The mosquito microbiome includes habitat-specific but rare symbionts

Microbial communities are known to influence mosquito lifestyles by modifying essential metabolic and behavioral processes that affect reproduction, development, immunity, digestion, egg survival, and ability to transmit pathogens. Many studies have used 16S rRNA gene amplicons to characterize mosquito microbiota and investigate factors that influence host-microbiota dynamics. However, a relatively low taxonomic resolution due to clustering methods based on arbitrary threshold and the overall dominance of Wolbachia or Asaia populations obscured the investigation of rare members of mosquito microbiota in previous studies. Here, we used high resolution Shannon entropy-based oligotyping approaches to analyze the microbiota of Culex pipiens, Culex quinquefasciatus and Aedes individuals from continental and overseas regions in Southern France and Guadeloupe as well as from laboratories with or without antibiotics treatment. Our experimental design that resulted in a series of mosquito samples with a gradient of Wolbachia density and relative abundance along with high-resolution analyses of amplicon sequences enabled the recovery of a robust signal from typically less accessible bacterial taxa. Our data confirm species-specific mosquito-bacteria associations with geography as a primary factor that influences bacterial community structure. But interestingly, they also reveal co-occurring symbiotic bacterial variants within single individuals for both Elizabethkingia and Erwinia genera, distinct and specific Asaia and Chryseobacterium in continental and overseas territories and a putative rare Wolbachia variant. Overall, our study reveals the presence of previously-overlooked microdiversity and multiple closely related symbiotic strains within mosquito individuals with a remarkable habitat-specificity.

High Incidence of Related Wolbachia across Unrelated Leaf-Mining Diptera

The maternally inherited endosymbiont, Wolbachia pipientis, plays an important role in the ecology and evolution of many of its hosts by affecting host reproduction and fitness. Here, we investigated 13 dipteran leaf-mining species to characterize Wolbachia infections and the potential for this endosymbiont in biocontrol. Wolbachia infections were present in 12 species, including 10 species where the Wolbachia infection was at or near fixation. A comparison of Wolbachia relatedness based on the wsp/MLST gene set showed that unrelated leaf-mining species often shared similar Wolbachia, suggesting common horizontal transfer. We established a colony of Liriomyza brassicae and found adult Wolbachia density was stable; although Wolbachia density differed between the sexes, with females having a 20-fold higher density than males. Wolbachia density increased during L. brassicae development, with higher densities in pupae than larvae. We removed Wolbachia using tetracycline and performed reciprocal crosses between Wolbachia-infected and uninfected individuals. Cured females crossed with infected males failed to produce offspring, indicating that Wolbachia induced complete cytoplasmic incompatibility in L. brassicae. The results highlight the potential of Wolbachia to suppress Liriomyza pests based on approaches such as the incompatible insect technique, where infected males are released into populations lacking Wolbachia or with a different incompatible infection.

Male age and Wolbachia dynamics: Determining how fast and why bacterial densities and cytoplasmic incompatibility strengths vary

Endosymbiotic Wolbachia bacteria infect divergent arthropod and nematode hosts. Many strains cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected embryos are protected from CI, promoting Wolbachia spread to high equilibrium frequencies balanced by imperfect maternal transmission. CI strength varies widely in nature and tends to decrease as males age. Understanding the causes of CI-strength variation is crucial to explain Wolbachia prevalence in host populations. Here, we investigate how fast and why CI strength decreases with male age in two model systems: wMel in Drosophila melanogaster and wRi in D. simulans. Average wMel CI strength decreases rapidly (19%/ day), and wRi CI strength decreases slowly (6%/ day) as males age; thus, within three days, wMel-infected males do not cause CI, whereas twelve-day-old wRi-infected males still cause minor, yet significant, CI. We tested if reductions in Wolbachia densities or CI gene expression as males age could explain this pattern. Indeed, wRi densities and CI gene expression decrease in testes as males age, but wMel densities and CI gene expression surprisingly increase with male age as CI strength decreases. Phage WO lytic activity and wMel Octomom copy number-an ampliconic gene region that influences wMel proliferation-do not explain age-dependent Wolbachia densities. However, the expression of Relish, an essential gene in the Drosophila immune deficiency pathway, strongly correlates with wMel densities. Together, these results suggest that testes-wide Wolbachia density and CI gene expression are insufficient to explain age-dependent CI strength across strains and that Wolbachia density is variably impacted by male age across Wolbachia-host associations. We hypothesize that host immunity may underlie variation in age-dependent density dynamics. More broadly, the rapid decline of wMel CI strength during the first week of D. melanogaster life likely contributes to wMel frequency variation observed on several continents.

