Genetic diversity of endosymbiotic bacteria Wolbachia infecting two mosquito species of the genus Eretmapodites occurring in sympatry in the Comoros archipelago

Introduction The influence of Wolbachia on mosquito reproduction and vector competence has led to renewed interest in studying the genetic diversity of these bacteria and the phenotypes they induced in mosquito vectors. In this study, we focused on two species of Eretmapodites, namely Eretmapodites quinquevittatus and Eretmapodites subsimplicipes, from three islands in the Comoros archipelago (in the Southwestern Indian Ocean). Methods Using the COI gene, we examined the mitochondrial genetic diversity of 879 Eretmapodites individuals from 54 sites. Additionally, we investigated the presence and genetic diversity of Wolbachia using the wsp marker and the diversity of five housekeeping genes commonly used for genotyping through Multiple Locus Sequence Typing (MLST). Results and discussion Overall, Er. quinquevittatus was the most abundant species in the three surveyed islands and both mosquito species occurred in sympatry in most of the investigated sites. We detected a higher mitochondrial genetic diversity in Er. quinquevittatus with 35 reported haplotypes (N = 615 specimens, Hd = 0.481 and π = 0.002) while 13 haplotypes were found in Er. subsimplicipes (N = 205 specimens, Hd = 0.338 and π = 0.001), this difference is likely due to the bias in sampling size between the two species. We report for the first time the presence of Wolbachia in these two Eretmapodites species. The prevalence of Wolbachia infection varied significantly between species, with a low prevalence recorded in Er. quinquevittatus (0.8%, N = 5/627) while infection was close to fixation in Er. subsimplicipes (87.7%, N = 221/252). Both male and female individuals of the two mosquito species appeared to be infected. The analysis of MLST genes revealed the presence of two Wolbachia strains corresponding to two new strain types (STs) within the supergroups A and B, which have been named wEretA and wEretB. These strains were found as mono-infections and are closely related, phylogenetically, to Wolbachia strains previously reported in Drosophila species. Finally, we demonstrate that maternal transmission of Wolbachia is imperfect in Er. subsimplicipes, which could explain the presence of a minority of uninfected individuals in the field.


Introduction:
The influence of Wolbachia on mosquito reproduction and vector competence has led to renewed interest in studying the genetic diversity of these bacteria and the phenotypes they induced in mosquito vectors.In this study, we focused on two species of Eretmapodites, namely Eretmapodites quinquevittatus and Eretmapodites subsimplicipes, from three islands in the Comoros archipelago (in the Southwestern Indian Ocean).
Methods: Using the COI gene, we examined the mitochondrial genetic diversity of Eretmapodites individuals from sites.Additionally, we investigated the presence and genetic diversity of Wolbachia using the wsp marker and the diversity of five housekeeping genes commonly used for genotyping through Multiple Locus Sequence Typing (MLST).
Results and discussion: Overall, Er. quinquevittatus was the most abundant species in the three surveyed islands and both mosquito species occurred in sympatry in most of the investigated sites.We detected a higher mitochondrial genetic diversity in Er. quinquevittatus with reported haplotypes (N = specimens, Hd = .and π = .
) while haplotypes were found in Er. subsimplicipes (N = specimens, Hd = .and π = . ), this di erence is likely due to the bias in sampling size between the two species.We report for the first time the presence of Wolbachia in these two Eretmapodites species.The prevalence of Wolbachia infection varied significantly between species, with a low prevalence recorded in Er. quinquevittatus ( .%, N = / ) while infection was close to fixation in Er. subsimplicipes ( .%, N = / ).Both male and female individuals of the two mosquito species appeared to be infected.The analysis of MLST genes revealed the presence of two Wolbachia strains corresponding to two new strain types (STs) within the supergroups A and B, which have been named wEretA and wEretB.These strains were found as mono-infections and are closely related, phylogenetically, to Wolbachia strains previously reported in Drosophila species.

