Wolbachia Infection in Wild Mosquitoes (Diptera: Culicidae): Implications for Transmission Modes and Host-Endosymbiont Associations

Background

Wolbachia is an intracellular endosymbiotic bacterium that alters host reproduction [1]. It is widespread in arthropods, infecting a wide range of insects, crustacean, and nematode species [2, 3]. In some cases, Wolbachia has a mutualistic relationship with their hosts [4–6]. However, Wolbachia is most often recognised as a reproductive manipulator that biases the sex-ratio of the host's offspring towards infected females [7, 8]. This reproductive manipulation is commonly achieved through four phenotypes – male-killing [9], feminisation [10, 11], parthenogenesis [12, 13], and cytoplasmic incompatibility [14, 15] – increasing the endosymbionts’ reproductive success [16]. Owing to their strong influence on host reproduction, an increasing amount of research is dedicated to exploring the impacts of reproductive endosymbiont on host population dynamics and evolution [17, 18], especially in medically important insects such as mosquitoes. The promise to alter both mosquito reproduction [19], and arboviral transmission [20], have prompted the deployment of novel Wolbachia-infected mosquitoes for population replacement and suppression [21].

Several countries, including Singapore, have started to employ Wolbachia as a biocontrol agent of mosquitoes [22–24], but most have not assessed the presence of naturally occurring infection in their wild mosquito populations. The release of artificially infected mosquitoes might have a profound impact on wild mosquito populations due to close interactions. The effect of endosymbiont on non-target species could potentially result in a population crash, opening a niche for other vector species [25]. Another implication of such a bio-control will be the increased likelihood of co-infections with naturally occurring Wolbachia strains or other endosymbionts, such as Cardinium, Rickettsia, and Spiroplasma, which can have a synergistic effect on host fitness and future transmission of endosymbionts [26–29]. Without a detailed characterisation of Wolbachia prevalence and diversity among wild mosquitoes, the ecological risk of releasing artificially infected mosquitoes might be overlooked. Therefore, with the precautionary principle in mind, it is important to investigate the natural occurrences of Wolbachia.

There is also a need to discern the mode of infection transmission among mosquitoes. Even though Wolbachia are mainly thought to be transmitted vertically [15, 30], there are accounts of horizontal transmissions into wild populations through parasitism [31, 32], or being near infected individuals [33]. Previous research also proposes that Wolbachia may not be strictly localised in the germline tissues and have been detected in somatic tissues such as the gastrointestinal tract and haemolymph [34–36]. Detection in the gastrointestinal tract suggests horizontal transmission through uptake from the environment or host sharing [34, 37, 38], whereas a detection in non-gastrointestinal somatic tissues, such as the jointed appendages, could be a case of horizontal bacterial genome integration into the host genome [36]. Currently, most research on the detection of Wolbachia in mosquitoes adopts conventional PCR methods which extract DNA from the entire individual or its abdomen [39–47]. This limits our ability to identify the exact location of endosymbiont infection within an individual. A tissue-specific screening of Wolbachia is necessary to provide insights into the extent of vertical and horizontal transmissions.

Given that vertical transmission is widely believed to be the main mode of Wolbachia transmission [15, 30], it has been proposed that host mitochondrial DNA (mtDNA) and Wolbachia are maternally co-transmitted within the cytoplasm [17, 48]. This results in congruency between host mtDNA and Wolbachia phylogenies – a consequence of cytoplasmic hitchhiking driven by endosymbiont transmission [17]. In insect systems such as bedbugs where vertical transmission has been established to be the main mode of transmission, Wolbachia exhibits clear patterns of co-phylogeny with its hosts, with few instances of host shifting or multiple infections within a single host species [49, 50]. In contrast, co-phylogeny is not apparent among nematodes and bees, and there are numerous acquisitions of Wolbachia infections through horizontal transmission as well as losses in these diversified host lineages [51, 52]. As the modes of Wolbachia transmission are not well-established among mosquitoes, the extent of host shifting or multiple infections within mosquito hosts are not well-explored.

Currently, there has yet been a comprehensive analysis of the evolutionary associations between Wolbachia and their mosquito host species. An understanding of host-endosymbiont association will not only further our ability to discern the mode of transmission which influences Wolbachia diversity, but it will also allow for evaluation of Wolbachia’s host specificity, speciation, and its ability to establish itself in new hosts. All of these are key to understanding Wolbachia’s diversity, ecology, and utility as a biocontrol method.

In this current study, we have three major research objectives: First, we examined the prevalence and diversity of Wolbachia among wild mosquitoes from Singapore. Second, using a tissue-specific PCR screening method, we also determined the localisation of Wolbachia infection within an individual. Finally, we reconstructed the evolutionary associations between Wolbachia and their mosquito hosts to provide a basis to understand host-endosymbiont evolution.

