Wolbachia-induced Cytoplasmic Incompatibility Research Articles

Rickettsia induces strong cytoplasmic incompatibility in a predatory insect.

Rickettsia, a group of intracellular bacteria found in eukaryotes, exhibits diverse lifestyles, with some acting as vertebrate pathogens transmitted by arthropod vectors and others serving as maternally transmitted arthropod endosymbionts, some of which manipulate host reproduction for their own benefit. Two phenotypes, namely male-killing and parthenogenesis induction are known as Rickettsia-induced host reproductive manipulations, but it remains unknown whether Rickettsia can induce other types of host manipulation. In this study, we discovered that Rickettsia induced strong cytoplasmic incompatibility (CI), in which uninfected females produce no offspring when mated with infected males, in the predatory insect Nesidiocoris tenuis (Hemiptera: Miridae). Molecular phylogenetic analysis revealed that the Rickettsia strain was related to Rickettsia bellii, a common insect endosymbiont. Notably, this strain carried plasmid-encoded homologues of the CI-inducing factors (namely cifA-like and cifB-like genes), typically found in Wolbachia, which are well-known CI-inducing endosymbionts. Protein domain prediction revealed that the cifB-like gene encodes PD-(D/E)XK nuclease and deubiquitinase domains, which are responsible for Wolbachia-induced CI, as well as ovarian tumour-like (OTU-like) cysteine protease and ankyrin repeat domains. These findings suggest that Rickettsia and Wolbachia endosymbionts share underlying mechanisms of CI and that CI-inducing ability was acquired by microbes through horizontal plasmid transfer.

Modelling suggests Wolbachia-induced cytoplasmic incompatibility in oak gall wasps with cyclical parthenogenesis.

Oak gall wasps typically exhibit a life cycle with one sexual and one asexual generation each year. These wasps can carry various endosymbionts, one of which is the maternally inherited bacterium Wolbachia that can induce several reproductive manipulations on its host. Cytoplasmic incompatibility (CI) has been described as the most prominent of these manipulations. CI leads to embryonic mortality in the hosts' offspring when infected males mate with either uninfected females or with females that harbour different Wolbachia strains. It has been hypothesized that Wolbachia can induce CI in oak gall wasps. To address this hypothesis, we derived a mathematical model to investigate the spread of a bacterial infection in naive populations and to determine the plausibility of CI occurrence. To validate our model, we used published data from Wolbachia-infected Belonocnema kinseyi populations in two approaches. Our first approach uses measurements of infection frequencies and maternal transmission in the sexual generation. For the second approach, we extended the model to compare predictions to estimates of mtDNA-haplotypes, which, like Wolbachia, are maternally inherited, and can therefore be associated with the infection. Both approaches indicate that CI is present in these populations. Our model can be generalized to investigate the occurrence of CI not only for oak gall wasps but also for other species.

The mechanism of cytoplasmic incompatibility is conserved in Wolbachia-infected Aedes aegypti mosquitoes deployed for arbovirus control.

The rising interest and success in deploying inherited microorganisms and cytoplasmic incompatibility (CI) for vector control strategies necessitate an explanation of the CI mechanism. Wolbachia-induced CI manifests in the form of embryonic lethality when sperm from Wolbachia-bearing testes fertilize eggs from uninfected females. Embryos from infected females however survive to sustain the maternally inherited symbiont. Previously in Drosophila melanogaster flies, we demonstrated that CI modifies chromatin integrity in developing sperm to bestow the embryonic lethality. Here, we validate these findings using wMel-transinfected Aedes aegypti mosquitoes released to control vector-borne diseases. Once again, the prophage WO CI proteins, CifA and CifB, target male gametic nuclei to modify chromatin integrity via an aberrant histone-to-protamine transition. Cifs are not detected in the embryo, and thus elicit CI via the nucleoprotein modifications established pre-fertilization. The rescue protein CifA in oogenesis localizes to stem cell, nurse cell, and oocyte nuclei, as well as embryonic DNA during embryogenesis. Discovery of the nuclear targeting Cifs and altered histone-to-protamine transition in both Aedes aegypti mosquitoes and D. melanogaster flies affirm the Host Modification Model of CI is conserved across these host species. The study also newly uncovers the cell biology of Cif proteins in the ovaries, CifA localization in the embryos, and an impaired histone-to-protamine transition during spermiogenesis of any mosquito species. Overall, these sperm modification findings may enable future optimization of CI efficacy in vectors or pests that are refractory to Wolbachia transinfections.

