Wolbachia Genes Research Articles

Counting rare Wolbachia endosymbionts using digital droplet PCR.

Wolbachia is the most widespread animal-associated intracellular microbe, living within the cells of over half of insect species. Since they can suppress pathogen replication and spread rapidly through insect populations, Wolbachia is at the vanguard of public health initiatives to control mosquito-borne diseases. Wolbachia's abilities to block pathogens and spread quickly are closely linked to their abundance in host tissues. The most common method for counting Wolbachia is quantitative polymerase chain reaction (qPCR), yet qPCR can be insufficient to count rare Wolbachia, necessitating tissue pooling and consequently compromising individual-level resolution of Wolbachia dynamics. Digital droplet PCR (ddPCR) offers superior sensitivity, enabling the detection of rare targets and eliminating the need for sample pooling. Here, we report three ddPCR assays to measure total Wolbachia abundance, Wolbachia abundance adjusted for DNA extraction efficiency, and Wolbachia density relative to host genome copies. Using Drosophila melanogaster with wMel Wolbachia as a model, we show these ddPCR assays can reliably detect as few as 7 to 12 Wolbachia gene copies in a 20 μL reaction. The designed oligos are homologous to sequences from at least 106 Wolbachia strains across Supergroup A and 53 host species from the Drosophila, Scaptomyza, and Zaprionus genera, suggesting broad utility. These highly sensitive ddPCR assays are expected to significantly advance Wolbachia-host interactions research by enabling the collection of molecular data from individual insect tissues. Their ability to detect rare Wolbachia will be especially valuable in applied and natural field settings where pooling samples could obscure important variation.

Incidence of endosymbiont bacteria Wolbachia in cowpea weevil Callosobruchus maculatus Fabricius (Coleoptera, Chrysomelidae).

This study focuses on the cowpea weevil, Callosobruchus maculatus, a globally distributed grain pest that affects cereals and pulses. Using chemicals to store grains can harm pest control and pose risks to consumers and the environment. The facultative intracellular symbiont bacteria Wolbachia can affect host's reproductive capacities in a variety of ways, which makes it useful in the management of pests such as C. maculatus. The main goal of the study was to identify Wolbachia diversity in the C. maculatus population. Phylogenetic analysis utilized mitochondrial COI and 12S rRNA genes to identify the host C. maculatus, while screening for Wolbachia was conducted using genes (wsp, coxA, and ftsZ) genes. Molecular phylogenetic analysis of the Wolbachia genes resulted in one new Wolbachia strain (wCmac1) in C. maculatus populations and contrasting already published data of other Callosobruchus strains. The study discussed the detection of Wolbachia and its phylogenetic comparison with other C. maculatus and Coleopteran populations. It is important to take these findings into account when considering host-pathogen interactions.

European Culex pipiens Populations Carry Different Strains of Wolbachia pipientis.

The mosquito Culex pipiens occurs in two ecotypes differing in their mating and overwintering behavior: pipiens mate in open environments and diapause, and molestus also mate in small spaces and is active throughout the year. Cx. pipiens carry Wolbachia endosymbionts of the wPip strain, but the frequency of infection differs between studied populations. Wolbachia infection affects the host reproductive success through cytoplasmic incompatibility. wPip Wolbachia is divided into five types, wPip I-V. The type of wPip carried varies among Cx. pipiens populations. In northern European locations different wPip types are found in the two ecotypes, whereas in southern locations, they often carry the same type, indicating differences in hybridization between ecotypes. In this study, Cx. pipiens specimens of both ecotypes were collected from Sweden and compared to specimens from Norway, England, Italy, and the Netherlands, as well as Cx. quinquefasciatus from Mali and Thailand. The abundance varied, but all specimens were infected by Wolbachia, while the tested specimens of other mosquito species were often uninfected. The wPip strains were determined through the sequence analysis of Wolbachia genes ank2 and pk1, showing that Cx. pipiens ecotypes in Scandinavia carry different wPip strains. The observed differences in wPip strains indicate that hybridization is not frequent and may contribute to barriers against hybridization of the ecotypes in Sweden and Norway.

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.

The prevalence of Wolbachia in multiple cockroach species and its implication for urban insect management.