Methods

Adult mosquito collection and identification

We collected mosquito samples from twelve localities across Singapore between March 2018 and November 2019 (Fig. 1A). We employed three methods to collect the samples: CO₂-baited Centers for Disease Control and Prevention (CDC) traps, sweep netting using handheld fan traps, and larval sampling [53]. For the latter, we carried out the dipping method at streams and ponds and used pipettes to collect larvae from various microhabitats including tree holes, plant axils, and artificial containers. Thereafter, we reared the field-collected larvae to adults in an incubator maintained at 26 °C, 70% RH, and under a photoperiod of 12:12 (day:night) hour diurnal cycle. We fed the larvae with pulverised fish food (TetraMin Granules) daily. Mosquitoes were identified using relevant taxonomic keys and descriptions [54–59]. We randomly selected a subset of individuals from commonly sampled species and preserved them in phosphate-buffered saline solution at -80 °C for the subsequent dissection step.

Tissue-specific Dissection

We carried out dissection on each adult mosquito sample and isolated the leg, gut, and reproductive tissues (Fig. 1B – Fig. 1D). To prevent the contamination of tissues, we dissected the leg first to prevent contamination by any bacteria residing within the mosquito. Between each dissection, all dissection equipment and microscope slides were thoroughly wiped with 70% ethanol before commencing dissection for the next sample. We placed the dissected tissues individually into a 96-well plate which was placed on ice to prevent DNA degradation.

Dna Extraction, Pcr Amplification, And Sequencing

We used 7µL of QuickExtract™ DNA Extraction Solution (Lucigen) to extract DNA from each of the dissected tissues. We placed the samples in a thermocycler with the following protocol: 65 °C for 18 minutes, followed by 98^oC for 2 minutes, and ending with cooling on ice for at least 10 minutes. All dissected tissues were screened for Wolbachia infections following single-primer PCR protocols described by Martin et al. [26] with slight modifications. We used the Wolbachia surface protein (wsp) general primers, wsp81F (5’- TGGTCCAATAAGTGATGAAGAAACTAGCT-3’) and wsp691R (5’- AAAAATTAAACGCTACTCCAGCTTCTGCAC-3’) [60]. We chose the wsp molecular marker as it can successfully detect Wolbachia infection across numerous taxa and it is also ideal for strain genotyping and evolutionary comparison between the detected Wolbachia strains [60]. In addition, we amplified a section of the cytochrome c oxidase subunit I (COI) gene of the mosquito hosts using primers LCO1498 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’- TAAACTTCAGGGTGACCAAAAAATCA-3’) [61]. This serves to confirm host identity and acts as an internal control. We used DNA from known Wolbachia-infected Nasonia specimens as positive controls for this study.

We performed all PCR procedures in reaction mixtures consisting of 12.5µL of GoTaq® G2 Green Mastermix (Promega), 1µL of 1 mg mL^⁻1 bovine serum albumin, 0.184µL of 25 mM magnesium chloride, 1.5µL of extracted DNA, and 1.5µL each of 5 µM wsp forward and reverse primers for Wolbachia PCR screens or 1.0µL each of 5 µM LCO1498 and HCO2198 primers for COI PCRs. We used double-distilled water (ddH₂O) to top up the reaction mixture to a final volume of 25µL. PCR amplification of positive and negative controls (by replacing DNA with ddH₂O) was also conducted simultaneously.

We used the following PCR conditions: 94 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min, with a final elongation step of 72 °C for 10 min. We separated the amplicons by gel electrophoresis on 2% agarose gel stained with GelRed® (Biotium) and visualised under a UV transilluminator (GeneSys). Amplicons were purified using SureClean Plus (Bioline) following the manufacturer’s protocol and sent for Sanger sequencing at First Base Laboratories (Axil Scientific Pte. Ltd., Singapore).

Using Geneious Prime (2019.2.3) (https://geneious.com), we conducted DNA sequence clean-up on all sequenced amplicons as follows: First, we trimmed the ends of the sequences using the default setting. Second, we created contiguous sequences with forward and reverse reads using de novo assemble at the highest sensitivity. Third, we visually confirmed the DNA bases. We checked the chromatogram for multiple peaks and corrected miscalled base pairs with “N”. Last, if it was not possible to generate a contiguous sequence with the forward and reverse reads, we chose the read with a higher percentage quality as the final consensus sequence. All consensus sequences were queried in National Center for Biotechnology Information BLAST [62].

Statistical Analyses

To identify significant differences in the infection across the different mosquito tissues, we carried out Cochran’s Q test. As a follow-up, we employed McNemar’s post-hoc test to identify the tissue pairs that differed significantly in infection. Individuals which had unsuccessful amplification of the internal control (COI gene) for any of the three dissected tissues were excluded from this statistical analysis. We also tested for the effect of sex on host infection using binary logistics regression with sex as a categorical dependent variable and infection outcome as a binary independent variable. We conducted a logistics regression on a subset which only includes species that have a roughly similar representation of both sexes i.e. for every species included, the number of individuals of the less common sex is at least 60% of the more common sex.

We determined the significance of the variables when the p-value is less than 0.05. We performed all statistical analyses in R version 3.6.2 [63], with packages nonpar [64], rcompanion [65], and ISLR [66].

