The cytoplasmic incompatibility Cif proteins from prophage WO modify sperm genome integrity

Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which sperms fertilize uninfected embryos that suffer catastrophic mitotic defects and lethality; however in infected females, CifA rescues the embryonic lethality and thus imparts a fitness advantage to Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here we use cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms of CI-causing males. Protamine-deficient sperms travel to the female reproductive tract together with Cif proteins. In female ovaries, CifA localizes to germ cell nuclei and overlaps with Wolbachia in the nurse cell cytoplasm and the oocyte, however Cifs are not present in late-stage oocytes and the embryo. Moreover, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Our results reveal a previously unrecognized phenomena in which prophage proteins invade animal gametic nuclei and modify the histone-protamine transition of spermatogenesis.

Cytoplasmic incompatibility in the semivoltine longicorn beetle Acalolepta fraudatrix (Coleoptera: Cerambycidae) double infected with Wolbachia

Wolbachia are obligatory endosymbiotic α-proteobacteria found in many arthropods. They are maternally inherited, and can induce reproductive alterations in the hosts. Despite considerable recent progress in studies on the associations between Wolbachia and various taxonomic groups of insects, none of the researches have revealed the effects of Wolbachia on longicorn beetles as the host insect. Acalolepta fraudatrix is a forest longicorn beetle that is distributed in East Asia. In this study, the relationship between Wolbachia and A. fraudatrix was investigated. Out of two populations of A. fraudatrix screened for Wolbachia using the genes ftsZ, wsp, and 16S rRNA, only one of the populations showed detection of all three genes indicating the presence of Wolbachia. Electron microscopy and fluorescent in situ hybridization also confirmed that the A. fraudatrix population was infected with Wolbachia. Sequencing the wsp genes derived from single insects revealed that two strains of Wolbachia coexisted in the insects based on the detection of two different sequences of the wsp gene. We designated these strains as wFra1 and wFra2. The bacterial titers of wFra1 were nearly 2-fold and 3-fold higher than wFra2 in the testes and ovaries, respectively. The two strains of Wolbachia in the insects were completely eliminated by rearing the insects on artificial diets containing 1% concentration of tetracycline for 1 generation. Reciprocal crosses between Wolbachia-infected and Wolbachia-uninfected A. fraudatrix demonstrated that only eggs produced by the crosses between Wolbachia-infected males and Wolbachia-uninfected females did not hatch, indicating that Wolbachia infecting A. fraudatrix causes cytoplasmic incompatibility in the host insect. This is the first report showing the effect of Wolbachia on reproductive function in a longicorn beetle, A. fraudatrix.

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

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

The Detection of Wolbachia in Tea Green Leafhopper (Empoasca onukii Matsuda) and Its Influence on the Host

Tea green leafhopper (Empoasca onukii Matsuda) is a critical pest in tea production. Wolbachia has attracted much attention as a new direction of pest biological control for its ability of manipulating the hosts’ reproductive biology. In this work, we focused on the detection of Wolbachia in tea green leafhopper and its effect on host reproduction and development. Polymerase chain reaction (PCR), real-time PCR, and fluorescence in situ hybridization (FISH) techniques were used to detect the distribution of Wolbachia in tea green leafhopper. Wolbachia infection levels were different in different organs of hosts in different insect stages. In addition, comparison between the infected populations and cured population (treated by tetracyclines) revealed that presence of Wolbachia apparently influenced the growth, life cycle, and other reproductive factors of tea green leafhopper, caused, for example, by cytoplasmic incompatibility (CI), thereby reducing number of offspring, shortening lifespan, and causing female-biased sex ratio. This research confirmed that the bacteria Wolbachia was of high incidence in tea leafhoppers and could significantly affect the hosts’ reproductive development and evolution.

Male Age and Wolbachia Dynamics: Investigating How Fast and Why Bacterial Densities and Cytoplasmic Incompatibility Strengths Vary

Wolbachia bacteria are the most common animal-associated endosymbionts due in large part to their manipulation of host reproduction. Many Wolbachia cause cytoplasmic incompatibility (CI) that kills uninfected host eggs.

Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector

Wolbachia, a maternally inherited intracellular bacterial species, can manipulate host insect reproduction by cytoplasmic incompatibility (CI), which results in embryo lethality in crosses between infected males and uninfected females. CI is encoded by two prophage genes, cifA and cifB. Wolbachia, coupled with the sterile insect technique, has been used in field trials to control populations of the dengue vector Aedes albopictus, but CI-inducing strains are not known to infect the malaria vector Anopheles gambiae. Here we show that cifA and cifB can induce conditional sterility in the malaria vector An. gambiae. We used transgenic expression of these Wolbachia-derived genes in the An. gambiae germline to show that cifB is sufficient to cause embryonic lethality and that cifB-induced sterility is rescued by cifA expression in females. When we co-expressed cifA and cifB in male mosquitoes, the CI phenotype was attenuated. In female mosquitoes, cifB impaired fertility, which was overcome by co-expression of cifA. Our findings pave the way towards using CI to control malaria mosquito vectors.

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

Many reproductive parasites such as Wolbachia spread within host populations by inducing cytoplasmic incompatibility (CI). CI occurs when parasite-modified sperm fertilizes uninfected eggs. In haplodiploid hosts, CI can lead to different phenotypes 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 strongly influences the stability of reproductive parasitism. Yet, despite the ubiquity of CI-inducing parasites in nature, there is no conclusive evidence for strong intraspecific host modulation of CI strength and phenotype. Here, we tested for intraspecific host modulation of Wolbachia-induced CI in haplodiploid Tetranychus spider mites. Using a single CI-inducing Wolbachia variant and mitochondrion, a Tetranychus urticae nuclear panel was created that consisted of infected and cured near-isogenic lines. We performed a highly replicated age-synchronized full diallel cross comprised of incompatible and compatible control crosses. We uncovered host modifier systems that strongly suppress CI strength when carried by infected T. urticae males. Interspecific crosses showed that the male modifier systems suppress CI strength across species boundaries. We also observed a continuum of CI phenotypes in our crosses and identified strong intraspecific female modulation of CI phenotype when paired with a specific male genotype. Crosses established a recessive genetic basis for the maternal effect and were consistent with polygenic Mendelian inheritance. Our findings identify spermatogenesis as an important target of selection for host suppression of CI strength and underscore the importance of maternal genetic effects for the CI phenotype. Both mechanisms interacted with the genotype of the mating partner, revealing that intraspecific host modulation of CI strength and phenotype is underpinned by complex genetic architectures.

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

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