Wolbachia Strains Research Articles

Chromosome structural rearrangements in invasive haplodiploid ambrosia beetles revealed by the genomes of Euwallacea fornicatus (Eichhoff) and Euwallacea similis (Ferrari) (Coleoptera, Curculionidae, Scolytinae).

Bark and ambrosia beetles are among the most ecologically and economically damaging introduced plant pests worldwide. Life history traits including polyphagy, haplodiploidy, inbreeding polygyny and symbiosis with fungi contribute to their dispersal and impact. Species vary in their interactions with host trees, with many attacking stressed or recently dead trees, such as the globally distributed E. similis (Ferrari). Other species, like the Polyphagous Shot Hole Borer (PSHB) Euwallacea fornicatus (Eichhoff), can attack over 680 host plants and is causing considerable economic damage in several countries. Despite their notoriety, publicly accessible genomic resources for Euwallacea Hopkins species are scarce, hampering our understanding of their invasive capabilities as well as modern control measures, surveillance and management. Using a combination of long and short read sequencing platforms we assembled and annotated high quality (BUSCO > 98% complete) pseudo-chromosome level genomes for these species. Comparative macro-synteny analysis identified an increased number of pseudo-chromosome scaffolds in the haplodiploid inbreeding species of Euwallacea compared to diploid outbred species, due to fission events. This suggests that life history traits can impact chromosome structure. Further, the genome of E. fornicatus had a higher relative proportion of repetitive elements, up to 17% more, than E. similis. Metagenomic assembly pipelines identified microbiota associated with both species including Fusarium fungal symbionts and a novel Wolbachia strain. These novel genomes of haplodiploid inbreeding species will contribute to the understanding of how life history traits are related to their evolution and to the management of these invasive pests.

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.

Draft Genome Resource of a Wolbachia Endosymbiont in Heterodera humuli

The bacterial endosymbiont Wolbachia is known to be associated with different arthropods and only two genera of plant-parasitic nematodes, Pratylenchus and Radopholus. This effort employed a genome skimming approach to discover the presence of endosymbionts in a population of Heterodera humuli sequenced with PacBio long-read sequencing. Wolbachia was found associated with the nematode. The genome of this Wolbachia is 1,051,007 bp and has a GC percentage (32.6%) within the expected range for the genus. A phylogenetic analysis placed the Wolbachia strain from H. humuli in a clade with another nematode-associated Wolbachia strain reported in Texas, with a bootstrap value of 1. To our knowledge, this is the first published report of Wolbachia associated with H. humuli, expanding the known association of this endosymbiont to three genera of plant-parasitic nematodes. This finding will enhance sequence resources for further comparisons of Wolbachia diversity. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Widespread Wolbachia infection is correlated with increased mtDNA diversity in native bees across the Fijian archipelago

AbstractInsects have a complex coevolutionary history with bacterial symbionts, among which Wolbachia pipientis stands out for its prevalence and role in reproductive manipulation. Wolbachia can induce cytoplasmic incompatibility, feminisation, male killing, and parthenogenesis, greatly influence the population genetics of their hosts and are potential drivers of invertebrate speciation. We might then assume that Wolbachia could act synergistically with other factors, such as niche conservation and isolation by distance, to drive speciation in hyper-diverse invertebrate taxa. The Fijian archipelago hosts a remarkable and recently diverged clade of Lasioglossum (Homalictus) bees. Some of these Lasioglossum have highly unusual species-level sex ratios and mitochondrial diversities, which can be hallmarks of infection by a bacterial reproductive manipulator. To examine the role of Wolbachia in Fijian Lasioglossum speciation, we screened the endemic Fijian bees for Wolbachia using the Wolbachia surface protein gene (wsp). We compare the distribution of Wolbachia infection across species with host mitochondrial haplotype diversity and conducted phylogenetic analyses to determine the relationship of host relatedness and symbiont infection status. We found that Wolbachia haplotypes probably span across supergroups A and B. In addition, we found multiple haplotypes were highly similar, with the most abundant group of wsp haplotypes being closely related with the drosophila Wolbachia strain wHa. We found evidence of mostly horizontal and limited vertical transmission, and little evidence for Wolbachia-host cospeciation. We show that, contrary to general patterns, Wolbachia-infected Lasioglossum species have higher mitochondrial diversity. Finally, we present evidence for the potential of multiple modes of host manipulation in this clade.