Phylogenetic Analyses

We carried out multiple alignment of consensus sequences using the ClustalW algorithm with default setting (‘‘gap penalty = 15’’; ‘‘gap extension penalty = 6.66’’) [67], in software MEGA X [68]. We aligned mosquito COI sequences generated in this study with 61 reference COI barcodes of identified local mosquitoes from Chan et al. [53]. For wsp sequences, we aligned the sequences with 54 available wsp sequences of known Wolbachia strains which were obtained from GenBank [69]. Short sequence reads (< 500 bp) were excluded.

We conducted objective clustering on COI sequences to sort mosquito samples into molecular clusters [70]. We treated terminal gaps as missing and determined species using a 3% threshold [71, 72]. In a previous study which conducted COI barcoding of mosquitoes from Singapore, researchers supported the cut-off of 3% to discriminate mosquito species [53]. The objective clustering, thus, serves to verify the morphological sorting done previously and to correct possible cases of misidentifications in the sample.

We also constructed neighbour-joining (NJ) phylogenetic trees for mosquito hosts and Wolbachia using the sequenced COI gene fragment and the wsp gene, respectively. We performed NJ tree reconstruction with Kimura 2-parameters as the nucleotide substitution model in MEGA X [68]. Internal gaps were treated as indels and terminal gaps as missing for wsp sequences. We estimated bootstrap probabilities by generating 1000 bootstrap replicates. We designated two biting midges species, Culicoides asiana (KJ162955.1) and Culicoides wadai (KT352425.1), as outgroups for the host NJ tree construction. Due to the lack of an appropriate endosymbiont outgroup [51], we midpoint rooted Wolbachia NJ tree instead.

When possible, we classified Wolbachia strains into supergroups and putative strains using 97% bootstrap probability as a threshold [60]. Wsp sequences that did not have 97% bootstrap support were evaluated on a case by case basis. For example, we deemed sequences which closely cluster together and have a relatively high support value (> 90%) as the same putative strain.

We categorized putative strains which are infectious to only one host species as “specialists” and those which infect two or more hosts as “generalists”. Then, we calculated the standardised phylogenetic host specificity (SPS) score of each generalist strain by adapting the method outlined by Poulin et al. [73] and Kembel et al. [74]. SPS measures the degree of phylogenetic relatedness among host species infected by the same endosymbiont strain. It also tests for significance by comparing it with null models generated with 999 replicates of random host-endosymbiont associations. A positive SPS value with high p-value (p > 0.95) indicates a high degree of host flexibility where Wolbachia infects hosts which are phylogenetically even. A negative SPS value with low p-value (p < 0.05) suggests a low degree of host flexibility where the infected hosts are phylogenetically clustered together. We calculated SPS score using R package picante [74].

Evolutionary analyses of the mosquito- Wolbachia relationship

Current literature adopts various approaches to explore correlation and evolution between hosts and their associated parasites or symbionts [51, 75–78]. Here, we used three distinct methods to explore the evolutionary associations between mosquito hosts and their Wolbachia endosymbionts. We carried out the analyses using pruned phylogenies where each species is represented by a single individual.

First, using the software TreeMap 3.0 [79], we created a tanglegram between host and endosymbiont NJ tree to visualise the mosquito-Wolbachia association. A tanglegram is useful as a pictorial representation of the interactions between two phylogenies [80]. TreeMap also seeks to minimise the entanglement between the two trees to provide a clearer visualisation of the phylogenetic relationship between host and endosymbiont [79].

Second, we employed a ParaFit Global test, a distance-based method, to quantitatively estimate congruence between the host and endosymbiont phylogenetic trees by comparing genetic distances among infected host species and the Wolbachia strains [81]. The null hypothesis (H₀) for this test states that the associations between host and endosymbiont trees are random, whereas the alternative hypothesis (H₁) suggests that there are strong associations with phylogenetic distances between hosts and parasites. We tested for significance by comparing the observed associations between host and endosymbiont with randomised associations generated with 5000 permutations. We also identified the respective host-endosymbiont associations which contributed significantly to the ParaFit Global statistics. We performed the ParaFit test with Caillliez correction to correct for negative eigenvalue generated [82] using R package ape [83].

Third, we performed an event-based analysis in Jane 4.0 [84] to map out potential evolutionary events of the endosymbiont in relation to the host phylogeny [85]. Five evolutionary events are considered: co-speciation (host and endosymbiont speciate simultaneously), duplication (intra-host speciation), duplication with host shift (endosymbiont host shifts), loss (host speciates but endosymbiont fails to establish in one of the new lineages), failure to diverge (host speciates and endosymbiont remains in both lineages). Due to the differing likelihood of each event, we attached different cost values to each of the events. Jane 4.0 determined the best reconstruction of evolutionary events by minimising the overall cost. We used the following cost-scheme regime with 100 generations and a population size of 300: co-speciation = 0, duplication = 1, duplication with host shift = 2, loss = 1, and failure to diverge = 1 [78]. As a follow-up, we carried out random tip mapping (randomisation of host-endosymbiont associations) for 50 iterations, to determine if the overall cost of reconstruction is significantly lower than expected by chance. If 5% or fewer of the random solutions have costs lower than the reconstructed coevolution phylogeny, there is support for the coevolution of the hosts and endosymbionts through co-speciation.