Insect population dynamics under Wolbachia-induced cytoplasmic incompatibility: Puzzle more than buzz in Drosophila suzukii.

In theory, the introduction of individuals infected with an incompatible strain of Wolbachia pipientis into a recipient host population should result in the symbiont invasion and reproductive failures caused by cytoplasmic incompatibility (CI). Modelling studies combining Wolbachia invasion and host population dynamics show that these two processes could interact to cause a transient population decline and, in some conditions, extinction. However, these effects could be sensitive to density dependence, with the Allee effect increasing the probability of extinction, and competition reducing the demographic impact of CI. We tested these predictions with laboratory experiments in the fruit fly Drosophila suzukii and the transinfected Wolbachia strain wTei. Surprisingly, the introduction of wTei into D. suzukii populations at carrying capacity did not result in the expected wTei invasion and transient population decline. In parallel, we found no Allee effect but strong negative density dependence. From these results, we propose that competition interacts in an antagonistic way with Wolbachia-induced cytoplasmic incompatibility on insect population dynamics. If future models and data support this hypothesis, pest management strategies using Wolbachia-induced CI should target populations with negligible competition but a potential Allee effect, for instance at the beginning of the reproductive season.

Transgenic expression of cif genes from Wolbachia strain wAlbB recapitulates cytoplasmic incompatibility in Aedes aegypti

The endosymbiotic bacteria Wolbachia can invade insect populations by modifying host reproduction through cytoplasmic incompatibility (CI), an effect that results in embryonic lethality when Wolbachia-carrying males mate with Wolbachia-free females. Here we describe a transgenic system for recreating CI in the major arbovirus vector Aedes aegypti using CI factor (cif) genes from wAlbB, a Wolbachia strain currently being deployed to reduce dengue transmission. CI-like sterility is induced when cifA and cifB are co-expressed in testes; this sterility is rescued by maternal cifA expression, thereby reproducing the pattern of Wolbachia-induced CI. Expression of cifB alone is associated with extensive DNA damage and disrupted spermatogenesis. The strength of rescue by maternal cifA expression is dependent on the comparative levels of cifA/cifB expression in males. These findings are consistent with CifB acting as a toxin and CifA as an antitoxin, with CifA attenuating CifB toxicity in both the male germline and in developing embryos. These findings provide important insights into the interactions between cif genes and their mechanism of activity and provide a foundation for the building of a cif gene-based drive system in Ae. aegypti.

Using Wolbachia to control rice planthopper populations: progress and challenges.

Wolbachia have been developed as a tool for protecting humans from mosquito populations and mosquito-borne diseases. The success of using Wolbachia relies on the facts that Wolbachia are maternally transmitted and that Wolbachia-induced cytoplasmic incompatibility provides a selective advantage to infected over uninfected females, ensuring that Wolbachia rapidly spread through the target pest population. Most transinfected Wolbachia exhibit a strong antiviral response in novel hosts, thus making it an extremely efficient technique. Although Wolbachia has only been used to control mosquitoes so far, great progress has been made in developing Wolbachia-based approaches to protect plants from rice pests and their associated diseases. Here, we synthesize the current knowledge about the important phenotypic effects of Wolbachia used to control mosquito populations and the literature on the interactions between Wolbachia and rice pest planthoppers. Our aim is to link findings from Wolbachia-mediated mosquito control programs to possible applications in planthoppers.

Why Wolbachia-induced cytoplasmic incompatibility is so common

Cytoplasmic incompatibility (CI) is the most common reproductive manipulation produced by Wolbachia, obligately intracellular alphaproteobacteria that infect approximately half of all insect species. Once infection frequencies within host populations approach 10%, intense CI can drive Wolbachia to near fixation within 10 generations. However, natural selection among Wolbachia variants within individual host populations does not favor enhanced CI. Indeed, variants that do not cause CI but increase host fitness or are more reliably maternally transmitted are expected to spread if infected females remain protected from CI. Nevertheless, approximately half of analyzed Wolbachia infections cause detectable CI. Why? The frequency and persistence of CI are more plausibly explained by preferential spread to new host species (clade selection) rather than by natural selection among variants within host populations. CI-causing Wolbachia lineages preferentially spread into new host species because 1) CI increases equilibrium Wolbachia frequencies within host populations, and 2) CI-causing variants can remain at high frequencies within populations even when conditions change so that initially beneficial Wolbachia infections become harmful. An epidemiological model describing Wolbachia acquisition and loss by host species and the loss of CI-induction within Wolbachia lineages yields simple expressions for the incidence of Wolbachia infections and the fraction of those infections causing CI. Supporting a determinative role for differential interspecific spread in maintaining CI, many Wolbachia infections were recently acquired by their host species, many show evidence for contemporary spatial spread or retreat, and rapid evolution of CI-inducing loci, especially degradation, is common.

Wolbachia wAlbB inhibit dengue and Zika infection in the mosquito Aedes aegypti with an Australian background.

Biological control of mosquito vectors using the endosymbiotic bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Aedes aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of wild populations of mosquitoes in the trial sites by exploiting the strain’s Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6–7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptible phenotype and genotype of native Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for population suppression programs, can also be effective in population replacement programs given its inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining the genotypes and phenotypes of the target population, shows the utility of this strain for controlling the Ae. aegypti mosquitoes and the pathogens they transmit.

Interacting host modifier systems control Wolbachia-induced cytoplasmic incompatibility in a haplodiploid mite.

Reproductive parasites such as Wolbachia spread within host populations by inducing cytoplasmic incompatibility (CI). CI occurs when parasite‐modified sperm fertilizes uninfected eggs and is typified by great variation in strength across biological systems. In haplodiploid hosts, CI has different phenotypic outcomes depending on whether the fertilized eggs die or develop into males. Genetic conflict theories predict the evolution of host modulation of CI, which in turn influences the stability of reproductive parasitism. However, despite the ubiquity of CI‐inducing parasites in nature, there is scarce evidence for intraspecific host modulation of CI strength and phenotype. Here, we tested for intraspecific host modulation of Wolbachia‐induced CI in haplodiploid Tetranychus urticae mites. Using a single CI‐inducing Wolbachia variant and mitochondrion, a nuclear panel was created that consisted of infected and cured near‐isogenic lines. We performed a highly replicated age‐synchronized full diallel cross composed of incompatible and compatible control crosses. We uncovered host modifier systems that cause striking variation in CI strength when carried by infected T. urticae males. We observed a continuum of CI phenotypes in our crosses and identified strong intraspecific female modulation of the CI phenotype. Crosses established a recessive genetic basis for the maternal effect and were consistent with polygenic Mendelian inheritance. Both male and female modulation interacted with the genotype of the mating partner. Our findings identify spermatogenesis as an important target of selection for host modulation of CI strength and underscore the importance of maternal genetic effects for the CI phenotype. Our findings reveal that intraspecific host modulation of CI is underpinned by complex genetic architectures and confirm that the evolution of reproductive parasitism is contingent on host genetics.

Why did theWolbachiatransinfection cross the road? drift, deterministic dynamics, and disease control.

Maternally inherited Wolbachia transinfections are being introduced into natural mosquito populations to reduce the transmission of dengue, Zika, and other arboviruses. Wolbachia-induced cytoplasmic incompatibility provides a frequency-dependent reproductive advantage to infected females that can spread transinfections within and among populations. However, because transinfections generally reduce host fitness, they tend to spread within populations only after their frequency exceeds a critical threshold. This produces bistability with stable equilibrium frequencies at both 0 and 1, analogous to the bistability produced by underdominance between alleles or karyotypes and by population dynamics under Allee effects. Here, we analyze how stochastic frequency variation produced by finite population size can facilitate the local spread of variants with bistable dynamics into areas where invasion is unexpected from deterministic models. Our exemplar is the establishment of wMel Wolbachia in the Aedes aegypti population of Pyramid Estates (PE), a small community in far north Queensland, Australia. In 2011, wMel was stably introduced into Gordonvale, separated from PE by barriers to A. aegypti dispersal. After nearly 6 years during which wMel was observed only at low frequencies in PE, corresponding to an apparent equilibrium between immigration and selection, wMel rose to fixation by 2018. Using analytic approximations and statistical analyses, we demonstrate that the observed fixation of wMel at PE is consistent with both stochastic transition past an unstable threshold frequency and deterministic transformation produced by steady immigration at a rate just above the threshold required for deterministic invasion. The indeterminacy results from a delicate balance of parameters needed to produce the delayed transition observed. Our analyses suggest that once Wolbachia transinfections are established locally through systematic introductions, stochastic "threshold crossing" is likely to only minimally enhance spatial spread, providing a local ratchet that slightly-but systematically-aids area-wide transformation of disease-vector populations in heterogeneous landscapes.

