Wolbachia Strain wMel Research Articles

Single-cell transcriptome sequencing reveals that Wolbachia induces gene expression changes in Drosophila ovary cells to favor its own maternal transmission.

Wolbachia is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of Wolbachia within the host's ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in Drosophila ovaries infected and uninfected with Wolbachia. Our findings indicate that Wolbachia significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of Wolbachia from extracellular to intracellular. Moreover, the genes nos and orb, which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with Wolbachia, revealing that Wolbachia can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of Wolbachia between host ovarian cells.IMPORTANCEWolbachia, an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host's eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from Drosophila melanogaster and observed the effects of Wolbachia (strain wMel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of Wolbachia within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with Wolbachia are significantly altered, which possibly favors the maternal transmission of Wolbachia. Meanwhile, we also discovered that Wolbachia may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host's ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of Wolbachia within the host.

Fertility differences between two wild-type Drosophila melanogaster lines correlate with differences in the expression of the Jheh1 gene, which codes for an enzyme degrading juvenile hormone.

Juvenile hormone plays a "status quo" role in Drosophila melanogaster larvae, preventing the untimely metamorphosis, and performs a gonadotropic function in imagoes, ensuring the ovaries' preparedness for vitellogenesis. The decreased level of juvenile hormone results in reproductive disorders in D. melanogaster females including a delay in the oviposition onset and a fertility decrease. Another factor that can affect the insect reproduction is an infection with the maternally inherited symbiotic α-proteobacterium Wolbachia. The present study is devoted to the analysis of the expression of two juvenile hormone metabolism genes encoding enzymes of its synthesis and degradation, juvenile hormone acid O-methyltransferase ( jhamt) and juvenile hormone epoxide hydrase (Jheh1), respectively, in four wild-type D. melanogaster lines, two of them being infected with Wolbachia. Lines w153 and Bi90 were both derived from an individual wild-caught females infected with Wolbachia, while lines w153T and Bi90T were derived from them by tetracycline treatment and are free of infection. Line Bi90 is known to be infected with the Wolbachia strain wMel, and line w153, with the Wolbachia strain wMelPlus belonging to the wMelCS genotype. It was found that infection with either Wolbachia strain does not affect the expression of the studied genes. At the same time, it was shown that the w153 and w153T lines differ from the Bi90 and Bi90T lines by an increased level of the Jheh1 gene expression and do not differ in the jhamt gene expression level. Analysis of the fertility of these four lines showed that it does not depend on Wolbachia infection either, but differs between lines with different nuclear genotypes: in w153 and w153T, it is significantly lower than in lines Bi90 and Bi90T. The data obtained allow us to reasonably propose that the inter-line D. melanogaster polymorphism in the metabolism of the juvenile hormone is determined by its degradation (not by its synthesis) and correlates with the fertility level.

Wolbachia strains wMel and wAlbB differentially affect Aedes aegypti traits related to fecundity.

Two Wolbachia strains, wMel and wAlbB, have been transinfected into Aedes aegypti mosquitoes for population replacement with the aim of reducing dengue transmission. Epidemiological data from various endemic sites suggest a pronounced decrease in dengue transmission after implementing this strategy. In this study, we investigated the impact of the Wolbachia strains wMel and wAlbB on Ae. aegypti fitness in a common genetic background. We found that Ae. aegypti females infected with the wMel strain exhibited several significant differences compared with those infected with the wAlbB strain. Specifically, wMel-infected females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on Ae. aegypti fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations in endemic settings. Further research is needed to better understand the underlying mechanisms responsible for these differences in fitness effects and their potential impact on the long-term efficacy of Wolbachia-based dengue control programs.IMPORTANCEThe transmission of arboviruses such as dengue, Zika, and chikungunya is on the rise globally. Among the most promising strategies to reduce arbovirus burden is the release of one out of two strains of Wolbachia-infected Aedes aegypti: wMel and wAlbB. One critical aspect of whether this approach will succeed involves the fitness cost of either Wolbachia strains on mosquito life history traits. For instance, we found that wMel-infected Ae. aegypti females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the wAlbB strain. Conversely, the wAlbB strain showed only mild negative effects when compared with Wolbachia-uninfected specimens. These differential effects on mosquito fitness following infection with either wMel or wAlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations.

