Diversity and function of bacterial microbiota in the mosquito holobiont

12 Pages·2013·1.3 MB·English

Minardetal.Parasites&Vectors2013,6:146
http://www.parasitesandvectors.com/content/6/1/146
REVIEW Open Access
Diversity and function of bacterial microbiota in
the mosquito holobiont
Guillaume Minard, Patrick Mavingui* and Claire Valiente Moro*
Abstract
Mosquitoes (Diptera: Culicidae) have been shown to host diverse bacterial communities thatvary depending on
thesex ofthe mosquito, thedevelopmental stage, and ecological factors. Some studies have suggesteda potential
roleof microbiota in thenutritional, developmental and reproductive biology of mosquitoes. Here, wepresent a
review of thediversity and functions of mosquito-associatedbacteria across multiple variationfactors,emphasizing
recent findings. Mosquito microbiota is considered inthe context of possible extended phenotypesconferred on
theinsect hosts that allow niche diversification and rapid adaptive evolution in other insects. These kinds of
observations have prompted therecent development ofnew mosquitocontrol methods based onthe use of
symbiotically-modifiedmosquitoesto interfere with pathogen transmission or reduce the host life span and
reproduction. Newopportunities for exploiting bacterial function for vector control are highlighted.
Keywords: Mosquito, Microbiome, Symbiont, Extended phenotype, Bacterial community
Introduction There are numerous examples of microbes influencing
Sustained relationships between prokaryotes and eukary- so-called extended phenotypes of different taxa, particu-
otes are known to be an important factor in the evolu- larly insects which establish association with microbial
tion and speciation of the interacting partners [1]. The communities ranging from parasitism to mutualism [7,8].
classic example of this evolutionary process is the mito- Bacterial endosymbionts are now known to play roles in
chondrion, an organelle essential for cell metabolism in manykeyinsectfunctionssuchasnutrition,reproduction,
eukaryotesthatderivesfromabacterialancestor[2].An- development or protection against enemies [8]. For ex-
other example is summed up in the coral probiotic hy- ample, the facultative bacterium Hamiltonella defensa
pothesis proposed by Reshef et al. (2006) which posits makes phytophagous aphids more resistant to parasitic
that corals can adapt to their environment by changing wasps [9], whereas the primary symbiont Buchnera
their symbiotic bacteria [3]. The symbiotic relationships aphidicolaprovidesessentialaminoacids[10].Thebacter-
between microbiota, whether algae, archaebacteria, eu- ium Wigglesworthia glossinidi is thought to provide vita-
bacteria, protozoa or viruses, and their invertebrate host minstothehematophagoustsetsefly,animportantvector
were shownto contribute tothe acquisition of resistance ofAfricantrypanosomiasisorsleepingsickness,andbene-
topathogensortolerancetoabiotic stresses[3,4].Rosen- fitsinreturnfromcarbonsourcesandprotectionfromthe
berg et al. (2007) recently proposed the hologenome the- insecthost[11,12].
orytoexplainsuchinteractionsbetweenhigherorganisms Mosquitoes, the Culicidae family, number more than
and microbiota [5]. The hologenome theory is based on 3,500 different species with a worldwide distribution [13].
the concept that higher organisms are not dissociable MostspeciesdescribedareinthegeneraAedes,Anopheles
fromtheirmicrobialpartners,andsotogetherformaunit and Culex including several blood feeding members able
of selection in which genes from the interacting partners to transmit pathogens to humans and animals, a great
arepooledfortheglobalfunctionoftheholobiont[6]. concern for public health [14]. Anopheles mostly transmit
parasites such as Plasmodium, whereas Aedes and Culex
*Correspondence:[email protected]; areresponsibleforthetransmissionofarbovirusesinclud-
[email protected]
ingDengue(Flavivirus),Chikungunya(Alphavirus)orJap-
UMRCNRS5557,USCINRA1364,VetAgroSup,EcologieMicrobienne,FR41
BioEnvironmentandHealth,UniversitédeLyon1,VilleurbanneF-69622, anese Encephalitis viruses, and filariases such as
France
©2013Minardetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Wuchereria bancrofti or Onchocerca volvulus. Despite in- keepinggenescanbedesignedtospecificallytestfortheir
tensive efforts, many mosquito-borne diseases (MBD) are prevalenceinmosquitoes[28,36,37].Whilethesemethods
increasing worldwide, partly due to the lack of effective are partially successful, they do not give complete over-
vaccines against etiological agents, but also due to global views of the mosquito-associated bacterial populations.
changes inhumanactivities, especiallyinternational travel High-throughput sequencing methods are now being
and trade, that have expanded the distribution of mos- implemented to reveal the previously underestimated mi-
quitospeciespreviouslyconfinedtoparticularregions.To crobialdiversity,andhowcertainfactorsimpact the com-
face these outbreaks, new control strategies based on ma- position and structure of these bacterial populations
nipulationofthemosquitohostsandtheirmicrobialpart- during the life cycle of mosquitoes [33-35]. Microbial
ners have been proposed recently [15]. A well-known communities may be influenced by host intrinsic factors
example of this in action is the use of the endosymbiotic (species, developmental stage, tissue tropism and genet-
bacterium Wolbachia [16,17]. This bacterium has a direct ics), the dynamics of intra- and inter-specific interactions
impact on the development of some mosquito species by andenvironmentalfactors.