Wolbachia modify host cell metabolite profiles in response to short-term temperature stress.

Wolbachia are common heritable endosymbionts that influence many aspects of ecology and evolution in various insects, yet Wolbachia-mediated intracellular metabolic responses to temperature stress have been largely overlooked. Here, we introduced the Wolbachia strain wLhui from the invasive Liriomyza huidobrensis (Blanchard) into a Drosophila Schneider 2 cell line (S2) and investigated the metabolite profile of wLhui-infected (S2_wLhui) and uninfected cell lines (S2_wu) under short-term exposure to either high (37°C), moderate (27°C), or low (7 and 17°C) temperatures. We find that Wolbachia infection, temperature stress, and their interactions significantly affect cellular metabolic profiles. Most significantly, when comparing the changes in metabolites between S2_wLhui and S2_wu, glycerophospholipids, amino acids, and fatty acids associated with metabolic pathways, microbial metabolism in diverse environments, and other pathways were significantly accumulated at either low or high temperatures. Our findings suggest Wolbachia-induced cellular physiological responses to short-term temperature stress, which may in turn affect the fitness and adaptive ability of its host as an invasive species.

Wolbachia infection in natural mosquito populations from Argentina.

The increasing spread of mosquito vectors has made mosquito-borne arboviral diseases a global threat to public health, leading to the urgent need for effective population control methods. Strategies based in the intracellular bacterium Wolbachia Hertig, 1936 are considered environmentally friendly, safe for humans, and potentially cost-effective for controlling arboviral diseases. To minimize undesirable side effects, it is relevant to assess whether Wolbachia is present in the area and understand the diversity associated with native infections before implementing these strategies. With this purpose, we investigated Wolbachia infection status, diversity, and prevalence in populations of Aedes albifasciatus (Macquart, 1838), Aedes fluviatilis (Lutz, 1904), and hybrids of the Culex pipiens (Linnaeus, 1758) complex from Argentina. Aedes albifasciatus and C. pipiens complex samples were collected in the province of Buenos Aires, and A. fluviatilis in the province of Misiones. Aedes albifasciatus was found to be uninfected, while infections with strains wFlu and wPip were detected in A. fluviatilis and hybrids of the C. pipiens complex, respectively. All strains were fixed or close to fixation and clustered within supergroup B. These finding provides valuable information on Wolbachia strains found in natural mosquito populations in Argentina that might be used in heterologous infections in the future or be considered when designing control strategies based on Wolbachia infection.

Wolbachia Natural Infection of Mosquitoes in French Guiana: Prevalence, Distribution, and Genotyping

Wolbachia are the most spread bacterial endosymbionts in the world. These bacteria can manipulate host reproduction or block virus transmission in mosquitoes. For this reason, Wolbachia-based strategies for vector control are seriously considered or have already been applied in several countries around the world. In South America, Wolbachia have been studied in human pathogen vectors such as sand flies and mosquitoes. In French Guiana, the diversity and distribution of Wolbachia are not well known in mosquitoes. In this study, we screened for Wolbachia natural infection in mosquitoes in French Guiana by using 16S rRNA, Wolbachia surface protein (WSP), and multi-locus sequence typing (MLST) molecular assays. A total of 29 out of 44 (65.9%) mosquito species were positive for natural Wolbachia infection according to the PCR results, and two Wolbachia strains co-infected three specimens of Mansonia titillans. Then, we analyzed the phylogenetic relationships among the Wolbachia detected. All of the tested specimens of Aedes aegypti, the major dengue vector of French Guiana, were negative. These results regarding Wolbachia strain, distribution, and prevalence in mosquitoes from French Guiana highlight Wolbachia–mosquito associations and pave the way for a future Wolbachia-based strategy for vector control in this Amazonian territory.

Discovery of a novel Wolbachia in Heterodera expands nematode host distribution.