Evidence of Differences in Cellular Regulation of Wolbachia-Mediated Viral Inhibition between Alphaviruses and Flaviviruses.

The intracellular bacterium Wolbachia is increasingly being utilised in control programs to limit the spread of arboviruses by Aedes mosquitoes. Achieving a better understanding of how Wolbachia strains can reduce viral replication/spread could be important for the long-term success of such programs. Previous studies have indicated that for some strains of Wolbachia, perturbations in lipid metabolism and cholesterol storage are vital in Wolbachia-mediated antiviral activity against the flaviviruses dengue and Zika; however, it has not yet been examined whether arboviruses in the alphavirus group are affected in the same way. Here, using the reporters for the alphavirus Semliki Forest virus (SFV) in Aedes albopictus cells, we found that Wolbachia strains wMel, wAu and wAlbB blocked viral replication/translation early in infection and that storage of cholesterol in lipid droplets is not key to this inhibition. Another alphavirus, o'nyong nyong virus (ONNV), was tested in both Aedes albopictus cells and in vivo in stable, transinfected Aedes aegypti mosquito lines. The strains wMel, wAu and wAlbB show strong antiviral activity against ONNV both in vitro and in vivo. Again, 2-hydroxypropyl-β-cyclodextrin (2HPCD) was not able to rescue ONNV replication in cell lines, suggesting that the release of stored cholesterol caused by wMel is not able to rescue blockage of ONNV. Taken together, this study shows that alphaviruses appear to be inhibited early in replication/translation and that there may be differences in how alphaviruses are inhibited by Wolbachia in comparison to flaviviruses.

Large-scale releases and establishment of wMel Wolbachia in Aedes aegypti mosquitoes throughout the Cities of Bello, Medellín and Itagüí, Colombia.

The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia. Local wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas. These results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas.

Wolbachia-mediated resistance to Zika virus infection in Aedes aegypti is dominated by diverse transcriptional regulation and weak evolutionary pressures.

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the wMel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of wMel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia-infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia. Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia-mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.

WMel Wolbachia alters female post-mating behaviors and physiology in the dengue vector mosquito Aedes aegypti

Globally invasive Aedes aegypti disseminate numerous arboviruses that impact human health. One promising method to control Ae. aegypti populations is transinfection with Wolbachia pipientis, which naturally infects ~40–52% of insects but not Ae. aegypti. Transinfection of Ae. aegypti with the wMel Wolbachia strain induces cytoplasmic incompatibility (CI), allows infected individuals to invade native populations, and inhibits transmission of medically relevant arboviruses by females. Female insects undergo post-mating physiological and behavioral changes—referred to as the female post-mating response (PMR)—required for optimal fertility. PMRs are typically elicited by male seminal fluid proteins (SFPs) transferred with sperm during mating but can be modified by other factors, including microbiome composition. Wolbachia has modest effects on Ae. aegypti fertility, but its influence on other PMRs is unknown. Here, we show that Wolbachia influences female fecundity, fertility, and re-mating incidence and significantly extends the longevity of virgin females. Using proteomic methods to examine the seminal proteome of infected males, we found that Wolbachia moderately affects SFP composition. However, we identified 125 paternally transferred Wolbachia proteins, but the CI factor proteins (Cifs) were not among them. Our findings indicate that Wolbachia infection of Ae. aegypti alters female PMRs, potentially influencing control programs that utilize Wolbachia-infected individuals.

Dengue Exposure and Wolbachia wMel Strain Affects the Fertility of Quiescent Eggs of Aedes aegypti.