Introduction
Endosymbiotic bacteria are of increasing interest due to their impact on the biology of arthropods.Some of these bacteria are known to be essential for the evolution of their hosts enabling them to adapt to new ecological niches (Douglas, 1998).Other bacteria provide selective advantages depending on the ecological contexts by providing for example protection against predators (Tsuchida et al., 2010) or pathogens (Oliver et al., 2003;Scarborough et al., 2005;Hedges et al., 2008;Teixeira et al., 2008;Jaenike et al., 2010).In addition to these positive effects, endosymbiotic bacteria are also selfish elements that can manipulate the reproduction of their hosts to increase their own fitness (Duron et al., 2008).This is the case for the bacteria Wolbachia which are associated with various reproductive manipulation phenotypes in arthropods (Werren et al., 2008).
Wolbachia are maternally inherited alpha-proteobacteria commonly found in arthropods and filarial nematodes (Werren et al., 2008).These bacteria are estimated to be present in up to 66% of insect species (Hilgenboecker et al., 2008;Zug and Hammerstein, 2012;Weinert et al., 2015), thus probably representing the most abundant endosymbiont described to date.Wolbachia exibit high genetic diversity and have been classified into 17 phylogenetic groups or supergroups (A to Q) (Baldo et al., 2006;Paraskevopoulos et al., 2006;Bordenstein et al., 2009;Ros et al., 2009;Glowska et al., 2015).The widespread distribution of Wolbachia is primarily attributed to their impact on the reproductive biology of their hosts.In arthropods, Wolbachia manipulate host reproduction by biasing the sex ratio toward females (the transmitting sex), or by causing sterility through a phenomenon known as cytoplasmic incompatibility (CI) (Werren et al., 2008).Cytoplasmic incompatibility results from spermegg incompatibility occuring when Wolbachia-infected males mate with either uninfected females or females infected with an incompatible Wolbachia strain, resulting in high embryonic mortality reaching up to 100% in certain mosquito species (Laven, 1951;Werren et al., 2008;Atyame et al., 2014).This phenotype is commonly observed in arthropods (Shropshire et al., 2020;Turelli et al., 2022) including in mosquito vectors (Sicard et al., 2019).
Aside from the manipulation of reproduction, Wolbachia can also impact the vector competence of mosquitoes, which refers to their ability to become infected with and transmit a pathogen.Wolbachia have shown to provide protection against major mosquito-borne pathogens like Dengue virus (DENV), Chikungunya virus (CHIKV) or Plasmodium infections (Moreira et al., 2009;Bian et al., 2010Bian et al., , 2013;;Hoffmann et al., 2011;Walker et al., 2011;Aliota et al., 2016;Dutra et al., 2016).
Mosquitoes from the Eretmapodites genus (Theobald, 1901) (subfamily: Culicinae; tribe: Aedini) are exclusively Afrotropical species occurring in continental Africa (Harbach, 2007), Madagascar (Tantely et al., 2016), and in the islands of the Comoros archipelago (composed of four volcanic islands: Grande Comore, Mohéli, Anjouan and Mayotte) within the Southwestern Indian Ocean (Le Goff et al., 2014;Boussès et al., 2018).A total of 51 Eretmapodites species have been described so far (https:// mosquito-taxonomic-inventory, accessed in November 2023) (Harbach, 2013), most (32 species) from Cameroon (Bamou et al., 2021), while only four species are known in Madagascar (Tantely et al., 2016), and two species are reported in the Comoros archipelago (Le Goff et al., 2014;Boussès et al., 2018).Eretmapodites species are mostly found in forested areas but some species are also adapted to rural and peri-urban environments (Le Goff et al., 2014;Boussès et al., 2018;Bamou et al., 2021).Along with their aggressive daytime biting behavior, these mosquitoes are known to bite both animals and humans (Musa et al., 2020); as a result they have the potential to serve as bridge vectors of pathogens between animals and humans.Different arboviruses such as Rift Valley fever virus (RVFV), Semliki Forest virus (SFV), or CHIKV have been detected and/or isolated from Eretmapodites mosquitoes [review in Bamou et al. (2021)].In addition, some studies have described the ability of Eretmapodites mosquitoes to transmit arboviruses under laboratory conditions.For example, Bauer (1928) showed that Eretmopodites chrysogaster is able to transmit the Yellow Fever virus (YFV).The study of Mclntosh  et al., 2023).Despite the medical interest of Eretmapodites species, the biology, ecology and genetics of these mosquitoes remain poorly investigated.
In this study we focused on two Eretmapodites species, namely Er. quinquevittatus and Er.subsimplicipes, from three islands in the Comoros archipelago: Grande Comore, Mohéli and Mayotte.We used molecular identification to determine the abundance of each mosquito species on the surveyed islands.Then, we examined the presence of Wolbachia in both Eretmapodites species through the presence/absence of the Wolbachia surface protein gene wsp (Braig et al., 1998).The genetic diversity of the detected Wolbachia was further characterized by sequencing the wsp gene and the five housekeeping genes developed for the Wolbachia typing (MLST) (Baldo et al., 2006).Finally, we examined the vertical transmission of Wolbachia in a laboratory colony of Er. subsimplicipes.The role of Wolbachia in the evolution of Eretmapodites species is discussed.

Mosquito sampling
Adult Eretmapodites specimens were collected in 2019 (March to May and November to December) from 54 natural breeding sites on three islands of the Comoros archipelago (in the Southwestern Indian Ocean): Grande Comore (18 sites), Mohéli (eight sites), and Mayotte (28 sites) (Figure 1; Supplementary Table 1).Larvae were also collected in the site Bambo Est on Mayotte in March 2019 and March 2022 to establish a laboratory colony for testing vertical transmission of Wolbachia (see below).Adult mosquitoes were captured using portable electric aspirators (BioQuip InsectaVac aspirator, Bioquip, CA).Collected adults were introduced in cages (16 × 16 × 16 cm) and brought to the laboratory where they were sorted by sex and individually stored in 1.5 ml tubes at −80 • C (for samples from Mayotte) or in 70% ethanol (for samples from Grande Comore and Mohéli) until morphological identification and molecular analyses.