Cockroach management relies heavily on the use of conventional insecticides in urban settings, which no longer provide the anticipated level of control. Knowledge of cockroach endosymbionts, like Wolbachia, might provide novel avenues for control. Therefore, we screened 16 cockroach species belonging to 3 families (Ectobiidae, Blattidae, and Blaberidae) for the presence of Wolbachia. We mapped the evolution of Wolbachia-cockroach relationships based on maximum likelihood phylogeny and phylogenetic species clustering on a multi-loci sequence dataset (i.e., coxA, virD4, hcpA, and gatB) of Wolbachia genes. We confirmed the previous report of Wolbachia in 1 Ectobiid species; Supella longipalpa (Fab.), and detected the presence of Wolbachia in 2 Ectobiid species; Balta notulata (Stål) and Pseudomops septentrionalis Hebard, and 1 Blaberid species; Gromphadorhina portentosa (Schaum). All cockroach-associated Wolbachia herein detected were clustered with the ancestor of F clade Wolbachia of Cimex lectularius L. (bed bugs). Since Wolbachia provision C. lectularius with biotin vitamins that confer reproductive fitness, we screened the cockroach-associated Wolbachia for the presence of biotin genes. In toto, our results reveal 2 important findings: (i) Wolbachia is relatively uncommon among cockroach species infecting about 25% of species investigated, and (ii) cockroach-associated Wolbachia have biotin genes that likely provide nutritional benefits to their hosts. Thus, we discuss the potential of exploring Wolbachia as a tool for urban insect management.

Diversity of Wolbachia infection and its influence on mitochondrial DNA variation in the diamondback moth, Plutella xylostella

Plutella xylostella is a pest that severely damages cruciferous vegetables worldwide and has been shown to be infected with the maternally inherited bacteria Wolbachia, with the main infected strain was plutWB1. In this study, we performed a large-scale global sampling of P. xylostella and amplified 3 mtDNA genes of P. xylostella and 6 Wolbachia genes to analyze the infection status, diversity of Wolbachia in P. xylostella, and its effect on mtDNA variation in P. xylostella. This study provides a conservative estimate of Wolbachia infection rates in P. xylostella, which was found to be 7% (104/1440). The ST 108 (plutWB1) was shared among butterfly species and the moth species P. xylostella, revealing that Wolbachia strain plutWB1 acquisition in P. xylostella may be through horizontal transmission. The Parafit analyses indicated a significant association between Wolbachia and Wolbachia-infected P. xylostella individuals, and individuals infected with plutWB1 tended to cluster in the basal positions of the phylogenetic tree based on the mtDNA data. Additionally, Wolbachia infections were associated with increased mtDNA polymorphism in the infected P. xylostella population. These data suggest that Wolbachia endosymbionts may have a potential effect on mtDNA variation of P. xylostella.

Analysing inhibition of dengue virus in Wolbachia-infected mosquito cells following the removal of Wolbachia

Wolbachia pipientis is known to block replication of positive sense RNA viruses. Previously, we created an Aedes aegypti Aag2 cell line (Aag2.wAlbB) transinfected with the wAlbB strain of Wolbachia and a matching tetracycline-cured Aag2.tet cell line. While dengue virus (DENV) was blocked in Aag2.wAlbB cells, we found significant inhibition of DENV in Aag2.tet cells. RNA-Seq analysis of the cells confirmed removal of Wolbachia and lack of expression of Wolbachia genes that could have been due to lateral gene transfer in Aag2.tet cells. However, we noticed a substantial increase in the abundance of phasi charoen-like virus (PCLV) in Aag2.tet cells. When RNAi was used to reduce the PCLV levels, DENV replication was significantly increased. Further, we found significant changes in the expression of antiviral and proviral genes in Aag2.tet cells. Overall, the results reveal an antagonistic interaction between DENV and PCLV and how PCLV-induced changes could contribute to DENV inhibition.

Wolbachia RNase HI contributes to virus blocking in the mosquito Aedes aegypti

The endosymbiotic bacterium Wolbachia pipientis blocks replication of several arboviruses in transinfected Aedes aegypti mosquitoes. However, the mechanism of virus blocking remains poorly understood. Here, we characterized an RNase HI gene from Wolbachia, which is rapidly induced in response to dengue virus (DENV) infection. Knocking down w RNase HI using antisense RNA in Wolbachia-transinfected mosquito cell lines and A.aegypti mosquitoes led to increased DENV replication. Furthermore, overexpression of wRNase HI, in the absence of Wolbachia, led to reduced replication of a positive sense RNA virus, but had no effect on a negative sense RNA virus, a familiar scenario in Wolbachia-infected cells. Altogether, our results provide compelling evidence for the missing link between early Wolbachia-mediated virus blocking and degradation of viral RNA. These findings and the successful pioneered knockdown of Wolbachia genes using antisense RNA in cell line and mosquitoes enable new ways to manipulate and study the complex endosymbiont-host interactions.

