilat virus host range restriction is present at multiple levels of the virus life cycle. J Virol. 2015;89:1404-18.

65. Erasmus JH, Auguste AJ, Kaelber JT, Luo H, Rossi SL, Fenton K, et al. A chikungunya fever vaccine utilizing an insect-specific virus platform. Nat Med. 2017;23:192-9.

66. Tree MO, McKellar DR, Kieft KJ, Watson AM, Ryman KD, Conway MJ. Insect-specific flavivirus infection is restricted by innate immunity in the vertebrate host. Virology. 2016;497:81-91.

67. Belda E, Coulibaly B, Fofana A, Beavogui AH, Traore SF, Gohl DM, et al. Preferential suppression of *Anopheles gambiae* host sequences allows detection of the mosquito eukaryotic microbiome. Sci Rep. 2017;7:3241.

68. Steyn A, Roets F, Botha A. Yeasts associated with *Culex pipiens* and *Culex theileri* mosquito larvae and the effect of selected yeast strains on the ontogeny of *Culex pipiens*. Microb Ecol. 2016;71:747-60.

69. Bozic J, Capone A, Pediconi D, Mensah P, Cappelli A, Valzano M, et al. Mosquitoes can harbour yeasts of clinical significance and contribute to their environmental dissemination. Environ Microbiol Rep. 2017;9(5):642-8.

70. Lavialle C, Cornelis G, Dupressoir A, Esnault C, Heidmann O, Vernochet C, et al. Paleovirology of "syncytins", retroviral env genes exapted for a role in placentation. Philos Trans R Soc Lond Ser B Biol Sci. 2013;368:20120507.

71. Mitraka E, Stathopoulos S, Siden-Kiamos I, Christophides GK, Louis C. *Asaia* accelerates larval development of *Anopheles gambiae*. Pathog Glob Health. 2013;107:305-11.
72. Díaz-Nieto LM, D Alessio C, Perotti MA, Berón CM. *Culex pipiens* development is greatly influenced by native bacteria and exogenous yeast. PLoS One. 2016;11:e0153133.

73. Vogel KJ, Valzania L, Coon KL, Brown MR, Strand MR. Transcriptome sequencing reveals large-scale changes in axenic *Aedes aegypti* larvae. PLoS Negl Trop Dis. 2017;11:e0005273.

74. Coon KL, Valzania L, McKinney DA, Vogel KJ, Brown MR, Strand MR. Bacteria-mediated hypoxia functions as a signal for mosquito development. Proc Natl Acad Sci U S A. 2017;114:E5362-9.

75. Valzania L, Coon KL, Vogel KJ, Brown MR, Strand MR. Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito *Aedes aegypti*. Proc Natl Acad Sci U S A. 2018;115:457-65.

76. Pennington MJ, Prager SM, Walton WE, Trumble JT. *Culex quinquefasciatus* larval microbiomes vary with instar and exposure to common wastewater contaminants. Sci Rep. 2016;6:21969.

77. Coon KL, Brown MR, Strand MR. Gut bacteria differentially affect egg production in the anautogenous mosquito *Aedes aegypti* and facultatively autogenous mosquito *Aedes atropalpus* (Diptera: Culicidae). Parasit Vectors. 2016;9:375.

78. Patil CD, Borase HP, Salunke BK, Patil SV. Alteration in *Bacillus thuringiensis* toxicity by curing gut flora: novel approach for mosquito resistance management. Parasitol Res. 2013;112:3283-8.

79. Caccia S, Di Lelio I, La Storia A, Marinelli A, Varricchio P, Franzetti E, et al. Midgut microbiota and host immunocompetence underlie *Bacillus thuringiensis* killing mechanism. Proc Natl Acad Sci U S A. 2016;113:9486-91.

80. Wei G, Lai Y, Wang G, Chen H, Li F, Wang S. Insect pathogenic fungus interacts with the gut microbiota to accelerate mosquito mortality. Proc Natl Acad Sci U S A. 2017;114:5994-9.

81. Minard G, Tran Van V, Tran FH, Melaun C, Klimpel S, Koch LK, et al. Identification of sympatric cryptic species of *Aedes albopictus* subgroup in Vietnam: new perspectives in phylosymbiosis of insect vector. Parasit Vectors. 2017;10:276.

82. Novakova E, Woodhams DC, Rodríguez-Ruano SM, Brucker RM, Leff JW, Maharaj A, et al. Mosquito microbiome dynamics, a background for prevalence and seasonality of West Nile virus. Front Microbiol. 2017;8:526.