Mosquito identification
The larvae and adults of Eretmapodites from the Bambo Est site (Mayotte) and collected in March 2019 and March 2022 were identified morphologically using taxonomic keys (Edwards, 1941;Hopkins, 1952;Service, 1990).The larvae of Er. quinquevittatus have a characteristic thick, heavily chitinized 3-VIII seta and, in adults, the scutum is decorated with five parallel bands of dark scales on a yellow-brown background.The larvae of Er. subsimplicipes are easily distinguished from those of Er. quinquevittatus by lateral setae of abdominal segments I-VI inserted on a sclerotized conical tubercle and, in adults, a more homogeneous scutum devoid of such dark bands.Molecular identification of species was realized on larvae and all collected adult specimens through PCR amplification and sequencing of a 658 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 encoding gene (COI) (Folmer et al., 1994) (primers listed in Supplementary Table 2).DNA extraction, PCR, and sequencing were performed as described below.

Wolbachia genotyping
Wolbachia detection was performed in all sampled adults using PCR targeting the surface protein gene wsp (Braig et al., 1998) (Supplementary Table 2) which is more variable than the slowly evolving 16S rRNA gene (Zhou et al., 1998).For wsppositive samples, Wolbachia were genotyped by sequencing the wsp gene and the five MLST loci: coxA, fbpA, ftsZ, gatB and hcpA (Baldo et al., 2006) (Supplementary Table 2).For all individuals in which Wolbachia DNA could not be amplified, the quality of the DNA template was checked by the amplification of the COI gene.

Sequences
All sequences were visually inspected and manually edited using Geneious Prime v.2022.2.2 (Kearse et al., 2012).For the COI gene, comparisons with public sequences were performed using basic local alignment search tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST,accessed on 28 July 2023) from GenBank.The mitochondrial haplotype diversity (Hd) and nucleotide diversity (π ) were calculated in the software DnaSP v6.12.03 (Rozas et al., 2017).For Wolbachia genes, the generated  sequences were compared with data available in the Wolbachia MLST database (https://pubmlst.org/organisms/wolbachia-spp,accessed on 03 August 2023) (Jolley et al., 2018).For each MLST gene, a new allele was considered if there was at least one nucleotide difference with alleles already present in the pubMLST database.Thereafter, the combination of alleles allowed identifying the Sequence Types (STs) among those existing or to propose new STs.

Phylogenetic analysis
Phylogenetic relationships were evaluated for Eretmapodites COI and Wolbachia genes.Only unique mitochondrial haplotypes and bacterial alleles were included in the analyses.For Wolbachia, phylogenetic analyses were conducted for each of the six sequenced genes and on all five MLST concatenated genes (coxA, fbpA, ftsZ, gatB, and hcpA, in this order).The phylogenies were constructed with data from Baldo et al. (2006).For each data set, the best-fitting model of sequence evolution was determined using jMoldelTest v.2.1.4(Darriba et al., 2012).Then, phylogenetic constructions were performed using MrBayes v.3.2.3 (Ronquist et al., 2012).For each phylogeny, the analysis corresponded to two independent runs of four incrementally heated Metropolis Coupled Markov Chain Monte Carlo (MCMCMC) starting from a random tree.The MCMCMC was run for 10 million generations with trees and associated model parameters sampled every 100 generations.The convergence level was accessed with an average standard deviation of split frequencies inferior to 0.05.The 10% initial trees for each run were discarded as burn-in and the phylogeny along with posterior probabilities were obtained from the remaining trees.The resulting Bayesian phylogeny trees were visualized and annotated with FigTree v.1.4.2 (Rambaut, 2014).

Vertical transmission of Wolbachia
Since only the Er.subsimplicipes species could be reared from larvae collected in the Bambo Est site (Mayotte) in March 2019, we used it to examine the vertical transmission of Wolbachia.Field larvae (F 0 generation) were brought to the laboratory and kept alive in the insectary where they were identified morphologically and maintained under standard rearing conditions (27 • C and 80% relative humidity with a 12h:12h photoperiod).Larvae were supplied every 2 days with yeast tablets and adults were fed with 10% sucrose solution.To get eggs and ensure the maintenance of mosquitoes, females were blood-fed using a Hemotek feeding system (Hemotek Limited, GreatHarwood, UK) with defibrinated cow blood.The eggs of the next generation (F 1 generation) were collected and reared to adulthood.The amplification process was performed over four generations (F 4 generation) to increase the number of females for the experiment.Then, the vertical transmission of Wolbachia was assessed using females and males from the established laboratory colony.Females of the F 4 generation were allowed to mate in the laboratory with males from the same colony.After mating, the females were blood-fed and individually isolated to lay eggs.Then, the presence of Wolbachia was tested for each female by PCR using the wsp gene as described above.The offsprings from each Wolbachia-infected female were kept alive until adulthood and males and females were screened for the presence of Wolbachia.

