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 , 26.4% in Sri Lanka , and 61.6% in Thailand . At the species level, past studies reported Wolbachia infection in 40% of all tested species in India , 18.2% in Sri Lanka , 51.7% in Taiwan , 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 . 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 , 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 , 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 . 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 , 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 . 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 . Although Ae. aegypti and Ae. albopictus belong to the same subgenus Stegomyia, and occupy similar ecological niches , 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 , grasshoppers , and gall-inducing aphids . 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 . 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 . 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 . 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 , 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 . 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.  and Ruang-Areerate et al. .
Host specificity is thought to be a characteristic of the ancestral Wolbachia strain, with host flexibility reported mainly in Wolbachia supergroup A and B . 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 . In bees, which harboured supergroup A Wolbachia, a mixture of host-specific and host flexible strains in the population has also been reported . 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 . 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 . 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 . 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 , 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 . 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 . 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 . 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 , 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 . 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 . 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 . 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 , 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 . 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 . 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 . 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.