Bioinformatics sequence data mining can reveal hidden microbial symbionts that might normally be filtered and removed as contaminants. Data mining can be helpful to detect Wolbachia, a widespread bacterial endosymbiont in insects and filarial nematodes whose distribution in plant-parasitic nematodes (PPNs) remains underexplored. To date, Wolbachia has only been reported a few PPNs, yet nematode-infecting Wolbachia may have been widespread in the evolutionary history of the phylum based on evidence of horizontal gene transfers, suggesting there may be undiscovered Wolbachia infections in PPNs. The goal of this study was to more broadly sample PPN Wolbachia strains in tylenchid nematodes to enable further comparative genomic analyses that may reveal Wolbachia's role and identify targets for biocontrol. Published whole-genome shotgun assemblies and their raw sequence data from 33 Meloidogyne spp. assemblies, seven Globodera spp. assemblies, and seven Heterodera spp. assemblies were analyzed to look for Wolbachia. No Wolbachia was found in Meloidogyne spp. and Globodera spp., but among seven genome assemblies for Heterodera spp., an H. schachtii assembly from the Netherlands was found to have a large Wolbachia-like sequence that, when re-assembled from reads, formed a complete, circular genome. Detailed analyses comparing read coverage, GC content, pseudogenes, and phylogenomic patterns clearly demonstrated that the H. schachtii Wolbachia represented a novel strain (hereafter, denoted wHet). Phylogenomic tree construction with PhyloBayes showed wHet was most closely related to another PPN Wolbachia, wTex, while 16S rRNA gene analysis showed it clustered with other Heterodera Wolbachia assembled from sequence databases. Pseudogenes in wHet suggested relatedness to the PPN clade, as did the lack of significantly enriched GO terms compared to PPN Wolbachia strains. It remains unclear whether the lack of Wolbachia in other published H. schachtii isolates represents the true absence of the endosymbiont from some hosts.

Prevalence of Wolbachia infection in field natural population of the apricot seed wasp Eurytoma samsonowi (Hymenoptera: Eurytomidae).

Obligate endosymbiont bacteria associated with insects are naturally providing their hosts with essential nutrients such as vitamins and amino acids and biological services including protection from pathogens. In this study, we aimed to investigate the presence of Wolbachia infection among males and females of the parasitic apricot seed wasp (ASW) Eurytoma samsonowi Vassiliev (Vassiliev Petrograd 11: 1-15, 1915) (Hymenoptera: Eurytomidae), a very harmful pest of apricot (Prunus armeniaca), in the oasis of Gafsa, Southern-West of Tunisia. The detection of Wolbachia infection was assessed based on the amplification of the Wolbachia surface protein (wsp) gene and a multilocus sequence typing (MLST) as a universal genotyping tool for Wolbachia involving the analyses of genes gatB, coxA, hcpA, fbpA, and ftsz. Confirming the screening results, Wolbachia was detected in the natural apricot wasp for the first time, with a significant difference between males (5%) and females (59%) based on wsp gene. All Wolbachia strains identified in E. samsonowi were clustered among supergroups B of Wolbachia.

Characterization of the microbiome of Aedes albopictus populations in different habitats from Spain and São Tomé

The mosquito microbiome significantly influences vector competence, including in Aedes albopictus, a globally invasive vector. Describing the microbiome and Wolbachia strains of Ae. albopictus from different regions can guide area-specific control strategies. Mosquito samples from Spain and São Tomé were analyzed using 16S rRNA gene sequencing and metagenomic sequencing. Wolbachia infection patterns were observed by sex and population. Female mosquitoes were blood-fed, a factor considered in analyzing their microbiota. Results revealed a dominance of dual Wolbachia infections, strains A and B, in the microbiome of both populations of Ae. albopictus, especially among females. Both populations shared a core microbiome, although 5 and 9 other genera were only present in Spain and São Tomé populations, respectively. Genera like Pelomonas and Nevskia were identified for the first time in Aedes mosquitoes. This study is the first to describe the Ae. albopictus bacteriome in Spain and São Tomé, offering insights for the development of targeted mosquito control strategies. Understanding the specific microbiome composition can help in designing more effective interventions, such as microbiome manipulation and Wolbachia-based approaches, to reduce vector competence and transmission potential of these mosquitoes.