83. Klasson L, Kambris Z, Cook PE, Walker T, Sinkins SP. Horizontal gene transfer between *Wolbachia* and the mosquito *Aedes aegypti*. BMC Genomics. 2009;10:33.

84. Hou Q, He J, Yu J, Ye Y, Zhou D, Sun Y, et al. A case of horizontal gene transfer from *Wolbachia* to *Aedes albopictus* C6/36 cell line. Mob Genet Elem. 2014;4:e28914.

85. Acuña R, Padilla BE, Flórez-Ramos CP, Rubio JD, Herrera JC, Benavides P, et al. Adaptive horizontal transfer of a bacterial gene to an invasive insect pest of coffee. Proc Natl Acad Sci U S A. 2012;109:4197-202.

86. Li C-X, Shi M, Tian J-H, Lin X-D, Kang Y-J, Chen L-J, et al. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. eLife. 2015;4:e05378.

87. Olson KE, Bonizzoni M. Nonretroviral integrated RNA viruses in arthropod vectors: an occasional event or something more? Curr Opin Insect Sci. 2017;22:45-53.

88. Audsley MD, Seleznev A, Joubert DA, Woolfit M, O'Neill SL, EA MG. Wolbachiainfection alters the relative abundance of resident bacteria in adult *Aedes aegypti* mosquitoes, but not larvae. Mol Ecol. 2018;27(1):297-309.

89. Werren JH, Baldo L, Clark ME. *Wolbachia*: master manipulators of invertebrate biology. Nat Rev Microbiol. 2008;6:741-51.

90. Hughes GL, Dodson BL, Johnson RM, Murdock CC, Tsujimoto H, Suzuki Y, et al. Native microbiome impedes vertical transmission of *Wolbachia* in *Anopheles* mosquitoes. Proc Natl Acad Sci U S A. 2014;111:12498-503.

91. Rossi P, Ricci I, Cappelli A, Damiani C, Ulissi U, Mancini MV, et al. Mutual exclusion of *Aisaia* and *Wolbachia* in the reproductive organs of mosquito vectors. Parasit Vectors. 2015;8:278.

92. Schnettler E, Sreenu VB, Mottram T, McFarlane M. *Wolbachia* restricts insect-specific flavivirus infection in *Aedes aegypti* cells. J Gen Virol. 2016;97:3024-9.

93. Cheng G, Liu Y, Wang P, Xiao X. Mosquito defense strategies against viral infection. Trends Parasitol. 2016;32:177-86.

94. Hegde S, Rasgon JL, Hughes GL. The microbiome modulates arbovirus transmission in mosquitoes. Curr Opin Virol. 2015;15:97-102.

95. Bartholomay LC, Michel K. Mosquito immunobiology: the intersection of vector health and vector competence. Annu Rev Entomol. 2018;63:145-67.

96. Saraiva RG, Kang S, Simões ML, Angleró-Rodríguez YI, Dimopoulos G. Mosquito gut antiparasitic and antiviral immunity. Dev Comp Immunol. 2016;64:53-64.

97. Weiss B, Aksoy S. Microbiome influences on insect host vector competence. Trends Parasitol. 2011;27:514-22.

98. Tchioffo MT, Boissière A, Churcher TS, Abate L, Gimonneau G, NSango SE, et al. Modulation of malaria infection in *Anopheles gambiae* mosquitoes exposed to natural midgut Bacteria. PLoS One. 2013;8(12):e81663.

99. Ramirez JL, Short SM, Bahia AC, Saraiva RG, Dong Y, Kang S, et al. *Chromobacterium* Csp_P reduces malaria and dengue infection in vector mosquitoes and has Entomopathogenic and in vitro anti-pathogen activities. PLoS Pathog. 2014;10(10):e1004398.

100. Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, et al. Natural microbe-mediated refractoriness to *Plasmodium* infection in *Anopheles gambiae*. Science. 2011;332:855-8.

101. Dennison NJ, Saraiva RG, Cirimotich CM, Mlambo G, Mongodin EF, Dimopoulos G. Functional genomic analyses of *Enterobacter*, *Anopheles* and *Plasmodium* reciprocal interactions that impact vector competence. Malar J. 2016;15(1):425.

102. Tchioffo MT, Abate L, Boissière A, NSango SE, Gimonneau G, Berry A, et al. An epidemiologically successful *Escherichia coli* sequence type modulates *Plasmodium falciparum* infection in the mosquito midgut. Infect Genet Evol. 2016;43:22-30.