Eretmapodites quinquevittatus is more abundant than Eretmapodites subsimplicipes
Larvae and adults collected in the site Bambo Est (Mayotte) in March 2019 and March 2022 were morphologically identified as Er.subsimplicipes.COI sequencing of these morphologically identified specimens showed a closed match with the published sequence of Er. subsimplicipes from Mozambique (GenBank accession number: LC664011, 99.8%−100.0%percentage of identity based on 633 bp), thus confirming the identification of our specimens.We then sequenced a total of 879 mosquitoes (655 females and 224 males) from Grande Comore (N = 327), Mohéli (N = 195), and Mayotte (N = 357).The comparison of the obtained COI sequences with the GenBank database indicated the presence of two Eretmapodites species: Er. quinquevittatus (GenBank accession number: LC664009, 98.4%−100.0%percentage of identity based on 629 bp) and Er.subsimplicipes (GenBank accession number: LC664011, 99.7%−100.0%percentage of identity based on 633 bp).Among the sequences, 71.3% (N = 627/879) and 28.7% (N = 252/879) belonged to Er. quinquevittatus and Er.subsimplicipes, respectively (Supplementary Tables 1, 3).Eretmapodites quinquevittatus appeared more common than Er.subsimplicipes in all three investigated islands (Figure 1A).Both Eretmapodites species were found in sympatry in 31 out of 54 sampled sites (seven sites in Grande Comore, seven sites in Mohéli and 17 sites in Mayotte), while Er.quinquevittatus was found alone in 22 sites (11 sites in Grande Comore, one site in Mohéli and ten sites in Mayotte) and Er.subsimplicipes alone at one site (Bambo Est) on Mayotte (Supplementary Table 1).

Higher mtDNA polymorphism in Er. quinquevittatus
Among the 627 Er. quinquevittatus specimens, COI sequences with good qualities (i.e., 658 bp with no ambiguities) were obtained for 615 samples leading to 35 haplotypes (Figure 2A; Supplementary Table 4).Pairwise nucleotide identity between the haplotypes ranged from 98.2% to 99.9%.The overall haplotype diversity (Hd) and nucleotide diversity (π ) values were 0.481 and 0.002, respectively.The most frequent haplotype [EQ_H01, found in 71.4% of sequences (N = 439/615)] was also the most widespread in all three islands (Figure 2A; Supplementary Table 4).The second most frequent haplotype (EQ_H27, scored in 43 specimens) was geographically restricted to Mayotte (Supplementary Table 4).Of the 35 haplotypes, four haplotypes were shared by all three islands (EQ_H01, EQ_H10, EQ_H11, and EQ_H14), two haplotypes (EQ_H02 and EQ_H07) were shared by Grande Comore and Mohéli, one haplotype (EQ_H16) was common to Grande Comore and Mayotte, and no common haplotype was detected between Mohéli and Mayotte (Supplementary Table 4).On Grande Comore, 18 haplotypes were found whereas the number of haplotypes was similar between Mohéli and Mayotte (14 haplotypes on each island).A total of 11, eight and nine haplotypes were unique on Grande Comore, Mohéli and Mayotte, respectively (Figure 2A; Supplementary Table 4).
COI good quality sequences were obtained for 205 of the 252 Er. subsimplicipes samples leading to 13 haplotypes (Figure 2B; Supplementary Table 5).Pairwise nucleotide identity between the haplotypes yielded values ranging from 99.4 to 99.9%.The overall Hd and π values were 0.338 and 0.001, respectively.The haplotype ES_H01 was the most frequently observed in the dataset, with 80.9% of specimens (N = 166/205) and the only one common to all three islands (Figure 2B; Supplementary Table 5).The number of haplotypes was higher in Mayotte (ten haplotypes), followed by Mohéli (four haplotypes), while the lowest diversity was observed in Grande Comore (two haplotypes).Unique haplotypes were only found in Mayotte (eight haplotypes) and Mohéli (three haplotypes) (Figure 2B; Supplementary Table 5).

Lower prevalence of Wolbachia in Er. quinquevittatus than in Er. subsimplicipes
The 879 Eretmapodites mosquitoes were screened for Wolbachia infection based on the detection of the wsp gene by PCR.The overall prevalence of Wolbachia was 25.7% (N = 226/879), with a significant lower prevalence detected in Er. quinquevittatus (0.8%, N = 5/627) as compared to Er. subsimplicipes (87.7%, N = 221/252) (Table 1) (Fisher's exact test, P < 0.001).Wolbachia infections were detected in both males and females of both mosquito species (Table 1).In Er. quinquevittatus, two out of the five Wolbachia infected mosquitoes were females and three were males.In contrast, the majority of the Wolbachia infected Er. subsimplicipes mosquitoes were females, with 201 females and 3 males out of a total of 221 infected mosquitoes.For Er. quinquevittatus, the bacterial infection prevalence between sites ranged from 0.0% to 7.7% with the five Wolbachia-infected specimens detected from five sites: two sites in Grande Comore, one site in Mohéli and two sites in Mayotte (Supplementary Table 1).For Er. subsimplicipes, Wolbachia-positive specimens were detected in all but one site (the Iconi site on Grande Comore, N = 32 sites with Er. subsimplicipes specimens) and infection prevalence ranged from 50.0% to 100.0% including in sites with a large number of samples (Figure 1B; Supplementary Table 1).For both mosquito species, Wolbachia infection prevalence did not significantly vary according to the sampled islands (Fisher's exact tests, all P > 0.7) (Supplementary Table 1).
Two Wolbachia A and B supergroups occurred in Er. quinquevittatus and Er.subsimplicipes The sequencing of the wsp gene in Er. quinquevittatus and Er.subsimplicipes revealed the presence of two Wolbachia supergroups A and B in each Eretmapodites species (Figure 3).For Er. quinquevittatus, three samples out of the five Wolbachiainfected were successfully sequenced and one sample belonged to supergroup A while two samples belonged to supergroup B (Figure 3; Supplementary Table 3).Concerning Er. subsimplicipes, the sequencing of the wsp gene was succeful for 218 out of the 221 samples.Almost all of these samples (N = 217/218) belonged to supergroup A and one sample to supergroup B. When comparing the 218 wsp sequences of supergroup A (217 sequences for Er.subsimplicipes and one sequence for Er.quinquevittatus), no polymorphism was noted, a unique wsp allele shared by the two mosquito species was observed (Figure 3; Supplementary Table 3).The analysis of the three wsp sequences from supergroup B (two sequences for Er.quinquevittatus and one sequence for Er. subsimplicipes) also revealed one wsp allele shared by both Eretmapodites species.
As the wsp gene alone is not relevant for a reliable genotyping of Wolbachia strains due to recombination in Wolbachia genomes (Jiggins et al., 2001;Bordenstein and Wernegreen, 2004;Baldo et al., 2006;Atyame et al., 2011), we sequenced the five Wolbachia MLST genes coxA, fbpA, ftsZ, gatB and hcpA.The sequences of the five MLST genes were not obtained systematically for each of the 226 Wolbachia-infected Eretmapodites mosquitoes.Indeed, PCR amplifications have failed for some genes (particularly fbpA) in certain samples, possibly due to mutations in the targeted primers sites.Additionally, since we used universal degenerated primers (Baldo et al., 2006), it may have been possible to improve our protocols to increase amplification success for Eretmapodites.Ultimately, we obtained 214 sequences for coxA, 114 sequences for fbpA, 210 sequences for gatB, 177 sequences for hcpA and 214 sequences for fstZ.We confirmed the presence of Wolbachia strains belonging to supergroups A and B with each of the five MLST genes (Supplementary Figures 1-5; Supplementary Table 3).We found two alleles for four of the five genes (coxA, fbpA, ftsZ and gatB), one allele belonging to supergroup A and the other one to supergroup B (Supplementary Figures 1-4; Supplementary Table 3).The most polymorphic locus was hcpA with three alleles, two alleles for supergroup A and one allele for supergroup B (Supplementary Figure 5; Supplementary Table 3).The two hcpA alleles falling in the supergroup A were genetically close, with 99.8% pairwise identity based on 476 bp.Our data do not support co-infection by Wolbachia strains from supergroups A and B. None of the five MLST genes could be amplified in the single Er.quinquevittatus sample infected with a Wolbachia strain from supergroup A. Therefore, using the MLST genes, we detected supergroup A only in Er. subsimplicipes and supergroup B in both Er.subsimplicipes and Er.quinquevittatus (Supplementary Figures 1-5; Supplementary Table 3).As observed

FIGURE
Bayesian phylogenic tree of Wolbachia strains based the wsp gene ( bp, sequences).The phylogenetic tree was built using the substitution model: GTR+I+G.Sequences in red and blue correspond to Wolbachia sequences detected in Er. subsimplicipes and Er.quinquevittatus, respectively.The accession numbers of Wolbachia strains identified in these mosquito species are also indicated.For each sequence the host species is indicated and the GenBank accession number is provided in brackets.The letters A, B and F correspond to Wolbachia supergroups.The tree is midpoint unrooted and the numbers associated with nodes correspond to posterior probability values.The scale bar is in units of substitutions/site.
with the wsp gene, all MLST alleles were shared by the two mosquito species within supergroup B (Supplementary Figures 1-5).
Comparison of allelic polymorphism with pubMLST database revealed that within the supergroup A, alleles identified in the present study for coxA, fbpA, ftsZ, and hcpA are new with the exception of the gatB allele matching with allele #49 (Supplementary Table 6).The coxA allele showed a close match with allele #173, the fbpA allele with allele #60, the ftsZ allele with allele #52, and the two hcpA alleles were genetically closely related to allele #11 (Supplementary Table 6).The combination of the five alleles resulted in a new Wolbachia strain type, which we named "wEretA."For supergroup B, all observed alleles for the five MLST genes are already present in the pubMLST database.Indeed, coxA, fbpA, ftsZ, gatB, and hcpA alleles matched with alleles #281, #453, #244, #283, and #309, respectively (Supplementary Table 6).However, no Wolbachia strain type was assigned to the combination of these five alleles in the pubMLST database.Hence, we considered this Wolbachia strain type as new and named it "wEretB."The MLST allelic profiles of wEretA and wEretB appeared genetically different from those of a Wolbachia strain previously described in the species Eretmapodites chrysogaster from Cameroon for which coxA matched with #275, ftsZ matched with #106, and fbpA matched with #6 (Osuna et al., 2023).Using complete MLST profiles obtained for 84 mosquitoes (83 Er. subsimplicipes and one Er.quinquevittatus), we performed a phylogenetic analysis based on the 2,079 bp concatenated sequences of the five MLST genes.It appears that Wolbachia strains wEretA (infecting Er. subsimplicipes) and wEretB (infecting both Er.subsimplicipes and Er.quinquevittatus) form two robust monophyletic clades within A and B supergroups, respectively (Figure 4).wEretA is genetically closely related to wDori and wDneo infecting Drosophila orientacea and Drosophila neotestacea, respectively (Figure 4).wEretB is closely related to wMa infecting Drosophila simulans (Figure 4).In summary, MLST data revealed that (i) wEretA is restricted to Er. subsimplicipes (83 complete MLST allelic profiles) and (ii) wEretB infects both Er.subsimplicipes and Er.quinquevittatus (1 complete MLST allelic profile each) (Figure 4; Supplementary Table 3).Finally, we examined the evolution of the diversity of Wolbachia in their hosts by comparing the concatenated MLST phylogeny and the COI phylogeny from different host species including in Er. subsimplicipes and Er.quinquevittatus.No congruence