shortening the insect life span, and has indirect effects
interfering with pathogen replication and dissemination Hostspecies
thataffectthevectortransmissionability[18,19]. Eachmosquitogenushasitsownpreferredhabitatandeco-
Other than Wolbachia, the interactions between mos- logical preference. Mosquitoes exhibit particular rhythmic
quitoes and their associated microbiota have yet to be behavioral patterns during their life cycle. For instance, the
investigated in depth. Most of the published studies de- majority of Anopheles and Culex species are nighttime
scribe bacterial diversity and how it varies according to biters,whereassomeAedesalsobiteinthedaytime.Anoph-
particular factors. Nevertheless, a common conclusion is eles mostly live in clear water exposed to sunlight whereas
that a more comprehensive analysis of symbiotic mos- CulexandAedesaremostlyfoundindarkortroubledwater
quito interactions is needed at evolutionary and func- containingalotoforganicmatter[38].InCulicoidesmidges
tional levels. Better knowledge of the biological impacts withinthesameinfraorderofCulicidae,ithasbeendemon-
will enable the development of efficient biocontrol ap- strated that the host species could explain 17% of the vari-
proaches for MBD. The present work provides an over- ability observed among their bacterial diversity [39].
view of the diversity of symbiotic bacteria and potential Surprisingly, there has been no exhaustive comparative
functions in the biology of mosquitoes, and highlights studyofbacterialdiversityacrossdifferentmosquitospecies.
the current and future applications in symbiont-based However, it is possible to compare some surveys and com-
mosquito control strategies. piletheinformationtohighlightspecificassociations.Forin-
stance, when the bacterial content of field populations of
Review adult females of Anopheles stephensi, Anopheles gambiae,
I-Bacterialdiversityandvariationinmosquitoes Aedes aegypti, Aedes triseriatus and Culex quinquefasciatus
Complementary approaches are needed for in-depth was screened with comparable molecular techniques, it
analyses of microbial communities in complex ecosys- emerged that Proteobacteria was the dominant phylum
tems. Both culture-dependent and culture-independent (Figure1);notablyGammaproteobacteriaclassrepresenting
techniques have been used to explore mosquito micro- 41%(forCx.quinquefasciatus)to86%(forAn.stephensi)of
biota.Somemicrofloracanbe culturedbyusing various the total sequences analyzed, while An. gambiae hosted
isolation procedures and media so that bacterial taxa mainly Alphaproteobacteria and Betaproteobacteria classes,
can be identified[20-31] (fordetailssee Additional file 1). possibly because the mosquito specimens were collected at
The main difficulty of the culture-dependent approach the larval stage and emerged under laboratory conditions
isinrecreatingthecomplexphysicochemicalenvironment [22,24,25,34]. Differences in the proportions of Firmicutes
of the insect body [32]. To overcome this limitation were observed as they account for 13% of sequences ana-
andmorethoroughlyidentifybacteriahostedbymosquito lyzed in Cx. quinquefasciatus, only 1% in An. stephensi and
populations, culture-independent methods such as were not detected at all in Ae. aegypti. Despite these varia-
Denaturating Gradient Gel Electrophoresis fingerprints, tions, the core bacterial genome present in mosquitoes
taxonomic microarrays, and meta-taxogenomics can be seems to be similar in different species. Some genera such
used (Additional file 1). For example, such molecular ap- as Pantoea, Acinetobacter or Asaia are very prevalent in
proaches, mainly based on analyzing the sequence of the mosquitoesandcapableofcross-colonizingdifferentspecies
16SribosomalRNAgene(rrs),haverepeatedlyshownthe [30,40,41].
dominance of phylum Proteobacteria in mosquitoes
[22-24,28,33-35]. Some bacterial taxa are often under- Localizationininsecthost
represented in results of these global methods, but Bacteria colonize different organs in mosquitoes, mainly
primerstargetingaparticularregionofrrsorotherhouse- the midgut and to a lesser extent salivary glands and
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0.3
Aedes mosquitoes (Ae. aegypti, Ae. albopictus, Ae. bromeliae, Ae. triseriatus)
Anopheles mosquitoes (An. albimanus, An. arabiensis, An. cousiani, An. funestus, An. gambiae, An maculipenis, An. stephensi)
Culex mosquitoes (Cx. quinquefasciatus, Cx. pipiens)
Mansonia mosquitoes (M. africana, M. uniformis)
Figure1BacterialgeneraidentifiedinCulicidae.Bacteriawereclassifiedaccordingtotheirphylabasedonbranchinginthe16SrRNA
sequencephylogenetictreewithnamesshownincolorasfollows:Proteobacteria(red),Bacteroidetes(blue),Actinobacteria(brown),[Firmicutes,
TenericutesandFusobacteria](green),Cyanobacteria(purple)andDeinococcus-Thermus(yellow).Themaximum-likelihoodtreewasbuiltwithan
HKYmodelusing100bootstraps.A16SrRNAsequencefromDesulfurococcus(Archaebacteria)wasusedasthetreeoutgroup.