Symbiotic Wolbachia bacteria in coccinellid parasitoids: genetic diversity, horizontal transfer, and recombination.

Parasitoids, which constitute about 25% of all insects and attack arthropods of virtually all taxa, are considered the most suitable vectors for horizontal transmission of the symbiotic bacterium Wolbachia among insects. The parasitoids studied in this article develop in the larvae and pupae of ladybirds. For the first time, Wolbachia was found in parasitic wasp species of the genus Homalotylus (Hymenoptera: Encyrtidae) and from the subfamily Tetrastichinae (Hymenoptera: Eulophidae). To characterize the Wolbachia strains, six bacterial housekeeping genes were examined and compared with previously published Wolbachia gene sequences. The same bacterial strains were found in all individuals of each species of parasitic wasps collected in different places and at different times, which indicates the absence of contamination and testifies to the heritability of the symbionts in the studied chalcids. No evidence was found that the parasitic wasps were infected with Wolbachia, identical to the symbionts of their ladybirds hosts. We found one Wolbachia strain, wHom-2, which is a product of bacterial recombination from unrelated insects, including ladybirds. The lack of correspondence between the molecular phylogenies of Wolbachia strains and mitochondrial DNA of their hosts indicates horizontal transfers of Wolbachia among parasitic wasps of the genus Homalotylus and from the subfamily Tetrastichinae.

Trans-Generational Symbiont Transmission Reduced at High Temperatures in a West Nile Virus Vector Mosquito Culex quinquefasciatus

The influence of environmental factors on the efficacy of the endosymbiont Wolbachia used in mosquito and pathogen control are poorly characterized and may be critical for disease control. We studied the vector mosquito Culex pipiens quinquefasciatus (Say) to determine the effect of temperature on the composition of the relative abundance of Wolbachia spp. and the microbiome, as well as key immune genes of interest in the Toll and IMD pathways. 16S barcode sequencing was used to determine the microbiome composition and qPCR was used to determine the relative abundance of Wolbachia spp. based on the highly utilized marker Wolbachia surface protein (wsp) gene. We found no effect of temperature within a single generation on the relative abundance of Wolbachia or immune gene expression, nor on the alpha or beta diversity of the microbiome. However, there was a significant difference in the abundance of Wolbachia between generations at high temperatures (≥ 28°C), but not at lower temperatures (≤ 23°C). These results support the idea that Wolbachia are reduced at higher temperatures between generations, which has an influence on the establishment of pathogens including West Nile Virus (WNV). Modulation of the Toll or IMD mosquito immune pathways was not indicated. Wolbachia endosymbiosis and trans-generation transmission appears especially sensitive to high temperatures, which may have implications for Wolbachia-based vector control strategies under climate change scenarios.

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.

Wolbachia Detection in Field-Collected Mosquitoes from Cameroon.

Simple SummaryWolbachia bacteria from different strains, carried by many insects and nematodes, can interact in many ways with their hosts by changing their biology in different ways, including by suppressing vector population and reducing parasite transmission. Consequently, Wolbachia play an important role in vector control strategies. This study assessed the prevalence of natural Wolbachia infections in mosquitoes collected in Cameroon. Despite the low prevalence that was revealed, Wolbachia spp. were found in eight species of field-collected mosquitoes and are closely related to clades A and B. Aedes aegypti and A. gambiae sl., the main vectors of dengue and malaria, respectively, were not infected in this study, while C. moucheti recorded a high prevalence (46.67%). Future characterisation of the Wolbachia bacteria obtained is needed.Wolbachia spp., known to be maternally inherited intracellular bacteria, are widespread among arthropods, including mosquitoes. Our study assessed the presence and prevalence of Wolbachia infection in wild mosquitoes collected in Cameroon, using the combination of 23s rRNA Anaplasmatacea and 16s rRNA Wolbachia genes. Mosquitoes that were positive for Wolbachia were sequenced for subsequent phylogenetic analysis. Out of a total of 1740 individual mosquitoes belonging to 22 species and five genera screened, 33 mosquitoes (1.87%) belonging to eight species (namely, Aedes albopictus, A. contigus, Culex quinquefasciatus, C. perfuscus, C. wigglesworthi, C. duttoni, Anopheles paludis and Coquillettidia sp.) were found to be positive for Wolbachia infections. Wolbachia spp. were absent in A. gambiae and A. aegypti, the main vectors of malaria and dengue, respectively. Phylogenetic analysis of the 16S RNA sequences showed they belong mainly to two distinct subgroups (A and B). This study reports the presence of Wolbachia in about eight species of mosquitoes in Cameroon and suggests that future characterisation of the strains is needed.