Genes associated with Wolbachia-induced cytoplasmic incompatibility in natural populations of Culex pipiens pallens: a preliminary study

To investigate the genes involved in Wolbachia-induced cytoplasmic incompatibility among three natural populations of Culex pipiens pallens in eastern China, so as to provide insights into the development of preventive and control measures for mosquito-borne diseases based on Wolbachia. The cytoplasmic incompatibility was tested among three natural populations of C. pipiens pallens collected from Nanjing and Wuxi of Jiangsu Province and Tangkou of Shandong Province using reciprocal crosses. Wolbachia infection was detected in C. pipiens pallens using a PCR assay, and the expression of Wolbachia wsp and WD0513 genes was quantified using a fluorescent quantitative real-time PCR (qPCR) assay. Bidirectional compatibility was found between the natural populations of C. pipiens pallens collected from Nanjing and Wuxi of Jiangsu Province (t = 0.57 and 0.15, both P values > 0.05), while bidirectional incompatibility was seen between the natural populations of C. pipiens pallens collected from Tangkou of Shandong Province and Wuxi of Jiangsu Province (t = 63.81 and 43.51, both P values < 0.01), and between the natural populations of C. pipiens pallens collected from Nanjing of Jiangsu Province and Tangkou of Shandong Province (t = 39.62 and 43.12, both P values < 0.01). Wolbachia wsp gene was amplified in all three natural populations of C. pipiens pallens, and qPCR assay detected no significant difference in the Wolbachia wsp gene expression among the three natural populations of C. pipiens pallens (F = 2.15, P > 0.05). In addition, there was no significant difference in the WD0513 gene expression between the natural populations of C. pipiens pallens collected from Tangkou of Shandong Province and Nanjing of Jiangsu Province (q = 8.42, P < 0.05) or between the natural populations of C. pipiens pallens collected from Tangkou of Shandong Province and Wuxi of Jiangsu Province (q = 7.84, P < 0.05); however, there was a significant difference detected in the WD0513 gene expression between the natural populations of C. pipiens pallens collected from Nanjing and Wuxi of Jiangsu Province (q = 0.40, P > 0.05). Different Wolbachia numbers are detected in natural populations of C. pipiens pallens collected from Nanjing and Wuxi of Jiangsu Province and Tangkou of Shandong Province, and WD0513 gene may be involved in the Wolbachia-induced cytoplasmic incompatibility among three natural populations of C. pipiens pallens.

Antagonistic interaction between male-killing and cytoplasmic incompatibility induced by Cardinium and Wolbachia in the whitefly, Bemisia tabaci.

Cardinium and Wolbachia are maternally inherited bacterial symbionts of arthropods that can manipulate host reproduction by increasing the fitness of infected females. Here, we report that Cardinium and Wolbachia coinfection induced male-killing and cytoplasmic incompatibility (CI) when they coexisted in a cryptic species of whitefly, Bemisia tabaci Asia II7. Cardinium and Wolbachia symbionts were either singly or simultaneously localized in the bacteriocytes placed in the abdomen of B. tabaci nymphs and adults. Cardinium-Wolbachia coinfection induced male-killing and resulted in a higher female sex ratio in the intraspecific amphigenetic progeny of Asia II7 ICWH and ICWL lines; interestingly, male-killing induction was enhanced with increased Cardinium titer. Moreover, single infection of Wolbachia induced partial CI in the Asia II7 IW line and resulted in reduced fecundity, higher embryonic mortality, and lower female sex ratio. The uninfected Asia II7 IU line had significantly higher fecundity, lower embryonic and nymphal mortalities, and a lower level of CI than both the Wolbachia-infected Asia II7 IW line and the Cardinium-Wolbachia-coinfected Asia II7 ICWH line. Our findings indicate that Cardinium-Wolbachia coinfection induced male-killing, which may have had antagonistic effects on Wolbachia-induced CI in the Asia II7 whiteflies. For the first time, our study revealed that B. tabaci Asia II7 reproduction is co-manipulated by Cardinium and Wolbachia endosymbionts.