reproductiveorgans[22,24,25,28,42,43].Asinmostanimal these organs were specifically screened for some bacterial
models, the insect gut is a key organ for nutrition and is endosymbionts. For example, the bacterium Asaia was
now considered as being immune-competent [24,32].The detected in salivary glands and reproductive systems of
gutisaninterfacewiththeexternalenvironmentandpro- different mosquitoes including Ae. aegypti, An. gambiae
vides resources and space that may be favorable to the and An. stephensi [36,40,47]. The endosymbiont
multiplicationofmicroorganismsingested[32].Activegut Wolbachia was also detected in the head, muscles, Mal-
bacteria contribute to mosquito digestion through the re- pighian tubules, ovaries and testes of Culex pipiens and
leaseoflytic enzymes[44].Insome insect species suchas Aedes albopictus [48,49]. Strikingly, Wolbachia was also
aphids, beetlesorcockroaches,specializedstructureshave found in Ae. albopictus hemolymph, a fluid which is gen-
evolved for microbial endosymbiosis called bacteriocytes erallyassumedtobebacteria-free[48].Ifmultiplecelltro-
or mycetocytes, which are known to be involved in func- pisms occur for bacterialpartners that are almost fixed in
tions including nutrition and immunity [10,45,46]. None thehostpopulation,itisnotunreasonabletoenvisagethat
of these structures has been described in mosquitoes. In- aphysiologicalroleisyettobediscovered.
sect salivary glands, ovaries and hemolymph are also
known to be key organs for virus or parasite replication, Sexofmosquito
but surprisingly the bacterial content of these organs in The sex of the mosquito is also an important factor that
mosquitoeshasnotbeenfullycharacterized.Nevertheless, affects bacterialmicrobiotacomposition.Male and female
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mosquitoes exhibit different ecological behaviors in terms diversity of bacteria present [24,33]. Zouache et al. (2011)
ofnutritionalanddispersalcapabilities.Bothsexesfeedon showed that around half of the bacterial diversity in field
nectar and plant saps and are able to hydrolyze sucrose, populations of Ae. albopictus was explained by the sex of
butfemalesare also hematophagous.Indeed,female mos- the mosquito with greater diversity observed in females
quitoes are anautogenous as they require blood for the [28]. The effect of the sex of the mosquito on bacterial di-
completion of their reproductive cycles [50]. In the mos- versitywasalsoreportedinfieldpopulationsofthemalaria
quito digestion process, different hydrolases are released vector An. stephensi; bacteria from genera Bacillus and
into the anterior and posterior midgut, which constitutes Staphylococcus were detected in males, whereas bacteria
a selective pressure for resident bacteria [51]. Conse- from genera Cryseobacterium, Pseudomonas and Serratia
quently, the composition and distribution of ingested nu- were present exclusively in females [24]. Considering all
trients themselves may also be a constraint for bacterial published data on mosquito-associated bacteria, it appears
communities.Forinstance,ahighconcentrationofcarbo- thatthemidgutoffemalesismostlycolonizedbymembers
hydrates and an acidic pH (from 5.2 to 6.5) occurring in of the Gammaproteobacteria, as is found in other blood-
the diverticulum structure are selective for certain bacter- feeding insects. Interestingly, the genera Pseudomonas,
ial taxa [52,53]. Blood digestion in females is also favored SerratiaandEnterobacterarefrequentlyassociatedwithfe-
by the selection of bacteria for their hemolytic ability males of several mosquito species [20-24,26,27,29,30,53].
[25,44].Moreover,afteramosquitoingestsabloodmeala In contrast, the midgut of males is dominated by bacteria
temperature burst occurs and oxidative stress and im- fromthephylumFirmicutesincludingthosefromStaphylo-
muneresponsesaredownregulated,whichleadstoanin- coccus, Bacillus, Paenibacillus and Micrococcus genera
creaseinthebacterialload[43,54,55]. (Figure2)[24].Finally,itwasalsoshownthatdiet,whether
As mosquito-associated bacteria rely on some of the nu- sugar or blood meals, significantly affects the bacterial
trients brought in the insect meal for growth, the nutrient populationstructure.Wangetal.(2011)demonstratedthat
composition of food sources may directly impact the bloodmealsdrasticallyreducedthecommunitydiversityin
A B
Figure2Phylogeneticdendrogramsofbacteriaidentifiedinmosquitoadults.Bacterialgeneraareclassifiedaccordingtomosquitosexof
Ae.albopictus(A)andAn.stephensi(B).Namesofbacteriaidentifiedonlyinmales(blue),onlyinfemales(red)orinbothmalesandfemales
(purple)aregiven.ThetreewasconstructedusingthemaximumlikelihoodmethodwithHKYmodelusing100bootstraps.Bootstrapvalues(60%
orabove)areshownatbranchpoints.Desulfurococcus(Archaebacteria)wasusedastheoutgroup.