FIGURE
Bayesian phylogenic tree of Wolbachia strains obtained from concatenated data set of MLST genes (coxA, fbpA, ftsZ, gatB, and hcpA) ( , bp, sequences).The phylogenetic tree was built using the substitution model: GTR+I+G.Sequences in red and blue correspond to Wolbachia sequences detected in Er. subsimplicipes and Er.quinquevittatus, respectively.The names of Wolbachia strains identified in these mosquito species (wEretA and wEretB) are indicated in brackets.The letters A, B, D, and F correspond to Wolbachia supergroups.The tree is midpoint unrooted and the numbers associated with nodes correspond to posterior probability values.The scale bar is in units of substitutions/site.
between Wolbachia and COI phylogenies was shown (Figure 5), demonstrating that Wolbachia mainly use horizontal transfers to spread in their hosts.

Wolbachia is maternally inherited in Er. subsimplicipes
To assess maternal transmission of Wolbachia in Eretmapodites mosquitoes, we focused on the species Er. subsimplicipes as it is the only species for which we currently have a laboratory colony.We examined the progeny of 30 wEretA infected laboratory females (see above) based on the sequencing of the wsp gene.In general, the number of eggs per female ranging from 4 to 61 (mean number of 32 eggs per female) (Table 2).The hatching rate of the eggs ranged from 5% to 100%, with a mean rate of 74%.It seems that the number of adults produced by each female is limited, as the mean rates for eggs becoming larvae and larvae reaching the adult stage are only 15% and 26%, respectively (Table 2).A total of 131 offspring (74 males and 57 females) from the 30 investigated females were then screened for the presence of Wolbachia.Sixty per cent (N = 78/131) were found infected (Table 2), leading to a maternal transmission of Wolbachia ranging from 0% to 100%.Among the 30 females, four females did not transmit Wolbachia to their offspring, the transmission of Wolbachia was imperfect (between 6% and 88%) for ten females while perfect maternal transmission of Wolbachia (100%) was recorded for 16 females (Table 2).

Discussion
Using morphological and molecular methods, we confirmed the presence of two Eretmapodites species, Er. quinquevittatus and Er.subsimplicipes, in three islands of the Comoros archipelago (Grande Comore, Mohéli and Mayotte) (Le Goff et al., 2014;Boussès et al., 2018).The two species occurred in sympatry in the majority of investigated sites but Er. quinquevittatus was most commonly found in the three islands.The higher abundance of Er. quinquevittatus observed in this study may be due to sampling biases related to the type of samples collected and the method used for collection.In contrast to a previous study