Each of the three methods has its own merits: (i) a tanglegram allows for clear visualisation of host-endosymbiont association without taking into account any evolutionary relationships, but there are calls for careful interpretation as the degree of entanglement may not necessarily represent phylogeny congruency [86]. (ii) A global ParaFit test seeks to address the limitation of the tanglegram by testing for global congruency in an unbiased, statistical approach [81]. (iii) An event-based method enables evaluation of potential evolutionary events that might have occurred throughout the endosymbiont’s evolutionary history such as co-speciation, duplication, and host shifting. Taken together, they allow for a well-round and comprehensive examination of the mosquito-Wolbachia association.

Results

Prevalence of Wolbachia in wild-caught mosquitoes

A total of 271 adult mosquitoes, representing 41 species and 14 genera, were collected from twelve localities in Singapore. Each specimen was dissected into three parts (leg, gut, and reproductive organ) and screened individually for the presence of Wolbachia using wsp primers [see Additional file 1]. Overall, infection prevalence was moderate with 119 out of 271 (43.9%) individuals screened positive for Wolbachia. In total, 21 (51.2%) species were positive for Wolbachia. Out of which, infection in eight species is reported here for the first time [Additional file 1]. All genera, except for Aedeomyia, Anopheles, and Mimomyia (i.e. 11 out of 14 genera; 78.6%), had positive detection of Wolbachia. Five out of seven Aedes species (71.4%) had positive detections of Wolbachia, while in the genus Culex, five out of 17 species (29.4%) are positive. Some of the positively screened species in the genera Aedes and Culex, such as Aedes albopictus and Culex quinquefasciatus, are medically important vector species which are responsible for the transmission of arboviral diseases and filariasis [87].

The infection rates vary across mosquito species. Notably, we observed variation in the percentage of infection between species that are epidemiologically related to each other. For instance, there was no Wolbachia infection detected in Aedes aegypti, the primary vector of dengue and Zika viruses [88]. However, infection was moderately high (56.8%) for Aedes albopictus which is considered as the secondary vector of dengue. We also observed a difference in the infection rate of two closely-related species Culex pseudovishnui (86.4%) and Culex vishnui (0%) [53], with the latter found to naturally harbour Japanese encephalitis virus in the Southeast Asian region [89].

Locality did not seem to play a part in the infection of mosquito hosts. Among species that have a wide range across Singapore, we observed that percentage infection was consistent in populations across different habitats. For example, the infection percentage was consistently high for Culex pseudovishnui, while consistently low for Malaya genurostris. Based on observation, species identity was better at predicting infection status than locality differences.

We explored the effect of sex on infection status using a binary logistics regression on a data subset containing 153 individuals (45.8% males) representing twelve species. We ran the regression on a data subset to ensure that both sexes of each species were similarly represented, preventing the introduction of bias due to unequal representation of sex. Sex was a significant explanatory variable and there was a significantly lower infection prevalence in males than females with an odds ratio of 0.434 (p = 0.013, D_f = 151).

Localisation of Wolbachia infection within a mosquito

Only individuals (n = 159) which had successful amplification of the COI fragment across all three tissues were included in the analyses. Wolbachia infection was mainly observed in the reproductive tissues. Among 159 reproductive tissues, 42.1% (n = 67) were infected. Percentage infection was lower among gut (5.7%, n = 9) and leg (3.1%, n = 5) tissues, respectively. The difference in percentage infection across the three dissected tissues was statistically significant (p < 0.001, D_f = 2). As a follow-up, percentage infection in the reproductive tissue was significantly higher than both the gut (p < 0.001) and leg tissue (p < 0.001). The difference in percentage infection between the gut and leg tissue was not significant (p = 1.0). Notably, the amplicon size of wsp in the gut and leg tend to be shorter than 400 base pairs.

Wolbachia diversity among mosquito fauna from Singapore

Following Zhou et al. [60], all wsp sequences obtained in this study can be broadly classified into A and B Wolbachia supergroups – out of 21 positively infected species, six were infected with supergroup A, ten with supergroup B, and one species, Aedes albopictus, were infected with both (Fig. 2). Infection of the remaining four species was unclassified due to either short sequences (< 400 bp) or sequence alignment issues during phylogenetic analyses. The analysed wsp sequences were also clustered into twelve putative strains which are labelled from “Wol 1” to “Wol 12” respectively. Four out of the twelve putative strains matched to previously typed strains [60, 90] – Wol 1, Wol 2, Wol 3, and Wol 8 were identified as wPip, wAlbB, wCra, and wRi strain respectively. Wolbachia strains from this study are also closely related to those from other insect groups (Fig. 2). For instance, Wol 9 and Wol 10 are closely related to the Wolbachia strains harboured by Drosophila spp. (bootstrap value > 99%).