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favor of enteric bacteria in the An. gambiae midgut, while Ecology
few changes were observed following sugar meals [33]. Studies of mosquito-associated bacteria often compare the
However, irrespective of the type of meal after 4 days the bacterial communities found in field and lab populations.
bacterial microflora reestablishes itself being dominated by However,resultsfromlab-rearedmosquitoeshaverevealed
the genus Elizabethkingia. Finally, male mosquitoes dis- the limits of such an approach. By cloning and analyzing
perse less than females and tend to remain close to breed- signature sequences, Rani et al. (2009) demonstrated that
ing sites which could be an additional factor constraining the bacterial diversity of midgut microflora in lab-reared
bacterialdiversity[50]. An. stephensi was less than in field-caught ones, both for
males(15versus27bacterialtaxa)andfemales(7versus36
bacterial taxa) [24]. Similarly, in An. gambiae 45 distinct
Stagesofmosquitodevelopment OTUwereidentifiedinlab-rearedmosquitoescomparedto
Mosquitoes are holometabola that undergo four gradual 155 in field-caught ones using a pyrosequencing approach
stages of metamorphosis - egg, larvae, nymph, and adult [34].AnotherstudyofAn.gambiaealsodemonstratedthat
- that are intimately connected to their respective bio- taxarichnessinfield-caughtmosquitoeswashigherthanin
topes. Eggs, larvae and nymphs are aquatic, whereas lab-rearedonesforanystageandnutritionalcondition[33].
adult mosquitoes live in terrestrial environments. The Bacterial taxa richness in field-caught mosquitoes shows
fraction of mosquito-associated microflora that is ac- the extent to which bacteria are acquired from the habitat.
quired from the surrounding environment is thus likely Environmental factors should be considered as important
to differ during the insect life cycle. At the larval stage, drivers impacting the load and composition of bacteria in
individualsconsumebacteriaandplanktonasnutritivere- mosquitoes.
sources. This allows a first stage of bacterial colonization As previously discussed,Culicidae usually live in highly
that adds to any inherited bacterial flora. Some of these contrasting environments where biotic (like competition
bacteria such as members of the genus Wolbachia are orthefoodchain)andabiotic(liketemperatureorhumid-
vertically acquired transovarially in Cx. pipiens, Cx. ity) factors can influence their microbiota [38]. The com-
quinquefasciatus or Ae. albopictus. Venereal transmission plexity of such ecosystems partly explains some of the
of the bacterium Asaia was reported in An. gambiae and conclusions drawn from the few existing studies of the
An. stephensi [47,56,57]. The midgut of mosquito larvae roleofenvironmentalfactorsinmodulatingbacterialcom-
alsocontainsmanyphotosyntheticcyanobacteriaacquired positioninfieldpopulationsofmosquitoes[24].Currently,
from breedingsites which are not found in adults [58,59]. the proportion of bacterial species acquired from the en-
Wang et al. (2011) showed that in the larval and pupal vironment is unknown [32]. Each mosquito species has
stages, cyanobacteria were very abundant accounting for ecological preferences that could determine its bacterial
40% of an entire microbial community in An. gambiae content. For instance, some of the adult microflora is ac-
[33]. During its metamorphosis, the mosquito anatomy quiredfromwaterduringmosquitoemergence[23].Plant
is radically modified. In particular, a first meconial and animal hosts are a major source of bacterial acquisi-
peritrophic matrix or membrane (MPM1) is formed early tion through feeding so have a direct impact on the bac-
in the pupal stadium and a second (MPM2) emerged terial colonization of mosquitoes [33]. In Culicoides
sometimesaroundthetimeofadultemergence[60].Are- sonorensis, biting midges which transmit viruses to ani-
cent study suggests that MPMs contribute to the mals, the bacterial flora is derived from soil, plant, bovine
sterilization of the adult midgut by sequestering microor- and ovine sources [39]. The bacterium Acinetobacter was
ganisms ingestedduring thelarval stage,which,along with showntobefrequentlyassociatedwithdifferentmosquito
remaining meconial material, are egested after adult emer- species, including Ae. aegypti, Ae. albopictus, Ae.
gence [60,61]. This phenomenon could explain why the triseriatus, An.stephensi,Cx.pipiens,Cx.quinquefasciatus
proportionsofdifferentbacterialclassesorphylaalterdras- and Psorophora columbiae. Interestingly, this bacterium
tically between immature and adult stages. For example, it was also found in mosquito larval breeding sites and in
was shown that the number of bacterial operational various imago food sources such as vertebrates or plants
taxonomic units (OTU) was 3 fold higher in larvae and [20,22,24,28,29,37,62,63]. In a similar way the genera
pupae than in imagos of An. gambiae [33]. To date, com- Asaia and Pantoea, whose natural habitat is the nectar of
parative studies of bacterial composition between stages tropical flowers, were also observed in mosquitoes
have only been done in Anopheles mosquitoes, in which [41,64,65].Therefore,theenvironmentmaystronglyaffect
transtadial maintenance of some bacterial genera such as thecompositionofmosquito-associatedbacteria.Wecon-
Acinetobacter, Bacillus, Enterobacter, Staphylococcus, firmed this recently by showing that Ae. albopictus indi-
Pseudomonas,Chryseobacterium and Serratia sp. has been viduals from urban areas of Madagascar with bush and
observed (Figure 3) [24,26,27,33]. Other mosquito genera fruit tree cover differed from those from suburban areas
shouldbestudiedinthesameway. with bamboo cover [28]. We also demonstrated that the
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Bacterial genera identified in adult Anopheles stephensi
Bacterial genera identified in larvae Anopheles stephensi
Bacterial genera identified in adult Anopheles gambiae
Bacterial genera identified in larvae Anopheles gambiae
Figure3PhylogeneticdendrogramofbacterialgeneraidentifiedinAn.stephensiandAn.gambiaeaccordingtomosquito
developmentalstage.Namesofbacteriaareshownincolorasfollows:Proteobacteria(red),Bacteroidetes(blue),Actinobacteria,Firmicutes,
Tenericutes(green),Cyanobacteria(purple),Fusobacteria(orange)andDeinococcus-Thermus(yellow).Thetreewasconstructedusingthe
maximumlikelihoodmethodwithHKYmodelusing100bootstraps.Desulfurococcus(Archaebacteria)wasusedastheoutgroup.