Transcriptional Response of Wolbachia to Dengue Virus Infection in Cells of the Mosquito Aedes aegypti.

ABSTRACTAedes aegypti transmits one of the most significant mosquito‐borne viruses, dengue virus (DENV). The absence of effective vaccines and clinical treatments and the emergence of insecticide resistance in A. aegypti necessitate novel vector control strategies. A new approach uses the endosymbiotic bacterium Wolbachia pipientis to reduce the spread of arboviruses. However, the Wolbachia-mediated antiviral mechanism is not well understood. To shed light on this mechanism, we investigated an unexplored aspect of Wolbachia-virus-mosquito interaction. We used RNA sequencing to examine the transcriptional response of Wolbachia to DENV infection in A. aegypti Aag2 cells transinfected with the wAlbB strain of Wolbachia. Our results suggest that genes encoding an endoribonuclease (RNase HI), a regulator of sigma 70-dependent gene transcription (6S RNA), essential cellular, transmembrane, and stress response functions and primary type I and IV secretion systems were upregulated, while a number of transport and binding proteins of Wolbachia, ribosome structure, and elongation factor-associated genes were downregulated due to DENV infection. Furthermore, bacterial retrotransposon, transposable, and phage-related elements were found among the up- and downregulated genes. We show that Wolbachia elicits a transcriptional response to virus infection and identify differentially expressed Wolbachia genes mostly at the early stages of virus infection. These findings highlight Wolbachia’s ability to alter its gene expression in response to DENV infection of the host cell.IMPORTANCE Aedes aegypti is a vector of several pathogenic viruses, including dengue, Zika, chikungunya, and yellow fever viruses, which are of importance to human health. Wolbachia is an endosymbiotic bacterium currently used in transinfected mosquitoes to suppress replication and transmission of dengue viruses. However, the mechanism of Wolbachia-mediated virus inhibition is not fully understood. While several studies have shown mosquitoes’ transcriptional responses to dengue virus infection, none have investigated these responses in Wolbachia, which may provide clues to the inhibition mechanism. Our results suggest changes in the expression of a number of functionally important Wolbachia genes upon dengue virus infection, including those involved in stress responses, providing insights into the endosymbiont’s reaction to virus infection.

Detection and characterization of wolbachia endosimbiont in wild bee (apis cerena) using the cytohrome C oxidase subunit I gene

Apis cerena; is as wild bee or honey bee, widespread in almost all regions of Indonesia, these bee economic value as the honey it produces. Indonesian people, especially in Tanjung Peropa Southeast Sulawesi who use bees as a honey-producing source maintain honey bees with a honeycomb claim system that lives in hives in forested trees or found in people’s homes. Until now, there is no breeding or breeding business. Wobachia is endosimbiont which was infected Artrophoda, including insects, which can influence the dynamics of these insects population and can reduce insect perform and viability. Therefore, molecular detection of the presence of Wolbachia in Apiscerena is important. The COI gene is a gene proposed as an animal barcode. There is evidence of primer use of the COI gene as a barcode which is often contaminated with Wolbachia COI gene which is not a target. The individual bee was extracted the genome DNA using the CTAB (cetyltrimethyl ammonium bromide) method, and then the cytochrome oxidase gene (COI) was umplified using an animal barcode primer. The amplification results are then sequenced and then characterized. Using Wolbachia COI gene data available on GenBank as a comparison, reconstruction of phylogenetic tree of Wobachian base on COI nucleotide sequences, so the position taxon of Wolbachia shall be determine. Phylogenetic tree reconstruction make with a Neiberjoining method with the Kimura 2-meter models and 1000x boostrapped. The results showed that the Wobachi COI gene was successfully amplified by these primer animal barcode, along 701bp. A proof that wild bees have been infected by Wolbachia. The findings of this study prove that PCR method are very sensitive to be used to detect existenceWolbachia in bees. This also means that the primer used to reveal animal barcodes, specifically vertebrates, can also amplify the Wolbachia gene; a Rickettsia. The gene has special characteristics, namely 633 base pairs (bp) conserved, and 24 bp variable nucleotides. Of these there are 12 nucleotides which unique for Wobachians who infected A.cerena from Tanjung Peropa, Kendari Southeast Sulawesi. Base N composition dominated of Timine. The phylogenetic tree showed that Wolbachia from Apis cerena, is closely related to Wolbachia endosymbiont Hymenoptera, with 99% of boostrapped value.

Potential co-infection of Wolbachia with Leishmania among sand fly vectors caught from endemic leishmaniasis foci in Fars province, southern Iran.

Leishmaniasis is one of the Neglected Tropical Diseases in the tropical region of many countries in the world. The etiological agents (Leishmania parasites) of the disease are transmitted to human and other vertebrate hosts by infectious bites of female phlebotomine sand flies. On the other hand, some symbiotic microorganisms such as Wolbachia (Rickettsiales: Anaplasmataceae) may be transmitted vertically in many arthropods and may cause synergistic or antagonistic effects on epidemiology of the vector-borne diseases. Hence, in the present study, potential coinfection of Wolbachia with Leishmania in the sand fly vectors will be examined by PCR technique in the important leishmaniasis foci of Fars province in southern Iran, as a new feature for the disease long-term control. Sand flies were collected by sticky traps from indoor and outdoor locations of 5 different areas of Fars province during 2018 and 2019. DNAs of sand flies were extracted and PCR method was performed based on primers which were designed from surface proteins (WSP) genome region for Wolbachia and minicircle kDNA gene for Leishmania detections. At last, PCR products were sequenced and recorded in the GenBank. Out of 1002 sand flies caught from 5 different foci of Fars province, 909 male and female and 386 female sand flies' DNAs were extracted for detection of Wolbachia and Leishmania by PCR, respectively. Accordingly, out of the total 44 pools prepared from sand flies, 6 out of 28 pools form P. papatasi female were positive for Wolbachia in Shiraz, Marvdasht and Kharameh. However, none of female sand flies were positive for Leishmania. The study also focused on monitoring of sand flies co-infection of Wolbachia with Leishmania, which was not found in any of the studied samples. The negative results may be due to control strategies implemented which were done against Leishmaniasis in the studied areas during last years.

Wolbachia affects mitochondrial population structure in two systems of closely related Palaearctic blue butterflies

The bacterium Wolbachia infects many insect species and spreads by diverse vertical and horizontal means. As co-inherited organisms, these bacteria often cause problems in mitochondrial phylogeny inference. The phylogenetic relationships of many closely related Palaearctic blue butterflies (Lepidoptera: Lycaenidae: Polyommatinae) are ambiguous. We considered the patterns of Wolbachia infection and mitochondrial diversity in two systems: Aricia agestis/Aricia artaxerxes and the Pseudophilotes baton species complex. We sampled butterflies across their distribution ranges and sequenced one butterfly mitochondrial gene and two Wolbachia genes. Both butterfly systems had uninfected and infected populations, and harboured several Wolbachia strains. Wolbachia was highly prevalent in A. artaxerxes and the host’s mitochondrial structure was shallow, in contrast to A. agestis. Similar bacterial alleles infected both Aricia species from nearby sites, pointing to a possible horizontal transfer. Mitochondrial history of the P. baton species complex mirrored its Wolbachia infection and not the taxonomical division. Pseudophilotes baton and P. vicrama formed a hybrid zone in Europe. Wolbachia could obscure mitochondrial history, but knowledge on the infection helps us to understand the observed patterns. Testing for Wolbachia should be routine in mitochondrial DNA studies.

Wolbachia

Ary Hoffman introduces Wolbachia.