Effects, interactions, and localization of Rickettsia and Wolbachia in the house fly parasitoid, Spalangia endius

Many insect species harbor facultative microbial symbionts that affect their biology in diverse ways. Here, we studied the effects, interactions, and localization of two bacterial symbionts-Wolbachia and Rickettsia-in the parasitoid Spalangia endius. We crossed between four S. endius colonies-Wolbachia only (W), Rickettsia only (R), both (WR), and none (aposymbiotic, APS) (16 possible crosses) and found that Wolbachia induces incomplete cytoplasmic incompatibility (CI), both when the males are W or WR. Rickettsia did not cause reproductive manipulations and did not rescue the Wolbachia-induced CI. However, when R females were crossed with W or WR males, significantly less offspring were produced compared with that of control crosses. In non-CI crosses, the presence of Wolbachia in males caused a significant reduction in offspring numbers. Females' developmental time was significantly prolonged in the R colony, with adults starting to emerge one day later than the other colonies. Other fitness parameters did not differ significantly between the colonies. Using fluorescence in situ hybridization microscopy in females, we found that Wolbachia is localized alongside Rickettsia inside oocytes, follicle cells, and nurse cells in the ovaries. However, Rickettsia is distributed also in muscle cells all over the body, in ganglia, and even in the brain.

Paternal Grandmother Age Affects the Strength of Wolbachia-Induced Cytoplasmic Incompatibility in Drosophila melanogaster.

Wolbachia are obligate intracellular bacteria that are globally distributed in half of all arthropod species. As the most abundant maternally inherited microbe in animals, Wolbachia manipulate host reproduction via reproductive parasitism strategies, including cytoplasmic incompatibility (CI). CI manifests as embryonic death when Wolbachia-modified sperm fertilize uninfected eggs but not maternally infected eggs. Thus, CI can provide a relative fitness advantage to Wolbachia-infected females and drive the infection through a population. In the genetic model Drosophila melanogaster, the Wolbachia strain wMel induces variable CI, making mechanistic studies in D. melanogaster cumbersome. Here, we demonstrate that sons of older paternal D. melanogaster grandmothers induce stronger CI than sons of younger paternal grandmothers, and we term this relationship the "paternal grandmother age effect" (PGAE). Moreover, the embryos and adult sons of older D. melanogaster grandmothers have higher Wolbachia densities, correlating with their ability to induce stronger CI. In addition, we report that Wolbachia density positively correlates with female age and decreases after mating, suggesting that females transmit Wolbachia loads that are proportional to their own titers. These findings reveal a transgenerational impact of age on wMel-induced CI, elucidate Wolbachia density dynamics in D. melanogaster, and provide a methodological advance to studies aimed at understanding wMel-induced CI in the D. melanogaster model.IMPORTANCE Unidirectional cytoplasmic incompatibility (CI) results in a postfertilization incompatibility between Wolbachia-infected males and uninfected females. CI contributes to reproductive isolation between closely related species and is used in worldwide vector control programs to drastically lower arboviral vector population sizes or to replace populations that transmit arboviruses with those resistant to transmission. Despite decades of research on the factors that influence CI, penetrance is often variable under controlled laboratory conditions in various arthropods, suggesting that additional variables influence CI strength. Here, we demonstrate that paternal D. melanogaster grandmother age influences the strength of CI induced by their sons. Older D. melanogaster females have higher Wolbachia densities and produce offspring with higher Wolbachia densities that associate with stronger CI. This work reveals a multigenerational impact of age on CI and expands our understanding of host-Wolbachia interactions and the biology of CI induced by the Wolbachia strain infecting the most widely used arthropod model, D. melanogaster.

Transcriptome of Tetranychus urticae embryos reveals insights into Wolbachia-induced cytoplasmic incompatibility.

The endosymbiont Wolbachia is known for manipulating host reproduction in selfish ways. However, the molecular mechanisms have not yet been investigated in embryos. Here, we found that Wolbachia had no effect on the number of deposited eggs in Tetranychus urticae Koch (Acari: Tetranychidae) but caused two types of reproductive manipulation: killing uninfected female embryos via cytoplasmic incompatibility (CI) and increasing the hatching ratio of infected female embryos. RNA sequencing analyses showed that 145 genes were differentially expressed between Wolbachia-infected (WI) and Wolbachia-uninfected (WU) embryos. Wolbachia infection down-regulated messenger RNA (mRNA) expression of glutathione S-transferase that could buffer oxidative stress. In addition, 1613 and 294 genes were identified as CI-specific up-/down-regulated genes. Compared to WU and WI embryos, embryos of CI cross strongly expressed genes involved in transcription, translation, tissue morphogenesis, DNA damage and mRNA surveillance. In contrast, most of the genes associated with energy production and metabolism were down-regulated in the CI embryos compared to the WU and WI embryos, which provides some clues as to the cause of death of CI embryos. These results identify several genes that could be candidates for explaining Wolbachia-induced CI. Our data form a basis to help elucidate the molecular consequences of CI in embryos.