prevalenceofAsaiainAe.albopictuswassignificantlycor- Interactionsbetweenmicrobialcommunities
relatedwiththeecologicalcharacteristicsofsamplingsites Bacterial interactions are important regulators of ecosys-
[37]. Generally, these observations support the idea that temcharacteristicsandspeciesdensity.Theseinteractions
field studies are necessary to get an integrated view of are ranged along the mutualism to parasitism continuum
mosquito-associated microbiota. However, studies of lab- and structure communities [66]. One interesting example
reared mosquitoes may be a more convenient alternative is the human gastrointestinal tract. The gut is naturally
to evaluate the impact of abiotic factors on the structure protected by a heterogeneous bacterial biofilm, a com-
and composition of bacterial communities. For example, munity of microorganisms living inside an adhesive
Wang and coworkers demonstrated that the main bacter- matrix that forms a mutual structure. Pathogen
ial families Enterobacteriaceae, Flavobacteriaceae and colonization directly alters (dysbiosis) the biofilm struc-
Pseudomonadaceaefoundinlab-rearedAn.gambiaewere ture [67]. Some recent studies focused on the positive
alsoidentifiedinfield-caughtindividualsfromKenya[33]. and negative interactions between bacteria inside insect
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hosts.Tereniusetal.(2012)testedbacterialinterspecies broad categories, namely primary and secondary symbi-
competition with isolates from Ae. aegypti and showed onts. Primary symbionts or obligate endosymbionts have
thatSerratiamarcescenscouldcreateaninhibitionzone co-evolved with their insect hosts while secondary symbi-
area on Sphingomonas and members of the family onts have become associated with their insect hosts more
Burkholderiaceae [30]. The authors suggested a poten- recently and are not obligate. As yet, there is no descrip-
tial link between the presence of S. marcescens and the tion of primary endosymbionts in mosquitoes; all studies
lowbacterialdiversityobservedinthemosquitomidgut. focusing on secondary symbionts and their potential role
Competitivecolonizationwaspreviouslyreportedinthe inhostbiology.
desert locust Schistocerca gregaria where bacterial di-
versity was shown to increase in the absence of
S. marcescens [68]. Recently, we found a statistically Nutrition
convincing association between the bacteria Asaia and Bacteria contribute to the nutrition of insects in different
Acinetobacter in Ae. albopictus [37]. Even though add- ways. Midgut bacteria can produce compounds that are
itionalanalysesarestill neededto betterunderstandthe directlyassimilatedbythehostortheycanimprovediges-
degree of interactions between the two genera, we tion by producing degradation enzymes which facilitate
showedthatbacterialinteractionseemstobesynergistic the assimilation of complex molecules. In phytophagous
because more Asaia-Acinetobacter double-infections insectsmicrobiotagenerallyprovidevitamins,aminoacids
were observed than would be expected if the bacteria and sterol that complement limited plant diets. The best
actedindependently. known example is the involvement of the bacterium
Bacterial symbionts associated with mosquito vectors Buchnera in providing essential amino acids to aphids
have recently been found to interact with pathogens they [10]. However, a role for bacteria in nutritional comple-
transmit,modifyingtheoutcomeofthemultipartiteinter- mentationinhematophagousinsectshasnotbeendemon-
actions.Forinstance,itwasshownthatremovingbacterial strated so unequivocally. One interesting example are the
communities from Anopheles gambiae increased its sus- bacteria that provide vitamin B which is not present in
ceptibility to Plasmodium falciparum infection [69]. On vertebrate blood, the sole nutrient source of Glossina tse-
the contrary,Boissièreet al. (2012) demonstratedthatthe tseflies[71].
presence of some bacteria could favor parasite infection, In mosquitoes, such a nutritional function has never
as they found a positive correlation between the abun- been formally demonstrated, but some evidence suggests
dance ofmembers of the Enterobacteriaceae familyinthe that bacteria could be involved in some processes. For
mosquito midgut and the Plasmodium infection status instance, Serratia and Enterobacter, which are known to
[34]. Conversely, Zouache et al. (2012) demonstrated that contain hemolytic enzymes, could play a role in blood
chikungunya virus infection could modify the diversity of digestion in hematophagous Diptera [25,39,44]. In Ae.
symbiotic bacteria in Ae. albopictus [34,70]. Indeed, taxo- albopictus, Acinetobacter baumannii and Acinetobacter
nomic microarray and quantitative PCR analyses showed johnsonii could be involved in both blood digestion and
that the abundance of Enterobacteriaceae increased with nectar assimilation [37].Theevidencefor this isthatun-
Chikungunya virus infection, whereas the abundance of like environmental Acinetobacter strains, mosquito iso-
some other bacterial genera such as Wolbachia and lates were able to metabolize the amino acids α-keto
Blattabacterium decreased [70]. All these results suggest -valeric acid and glycine, which are blood components,
thatcomplexmicrobialinteractions(directorindirect,co- as well as 4-hydroxy-benzoic acid and xylose, which are
operation or competition) occur between pathogens and common constituents of plant sap. The bacterial species
microbiota that may affect mosquito traits such as vector Asaia bogorensis isolated from An. stephensi was shown
competence. to be prototrophic with respect to vitamins suggesting it
mayprovidethemosquitowithvitamins[40].