Host specificity of Wolbachia strains

The degree of host specificity varied across the twelve putative strains. We considered seven out of the twelve strains (Wol 2, Wol 4, Wol 5, Wol 6, Wol 8, Wol 10, and Wol 12) as specialists. These strains were host specific and were only detected in one host species each (Fig. 3). The remaining five strains were considered as generalists as they were found in more than one host. Wol 3 was found in three host species, Coquillettidia crassipes, Mansonia sp. 1, and Culex sitiens, the most out of the generalists. Standardised phylogenetic host specificity (SPS) score was calculated for each generalist strain (Table 1). The SPS scores revealed that Wol 1 had the lowest degree of host flexibility (z = − 1.41) and this was significant (p = 0.049). On the other hand, Wol 7 had the highest degree of host flexibility (z = 0.07), but this was not significant (p = 0.779)

Table 1

Standardised phylogenetic host specificity score of putative *Wolbachia* generalists.
Wolbachia putative strain	Number of infected hosts	Phylogenetic host specificity score	Standardised phylogenetic host specificity score	p-value
Wol 1	2	0.281	–1.41	0.049*
Wol 3	3	0.391	–0.162	0.421
Wol 7	2	0.281	0.068	0.779
Wol 9	2	0.281	–0.234	0.249
Wol 11	2	0.281	–0.817	0.157

Evolutionary relationship between mosquitoes and Wolbachia

We recorded 18 counts of mosquito-Wolbachia associations in wild-caught mosquitoes from Singapore. A visualisation of these associations using a tanglegram showed patterns of broad associations (Fig. 3). For instance, the clade which consists of Aedes species was observed to be mostly associated with Wolbachia supergroup A. In contrast, other species, especially the clade representing various Culex species, had numerous associations with Wolbachia supergroup B. Nevertheless, there were several one-to-many and many-to-one host-endosymbiont associations which complicated the general mosquito-Wolbachia association pattern.

The distance-based quantitative test showed that mosquito and Wolbachia phylogenies were weakly congruent at the global level (ParaFit Global = 0.006, p = 0.048). Among the numerous host-endosymbiont links, only the association between Mansonia sp. 1 and Wol 3 was statistically significant (p = 0.031) (Fig. 3).

The event-based analysis between mosquito and Wolbachia phylogenies resulted in a reconstructed output of one co-speciation event, four counts of duplication, six counts of duplication with host shift, 31 losses, and six counts of failure to diverge, amounting to a total cost of 53 (Fig. 4). Interestingly, the number of duplications with a host shift and losses was much greater than co-speciation events. Notably, multiple host shift events tend to follow after loss events occurring earlier in the evolutionary history of the endosymbiont. For example, we see instances of consecutive host shifts to new hosts that were not previously infected (red arrows in Fig. 4). Additionally, based on random tip mapping, 14% of random solutions have costs lower than the reconstructed output. Overall, there was support for multiple host shift events and losses of Wolbachia among the mosquitoes, and there was no clear signal for mosquito-Wolbachia co-phylogeny.

Discussion

Detection of Wolbachia infection and distribution in wild mosquitoes

Using PCR-based Wolbachia screening, we successfully detected Wolbachia infections in wild-caught mosquitoes. The PCR-based method has a high positive detection rate with 86.3% of all sequenced amplicons having successful BLAST matches to Wolbachia. This suggests that the conventional PCR method is adequate in detecting Wolbachia infection. Even if the study was to proceed without the additional DNA sequencing step to confirm infection, an observation of an amplicon band would likely indicate a true positive Wolbachia infection.

Based on our results, Wolbachia is highly widespread across members of the Culicidae family. Here, we report infection in eight mosquito species that have not been previously described for harbouring Wolbachia. Overall, the percentage infection of screened individuals was 43.9% which was largely congruent with percentages reported in past studies from the Oriental region: 31% infection in Malaysia [91], 26.4% in Sri Lanka [39], and 61.6% in Thailand [92]. At the species level, past studies reported Wolbachia infection in 40% of all tested species in India [93], 18.2% in Sri Lanka [39], 51.7% in Taiwan [94], and between 28.1% and 37.8% in Thailand [92, 95]. Our study showed that 51.2% of all tested species were infected with Wolbachia which is generally higher than previous studies. This is likely attributed to the broad range of species tested in this study, including species that are usually not included in these studies – species from the Malaya, Verrallina, and Zeugnomyia genera [95]. It is also possible that infection prevalence may vary across geographical regions.