(1) Background: The deployment of the bacterium Wolbachia to reduce arbovirus transmission is ongoing in several countries worldwide. When Wolbachia-carrying Aedes aegypti are released and established in the field, females may feed on dengue-infected hosts. The effects of simultaneous exposure on life-history traits of Ae. aegypti to Wolbachia wMel strain and dengue-1 virus DENV-1 remain unclear. (2) Methods: We monitored 4 groups (mosquitoes with either DENV-1 or Wolbachia, coinfected with DENV-1 and Wolbachia, as well as negative controls) to estimate Ae. aegypti survival, oviposition success, fecundity, collapsing and fertility of quiescent eggs for 12 weeks. (3) Results: Neither DENV-1 nor Wolbachia had a significant impact on mosquito survival nor on mosquito fecundity, although the last parameter showed a tendency to decrease with ageing. There was a significant decrease in oviposition success in individuals carrying Wolbachia. Wolbachia infection and storage time significantly increased egg collapse parameter on the egg viability assay, while DENV-1 had a slight protective effect on the first four weeks of storage. (4) Conclusions: Despite limitations, our results contribute to better understanding of the tripartite interaction of virus, bacteria and mosquito that may take place in field conditions and aid in guaranteeing the Wolbachia strategy success.

Genome evolution of dengue virus serotype 1 under selection by Wolbachia pipientis in Aedes aegypti mosquitoes.

The introgression of antiviral strains of Wolbachia into Aedes aegypti mosquito populations is a public health intervention for the control of dengue. Plausibly, dengue virus (DENV) could evolve to bypass the antiviral effects of Wolbachia and undermine this approach. Here, we established a serial-passage system to investigate the evolution of DENV in Ae. aegypti mosquitoes infected with the wMel strain of Wolbachia. Using this system, we report on virus genetic outcomes after twenty passages of serotype 1 of DENV (DENV-1). An amino acid substitution, E203K, in the DENV-1 envelope protein was more frequently detected in the consensus sequence of virus populations passaged in wMel-infected Ae. aegypti than wild-type counterparts. Positive selection at residue 203 was reproducible; it occurred in passaged virus populations from independent DENV-1-infected patients and also in a second, independent experimental system. In wild-type mosquitoes and human cells, the 203K variant was rapidly replaced by the progenitor sequence. These findings provide proof of concept that wMel-associated selection of virus populations can occur in experimental conditions. Field-based studies are needed to explore whether wMel imparts selective pressure on DENV evolution in locations where wMel is established.

Fitness costs of Wolbachia shift in locally-adapted Aedes aegypti mosquitoes.

Aedes aegypti mosquito eggs can remain quiescent for many months before hatching, allowing populations to persist through unfavourable conditions. A. aegypti infected with the Wolbachia strain wMel have been released in tropical and subtropical regions for dengue control. wMel reduces the viability of quiescent eggs, but this physiological cost might be expected to evolve in natural mosquito populations that frequently experience stressful conditions. We found that the cost of wMel infection differed consistently between mosquitoes collected from different locations and became weaker across laboratory generations, suggesting environment-specific adaptation of mosquitoes to the wMel infection. Reciprocal crossing experiments show that differences in the cost of wMel to quiescent egg viability were mainly due to mosquito genetic background and not Wolbachia origin. wMel-infected mosquitoes hatching from long-term quiescent eggs showed partial loss of cytoplasmic incompatibility and female infertility, highlighting additional costs of long-term quiescence. Our study provides the first evidence for a shift in Wolbachia phenotypic effects following deliberate field release and establishment and it highlights interactions between Wolbachia infections and mosquito genetic backgrounds. The unexpected changes in fitness costs observed here suggest potential tradeoffs with undescribed fitness benefits of the wMel infection.