II-Putativeimpactofbacteriaonmosquitobiology Bacteria are involved in nutrition through the release of
The huge bacterial diversity associated with insects and various compounds useful for mosquito larval develop-
the complexity of potential interactions between symbi- ment. For instance, it has been demonstrated that a high
otic microorganisms and their hosts pose a significant level of Pseudomonas aeruginosa improved larval growth
challenge to understanding extended phenotypes in of Cx. quinquefasciatus in a phosphorus-rich medium
mosquitoes. Current technologies are not sufficient to whilethatofCx.tarsaliswassloweddown[72].Thelevel
pinpoint all the fluxes of matter and energy between mi- of phosphorus in breeding sites could be a factor
croorganisms and their hosts. However, some beneficial explaining how mosquitoes can adapt to a specific condi-
functions provided by bacteria, especially those living tion according to their bacterial load, possibly with a
intracellularly, the endosymbionts, have been deciphered. trade-off between the nutritional and toxic roles of bac-
Generally, insect-associated bacteria are classified in two teria. Differential tolerance of larvae to putative toxins
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present in P. aeruginosa could explain why the two mos- that this bacterium plays a beneficial role in the develop-
quitospeciesarenotfoundinthesameaquatichabitat. mentofthemosquitoes[78].Eventhoughthemechanism
remains to be identified, the high prevalence of Asaia
Reproduction combined with their ability to be transmitted both hori-
As previously shown, some bacteria colonize the repro- zontally and vertically provide evidence of the biological
ductive organs of insects allowing them to manipulate roleofbacteriuminthesemosquitoes[36,47,79].
host reproduction, allowing them to spread considerably
throughhostpopulations.ThegenusWolbachiaisableto III-Potentialapplicationsofbacteriaagainstmosquitovectors
control mosquito mating by a phenomenon called cyto- Application of chemical insecticides is still the most
plasmic incompatibility. This process prevents infected common method for mosquito vector control. However,
males from producing viable progeny when mating with negative consequences like the emergence of insecticide-
an uninfected female or a female infected with an incom- resistant mosquitoes, environmental contamination and
patible Wolbachia strain. In this way, certain mosquito damage to non-target organisms have called chemical-
species of Aedes and Culex are dependent on Wolbachia based methods of control into question [80]. The use of
toproduceviableoffspring.BesidesWolbachia,otherbac- bacteria to biologically control mosquito vectors has be-
teria could playaroleinreproduction,suchasthegenera comeapromisingstrategy.
BacillusandStaphylococcussuspectedtoaffectthefertility
ofthemosquitoCx.pipiens,althoughthemechanismsre- Cytoplasmicincompatibility
maintobedetermined[73]. Inthelastdecade,oneofthefirstefficientstrategiestore-
duce crop insect pests was the introduction of sterile
Otherpotentialfunctions males into a population that, for instance, succeeded in
Bacteria occurring in the environment where mosquitoes limiting the expansion of the fruit fly Ceratitis capitata
maturemayalsoimpactontheirbehavior.Thisisthecase [81]. The major difficulty of applying the sterile insect
for bacteria producing specific odorant compounds that technique (SIT) in mosquito populations was the loss of
can act as attractants towards mosquitoes. It was demon- fitnessobservedinsterilizedmales[82-84].RelatedtoSIT,
stratedthatthecompositionofskinmicrobiotaaffectsthe the incompatible insect technique (IIT) was developed
degree of attractiveness of humans to mosquito species basedonWolbachia-mediatedcytoplasmicincompatibility
[74]. For example, Corynebacterium minutissimum pro- [85].Thetrans-infectionofAe.albopictuswithWolbachia
duces volatile compounds such as lactic acid or butyl bu- strains wRi and wPip Istambul originating from Drosoph-
tyrate that attract An. gambiae [74]. Moreover, bacteria ilasimulansandCx.pipiens,respectively,causedasignifi-
from breeding sites or water-soluble compounds secreted cant reduction in hatching rates [86,87]. Interestingly,
by those bacteria are able to stimulate the hatchingof Ae. when Cx. pipiens was trans-infected with strain wPip
aegypti eggs [75]. Some studies demonstrated a link be- Istambul, no impact was observed on the mosquito’s
tween the presence of bacteria in insect hosts and their fitness, making this a more promising approach than
ability to degrade some insecticide molecules. For in- SIT[82,83,87].
stance, the stinkbugwhich lives on sugarcane may harbor
some fenithrotion-resistant Burkholderia which are ac- Paratransgenesis
quired from the environment [76]. The acquisition of Transgenesishasalsobeenproposedasavaluablemethod
thesebacteriabyeachgenerationcouldbeaneasywayfor for controlling mosquito populations. This method is
the insect to detoxify itself from the insecticide without based on the introduction of a transgene in insectvectors
any genetic cost. As yet, very few studies have described which can directly impact on their life history traits or
theroleof bacteriainthedegradationofxenobioticmole- vector competence or indirectly interfere with pathogen
cules, though this could be important in understanding replication and transmission [88]. One inconvenience of
why the number of insecticide-resistant mosquitoes is using transgenic mosquitoes is the cost to mosquito fit-
growing. The load of Wolbachia in Cx. pipiens seems ness as they are much less competitive [89]. Rather than
to be positively correlated with insecticide resistance modifyingtheinsectgenomeper se, acomplementaryap-
mediated by esterase genes at some metabolic cost to proach called paratransgenesis was proposed, which con-
mosquitoes[77]. sists of using a genetically-modified symbiont known to
Finally,theeffectsofexperimentaldepletionofthesym- haveanimpactoninsectlifehistorytraits[90,91].Recently,
biotic strain Asaia SF2.1 in An. stephensi larvae strongly Asaiawasproposedasapromisingsymbioticcontrolagent
suggest that the bacterium is a beneficial symbiont of this for paratransgenesisasthis bacterium is transformable and
insect. Indeed, the observation of a delay in the develop- can be used to express candidate genes in key organs of
ment inlarvae after antibiotic treatment inparallelwitha infectedmosquitospecies.Similarly,Pantoea,anewlyiden-
dramaticreductionofAsaia burden,led tothehypothesis tified mosquito symbiont that cross-colonizes several
Minardetal.Parasites&Vectors2013,6:146 Page9of12
http://www.parasitesandvectors.com/content/6/1/146
mosquito species and can be transformed and cultured, Bacteriausedasinsecticides
wasalso proposed for paratransgenic applications [27]. Re- Some bacterial strains are able to produce insecticidal
cently,transgenicstrainsofPantoeaagglomeransweregen- compoundsthatactlikenaturalpesticides.Thebacterium
erated by transformation with a plasmid expressing Bacillusthuringiensisserovar israelensisproducestwodif-
antiplasmodialcompounds[92]. ferent toxins encoded by the cry and cyt genes located on
aplasmidreplicon[102].TheCrytoxinsactonalargein-
Modificationofvectorcompetence sect spectrum (Coleoptera, Lepidoptera, Hymenoptera
Vector competence is the ability of a vector to transmit a and Diptera), whereas the Cyt toxins act specifically on
pathogen,i.e.theintrinsicpermissivenessofavectortobe Diptera[103].BothtoxinsareactivatedbythealkalinepH
infected,thentoreplicateandtotransmitapathogen[93]. of the larval gut and are able to degrade the midgut
One strategy used to fight vector-borne pathogens is to membrane causing larvae to die [104]. The Firmicute
decrease vector competence. There is a growing interest LysinibacillussphaericusalsocontainstheinsecticidalMtx
indiscoveringhowbacteriainterferewithpathogentrans- andBintoxinsthatarehighlyactiveagainstmosquitolar-
mission. In particular, several studies have shown that vae [105]. These toxins paralyze the digestive system and
Wolbachiacandecreaseorinhibitpathogenreplicationor disrupt the insect nervous system. These two classes of
transmission in different mosquito species. In general, larvicidalbacteriaaremajormosquitocidalcandidatesand
bacteriasuccessfullyinterferedwithpathogenswhenmos- were successfully used in the field to reduce An. gambiae
quitoes were trans-infected with strains isolated from a populations responsible for malaria outbreaks in Gambia
differenthost. This isthe case for Ae. aegyptiand Anoph- and Ghana [106,107].Finally, larvicidal toxins of Clostrid-
eles which are not naturally infected with Wolbachia. In iumbifermentansserovarMalaysiaandthepupicidaltoxin
such artificial systems, a significant reduction in life span of Bacillus subtilis subspecies subtilis are also potential
and pathogen load (including viruses such as Dengue and candidates as agents in biological control of mosquito
Chikungunyaorparasitessuchasplasmodiumsandfilari- populations[108,109].
ases) has been observed [16,18,19,94,95]. In Cx. pipiens
which is naturally infected by Wolbachia, the West Conclusions
Nile virus load was reduced only 2–3 fold compared to Even though information on the nature of mosquito-
individualslackingWolbachia[96].Morerecently,theAe. associatedbacteriaisincreasing,theirfunctionsandgenetic
albopictus ALPROV line naturally harboring two potential are still underexplored. This is partly due to the
Wolbachia strains, wAlbA and wAlbB, was shown to effi- complexity of interactions in terms of bacterial population
ciently replicate the dengue virus but transmission, as dynamics influenced by different biotic and abiotic factors.
measured by the amount of genomic RNA and infectious In the near future, the application of next generation se-
particlesinsalivaryglands,wassignificantlyreducedcom- quencing should improve our knowledge of the essential
pared tothe Wolbachia-uninfected line [97]. Mechanisms microbial partners and their roles in mosquito biology.
of bacterialinterferenceof vectorcompetencestillremain Interestingly,therecentdevelopmentoftechniquessuchas
to be deciphered, but some hypotheses have been sug- metagenomics, metatranscriptomics, metaproteomics and
gested. As bacteria and pathogens can invade similar tis- metabolomics is opening up the possibility of more com-
sues or even the same cells, a theoretical assumption is prehensive descriptions of molecular foundations and sig-
that they could directly compete for resources and space natures of the relationships between insects and their
[98]. The presence of bacteria could also induce the im- microbiomes. Such high-throughput analysis will allow a
mune system by producing specific compounds that dir- better understanding of the dynamics and function of the
ectly interact with pathogens like antiviral or antiparasitic mosquito-associated microbiota. It will be possible to ex-
compounds.Recently,Panetal.(2012)demonstratedthat plore bacterial communities in an unprecedented way by
the inhibition of dengue virus in the presence of highlighting metabolically active bacteria and discovering
Wolbachia was correlated with the induction of oxidative novel bacterial genes that play important roles in chemical
stress in the mosquito Ae. aegypti [99]. This response and biological processes of the insect host. Moreover, with
resulted in an activation of theToll pathway allowing the globalchangesthathavegreatlycontributedtoincreasethe
production of antioxidant molecules and anti-microbial densityandgeographicexpansionofmosquitopopulations,
peptides(defensin and cecropins) against dengue virus.In questions are now raised about the possible scenarios of
An. gambiae, it was shown that oxidative compounds se- emergence or re-emergence of mosquito-borne diseases
creted by the strain Enterobacter Esp_Z. induced a large worldwide.Asmicrobialsymbiontsofinsectsoftenmediate
decrease in Plasmodium in mosquitoes [100]. Joyce et al. or constrain adaptation to environmental fluctuations, bet-
(2011) showed that half of the bacterial species isolated ter knowledge of mosquito-associated bacterial communi-
from Ae. albopictus midguts decreased the infectivity of ties will be an important aspect of understanding what
theLaCrossevirusinanimalcells[101]. drivesmosquitoadaptation.
Minardetal.Parasites&Vectors2013,6:146 Page10of12
http://www.parasitesandvectors.com/content/6/1/146
Additional file suppressionstrategyagainstthediseasevectorAedesalbopictus.
Parasites&Vectors2012,5:254.
18. McMenimanCJ,LaneRV,CassBN,FongAWC,SidhuM,WangY-F,O’Neill
Additionalfile1:Bacterialgeneraidentifiedinmosquitoesand
SL:Stableintroductionofalife-shorteningWolbachiainfectionintothe
theirhabitats.Anindexofbacterialgeneraidentifiedinmosquitoesand
mosquitoAedesaegypti.Science2009,323:141–144.
theirhabitatsispresentedaccordingtomosquitospecies,stage,sex,
19. MoreiraLA,Iturbe-OrmaetxeI,JefferyJA,LuG,PykeAT,HedgesLM,Rocha
nutritiontype,samplinglocality,populationtypeandexperimental
BC,Hall-MendelinS,DayA,RieglerM,HugoLE,JohnsonKN,KayBH,
approach(N/A=NotApplicable).
McGrawEA,vandenHurkAF,RyanPA,O’NeillSL:AWolbachiasymbiont
inAedesaegyptilimitsinfectionwithdengue,Chikungunya,and
Plasmodium.Cell2009,139:1268–1278.
Competinginterests
20. DemaioJ,PumpuniCB,KentM,BeierJC:Themidgutbacterialfloraof
Theauthorsdeclarethattheyhavenocompetinginterests.
wildAedestriseriatus,Culexpipiens,andPsorophoracolumbiae
mosquitoes.AmJTropMedHyg1996,54:219–223.
Authors’contributions 21. Gonzalez-CeronL,SantillanF,RodriguezMH,MendezD,Hernandez-AvilaJE:
GMmadethealignments,builtthephylogenictreesanddraftedthe Bacteriainmidgutsoffield-collectedAnophelesalbimanusblockPlasmodium
manuscript,CVMandPMhelpedtodraftthemanuscript.Allauthorsread vivaxsporogonicdevelopment.JMedEntomol2003,40:371–374.
andapprovedthefinalmanuscript. 22. PidiyarVJ,JangidK,PatoleMS,ShoucheYS:Studiesonculturedand
unculturedmicrobiotaofwildCulexquinquefasciatusmosquitomidgutbased
Acknowledgements on16sribosomalRNAgeneanalysis.AmJTropMedHyg2004,70:597–603.
Wethankanonymousreviewersforhelpfulcomments.GMwassupported 23. LindhJM,TereniusO,FayeI:16SrRNAgene-basedidentificationof
byagrantfromAcademicResearchCluster1ofRhône-AlpesRegion.This midgutbacteriafromfield-caughtAnophelesgambiaesensulatoandA.
workwaspartiallyfundedbythegrantEC2COCNRS,andwascarriedout funestusmosquitoesrevealsnewspeciesrelatedtoknowninsect
withthesupportofCMIRACoopéraRhône-Alpesandwithintheframeworks symbionts.ApplEnvironMicrobiol2005,71:7217–7223.
ofGDRIs“BiodiversitéetDéveloppementDurableàMadagascar”and 24. RaniA,SharmaA,RajagopalR,AdakT,BhatnagarRK:Bacterialdiversity
“BiodiversityandInfectiousDiseases”aswellasCOSTactionF0701 analysisoflarvaeandadultmidgutmicroflorausingculture-dependent
‘ArthropodSymbioses:fromfundamentaltopestdiseasemanagement’. andculture-independentmethodsinlab-rearedandfield-collected
Anophelesstephensi-anAsianmalarialvector.BMCMicrobiol2009,9:96.
Received:19March2013Accepted:8May2013 25. GusmãoDS,SantosAV,MariniDC,BacciMJr,Berbert-MolinaMA,Lemos
Published:20May2013 FJA:Culture-dependentandculture-independentcharacterizationof
microorganismsassociatedwithAedesaegypti(Diptera:Culicidae)(L.)
anddynamicsofbacterialcolonizationinthemidgut.ActaTrop2010,
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