Wolbachia detection in three medically important mosquito genera, Culex, Anopheles, and Aedes, was highly consistent with past studies. Among the Culex mosquitoes, Wolbachia infection has been reported to be variable across its member species [39, 46, 92, 94]. Similarly, we observed infection only in five out of 17 Culex species. We noticed moderately high Wolbachia infection in Culex quinquefasciatus which is a member of the Culex pipiens complex responsible for the transmission of filariasis worm disease in Singapore [87, 88]. Surprisingly, between closely-related species, Culex pseudovishnui and Culex vishnui [96], we only found high percentage infection in the former. However, previous studies in India and Thailand showed a reverse pattern – infection in Cx. vishnui and not in Cx. psuedovishnui [39, 95]. Even though the two species are morphologically similar [53], species misidentification in our study was unlikely as we had carried out DNA barcoding to verify the identification of the species. This lends further support that infection prevalence may vary between populations that are found in geographically distal regions.

We did not detect Wolbachia in any of the wild-caught Anopheles species (18 individuals representing three species) examined in this study, many of which are potential malaria vectors [87]. This is largely consistent with previous reports published globally [39, 97, 98]. The absence of Wolbachia in Anopheles mosquitoes is thought to be due to the unsuitability of Anopheles reproductive tissues for Wolbachia establishment [94, 95]. However, in recent years, there are reports of sporadic Wolbachia detection in field Anopheles mosquitoes [42, 99, 100]. Knowledge of natural Wolbachia infections in Anopheles mosquitoes has implications on malaria control strategies [100], hence more wild-caught Anopheles samples should be screened to determine the infection status in Singapore more accurately.

We did not detect Wolbachia infection in Aedes aegypti, the primary vector of dengue in the Southeast Asian region [88]. Conversely, Wolbachia infection was moderately high in the secondary vector Aedes albopictus. This pattern is highly consistent with past studies that found an absence of infection in wild Ae. aegypti [21, 101], but stable infection in wild Ae. albopictus [102]. Although Ae. aegypti and Ae. albopictus belong to the same subgenus Stegomyia, and occupy similar ecological niches [103], they are rarely found in the same locality which was likewise observed in this study [43, 104, 105]. This could imply a certain degree of competitive exclusion between the two species, preventing them from occupying the same space. There is evidence showing that symbionts may influence host’s resource acquisition and specificity which ultimately lead to competitive exclusion between closely related host species with differing symbiont infections [106, 107]. However, research on Wolbachia-induced competitive exclusion is scarce except for a few studies that have looked at heterogonic gall wasps [108], grasshoppers [109], and gall-inducing aphids [110]. Given the widespread influence of Wolbachia, future research can explore potential cases of Wolbachia-induced competitive exclusion between closely related species which have a huge implication on understanding symbiosis and speciation.

Additionally, given the frequent artificial Wolbachia infection into Ae. aegypti for bio-control purposes [111–115], our finding could suggest that Ae. aegypti might not be stably maintained in the wild. This can be advantageous for vector population suppression as the cytoplasmic-incompatibility effect of any artificially introduced Wolbachia strain will likely be fully manifested in the uninfected native population [21]. However, this also implies that such a bio-control method may have low long-term effectiveness if the infection cannot be naturally sustained in the wild population. Natural Wolbachia infection in wild Ae. aegypti, therefore, has a huge implication on vector control programmes [21]. Not only does it inform the selection of Wolbachia strain prior to its field-release, but it can also be used to gauge the long-term effectiveness of the programme.

Interestingly, there was an effect of sex on infection status. From our study, females had higher odds of being infected than males. This could be an artefact of the various reproductive phenotypes induced by Wolbachia such as parthenogenesis and male-killing. They result in offspring that are largely female or end up female only because males are killed [15]. Over multiple generations with vertical Wolbachia transmission, one would observe an increasing proportion of females that are infected. Hence, the phenomenon could be a consequence of Wolbachia’s reproductive manipulation and vertical transmission.

We were unable to statistically test for the effects of locality on infection status due to uneven and small sample sizes of the respective species across different localities. Nevertheless, we documented that infection status was consistent across localities for widespread species such as Culex pseudovishnui and Malaya genurostris. This implies that mosquitoes found in localities across Singapore have roughly equal chances of having Wolbachia. This also suggests that underlying physiological factors and phylogenetic relatedness in mosquitoes contributed more to the Wolbachia infections than the habitat which they are found in.

The reproductive effect of Wolbachia can be masked or enhanced by other endosymbionts and there are studies which looked at infections with other reproductive endosymbionts such as Cardinium, Rickettsia, and Spiroplasma [7, 26–29]. Unfortunately, in our preliminary screening, we were unable to detect those endosymbionts due to a high degree of false positives using PCR-based screening methods [Additional file 2]. This is likely attributed to primers which were not optimised for mosquito screening [116–118]. As a result, we were unable to identify co-infections of various reproductive endosymbionts among wild mosquitoes which would have provided greater insights into the synergistic effects of co-infections on mosquito evolution. There is, hence, a need to develop and optimise alternative screening methods, such as multilocus sequence typing (MLST) techniques, especially for the detection of Cardinium, Rickettsia, and Spiroplasma in mosquitoes.