The Biochemistry of Cytoplasmic Incompatibility Caused by Endosymbiotic Bacteria

Many species of arthropods carry maternally inherited bacterial endosymbionts that can influence host sexual reproduction to benefit the bacterium. The most well-known of such reproductive parasites is Wolbachia pipientis. Wolbachia are obligate intracellular α-proteobacteria found in nearly half of all arthropod species. This success has been attributed in part to their ability to manipulate host reproduction to favor infected females. Cytoplasmic incompatibility (CI), a phenomenon wherein Wolbachia infection renders males sterile when they mate with uninfected females, but not infected females (the rescue mating), appears to be the most common. CI provides a reproductive advantage to infected females in the presence of a threshold level of infected males. The molecular mechanisms of CI and other reproductive manipulations, such as male killing, parthenogenesis, and feminization, have remained mysterious for many decades. It had been proposed by Werren more than two decades ago that CI is caused by a Wolbachia-mediated sperm modification and that rescue is achieved by a Wolbachia-encoded rescue factor in the infected egg. In the past few years, new research has highlighted a set of syntenic Wolbachia gene pairs encoding CI-inducing factors (Cifs) as the key players for the induction of CI and its rescue. Within each Cif pair, the protein encoded by the upstream gene is denoted A and the downstream gene B. To date, two types of Cifs have been characterized based on the enzymatic activity identified in the B protein of each protein pair; one type encodes a deubiquitylase (thus named CI-inducing deubiquitylase or cid), and a second type encodes a nuclease (named CI-inducing nuclease or cin). The CidA and CinA proteins bind tightly and specifically to their respective CidB and CinB partners. In transgenic Drosophila melanogaster, the expression of either the Cid or Cin protein pair in the male germline induces CI and the expression of the cognate A protein in females is sufficient for rescue. With the identity of the Wolbachia CI induction and rescue factors now known, research in the field has turned to directed studies on the molecular mechanisms of CI, which we review here.

Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines.

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.

Detection and characterization of bacterial endosymbionts inSoutheast Asian tephritid fruit fly populations

BackgroundVarious endosymbiotic bacteria, including Wolbachia of the Alphaproteobacteria, infect a wide range ofinsects and are capable of inducing reproductive abnormalities to their hostssuch as cytoplasmic incompatibility (CI), parthenogenesis, feminization andmale-killing. These extended phenotypes can be potentially exploited inenhancing environmentally friendly methods, such as the sterile insect technique(SIT), for controlling natural populations of agricultural pests. The goal ofthe present study is to investigate the presence of Wolbachia, Spiroplasma,Arsenophonus and Cardinium among Bactrocera,Dacus and Zeugodacus flies of Southeast Asian populations, and to genotypeany detected Wolbachia strains.ResultsA specific 16S rRNA PCR assay wasused to investigate the presence of reproductive parasites in naturalpopulations of nine different tephritid species originating from three Asiancountries, Bangladesh, China and India. Wolbachia infections were identified in Bactrocera dorsalis, B.correcta, B. scutellaris andB. zonata, with 12.2–42.9% occurrence,Entomoplasmatales in B. dorsalis, B. correcta, B.scutellaris, B. zonata,Zeugodacus cucurbitae and Z. tau (0.8–14.3%) and Cardinium in B. dorsalis andZ. tau (0.9–5.8%), while none of thespecies tested, harbored infections with Arsenophonus. Infected populations showed a medium (between 10and 90%) or low (< 10%) prevalence, ranging from 3 to 80% for Wolbachia, 2 to 33% for Entomoplasmatales and 5 to45% for Cardinium. Wolbachia and Entomoplasmatales infections were found both intropical and subtropical populations, the former mostly in India and the latterin various regions of India and Bangladesh. Cardinium infections were identified in both countries but onlyin subtropical populations. Phylogenetic analysis revealed the presence ofWolbachia with some strains belongingeither to supergroup B or supergroup A. Sequence analysis revealed deletions ofvariable length and nucleotide variation in three Wolbachia genes. Spiroplasmastrains were characterized as citri–chrysopicola–mirum and ixodetis strainswhile the remaining Entomoplasmatales to the Mycoides–Entomoplasmataceae clade.Cardinium strains were characterized asgroup A, similar to strains infecting Encarsiapergandiella.ConclusionsOur results indicated that in the Southeast natural populationsexamined, supergroup A Wolbachia straininfections were the most common, followed by Entomoplasmatales and Cardinium. In terms of diversity, most strains ofeach bacterial genus detected clustered in a common group. Interestingly, thedeletions detected in three Wolbachia geneswere either new or similar to those of previously identified pseudogenes thatwere integrated in the host genome indicating putative horizontal gene transferevents in B. dorsalis, B. correcta and B.zonata.