103. Gendrin M, Rodgers FH, Yerbanga RS, Ouédraogo JB, Basáñez M-G, Cohuet A, et al. Antibiotics in ingested human blood affect the mosquito microbiota and capacity to transmit malaria. Nat Commun. 2015;6:5921.

104. Gendrin M, Yerbanga RS, Ouédraogo JB, Lefèvre T, Cohuet A, Christophides GK. Differential effects of azithromycin, doxycycline, and cotrimoxazole in ingested blood on the vectorial capacity of malaria mosquitoes. Open Forum Infect Dis. 2016;3:ofw074.

105. Rodgers FH, Gendrin M, Wyer CAS, Christophides GK. Microbiota-induced peripertic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes. PLoS Pathog. 2017;13(5):e1006391.

106. Stathopoulos S, Neafsey DE, Lawniczak MKN, Muskavitch MAT, Christophides GK. Genetic dissection of *Anopheles gambiae* gut epithelial responses to *Serratia marcescens*. PLoS Pathog. 2014;10(3):e1003897.

107. Gendrin M, Zaidman-Rémy A, Broderick NA, Paredes J, Poidevin M, Roussel A, et al. Functional analysis of PGRP-LA in Drosophila immunity. PLoS One. 2013;8:e69742.

108. Gendrin M, Turlure F, Rodgers FH, Cohuet A, Morlais I, Christophides GK. The peptidoglycan recognition proteins PGRP LA and PGRP LB regulate *Anopheles* immunity to Bacteria and affect infection by *Plasmodium*. J Innate Immun. 2017;9:333-42.

109. Wang Y, Wang Y, Zhang J, Xu W, Zhang J, Huang FS. Ability of TEP1 in intestinal flora to modulate natural resistance of *Anopheles dirus*. Exp Parasitol. 2013;134:460-5.

110. Dennison NJ, BenMarzouk-Hidalgo OJ, Dimopoulos G. MicroRNA-regulation of *Anopheles gambiae* immunity to *Plasmodium falciparum* infection and midgut microbiota. Dev Comp Immunol. 2015;49:170-8.

111. Kamthum-Tatuene J, Makepeace BL, Benjamin L, Baylis M, Solomon T. The potential role of *Wolbachia* in controlling the transmission of emerging human arboviral infections. Curr Opin Infect Dis. 2017;30:108-16.

112. Audsley MD, Ye YH, McGraw EA. The microbiome composition of *Aedes aegypti* is not critical for *Wolbachia*-mediated inhibition of dengue virus. PLoS Negl Trop Dis. 2017;11:e0005426.

113. Dickson LB, Jiolle D, Minard G, Moltini-Conclois J, Volant S, Ghozlane A, et al. Carryover effects of larval exposure to different environmental bacteria drive adult trait variation in a mosquito vector. Sci Adv. 2017;3:e1700585.

114. Apte-Deshpande A, Paingankar M, Gokhale MD, Deobagkar DN. *Serratia odorifera* a midgut inhabitant of *Aedes aegypti* mosquito enhances its susceptibility to dengue-2 virus. PLoS One. 2012;7:e40401.

115. Apte-Deshpande AD, Paingankar MS, Gokhale MD, Deobagkar DN. *Serratia odorifera* mediated enhancement in susceptibility of *Aedes aegypti* for chikungunya virus. Indian J Med Res. 2014;139:762-8.

116. Dennison NJ, Jupatanakul N, Dimopoulos G. The mosquito microbiota influences vector competence for human pathogens. Curr Opin Insect Sci. 2014;3:6-13.

117. Vega-Rua A, Zouache K, Caro V, Diancourt L, Delaunay P, Grandadam M, et al. High efficiency of temperate *Aedes albopictus* to transmit chikungunya and dengue viruses in the southeast of France. PLoS One. 2013;8(3):e59716.

118. Zouache K, Fontaine A, Vega-Rua A, Mousson L, Thiberge J-M, Lourenco-De-Oliveira R, Caro V, Lambrechts L, Failoux A-B. Three-way interactions between mosquito population, viral strain and temperature underlying chikungunya virus transmission potential. Proc R Soc B Biol Sci. 2014;281(1792).

119. Sim S, Jupatanakul N, Dimopoulos G. Mosquito immunity against arboviruses. Viruses. 2014;6:4479-504.
120. Blair CD, Olson KE. The role of RNA interference (RNAi) in arbovirus-vector interactions. Viruses. 2015;7:820–43.