A Wolbachia nuclease and its binding partner provide a distinct mechanism for cytoplasmic incompatibility

Wolbachia are endosymbiotic bacteria that infect nearly half of all arthropod species. This pandemic is due in part to their ability to increase their transmission through the female germline, most commonly by a mechanism called cytoplasmic incompatibility (CI). The Wolbachia cid operon, encoding 2 proteins, CidA and CidB, the latter a deubiquitylating enzyme (DUB), recapitulates CI in transgenic Drosophila melanogaster However, some CI-inducing Wolbachia strains lack a DUB-encoding cid operon; it was therefore proposed that the related cin operon codes for an alternative CI system. Here we show that the Wolbachia cin operon encodes a nuclease, CinB, and a second protein, CinA, that tightly binds CinB. Recombinant CinB has nuclease activity against both single-stranded and double-stranded DNA but not RNA under the conditions tested. Expression of the cin operon in transgenic male flies induces male sterility and embryonic defects typical of CI. Importantly, transgenic CinA can rescue defects in egg-hatch rates when expressed in females. Expression of CinA also rescues CinB-induced growth defects in yeast. CinB has 2 PD-(D/E)xK nuclease domains, and both are required for nuclease activity and for toxicity in yeast and flies. Our data suggest a distinct mechanism for CI involving a nuclease toxin and highlight the central role of toxin-antidote operons in Wolbachia-induced cytoplasmic incompatibility.

Small RNA analysis provides new insights into cytoplasmic incompatibility in Drosophila melanogaster induced by Wolbachia

Wolbachia is a genus of endosymbiotic bacteria that induce a wide range of effects on their insect hosts. Cytoplasmic incompatibility (CI) is the most common phenotype mediated by Wolbachia and results in embryonic lethality when Wolbachia-infected males mate with uninfected females. Studies have revealed that bacteria can regulate many cellular processes in their hosts using small non-coding RNAs, so we investigated the involvement of small RNAs (sRNAs) in CI. Comparison of sRNA libraries between Wolbachia-infected and uninfected Drosophila melanogaster testes revealed 18 novel microRNAs (miRNAs), of which 12 were expressed specifically in Wolbachia-infected flies and one specifically in Wolbachia-uninfected flies. Furthermore, ten miRNAs showed differential expression, with four upregulated and six downregulated in Wolbachia-infected flies. Of the upregulated miRNAs, nov-miR-12 exhibited the highest upregulation in the testes of D. melanogaster. We then identified pipsqueak (psq) as the target gene of nov-miR-12 with the greatest complementarity in its 3′ untranslated region (UTR). Wolbachia infection was correlated with reduced psq expression in D. melanogaster, and luciferase assays demonstrated that nov-miR-12 could downregulate psq through binding to its 3′UTR region. Knockdown of psq in Wolbachia-free fly testes significantly reduced egg hatching rate and mimicked the cellular abnormalities of Wolbachia-induced CI in embryos, including asynchronous nuclear division, chromatin bridging, and chromatin fragmentation. These results suggest that Wolbachia may induce CI in insect hosts by miRNA-mediated changes in host gene expression. Moreover, these findings reveal a potential molecular strategy for elucidating the complex interactions between endosymbionts and their insect hosts, such as Wolbachia-driven CI.