FIGURE
Comparisons between phylogeny of Wolbachia and phylogeny of arthropod hosts mitochondria (COI gene).The phylogeny of Wolbachia was constructed using concatenated sequences of the five MLST genes (coxA, fbpA, ftsZ, gatB, and hcpA) ( , bp, sequences).The mitochondrial phylogeny was constructed using sequences of the COI gene ( bp, sequences).Only Wolbachia strains for which the COI sequences of the associated host species could be found in public database were used.The letters A, B, D and F correspond to Wolbachia supergroups.For COI phylogeny, the GenBank or BOLD accession numbers are indicated in brackets.The two phylogenies were built with Bayesian inference using the substitution model: GTR+I+G.The phylogenies were midpoint unrooted and the numbers associated with nodes correspond to posterior probability values.The scale bar is in units of substitutions/site.conducted between 2008 and 2012 in Mayotte, which found Er. subsimplicipes to be the most frequently encountered mosquito species on the island (Le Goff et al., 2014), our observations were based on adult mosquitoes.The difference between our findings and the previous study could be attributed to the fact that larvae were sampled in the study of Le Goff et al. (2014), whereas we focused on adult collection.It is possible that breeding sites of Er. quinquevittatus are less accessible compared to those of Er. subsimplicipes, which could result in sampling bias when working with adults that have the ability to fly far away from their breeding sites.However, it is also plausible that the distribution area of Er. quinquevittatus in Mayotte has increased over the last past 10 years.Additionally, the sampling method used in our study, which involved portable electric aspirators to collect resting adult mosquitoes in vegetation and flying adults around manipulators, may have better suited the collection of Er. quinquevittatus adults compared to Er. subsimplicipes.Since the biology and ecology of both species in the field are not well understood, it is possible that this methodological difference influenced our findings.It would be interesting in future investigations to compare the distribution area of both Eretmapodites species in Mayotte, but also in the other islands of the Comoros archipelago, using both larval and adult sampling.
The mtDNA polymorphism based on the COI gene revealed 13 and 35 haplotypes in Er. subsimplicipes and Er.quinquevittatus, respectively.In both species, we found unique haplotypes (i.e., encountered in only one island), suggesting different colonization events probably from Madagascar or the east coast of Africa, regions geographically close to the Comoros archipelago and where both Eretmapodites species have been also identified (Harbach, 2007;Tantely et al., 2016).Other mtDNA haplotypes were shared by different islands and could be the result of a single colonization event of Eretmapodites mosquitoes (from Madagascar or Africa) either to different islands, or to one island followed by a secondary dispersion event in a stepping stone mode.Such dispersion from a nearby island can be facilitated by frequent trade between the islands of the Comoros archipelago (Roger et al., 2014).For example, it is well known that the spread of the Asian tiger mosquito Ae. albopictus worldwide has been facilitated by the international trade of used tires (Reiter and Sprenger, 1987).
The mitochondrial haplotype diversity was higher in Er. quinquevittatus (35 haplotypes, Hd = 0.481 and π = 0.002, for N = 615 samples) than in Er. subsimplicipes (13 haplotypes, Hd = 0.338 and π = 0.001, for N = 205 samples).The difference between the two species can be explained by the sampling sizes as we found more Er.quinquevittatus specimens in our dataset.Alternatively, a higher mtDNA diversity in Er. quinquevittatus could result from a low prevalence of Wolbachia infection.Indeed, mitochondria and Wolbachia are in linkage disequilibrium, both cytoplasmic elements being linked through maternal cotransmission within egg cytoplasm's (Rasgon et al., 2006;Atyame et al., 2011;Dumas et al., 2013).Therefore, the spread of Wolbachia in host populations should result in an indirect selective sweep of the mtDNA leading to a reduction of mitochondrial diversity in Wolbachia infected host populations (Rasgon et al., 2006;Atyame et al., 2011;Dumas et al., 2013).In this study, we detected Wolbachia for the first time in both Er.quinquevittatus and Er.subsimplicipes, the lowest Wolbachia prevalence occurring in Er. quinquevittatus (0.8% vs. 87.7% in Er. subsimplicipes).The presence of Wolbachia in females and their offspring was examined through the presence/absence of the wsp gene.For each female, the number of eggs, the number of larvae (first instar larvae), the egg hatching rate (%), the percentage of emerging adults, the percentage of egg reaching to adult and the number of emerging adults (males and females) were provided.Finally, the rate of maternal transmission of Wolbachia was determined from adult progeny of each female.N = total number of mosquitoes examined (eggs or larvae).
Wolbachia infection is not fixed in any of the Eretmapodites field populations, with Wolbachia-infected and uninfected specimens found within the same sampling sites.The presence of Wolbachia-infected and uninfected specimens is commonly observed in field populations of other arthropod species and can be associated with low phenotypic manipulation but also to imperfect maternal transmission (Werren et al., 2008).We have monitored maternal transmission of (the most frequent) wEretA using a laboratory colony of Er. subsimplicipes.It should be noted that it was challenging to rear Er. subsimplicipes species under insectary conditions and a reduced number of adult offspring was obtained for each female.Despite this challenge, our results show that maternal transmission of Wolbachia is imperfect or non-existent in some females, which could explain why Wolbachia infection is not fixed in Er. subsimplicipes field populations.COI sequencing data is also consistent with imperfect maternal transmission in Er. subsimplicipes since identical mtDNA haplotypes are shared by Wolbachia-infected and uninfected mosquitoes (Figure 2C).
The examined phylogenies of wsp and each of the five MLST genes showed that both Er.quinquevittatus and Er.subsimplicipes are infected with two Wolbachia supergroups A and B. Within each Wolbachia supergroup, the genetic diversity was low, only one allele being detected for almost all loci (except for hcpA).The concatenated phylogeny of the five MLST genes also confirmed the presence of two Wolbachia supergroups A and B strains (namely wEretA and wEretB, respectively) in our dataset.In Er. subsimplicipes, mosquitoes were infected with either wEretA or wEretB, although more higher infections by wEretA than wEretB were observed; while only wEretB was observed in Er. quinquevittatus.The presence of two divergent Wolbachia strains in Er. subsimplicipes can be explained by horizontal transfer events from other arhtropod species infected with genetically related Wolbachia such as Drosophila spp.which appeared to be infected with Wolbachia strains closely related to the strains wEretA and wEretB (Figure 4).Interestingly, the strain wEretB was shared by Er. subsimplicipes and Er.quinquevittatus.Several hypotheses can be proposed to explain this pattern.The wEretB strain might have been present in the common ancestor of both Eretmapodites species, and this Wolbachia strain was maintained in both species after their divergence, but the absence of nucleotide diversity between wEretB infecting both mosquito species does not support this assumption.For instance, some difference exists between the Wolbachia strains Dinn_A and Drec_A (within the supergroup A) infecting the genetically closely related Drosphila species D. innibula and D. recens (Figure 4).Another possibility would be horizontal transfers of Wolbachia between both mosquito species or from other host species.The widespread distribution of Wolbachia in arthropods is commonly associated with horizontal transfers occurring between closely related or genetically divergent host species (Heath et al., 1999;Ahmed et al., 2016;Tolley et al., 2019).These transfers would take place through mechanisms such as contamination, predation, or parasitism, particularly among species sharing the same ecological niches.Although evidence of horizontal transfers is rare, studies have shown that such transfers can occur in host-parasitoid associations (Huigens et al., 2004;Ahmed et al., 2015).The lack of congruence between Wolbachia and hosts phylogenies also support the possibility of horizontal transfers of Wolbachia between species (Tolley et al., 2019).In our Frontiers in Microbiology frontiersin.orgstudy, we compared the phylogenies of concatenated MLST genes and COI, and no congruence was found (Figure 5), confirming the potential for horizontal transfers of Wolbachia between host species.Furthermore, the success of interspecific transfers of Wolbachia via embryonic microinjections (Sasaki and Ishikawa, 2000;McMeniman et al., 2009;Hughes and Rasgon, 2014) also supports the hypothesis of horizontal transfers of Wolbachia.
Assuming a horizontal transfer of wEretB from Er. subsimplicipes to Er. quinquevittatus, the low prevalence of Wolbachia in Er. quinquevittatus could be explained either by a recent transfer of wEretB, or by differences in phenotypes induced by this Wolbachia strain when infecting each mosquito species.This change in phenotype expression of the same Wolbachia strain when infecting different host species has been previously described in Drosophila recens and Drosophila subquinaria (Jaenike, 2007).
It would be interesting for future investigations to examine the phenotypes induced by the Wolbachia strains wEretA and wEretB in Er. subsimplicipes and Er.quinquevittatus to better understand the dynamics of these bacteria in the field.Lastly, a horizontal transfer of wEretB might have happened through introgression between both Eretmapodites species.Introgressions of Wolbachia have been observed in various subspecies of mosquitoes in the Culex pipiens complex, such as Culex pipiens pipiens and Culex pipiens quinquefasciatus, which hybridize in natural environments.This hybridization leads to subspecies sharing the same Wolbachia strains, as determined through the Wolbachia MLST genotyping method (Atyame et al., 2011;Dumas et al., 2013) (see also Figure 4).This hypothesis could be tested in the future by comparing the polymorphism in nuclear genomes of Er. subsimplicipes and Er.quinquevittatus mosquitoes.

Conclusion
In the present study, we characterized the mitochondrial genetic diversity of Er. quinquevittatus and Er.subsimplicipes occurring in sympatry in three islands of the Comoros archipelago.We also characterized the genetic diversity of Wolbachia infecting both mosquito species and identified two new Wolbachia strains, which have been named wEretA and wEretB.Experimental rearing of Er. subsimplicipes revealed imperfect maternal transmission of Wolbachia that might explain the infection patterns found in the field.Future studies will examine the phenotypes induced by these Wolbachia in Er. quinquevittatus and Er.subsimplicipes to better understand their dynamics in natura.As Eretmapodites mosquitoes are competent vectors for the transmission of arboviruses (Bamou et al., 2021;Cêtre-Sossah et al., 2023), future investigations should also consider the effects of wEretA and wEretB on vector competence.

FIGURE
FIGURE Maps showing the abundance of Eretmapodites quinquevittatus and Eretmapodites subsimplicipes (A) and the prevalence of Wolbachia in field populations of Er. subsimplicipes (B) in three islands of the Comoros archipelago (Grande Comore, Mohéli and Mayotte).Mosquitoes were sampled as adults and identified with the sequencing of the mitochondrial COI gene.The prevalence of Wolbachia is based on the presence/absence of the wsp gene.Only sites with more than five individuals are shown (see Supplementary Tableformore details).

FIGURE
FIGURE (A) Heatmap showing the distribution of COI haplotypes in Eretmapodites quinquevittatus from three islands of Comoros archipelago; (B) Heatmap showing the distribution of the COI haplotypes in Eretmapodites subsimplicipes from the three surveyed islands; (C) Bayesian phylogenetic tree based on COI gene ( bp, sequences) showing relationships between Er. subsimplicipes (in red) Er. quinquevittatus (in blue) mosquitoes.The phylogenetic tree was built using the substitution model: GTR+I+G.Only unique mtDNA haplotypes are shown: ES_H to ES_H for Er.subsimplicipes and EQ_H to EQ_H for Er.quinquevittatus.Asterisks indicate mitochondrial haplotypes detected in both Wolbachia infected and uninfected specimens.Names in bold indicate specimens morphologically identified as Er.subsimplicipes in this study.GenBank accession numbers are indicated in brackets.The tree was rooted on Toxorhynchites splendens and the numbers associated with nodes indicate the posterior probability.The scale bar is in units of substitutions/site.
McIntosh et al., 1980)demonstrated the ability of Eretmapodites quinquevittatus to transmit the RVFV.Likewise, it has been recently shown that Eretmapodites subsimplicipes is a competent vector for the transmission of RVFV (Cêtre-Sossah Table for more details).

TABLE Prevalence of
Wolbachia in Eretmapodites quinquevittatus and Eretmapodites subsimplicipes in three islands of the Comoros archipelago, based on presence/absence of the wsp gene and according to the sex of mosquitoes.
N = total number of mosquitoes examined.
TABLE Maternal transmission of Wolbachia in Eretmapodites subsimplicipes.