121. Etebari K, Hegde S, Saldaña MA, Widen SG, Wood TG, Asgari S, et al. Global transcriptome analysis of Aedes aegypti mosquitoes in response to Zika virus infection. mSphere. 2017;2:e00456–17.

122. Pan X, Zhou G, Wu J, Bian G, Lu P, Raikhel AS, et al. *Wolbachia* induces reactive oxygen species (ROS)-dependent activation of the toll pathway to control dengue virus in the mosquito *Aedes aegypti*. Proc Natl Acad Sci U S A. 2012;109:E23–31.

123. Pan X, Thiem S, Xi Z. Chapter 3—Wolbachia-mediated immunity induction in mosquito vectors. In: Wikel SK, Aksoy S, Dimopoulos G, editors. Arthropod Vector: Controller of Disease Transmission. Vol. 1. Cambridge: Academic Press. 2017. p. 35–58.

124. Carissimo G, Pondeville E, McFarlane M, Dietrich I, Mitri C, Bischoff E, et al. Antiviral immunity of *Anopheles gambiae* is highly compartmentalized, with distinct roles for RNA interference and gut microbiota. Proc Natl Acad Sci U S A. 2015;112:E176–85.

125. Vayssier-Taussat M, Albina E, Citti C, Cosson J-F, Jacques M-A, Lebrun M-H, et al. Shifting the paradigm from pathogens to pathobiome: new concepts in the light of meta-omics. Front Cell Infect Microbiol. 2014;4:29.

126. Ryu J-H, Kim S-H, Lee H-Y, Bai JY, Nam Y-D, Bae J-W, et al. Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in *Drosophila*. Science. 2008;319:777–82.

127. Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C. Hemocyte differentiation mediates innate immune memory in *Anopheles gambiae* mosquitoes. Science. 2010;329:1353–5.

128. Wu S-C, Liao C-W, Pan R-L, Juang J-L. Infection-induced intestinal oxidative stress triggers organ-to-organ immunological communication in *Drosophila*. Cell Host Microbe. 2012;11:410–7.

129. Valzano M, Cecarini V, Cappelli A, Capone A, Bozic J, Cuccioloni M, et al. A yeast strain associated to *Anopheles* mosquitoes produces a toxin able to kill malaria parasites. Malar J. 2016;15:21.

130. Angleró-Rodríguez YI, Blumberg BJ, Dong Y, Sandiford SL, Pike A, Clayton AM, et al. A natural *Anopheles*-associated *Penicillium chrysogenum* enhances mosquito susceptibility to *Plasmodium* infection. Sci Rep. 2016;6:34084.

131. Angleró-Rodríguez YI, Talyuli OA, Blumberg BJ, Kang S, Demby C, Shields A, et al. An *Aedes* aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity. elife. 2017;6:e28844.

132. Kent RJ, Crabtree MB, Miller BR. Transmission of West Nile virus by *Culex quinquefasciatus* say infected with *Culex* Flavivirus Izabal. PLoS Negl Trop Dis. 2010;4:e671.

133. Kuwata R, Isawa H, Hoshino K, Sasaki T, Kobayashi M, Maeda K, et al. Analysis of mosquito-borne Flavivirus superinfection in *Culex tritaeniorhynchus* (Diptera: Culicidae) cells persistently infected with *Culex Flavivirus* (Flaviviridae). J Med Entomol. 2015;52:222–9.

134. Kenney JL, Solberg OD, Langevin SA, Brault AC. Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses. J Gen Virol. 2014;95:2796–808.

135. Goenaga S, Kenney JL, Duggal NK, Delorey M, Ebel GD, Zhang B, et al. Potential for co-infection of a mosquito-specific Flavivirus, Nhumirim virus, to block West Nile virus transmission in mosquitoes. Viruses. 2015;7:5801–12.

136. Gabrieli P, Smidler A, Catteruccia F. Engineering the control of mosquito-borne infectious diseases. Genome Biol. 2014;15:535.

137. Wilke ABB, Marrelli MT. Paratransgenesis: a promising new strategy for mosquito vector control. Parasit Vectors. 2015;8:342.

138. Wang S, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M. Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc Natl Acad Sci U S A. 2012;109:12734–9.

139. Mancini MV, Spaccapelo R, Damiani C, Accoti A, Tallarita M, Petraglia E, et al. Paratransgenesis to control malaria vectors: a semi-field pilot study. Parasit Vectors. 2016;9:140.

140. Bongio NJ, Lampe DJ. Inhibition of *Plasmodium berghei* development in mosquitoes by effector proteins secreted from *Asaia* sp. Bacteria Using a Novel Native Secretion Signal. PloS One. 2015;10:e0143541.