Localisation of Wolbachia infection in mosquitoes

In this study, we detected Wolbachia mainly in the reproductive tissues which is in line with past studies across multiple insect groups [15, 94, 119]. This suggests the vertical transmission of Wolbachia. Interestingly, through the course of this study, we noticed a significant variation in the size of reproductive traits (testis and ovary length) across and within species. These reproductive traits did not vary significantly with Wolbachia infection status, even after accounting for phylogenetic relatedness [see Additional file 3].

We also detected infection in the gut and leg tissues, albeit infrequently. This is not surprising as previous reports have detected Wolbachia in those tissues [34–36, 120]. Interestingly, the nucleotide sequences from gut and leg infections tend to be shorter in length. Considering that Wolbachia is unlikely to survive extracellularly for a long duration [35], the small amplicon size suggests potential horizontal integration of the Wolbachia genome into the host genome for a few species. This phenomenon has been observed in several Wolbachia hosts [121, 122], and mosquito species such as Aedes aegypti and Culex quinquefasciatus [123, 124]. For instance, a recent study showed that horizontal integration of Wolbachia genome into the host genome can have sex determination and evolution implications. This is evident in the common pillbug Armadillidium vulgare, resulting in the formation of a new sex chromosome [125]. Researchers have also proposed that horizontal gene transfer between endosymbiont and host can result in evolutionary innovation where new functional genes arise for both host and bacteria [123, 124].

Future research should explore the relative importance of each transmission method with relation to host-endosymbiont ecology and evolution. Such tissue-specific screening methods can be used in other arthropods especially when the mode of transmission is not clear. Currently, most Wolbachia screening is conducted on ground specimens or specimens in their entirety [39–41]. By doing so, researchers would be unable to localise Wolbachia infection within an individual which could have provided clues to its mode of transmission. In this context, adopting tissue-specific screening methods can seek to verify or refute the assumption that Wolbachia is transmitted vertically which is common in literature [15, 30].

Diversity and host-specificity of Wolbachia strains

Wolbachia wsp sequences generated in this study were clustered into twelve putative Wolbachia strains falling with the supergroup A or B which is consistent with previous studies that looked at Wolbachia infections in mosquitoes [39, 90, 95]. Each mosquito host species was only infected by strains belonging to A or B, with the exception of Aedes albopictus which harboured both. Infection of more than one strain (superinfection of wild Ae. albopictus with Wolbachia supergroup A and B) has been previously reported, and this phenomenon was commonly observed to be fixed in those examined populations due to strong cytoplasmic incompatibility effects [126, 127]. This suggests stable vertical transmission of both strains in Ae. albopictus. Additionally, only four out of twelve putative strains were identified to previously typed Wolbachia strains reported by Zhou et al. [60] and Ruang-Areerate et al. [90].

Host specificity is thought to be a characteristic of the ancestral Wolbachia strain, with host flexibility reported mainly in Wolbachia supergroup A and B [128]. In our study, we found a combination of specialists and generalists with more counts of the former. A study of mosquitoes from Taiwan showed a similar pattern [94]. In bees, which harboured supergroup A Wolbachia, a mixture of host-specific and host flexible strains in the population has also been reported [49]. While our estimates of specialists and generalists could vary with greater sampling effort, the higher numbers of specialists observed can be explained by the process of reciprocal selection between host and endosymbiont over evolutionary time [75]. This is also known as the “Red Queen” dynamics, where the endosymbiont constantly adapts to its host to ensure continued establishment in the same host [129]. An alternative strategy of being a generalist can also be maintained in a population. It ensures survivorship in an environment where resources (i.e. hosts) are rarely found [75]. However, there are generally more instances of host specialists than generalists across numerous parasitic and endosymbiotic taxa [130–132].

The standardised phylogenetic host specificity scores revealed that host flexibility among generalists varied greatly. Wol 1 had the lowest degree of host flexibility and was shown to infect mosquito hosts that are closely related: Culex pseudovishnui and Culex quinquefasciatus. Although Wol 3 infected the greatest number of mosquito species, Wol 7 was the most host flexible strain as it infected distally related Uranotaenia trilineata and Malaya genurostris. Understanding Wolbachia host specificity has huge implications especially for the optimisation of Wolbachia biocontrol strategy. Not only should researchers select strains that can limit pathogen replication [133], they should also select strains for their host specificity. This would not be possible without the screening of a wide variety of species or closely related species which was achieved in this study. A host-specific strain will decrease the likelihood of infection host shift to non-target species, thereby minimising the strategy’s overall ecological risk.

Evolutionary relationship between mosquito and Wolbachia

Host-Wolbachia relationships are often understudied and limited to a few taxa [52]. Current attempts to reconstruct host-Wolbachia evolutionary association have found co-phylogeny patterns in beetles and bedbugs [49, 50]; co-speciation with infrequent horizontal acquisitions patterns in filarial nematodes and bees [51, 52]; and evidence for host shifting across distantly related species in butterflies and moths [134]. Hence, patterns for Wolbachia transmission and diversification tend to vary across the various taxa. In our study, the relationship between mosquito hosts and Wolbachia is highly complex, with neither co-speciation nor host shifting fully accounting for the evolutionary association in these lineages.