Parthenogenesis in weevils of the tribe Naupactini (Coleoptera, Curculionidae): a Wolbachia-density dependent trait?

The intracellular bacteria Wolbachia pipientis can manipulate host reproduction to enhance their vertical transmission. It has been reported an association between parthenogenesis and Wolbachia infection in weevils from the tribe Naupactini. A curing experiment suggested that a threshold density of Wolbachia is required for parthenogenesis to occur. The aim of this study was to analyze Wolbachia infection status in the bisexual species Naupactus xanthographus and Naupactus dissimulator.Wolbachia infection was detected in both species from some geographic locations, not being fixed. In all positive cases, faint PCR bands were observed. Quantification through real time PCR confirmed that Wolbachia loads in bisexual species were significantly lower than in parthenogenetic ones; this strengthens the hypothesis of a threshold level. Strain typing showed that both species carry wNau1, the most frequent in parthenogenetic Naupactini weevils. These infections seem to be recently acquired by horizontal transfer. Wolbachia was located throughout the whole body, which reinforce the idea of recent transmission. Moreover, we demonstrated that this strain carries the WO phage.Finally, the analysis of eubacterial 16S rRNA gene showed intense PCR bands for both bisexual species, suggesting –the presence of additional bacteria. Interspecific competition might explain why the parthenogenetic phenotype is not triggered.

Impacts of Low Temperatures on Wolbachia (Rickettsiales: Rickettsiaceae)-Infected Aedes aegypti (Diptera: Culicidae)

In recent decades, the occurrence and distribution of arboviral diseases transmitted by Aedes aegypti mosquitoes has increased. In a new control strategy, populations of mosquitoes infected with Wolbachia are being released to replace existing populations and suppress arboviral disease transmission. The success of this strategy can be affected by high temperature exposure, but the impact of low temperatures on Wolbachia-infected Ae. aegypti is unclear, even though low temperatures restrict the abundance and distribution of this species. In this study, we considered low temperature cycles relevant to the spring season that are close to the distribution limits of Ae. aegypti, and tested the effects of these temperature cycles on Ae. aegypti, Wolbachia strains wMel and wAlbB, and Wolbachia phage WO. Low temperatures influenced Ae. aegypti life-history traits, including pupation, adult eclosion, and fertility. The Wolbachia-infected mosquitoes, especially wAlbB, performed better than uninfected mosquitoes. Temperature shift experiments revealed that low temperature effects on life history and Wolbachia density depended on the life stage of exposure. Wolbachia density was suppressed at low temperatures but densities recovered with adult age. In wMel Wolbachia there were no low temperature effects specific to Wolbachia phage WO. The findings suggest that Wolbachia-infected Ae. aegypti are not adversely affected by low temperatures, indicating that the Wolbachia replacement strategy is suitable for areas experiencing cool temperatures seasonally.

Environmental Concentrations of Antibiotics May Diminish Wolbachia infections in Aedes aegypti (Diptera: Culicidae)

Wolbachia-infected Aedes aegypti (L.) mosquitoes for control of dengue transmission are being released experimentally in tropical regions of Australia, south-east Asia, and South America. To become established, the Wolbachia Hertig (Rickettsiales: Rickettsiaceae) strains used must induce expression of cytoplasmic incompatibility (CI) in matings between infected males and uninfected females so that infected females have a reproductive advantage, which will drive the infection through field populations. Wolbachia is a Rickettsia-like alphaproteobacterium which can be affected by tetracycline antibiotics. We investigated whether exposure of Wolbachia-infected mosquitoes to chlortetracycline at environmentally relevant levels during their aquatic development resulted in loss or reduction of infection in three strains, wAlbB, wMel, and wMelPop. Wolbachia density was reduced for all three strains at the tested chlortetracycline concentrations of 5 and 50 µg/liter. Two of the strains, wMel and wMelPop, showed a breakdown in CI. The wAlbB strain maintained CI and may be useful at breeding sites where tetracycline contamination has occurred. This may include drier regions where Ae. aegypti can utilize subterranean water sources and septic tanks as breeding sites.