141. Touré YT, Oduola A, Sommerfeld J, Morel C. Biosafety and risk assessment in the use of genetically modified mosquitoes for disease control. In: Takken W, Scott TW, editors. Ecological Aspects for Application of Genetically Modified Mosquitoes. Wageningen: Wageningen UR Frontis Series. 2002;2:217–22.

142. Christodoulou M. Biological vector control of mosquito-borne diseases. Lancet Infect Dis. 2011;11:84–5.

143. Kean J, Rainey SM, McFarlane M, Donald CL, Schnettler E, Kohl A, et al. Fighting arbovirus transmission: natural and engineered control of vector competence in *Aedes* mosquitoes. Insects. 2015;6:236–78.

144. Carrington LB, Tran BCN, Le NTH, Luong TTH, Nguyen TT, Nguyen PT, et al. Field- and clinically derived estimates of *Wolbachia*-mediated blocking of dengue virus transmission potential in *Aedes aegypti* mosquitoes. Proc Natl Acad Sci U S A. 2018;115:361–6.

145. Baldini F, Segata N, Pompon J, Marcenac P, Shaw WR, Dabiré RK, et al. Evidence of natural *Wolbachia* infections in field populations of *Anopheles gambiae*. Nat Commun. 2014;5:3985.

146. Shaw WR, Marcenac P, Childs LM, Buckee CO, Baldini F, Sawadogo SP, et al. *Wolbachia* infections in natural *Anopheles* populations affect egg laying and negatively correlate with *Plasmodium* development. Nat Commun. 2016;7:11772.

147. Zhang D, Lees RS, Xi Z, Bourtzis K, Gilles JRL. Combining the sterile insect technique with the incompatible insect technique: ill-robust mating competitiveness of irradiated triple *Wolbachia*-infected *Aedes albopictus* males under semi-field conditions. PLoS One. 2016;11:e0151864.

148. Zhang D, Zheng X, Xi Z, Bourtzis K, Gilles JRL. Combining the sterile insect technique with the incompatible insect technique: I-impact of *wolbachia* infection on the fitness of triple- and double-infected strains of *Aedes albopictus*. PLoS One. 2015;10:e0121126.

149. Dittmer J, van Opstal EJ, Shropshire JD, Bordenstein SR, Hurst GDD, Brucker RM. Disentangling a Holobiont - recent advances and perspectives in *Nasonia* wasps. Front Microbiol. 2016;7:1478.

150. Brucker RM, Bordenstein SR. The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus *Nasonia*. Science. 2013;341:667–9.

151. Kikuchi Y, Hayatsu M, Hosokawa T, Nagayama A, Tago K, Fukatsu T. Symbiont-mediated insecticide resistance. Proc Natl Acad Sci U S A. 2012; 109:8618–22.

152. Dunbar HE, Wilson ACC, Ferguson NR, Moran NA. Aphid thermal tolerance is governed by a point mutation in bacterial symbionts. PLoS Biol. 2007;5:e96.

153. Bourtzis K, Dobson SL, Xi Z, Rasgon JL, Calvitti M, Moreira LA, et al. Harnessing mosquito-*Wolbachia* symbiosis for vector and disease control. Acta Trop. 2014;132(Suppl):S150–63.

154. Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Terenius O. Isolation and identification of culturable bacteria from wild *Anopheles culicifacies*, a first step in a paratransgenesis approach. Parasit Vectors. 2014;7:419.

155. Sharma P, Sharma S, Maurya RK, De TD, Thomas T, Lata S, et al. Salivary glands harbor more diverse microbial communities than gut in *Anopheles culicifacies*. Parasit Vectors. 2014;7:235.

156. Thongsripong P, Chandler JA, Green AB, Kittayapong P, Wilcox BA, Kapan DD, et al. Mosquito vector-associated microbiota: metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases. Ecol Evol. 2018;8:1352–68.

157. Mwadondo EM, Ghilamicael A, Alakonya AE, Kasili RW. Midgut bacterial diversity analysis of laboratory reared and wild *Anopheles gambiae* and *Culex quinquefasciatus* mosquitoes in Kenya. Afr J Microbiol Res. 2017;11:1171–83.

Submit your next manuscript to BioMed Central and we will help you at every step:

* We accept pre-submission inquiries

* Our selector tool helps you to find the most relevant journal

* We provide round the clock customer support

* Convenient online submission

* Thorough peer review

* Inclusion in PubMed and all major indexing services

* Maximum visibility for your research

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central