Wolbachia genetic similarity in different insect host species: Drosophila melanogaster and Yogyakarta’s (Indonesia) Aedes aegypti as a novel host

Abstract. Rovik A, Daniwijaya EW, Supriyati E, Rahayu A, Kumalawati DA, Saraswati U, Handayaningsih AE, Rachman MP, Oktriani R, Kurniasari I, Candrasari DS, Nurhayati I, Sholeh R, Arianto B, Tantowijoyo W, Ahmad RA, Utarini A, Arguni E. 2022. Wolbachia genetic similarity in different insect host species: Drosophila melanogaster and Yogyakarta’s (Indonesia) Aedes aegypti as a novel host. Biodiversitas 23: 2321-2328. Wolbachia naturally presents in a large number of insects and other arthropod species. The Wolbachia strain wMel from Drosophila melanogaster has been stably transinfected into Aedes aegypti where it stops the mosquito host from being infected with medically important arbovirus like dengue. Consequently, Ae. aegypti infected with wMel have been released in Indonesia as a public health intervention against dengue. This study genetically compared wMel from Yogya field-caught D. melanogaster and the wMel in stably transfected Ae. aegypti used for field releases in Yogyakarta, Indonesia. The genetic similarity between wMel Wolbachia was evaluated by sequencing of Wolbachia surface protein (wsp) gene and some polymorphic genomic regions of insertion sites (IS) and variable number tandem repeats (VNTR) loci. The sequence of the Wolbachia surface protein (wsp) gene was 100% identical between hosts. There is no insertion sequence among specimens. The insertion sequence IS-WD1310 was identical between wMel from both hosts and among other strains, as well as the IS-WD516/7. The VNTR-141 period was identical within wMel from both hosts and among other strains, the VNTR-105 as well. Wolbachia Yogya field-caught D. melanogaster and Wolbachia strain wMel present in Ae. aegypti used for bio-control of dengue were genetically identical. These findings provide beneficial understanding to answer the public attention on safety issues, especially on the genetic similarity between Wolbachia strain in the natural and transfected hosts of this novel technology for dengue control.

Environmental factors influence the local establishment of Wolbachia in Aedes aegypti mosquitoes in two small communities in central Vietnam.

Background: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and subsequently shown to reduce transmission of dengue and other pathogens, under both laboratory and field conditions. Here we describe the entomological outcomes of wMel Wolbachia mosquito releases in two small communities in Nha Trang City in central Vietnam. Methods: The wMel strain of Wolbachia was backcrossed into local Aedes aegypti genotype and mosquito releases were undertaken by community members or by staff. Field monitoring was undertaken to track Wolbachia establishment in local Ae. aegypti mosquito populations. Ecological studies were undertaken to assess relationships between environmental factors and the spatial and temporal variability in Wolbachia infection prevalence in mosquitoes. Results: Releases of wMel Wolbachia Ae. aegypti mosquitoes in two small communities in Nha Trang City resulted in the initial establishment of Wolbachia in the local Ae. aegypti mosquito populations, followed by seasonal fluctuations in Wolbachia prevalence. There was significant small-scale spatial heterogeneity in Wolbachia infection prevalence in the Tri Nguyen Village site, resulting in the loss of wMel Wolbachia infection in mosquitoes in north and center areas, despite Wolbachia prevalence remaining high in mosquitoes in the south area. In the second site, Vinh Luong Ward, Wolbachia has persisted at a high level in mosquitoes throughout this site despite similar seasonal fluctuations in wMel Wolbachia prevalence. Conclusion: Seasonal variation in Wolbachia infection prevalence in mosquitoes was associated with elevated temperature conditions, and was possibly due to imperfect maternal transmission of Wolbachia. Heterogeneity in Wolbachia infection prevalence was found throughout one site, and indicates additional factors may influence Wolbachia establishment.

Crystal Structures of Wolbachia CidA and CidB Reveal Determinants of Bacteria-induced Cytoplasmic Incompatibility and Rescue

Cytoplasmic incompatibility (CI) results when Wolbachia bacteria-infected male insects mate with uninfected females, leading to embryonic lethality. “Rescue” of viability occurs if the female harbors the same Wolbachia strain. CI is caused by linked pairs of Wolbachia genes called CI factors (CifA and CifB). The co-evolution of CifA-CifB pairs may account in part for the incompatibility patterns documented in insects infected with different Wolbachia strains, but the molecular mechanisms remain elusive. Here, we use X-ray crystallography and AlphaFold to analyze the CI factors from Wolbachia strain wMel called CidAwMel and CidBwMel. Substituting CidAwMel interface residues with those from CidAwPip (from strain wPip) enables the mutant protein to bind CidBwPip and rescue CidBwPip-induced yeast growth defects, supporting the importance of CifA-CifB interaction in CI rescue. Sequence divergence in CidAwPip and CidBwPip proteins affects their pairwise interactions, which may help explain the complex incompatibility patterns of mosquitoes infected with different wPip strains.