Cross-Generational Effects of Heat Stress on Fitness and Wolbachia Density in Aedes aegypti Mosquitoes

Aedes aegypti mosquitoes infected with Wolbachia symbionts are now being released into the field to control the spread of pathogenic human arboviruses. Wolbachia can spread throughout vector populations by inducing cytoplasmic incompatibility and can reduce disease transmission by interfering with virus replication. The success of this strategy depends on the effects of Wolbachia on mosquito fitness and the stability of Wolbachia infections across generations. Wolbachia infections are vulnerable to heat stress, and sustained periods of hot weather in the field may influence their utility as disease control agents, particularly if temperature effects persist across generations. To investigate the cross-generational effects of heat stress on Wolbachia density and mosquito fitness, we subjected Ae. aegypti with two different Wolbachia infection types (wMel, wAlbB) and uninfected controls to cyclical heat stress during larval development over two generations. We then tested adult starvation tolerance and wing length as measures of fitness and measured the density of wMel in adults. Both heat stress and Wolbachia infection reduced adult starvation tolerance. wMel Wolbachia density in female offspring was lower when mothers experienced heat stress, but male Wolbachia density did not depend on the rearing temperature of the previous generation. We also found cross-generational effects of heat stress on female starvation tolerance, but there was no cross-generational effect on wing length. Fitness costs of Wolbachia infections and cross-generational effects of heat stress on Wolbachia density may reduce the ability of Wolbachia to invade populations and control arbovirus transmission under specific environmental conditions.

Effects of Alternative Blood Sources on Wolbachia Infected Aedes aegypti Females within and across Generations

Wolbachia bacteria have been identified as a tool for reducing the transmission of arboviruses transmitted by Aedes aegypti. Research groups around the world are now mass rearing Wolbachia-infected Ae. aegypti for deliberate release. We investigated the fitness impact of a crucial element of mass rearing: the blood meal required by female Ae. aegypti to lay eggs. Although Ae. aegypti almost exclusively feed on human blood, it is often difficult to use human blood in disease-endemic settings. When females were fed on sheep or pig blood rather than human blood, egg hatch rates decreased in all three lines tested (uninfected, or infected by wMel, or wAlbB Wolbachia). This finding was particularly pronounced when fed on sheep blood, although fecundity was not affected. Some of these effects persisted after an additional generation on human blood. Attempts to keep populations on sheep and pig blood sources only partly succeeded, suggesting that strong adaptation is required to develop a stably infected line on an alternative blood source. There was a decrease in Wolbachia density when Ae. aegypti were fed on non-human blood sources. Density increased in lines kept for multiple generations on the alternate sources but was still reduced relative to lines kept on human blood. These findings suggest that sheep and pig blood will entail a cost when used for maintaining Wolbachia-infected Ae. aegypti. These costs should be taken into account when planning mass release programs.

The maternal effect gene Wds controls Wolbachia titer in Nasonia

SUMMARYMaternal transmission of intracellular microbes is pivotal in establishing long-term, intimate symbioses. For germline microbes that exert negative reproductive effects on their hosts, selection can theoretically favor the spread of host genes that counteract the microbe’s harmful effects. Here, we leverage a major difference in bacterial (Wolbachia pipientis) titers between closely-related wasp species with forward genetic, transcriptomic, and cytological approaches to map two quantitative trait loci that suppress bacterial titers via a maternal effect. Fine mapping and knockdown experiments identify the gene Wolbachia density suppressor (Wds), which dominantly suppresses bacterial transmission from mother to embryo. Wds evolved by lineage-specific non-synonymous changes driven by positive selection. Collectively, our findings demonstrate that a genetically simple change arose by Darwinian selection in less than a million years to regulate maternally transmitted bacteria via a dominant, maternal effect gene.