Wolbachia strain diversity in a complex group of sympatric cryptic parasitoid wasp species

BackgroundMaternally-inherited symbionts can induce pre-mating and/or post-mating reproductive isolation between sympatric host lineages, and speciation, by modifying host reproductive phenotypes. The large parasitoid wasp genus Cotesia (Braconidae) includes a diversity of cryptic species, each specialized in parasitizing one to few related Lepidoptera host species. Here, we characterized the infection status of an assemblage of 21 Cotesia species from 15 countries by several microbial symbionts, as a first step toward investigating whether symbionts may provide a barrier to gene flow between these parasitoid host lineages.ResultsThe symbiotic microbes Arsenophonus, Cardinium, Microsporidium and Spiroplasma were not detected in the Cotesia wasps. However, the endosymbiotic bacterium Wolbachia was present in at least eight Cotesia species, and hence we concentrated on it upon screening additional DNA extracts and SRAs from NCBI. Some of the closely related Cotesia species carry similar Wolbachia strains, but most Wolbachia strains showed patterns of horizontal transfer between phylogenetically distant host lineages.ConclusionsThe lack of co-phylogenetic signal between Wolbachia and Cotesia suggests that the symbiont and hosts have not coevolved to an extent that would drive species divergence between the Cotesia host lineages. However, as the most common facultative symbiont of Cotesia species, Wolbachia may still function as a key-player in the biology of the parasitoid wasps. Its precise role in the evolution of this complex clade of cryptic species remains to be experimentally investigated.

Optimization approaches to Wolbachia-based biocontrol

This paper proposes two realistic and biologically viable methods for Wolbachia-based biocontrol of Aedes aegypti mosquitoes, assuming imperfect maternal transmission of the Wolbachia bacterium, incomplete cytoplasmic incompatibility, and direct loss of Wolbachia infection caused by thermal stress. Both methods are based on optimization approaches and allow for the stable persistence of Wolbachia-infected mosquitoes in the wild Ae. aegypti populations in a minimum time and using the smallest quantity of Wolbachia-carrying insects to release. The first method stems from the continuous-time optimal release strategy, which is further transformed into a sequence of suboptimal impulses mimicking instantaneous releases of Wolbachia-carrying insects. The second method constitutes a novel alternative to all existing techniques aimed at the design of release strategies. It is developed using metaheuristics (ϵ-constraint method combined with the genetic algorithm) and directly produces a discrete sequence of decisions, where each element represents the quantity of Wolbachia-carrying mosquitoes to be released instantaneously and only once per a specified time unit. It turns out that a direct discrete-time optimization (second method) renders better quantifiable results compared to transforming a continuous-time optimal release function into a sequence of suboptimal impulses (first method). As an illustration, we provide examples of daily, weekly, and fortnightly release strategies designed by both methods for two Wolbachia strains, wMel and wMelPop.

Cell-based assays and comparative genomics revealed the conserved and hidden effects of Wolbachia on insect sex determination.

It is advantageous for maternally transmitted endosymbionts to skew the sex ratio of their hosts toward females. Some endosymbiotic bacteria, such as Wolbachia, cause their insect hosts to exclusively produce female offspring through male killing (MK) or feminization. In some lepidopteran insects, MK is achieved by affecting the sex-determining process in males, and a unique mechanism of MK and its functional link with feminization have been implicated. However, comparative analysis of these phenotypes is often difficult because they have been analyzed in different host-symbiont systems, and transinfection of Wolbachia across different hosts is often challenging. In this study, we demonstrated the effects of nine Wolbachia strains on the splicing of sex-determining genes in Lepidoptera by fixing the host genetic background using a cell culture system. Cell transinfection assays confirmed that three MK-inducing Wolbachia strains and one feminization-inducing Wolbachia strain increased the female-type splicing products of the core sex-determining genes doublesex, masculinizer, and zinc finger protein 2. Regarding Wolbachia strains that do not induce MK/feminization, three had no effect on these sex-determining genes, whereas two strains induced female-type splicing of masculinizer and doublesex but not zinc finger protein 2. Comparative genomics confirmed that homologs of oscar, the Wolbachia gene responsible for MK in Ostrinia, were encoded by four MK/feminizing Wolbachia strains, but not by five non-MK/nonfeminizing strains. These results support the conserved effects underlying MK and feminization induced by oscar-bearing Wolbachia and suggested other potential mechanisms that Wolbachia might employ to manipulate host sex.