A broad association pattern between mosquitoes and Wolbachia strains was observed (Fig. 3). Aedes mosquitoes tend to be associated with supergroup A Wolbachia, while other clades, particularly the genus Culex, were largely associated with supergroup B Wolbachia. This showed that closely related Wolbachia strains were likely to establish themselves in related hosts. There might have been radiation of Wolbachia in these clades after their respective initial establishments. However, without information about the time of divergence, this could be an ecological event where closely related Wolbachia strains occupied similar niches.

The ParaFit analysis showed weak support for congruency between host and endosymbiont phylogenies. Among the 18 host-Wolbachia associations, only the link between Mansonia sp. 1 and Wol 3 showed a significant association (Fig. 3). This was interesting considering that Wol 3 was largely host flexible. Given that this was the only significant association, a genus-specific study on Mansonia spp. is worthy of further exploration to elucidate coevolutionary patterns within a group of closely related mosquito species. Perhaps, the degree to which Wolbachia coevolves with its mosquito host can vary across phylogenetic resolution [81]. The analyses thus far suggest that mosquito-Wolbachia associations are likely random at higher taxonomic levels with occasions of mosquito-Wolbachia co-speciation at finer phylogenetic resolution (i.e. like patterns seen in diffuse coevolution).

Referring to the least cost coevolutionary construction by Jane 4.0 (Fig. 4), co-speciation events were infrequent as compared to other evolutionary events. We noticed a greater proportion of host shifts and numerous losses. Interestingly, Jane indicated multiple consecutive host shifts occurring near the tips of the cladogram. This suggests that co-speciation does not fully explain the evolutionary association between mosquito hosts and Wolbachia. Instead, recent host shifting through horizontal transmission seems to promote Wolbachia diversification. This lends greater support that horizontal transmission between distantly related species is possible [32, 33, 135]. The consecutive host shifts are possible cases of Wolbachia radiation by host shift and that horizontal transmission is likely to better explain the observed Wolbachia diversity than vertical transmission. However, an absence of host shifts during early parts of Wolbachia's evolutionary history questions the effectiveness of horizontal transmission in maintaining infection over generations.

Furthermore, losses, which represent endosymbiont extinction events that occurred upon host speciation, seemed to dominate the evolutionary history of Wolbachia. Extinction events are believed to be frequent in host-endosymbiont systems [75], due to either evolution of resistance in the host or declining host population size which results in the inability for highly specialised endosymbionts to establish themselves [136, 137]. Additionally, losses could potentially influence endosymbiont evolution through the creation of vacant niches [136]. The observed losses followed by host shifts in the mosquito-Wolbachia relationship are possible consequences of vacant niche exploitation by generalists. Perhaps, this enabled successful endosymbiont invasion due to minimal intra-strain competition. Therefore, horizontal Wolbachia transmission and losses may play a bigger role in accounting for Wolbachia diversity than previously expected.

Based on our study, it is difficult to determine the mechanism which explains Wolbachia’s diversity and evolutionary association. The presence of numerous specialists could be a sign of mosquito-Wolbachia coevolution since coevolution is fundamentally reciprocal selection between host and endosymbiont which gives rise to micro-evolutionary changes [138]. Being highly adapted to their hosts could imply strain level evolution. The numerous host shifts and losses might have, however, blurred the effects of vertical transmission over the long evolutionary period [52]. Thus, we propose that co-speciation might have occurred within smaller clades, but at the broader perspective, horizontal transmission and loss events are more likely the prominent force driving Wolbachia evolution.

The Wolbachia wsp gene has been shown to provide phylogenies with a good resolution [60], and our study provides an exploratory snapshot of the evolutionary associations between mosquito hosts and their Wolbachia endosymbionts. Of course, this is a potential caveat since we only used a single gene each to construct the respective phylogenetic trees. To obtain a more accurate phylogeny, future studies can adopt MLST [17, 51], or whole-genome shotgun sequencing in their methods [52]. The former could potentially characterise putative Wolbachia strains that cannot be distinguished with wsp gene primers.

A limitation of an event-based analysis (Jane 4.0) is that such a method cannot distinguish between topological congruence and an evolutionary event [75]. It is ecologically likely that there might be a co-phylogenetic coincidence in a eukaryote-bacteria relationship given that bacterial lineages often evolve faster than the hosts [139, 140], and that host shifts among closely related species are highly likely [138]. In other words, our event-based analysis does not take into account the time of divergences for both symbiont and host and is, thus, unable to accurately differentiate co-phylogeny and co-speciation.

Notwithstanding the limitations, the employment of various analytical methods allows for a comprehensive study of the evolutionary association between Wolbachia and mosquito hosts which are lacking in current literature. Using single genes to reconstruct evolutionary trees, this study serves as an initial exploratory study which examined mosquito-Wolbachia evolutionary associations across a wide range of host mosquito species. Future studies interested in the evolution of medically important vector species could narrow their scope on the Aedini tribe which will provide greater statistical power for the examination of mosquito-endosymbiont association.

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