A wMel Wolbachia variant in Aedes aegypti from field-collected Drosophila melanogaster with increased phenotypic stability under heat stress.

SummaryMosquito‐borne diseases remain a major cause of morbidity and mortality. Population replacement strategies involving the wMel strain of Wolbachia are being used widely to control mosquito‐borne diseases. However, these strategies may be influenced by temperature because wMel is vulnerable to heat. wMel infections in Drosophila melanogaster are genetically diverse, but few transinfections of wMel variants have been generated in Aedes aegypti. Here, we successfully transferred a wMel variant (termed wMelM) originating from a field‐collected D. melanogaster into Ae. aegypti. The new wMelM variant (clade I) is genetically distinct from the original wMel transinfection (clade III), and there are no genomic differences between wMelM in its original and transinfected host. We compared wMelM with wMel in its effects on host fitness, temperature tolerance, Wolbachia density, vector competence, cytoplasmic incompatibility and maternal transmission under heat stress in a controlled background. wMelM showed a higher heat tolerance than wMel, likely due to higher overall densities within the mosquito. Both wMel variants had minimal host fitness costs, complete cytoplasmic incompatibility and maternal transmission, and dengue virus blocking under laboratory conditions. Our results highlight phenotypic differences between Wolbachia variants and wMelM shows potential as an alternative strain in areas with strong seasonal temperature fluctuations.

A decade of stability for wMel Wolbachia in natural Aedes aegypti populations.

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes.

Environmental factors influence the local establishment of Wolbachia in Aedes aegypti mosquitoes in two small communities in central Vietnam

Background: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and subsequently shown to reduce transmission of dengue and other pathogens, under both laboratory and field conditions. Here we describe the entomological outcomes of wMel Wolbachia mosquito releases in two small communities in Nha Trang City in central Vietnam. Methods: The wMel strain of Wolbachia was backcrossed into local Aedes aegypti genotype and mosquito releases were undertaken by community members or by staff. Field monitoring was undertaken to track Wolbachia establishment in local Ae. aegypti mosquito populations. Ecological studies were undertaken to assess relationships between environmental factors and the spatial and temporal variability in Wolbachia infection prevalence in mosquitoes. Results: Releases of wMel Wolbachia Ae. aegypti mosquitoes in two small communities in Nha Trang City resulted in the initial establishment of Wolbachia in the local Ae. aegypti mosquito populations, followed by seasonal fluctuations in Wolbachia prevalence. There was significant small-scale spatial heterogeneity in Wolbachia infection prevalence in the Tri Nguyen Village site, resulting in the loss of wMel Wolbachia infection in mosquitoes in north and center areas, despite Wolbachia prevalence remaining high in mosquitoes in the south area. In the second site, Vinh Luong Ward, Wolbachia has persisted at a high level in mosquitoes throughout this site despite similar seasonal fluctuations in wMel Wolbachia prevalence. Conclusion: Seasonal variation in Wolbachia infection prevalence in mosquitoes was associated with elevated temperature conditions, and was possibly due to imperfect maternal transmission of Wolbachia. Heterogeneity in Wolbachia infection prevalence was found throughout one site, and indicates additional factors may influence Wolbachia establishment.

Environmental factors influence the local establishment of Wolbachia in Aedes aegypti mosquitoes in two small communities in central Vietnam

Background: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and subsequently shown to reduce transmission of dengue and other pathogens, under both laboratory and field conditions. Here we describe the entomological outcomes of wMel Wolbachia mosquito releases in two small communities in Nha Trang City in central Vietnam. Methods: The wMel strain of Wolbachia was backcrossed into local Aedes aegypti genotype and mosquito releases were undertaken by community members or by staff. Field monitoring was undertaken to track Wolbachia establishment in local Ae. aegypti mosquito populations. Ecological studies were undertaken to assess relationships between environmental factors and the spatial and temporal variability in Wolbachia infection prevalence in mosquitoes. Results: Releases of wMel Wolbachia Ae. aegypti mosquitoes in two small communities in Nha Trang City resulted in the initial establishment of Wolbachia in the local Ae. aegypti mosquito populations, followed by seasonal fluctuations in Wolbachia prevalence. There was significant small-scale spatial heterogeneity in Wolbachia infection prevalence in the Tri Nguyen Village site, resulting in the loss of wMel Wolbachia infection in mosquitoes in north and center areas, despite Wolbachia prevalence remaining high in mosquitoes in the south area. In the second site, Vinh Luong Ward, Wolbachia has persisted at a high level in mosquitoes throughout this site despite similar seasonal fluctuations in wMel Wolbachia prevalence. Conclusion: Seasonal variation in Wolbachia infection prevalence in mosquitoes was associated with elevated temperature conditions, and was possibly due to imperfect maternal transmission of Wolbachia. Heterogeneity in Wolbachia infection prevalence was found throughout one site, and indicates additional factors may influence Wolbachia establishment.

Differential gene expression in Drosophilamelanogaster and D.nigrosparsa infected with the same Wolbachia strain

Wolbachia are maternally inherited endosymbionts that infect nearly half of all arthropod species. Wolbachia manipulate their hosts to maximize their transmission, but they can also provide benefits such as nutrients and resistance against viruses to their hosts. The Wolbachia strain wMel was recently found to increase locomotor activities and possibly trigger cytoplasmic incompatibility in the transinfected fly Drosophilanigrosparsa. Here, we investigated, in females of both D.melanogaster and D.nigrosparsa, the gene expression between animals uninfected and infected with wMel, using RNA sequencing to see if the two Drosophila species respond to the infection in the same or different ways. A total of 2164 orthologous genes were used. The two fly species responded to the infection in different ways. Significant changes shared by the fly species belong to the expression of genes involved in processes such as oxidation–reduction process, iron-ion binding, and voltage-gated potassium-channel activity. We discuss our findings also in the light of how Wolbachia survive within both the native and the novel host.

Transmission of the wMel Wolbachia strain is modulated by its titre and by immune genes in Drosophila melanogaster (Wolbachia density and transmission)

Wolbachia are common intracellular endosymbionts of arthropods, but the interactions between Wolbachia and arthropods are only partially understood. The fruit fly Drosophila melanogaster is a model insect for understanding Wolbachia-host interactions. Here the native wMel strain of D. melanogaster was isolated and then different initial titres of wMel were artificially transferred back into antibiotics-treated fruit flies. Our purpose was to examine the interactions between the injected wMel in a density gradient and the recipient host during trans-generational transmission. The results showed that the trans-generational transmission rates of wMel and titres of wMel exhibited a fluctuating trend over nine generations, and the titres of wMel displayed a similar fluctuating trans-generational trend. There was a significant positive correlation between the transmission rate and the titre of wMel. Reciprocal crossings between wMel-transinfected and uninfected fruit flies revealed that wMel could induce cytoplasmic incompatibility (CI) at different initial titres, but the intensity of CI was not significantly correlated with the initial titre of wMel. Quantitative PCR analysis showed that the immune genes Drsl5 and Spn38F displayed a significant transcriptional response to wMel transfection, with an obvious negative correlation with the titre of wMel at the 3rd and 4th generations. Furthermore, RNA interference-mediated knockdown of Drsl5 and Spn38F elicited a drastic increase in the titre of wMel. In combination, our study suggests that the trans-generational transmission of wMel is modulated by its density, and the immune genes are involved in the regulation of Wolbachia density.

Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics.

The endosymbiotic bacterium Wolbachia shows viral blocking in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11–19°C and 22–30°C. Eggs stored at 11–19°C had higher hatch proportions than those stored at 22–30°C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22–30°C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected A. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.