Proteomic analysis of Laodelphax striatellus gonads reveals proteins that may manipulate host reproduction by Wolbachia

Wolbachia are intracellular bacteria that manipulate host reproduction by several mechanisms including cytoplasmic incompatibility (CI). However, the underlying mechanisms of Wolbachia-induced CI are not entirely clear. Here, we monitored the Wolbachia distribution in the male gonads of the small brown planthopper (Laodelphax striatellus, SBPH) at different development stages, and investigated the influence of Wolbachia on male gonads by a quantitative proteomic analysis. A total of 276 differentially expressed proteins were identified, with the majority of them participating in metabolism, modification, and reproduction. Knocking down the expression of outer dense fiber protein (ODFP) and venom allergen 5-like (VA5L) showed decreased egg reproduction, and these two genes might be responsible for Wolbachia improved fecundity in infected L. striatellus; whereas knocking down the expression of cytosol amino-peptidase-like (CAL) significantly decreased the egg hatch rate in Wolbachia-uninfected L. striatellus, but not in the Wolbachia-infected one. Considering that the mRNA/protein level of CAL was downregulated by Wolbachia infection and dsCAL treatment closely mimicked Wolbachia-induced CI, we presumed that CAL might be one of the factors determining the CI phenotype.

Tetracycline reduces feeding and reproduction of the parthenogenetic springtail, Folsomia candida

Treatment with tetracycline or rifampicin to eliminate the obligate intracellular bacterium, Wolbachia pipientis, provides an important tool for validating Wolbachia’s diverse effects on arthropod reproduction, including cytoplasmic incompatibility, feminization, male killing and parthenogenesis. For the soil collembolan, Folsomia candida (Entomobryomorpha: Isotomidae) efforts to establish the role of Wolbachia in parthenogenesis using tetracycline have been ambiguous, possibly reflecting variation in overall experimental design, mode of antibiotic application and duration of treatment. By maintaining F. candida populations on agar plates containing dissolved antibiotics we show that the EC50 (effective concentration for 50% reduction in biomass) for tetracycline is 50-fold higher than for rifampicin. Experiments with the fluorescent dye rhodamine further show that tetracycline deters feeding. Using individual Collembola, we show that reproductively mature F. candida survive more than 60 days exposure to tetracycline at concentrations 4-fold higher than the EC50 and that its effect on egg production is reversible, consistent with persistence of Wolbachia during tetracycline treatment. As has been shown with other microbes, our results suggest that in F. candida, Wolbachia survives antibiotic exposure by entering a metabolically dormant persister state, from which it can recover under favorable conditions. This possibility is of particular interest because persister cells are induced by toxin-antitoxin (TA) gene pairs, which have recently been associated with Wolbachia-induced cytoplasmic incompatibility in Culex pipiens mosquitoes.

Combined sterile insect technique and incompatible insect technique: sex separation and quality of sterile Aedes aegypti male mosquitoes released in a pilot population suppression trial in Thailand

ABSTRACTBackgroundThe sterile insect technique (SIT), which is based on irradiation-induced sterility, and incompatible insect technique (IIT), which is based on Wolbachia-induced cytoplasmic incompatibility (a kind of male sterility), have been used as alternative methods to reduce mosquito vector populations. Both methods require the release of males to reduce fertile females and suppress the number of natural populations. Different techniques of sex separation to obtain only males have been investigated previously. Our work involves an application of mechanical larval-pupal glass separators to separate Wolbachia-infected Aedes aegypti males from females at the pupal stage, prior to irradiation, and for use in a pilot field release and to assess the quality of males and females before and after sex separation and sterilization.ResultsThis study was the first to demonstrate the efficiency of mechanical glass separators in separating males for use in an Ae. aegypti suppression trial by a combined SIT/IIT approach. Our results indicated that male and female pupae of Wolbachia-infected Ae. aegypti mosquitoes were significantly different (p < 0.05) in weight, size, and emergence-time, which made it easier for sex separation by this mechanical method. During the pilot field release, the percentage of female contamination was detected to be quite low and significantly different between the first (0.10 ± 0.13) and the second (0.02 ± 0.02) twelve-week period. Both males and females were almost completely sterile after exposure to 70 Gy irradiation dose. We observed that both irradiated Wolbachia-infected males and females survived and lived longer than two weeks, but males could live longer than females (p < 0.05) when they were irradiated at the same irradiation dose. When comparing irradiated mosquitoes with non-irradiated ones, there was no significant difference in longevity and survival-rate between those males, but non-irradiated females lived longer than irradiated ones (p < 0.05).ConclusionMechanical sex separation by using a larval-pupal glass separator was practically applied to obtain only males for further sterilization and open field release in a pilot population suppression trial of Ae. aegypti in Thailand. Female contamination was detected to be quite low, and skilled personnel can reduce the risk for female release. The irradiated Wolbachia-infected females accidentally released were found to be completely sterile, with shorter life span than males.