Bunyamwera Virus Infection of Wolbachia-Carrying Aedes aegypti Mosquitoes Reduces Wolbachia Density.

Wolbachia symbionts introduced into Aedes mosquitoes provide a highly effective dengue virus transmission control strategy, increasingly utilised in many countries in an attempt to reduce disease burden. Whilst highly effective against dengue and other positive-sense RNA viruses, it remains unclear how effective Wolbachia is against negative-sense RNA viruses. Therefore, the effect of Wolbachia on Bunyamwera virus (BUNV) infection in Aedes aegypti was investigated using wMel and wAlbB, two strains currently used in Wolbachia releases for dengue control, as well as wAu, a strain that typically persists at a high density and is an extremely efficient blocker of positive-sense viruses. Wolbachia was found to reduce BUNV infection in vitro but not in vivo. Instead, BUNV caused significant impacts on density of all three Wolbachia strains following infection of Ae. aegypti mosquitoes. The ability of Wolbachia to successfully persist within the mosquito and block virus transmission is partially dependent on its intracellular density. However, reduction in Wolbachia density was not observed in offspring of infected mothers. This could be due in part to a lack of transovarial transmission of BUNV observed. The results highlight the importance of understanding the complex interactions between multiple arboviruses, mosquitoes and Wolbachia in natural environments, the impact this can have on maintaining protection against diseases, and the necessity for monitoring Wolbachia prevalence at release sites.

Temporal stability of sex ratio distorter prevalence in natural populations of the isopod Armadillidium vulgare.

In the terrestrial isopod Armadillidium vulgare, many females produce progenies with female-biased sex ratios due to two feminizing sex ratio distorters (SRD): Wolbachia endosymbionts and a nuclear non-mendelian locus called the f element. To investigate the potential impact of these SRD on the evolution of host sex determination, we analyzed their temporal distribution in six A. vulgare populations sampled between 2003 and 2017, for a total of 29 time points. SRD distribution was heterogeneous among populations despite their close geographic locations, so that when one SRD was frequent in a population, the other SRD was rare. In contrast with spatial heterogeneity, our results overall did not reveal substantial temporal variability in SRD prevalence within populations, suggesting equilibria in SRD evolutionary dynamics may have been reached or nearly so. Temporal stability was also generally reflected in mitochondrial and nuclear variation. Nevertheless, in a population, a Wolbachia strain replacement coincided with changes in mitochondrial composition but no change in nuclear composition, thus constituting a typical example of mitochondrial sweep caused by endosymbiont rise in frequency. Rare incongruence between Wolbachia strains and mitochondrial haplotypes suggested the occurrence of intraspecific horizontal transmission, making it a biologically relevant parameter for Wolbachia evolutionary dynamics in A. vulgare. Overall, our results provide an empirical basis for future studies on SRD evolutionary dynamics in the context of multiple sex determination factors co-existing within a single species, to ultimately evaluate the impact of SRD on the evolution of host sex determination mechanisms and sex chromosomes.

Assessing Wolbachia-mediated sterility for dengue control: emulation of a cluster-randomized target trial in Singapore.

Matings between male Aedes aegypti mosquitoes infected with wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected Ae. aegypti male mosquitoes to suppress dengue. We specified the protocol of a two-arm cluster-randomized test-negative controlled trial (cRCT) and emulated it using a nationally representative test-negative/positive database of individuals reporting for febrile illness to any public hospital, general practitioner or polyclinic. We retrospectively built a cohort of individuals who reside in Wolbachia locations vs a comparator control group who do not reside in Wolbachia locations, using a nationally representative database of all individuals whom report for febrile illness and were tested for dengue at the Environmental Health Institute/hospital laboratories/commercial diagnostic laboratories, through general practitioner clinic, polyclinic or public/private hospital from epidemiological week (EW) 1 2019 to EW26 2022. We emulated a constrained randomization protocol used in cRCTs to balance dengue risk between intervention and control arms in the pre-intervention period. We used the inverse probability weighting approach to further balance the intervention and control groups using a battery of algorithmically selected sociodemographic, environmental and anthropogenic variables. Intention-to-treat analyses were conducted to estimate the risk reduction of dengue given Wolbachia exposure. Intention-to-treat analyses revealed that, compared with controls, Wolbachia releases for 3, 6 and ≥12 months was associated to 47% (95% confidence interval: 25-69%), 44% (33-77%) and 61% (38-78%) protective efficacy against dengue, respectively. When exposed to ≥12 months of Wolbachia releases, protective efficacies ranged from 49% (13-72%) to 77% (60-94%) across years. The proportion of virologically confirmed dengue cases was lower overall in the intervention arm. Protective efficacies were found across all years, age and sex subgroups, with higher durations of Wolbachia exposure associated to greater risk reductions of dengue. Results demonstrated that Wolbachia-mediated sterility can strengthen dengue control in tropical cities, where dengue burden is the greatest.

Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes does not alter vector competence for Hawaiian avian malaria (Plasmodium relictum GRW4).

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes.

Wolbachia confers protection against the entomopathogenic fungus Metarhizium pingshaense in African Aedes aegypti.

Symbiotic and pathogenic microorganisms such as bacteria and fungi represent promising alternatives to chemical insecticides to respond to the rapid increase of insecticide resistance and vector-borne disease outbreaks. This study investigated the interaction of two strains of Wolbachia, wAlbB and wAu, with the natural entomopathogenic fungi from Burkina Faso Metarhizium pingshaense, known to be lethal against Anopheles mosquitoes. In addition to showing the potential of Metarhizium against African Aedes aegypti wild-type populations, our study shows that the wAlbB and wAu provide a protective advantage against entomopathogenic fungal infections. Compared to controls, fungal-infected wAu and wAlbB-carrying mosquitoes showed higher longevity, without any significant impact on fecundity and fertility phenotypes. This study provides new insights into the complex multipartite interaction among the mosquito host, the Wolbachia endosymbiont and the entomopathogenic fungus that might be employed to control mosquito populations. Future research should investigate the fitness costs of Wolbachia, as well as its spread and prevalence within mosquito populations. Additionally, evaluating the impact of Wolbachia on interventions involving Metarhizium pingshaense through laboratory and semi-field population studies will provide valuable insights into the effectiveness of this combined approach.

Complete de novo assembly of Wolbachia endosymbiont of Drosophila willistoni using long-read genome sequencing

Wolbachia is an obligate intracellular α-proteobacterium, which commonly infects arthropods and filarial nematodes. Different strains of Wolbachia are capable of a wide range of regulatory manipulations in their diverse hosts, including the modulation of host cellular differentiation to influence host reproduction. The genetic basis for the majority of these phenotypes is unknown. The wWil strain from the neotropical fruit fly, Drosophila willistoni, exhibits a remarkably high affinity for host germline-derived cells relative to the somatic cells. This trait could be leveraged for understanding how Wolbachia influences the host germline and for controlling host populations in the field. To further the use of this strain in biological and biomedical research, we sequenced the genome of the wWil strain isolated from host cell culture cells. Here, we present the first high quality Nanopore assembly of wWil, the Wolbachia endosymbiont of D. willistoni. Our assembly resulted in a circular genome of 1.27 Mb with a BUSCO completeness score of 99.7%. Consistent with other insect-associated Wolbachia strains, comparative genomic analysis revealed that wWil has a highly mosaic genome relative to the closely related wMel and wAu strains from Drosophila melanogaster and Drosophila simulans, respectively.

Mixed Wolbachia infections resolve rapidly during in vitro evolution.

The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between the two strains in cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems.