Prophage proteins alter long noncoding RNA and DNA of developing sperm to induce a paternal-effect lethality.

The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA) and B (CifB) proteins, encoded by prophage WO of the endosymbiont Wolbachia, alter long noncoding RNA (lncRNA) and DNA during Drosophila sperm development to establish a paternal-effect embryonic lethality known as cytoplasmic incompatibility (CI). CifA is a ribonuclease (RNase) that depletes a spermatocyte lncRNA important for the histone-to-protamine transition of spermiogenesis. Both CifA and CifB are deoxyribonucleases (DNases) that elevate DNA damage in late spermiogenesis. lncRNA knockdown enhances CI, and mutagenesis links lncRNA depletion and subsequent sperm chromatin integrity changes to embryonic DNA damage and CI. Hence, prophage proteins interact with eukaryotic macromolecules during gametogenesis to create a symbiosis that is fundamental to insect evolution and vector control.

Rapid turnover of pathogen-blocking Wolbachia and their incompatibility loci.

At least half of all insect species carry maternally inherited Wolbachia alphaproteobacteria, making Wolbachia the most common endosymbionts in nature. Wolbachia spread to high frequencies is often due to cytoplasmic incompatibility (CI), a Wolbachia-induced sperm modification that kills embryos without Wolbachia. Several CI-causing Wolbachia variants, including wMel from Drosophila melanogaster, also block viruses. Establishing pathogen-blocking wMel in natural Aedes aegypti mosquito populations has reduced dengue disease incidence, with one study reporting about 85% reduction when wMel frequency is high. However, wMel transinfection establishment is challenging in many environments, highlighting the importance of identifying CI-causing Wolbachia variants that stably persist in diverse hosts and habitats. We demonstrate that wMel-like variants have naturally established in widely distributed holometabolous dipteran and hymenopteran insects that diverged approximately 350 million years ago, with wMel variants spreading rapidly among these hosts over only the last 100,000 years. Wolbachia genomes contain prophages that encode CI-causing operons (cifs). These cifs move among Wolbachia genomes - with and without prophages - even more rapidly than Wolbachia move among insect hosts. Our results shed light on how rapid host switching and horizontal gene transfer contribute to Wolbachia and cif diversity in nature. The diverse wMel variants we report here from hosts present in different climates offer many new options for broadening Wolbachia-based biocontrol of diseases and pests.

cifB-transcript levels largely explain cytoplasmic incompatibility variation across divergent Wolbachia.

Divergent hosts often associate with intracellular microbes that influence their fitness. Maternally transmitted Wolbachia bacteria are the most common of these endosymbionts, due largely to cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-infected males. Closely related infections in females rescue CI, providing a relative fitness advantage that drives Wolbachia to high frequencies. One prophage-associated gene (cifA) governs rescue, and two contribute to CI (cifA and cifB), but CI strength ranges from very strong to very weak for unknown reasons. Here, we investigate CI-strength variation and its mechanistic underpinnings in a phylogenetic context across 20 million years (MY) of Wolbachia evolution in Drosophila hosts diverged up to 50 MY. These Wolbachia encode diverse Cif proteins (100% to 7.4% pairwise similarity), and AlphaFold structural analyses suggest that CifB sequence similarities do not predict structural similarities. We demonstrate that cifB-transcript levels in testes explain CI strength across all but two focal systems. Despite phylogenetic discordance among cifs and the bulk of the Wolbachia genome, closely related Wolbachia tend to cause similar CI strengths and transcribe cifB at similar levels. This indicates that other non-cif regions of the Wolbachia genome modulate cif-transcript levels. CI strength also increases with the length of the host's larval life stage, presumably due to prolonged cif action. Our findings reveal that cifB-transcript levels largely explain CI strength, while highlighting other covariates. Elucidating CI's mechanism contributes to our understanding of Wolbachia spread in natural systems and to improving the efficacy of CI-based biocontrol of arboviruses and agricultural pests globally.

Temperature effects on cellular host-microbe interactions explain continent-wide endosymbiont prevalence.

Endosymbioses influence host physiology, reproduction, and fitness, but these relationships require efficient microbe transmission between host generations to persist. Maternally transmitted Wolbachia are the most common known endosymbionts,1 but their frequencies vary widely within and among host populations for unknown reasons.2,3 Here, we integrate genomic, cellular, and phenotypic analyses with mathematical models to provide an unexpectedly simple explanation for global wMel Wolbachia prevalence in Drosophila melanogaster. Cooling temperatures decrease wMel cellular abundance at a key stage of host oogenesis, producing temperature-dependent variation in maternal transmission that plausibly explains latitudinal clines of wMel frequencies on multiple continents. wMel sampled from a temperate climate targets the germline more efficiently in the cold than a recently differentiated tropical variant (∼2,200 years ago), indicative of rapid wMel adaptation to climate. Genomic analyses identify a very narrow list of wMel alleles-most notably, a derived stop codon in the major Wolbachia surface protein WspB-that underlie thermal sensitivity of cellular Wolbachia abundance and covary with temperature globally. Decoupling temperate wMel and host genomes further reduces transmission in the cold, a pattern that is characteristic of host-microbe co-adaptation to a temperate climate. Complex interactions among Wolbachia, hosts, and the environment (GxGxE) mediate wMel cellular abundance and maternal transmission, implicating temperature as a key determinant of Wolbachia spread and equilibrium frequencies, in conjunction with Wolbachia effects on host fitness and reproduction.4,5 Our results motivate the strategic use of locally selected wMel variants for Wolbachia-based biocontrol efforts, which protect millions of individuals from arboviruses that cause human disease.6.

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

Endosymbionts can influence host reproduction and fitness to favor their maternal transmission. For example, endosymbiotic Wolbachia bacteria often cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-modified sperm. Infected females can rescue CI, providing them a relative fitness advantage. Wolbachia-induced CI strength varies widely and tends to decrease as host males age. Since strong CI drives Wolbachia to high equilibrium frequencies, understanding how fast and why CI strength declines with male age is crucial to explaining age-dependent CI's influence on Wolbachia prevalence. Here, we investigate if Wolbachia densities and/or CI gene (cif) expression covary with CI-strength variation and explore covariates of age-dependent Wolbachia-density variation in two classic CI systems. wRi CI strength decreases slowly with Drosophila simulans male age (6%/day), but wMel CI strength decreases very rapidly (19%/day), yielding statistically insignificant CI after only 3 days of Drosophila melanogaster adult emergence. Wolbachia densities and cif expression in testes decrease as wRi-infected males age, but both surprisingly increase as wMel-infected males age, and CI strength declines. We then tested if phage lysis, Octomom copy number (which impacts wMel density), or host immune expression covary with age-dependent wMel densities. Only host immune expression correlated with density. Together, our results identify how fast CI strength declines with male age in two model systems and reveal unique relationships between male age, Wolbachia densities, cif expression, and host immunity. We discuss new hypotheses about the basis of age-dependent CI strength and its contributions to Wolbachia prevalence. IMPORTANCEWolbachia 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. Infected eggs are protected from CI, favoring Wolbachia spread in natural systems and in transinfected mosquito populations where vector-control groups use strong CI to maintain pathogen-blocking Wolbachia at high frequencies for biocontrol of arboviruses. CI strength varies considerably in nature and declines as males age for unknown reasons. Here, we determine that CI strength weakens at different rates with age in two model symbioses. Wolbachia density and CI gene expression covary with wRi-induced CI strength in Drosophila simulans, but neither explain rapidly declining wMel-induced CI in aging D. melanogaster males. Patterns of host immune gene expression suggest a candidate mechanism behind age-dependent wMel densities. These findings inform how age-dependent CI may contribute to Wolbachia prevalence in natural systems and potentially in transinfected systems.

Living in the endosymbiotic world of Wolbachia: A centennial review.

The most widespread intracellular bacteria in the animal kingdom are maternally inherited endosymbionts of the genus Wolbachia. Their prevalence in arthropods and nematodes worldwide and stunning arsenal of parasitic and mutualistic adaptations make these bacteria a biological archetype for basic studies of symbiosis and applied outcomes for curbing human and agricultural diseases. Here, we conduct a summative, centennial analysis of living in the Wolbachia world. We synthesize literature on Wolbachia's host range, phylogenetic diversity, genomics, cell biology, and applications to filarial, arboviral, and agricultural diseases. We also review the mobilome of Wolbachia including phage WO and its essentiality to hallmark reproductive phenotypes in arthropods. Finally, the Wolbachia system is an exemplar for discovery-based science education using biodiversity, biotechnology, and bioinformatics lessons. As we approach a century of Wolbachia research, the interdisciplinary science of this symbiosis stands as a model for consolidating and teaching the integrative rules of endosymbiotic life.

The impacts of cytoplasmic incompatibility factor (cifA and cifB) genetic variation on phenotypes.

Wolbachia are maternally transmitted, intracellular bacteria that can often selfishly spread through arthropod populations via cytoplasmic incompatibility (CI). CI manifests as embryonic death when males expressing prophage WO genes cifA and cifB mate with uninfected females or females harboring an incompatible Wolbachia strain. Females with a compatible cifA-expressing strain rescue CI. Thus, cif-mediated CI confers a relative fitness advantage to females transmitting Wolbachia. However, whether cif sequence variation underpins incompatibilities between Wolbachia strains and variation in CI penetrance remains unknown. Here, we engineer Drosophila melanogaster to transgenically express cognate and non-cognate cif homologs and assess their CI and rescue capability. Cognate expression revealed that cifA;B native to D. melanogaster causes strong CI, and cognate cifA;B homologs from two other Drosophila-associated Wolbachia cause weak transgenic CI, including the first demonstration of phylogenetic type 2 cifA;B CI. Intriguingly, non-cognate expression of cifA and cifB alleles from different strains revealed that cifA homologs generally contribute to strong transgenic CI and interchangeable rescue despite their evolutionary divergence, and cifB genetic divergence contributes to weak or no transgenic CI. Finally, we find that a type 1 cifA can rescue CI caused by a genetically divergent type 2 cifA;B in a manner consistent with unidirectional incompatibility. By genetically dissecting individual CI functions for type 1 and 2 cifA and cifB, this work illuminates new relationships between cif genotype and CI phenotype. We discuss the relevance of these findings to CI's genetic basis, phenotypic variation patterns, and mechanism.

Symbiont-mediated cytoplasmic incompatibility: what have we learned in 50 years?

Cytoplasmic incompatibility (CI) is the most common symbiont-induced reproductive manipulation. Specifically, symbiont-induced sperm modifications cause catastrophic mitotic defects in the fertilized embryo and ensuing lethality in crosses between symbiotic males and either aposymbiotic females or females harboring a different symbiont strain. However, if the female carries the same symbiont strain, then embryos develop properly, thereby imparting a relative fitness benefit to symbiont-transmitting mothers. Thus, CI drives maternally-transmitted bacteria to high frequencies in arthropods worldwide. In the past two decades, CI experienced a boom in interest due to its (i) deployment in worldwide efforts to curb mosquito-borne diseases, (ii) causation by bacteriophage genes, cifA and cifB, that modify sexual reproduction, and (iii) important impacts on arthropod speciation. This review serves as a gateway to experimental, conceptual, and quantitative themes of CI and outlines significant gaps in understanding CI's mechanism that are ripe for investigation from diverse subdisciplines in the life sciences.

Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifA's complex functional repertoire and new essential regions in CifB.

Wolbachia are the world's most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due, in part, to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifA's N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifA's putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be crucial in rescue are correspondingly crucial in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all crucial for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI.

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.

Two-By-One model of cytoplasmic incompatibility: Synthetic recapitulation by transgenic expression of cifA and cifB in Drosophila.

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.

One prophage WO gene rescues cytoplasmic incompatibility in Drosophila melanogaster.

Wolbachia are maternally inherited, intracellular bacteria at the forefront of vector control efforts to curb arbovirus transmission. In international field trials, the cytoplasmic incompatibility (CI) drive system of wMel Wolbachia is deployed to replace target vector populations, whereby a Wolbachia-induced modification of the sperm genome kills embryos. However, Wolbachia in the embryo rescue the sperm genome impairment, and therefore CI results in a strong fitness advantage for infected females that transmit the bacteria to offspring. The two genes responsible for the wMel-induced sperm modification of CI, cifA and cifB, were recently identified in the eukaryotic association module of prophage WO, but the genetic basis of rescue is unresolved. Here we use transgenic and cytological approaches to demonstrate that maternal cifA expression independently rescues CI and nullifies embryonic death caused by wMel Wolbachia in Drosophila melanogaster Discovery of cifA as the rescue gene and previously one of two CI induction genes establishes a "Two-by-One" model that underpins the genetic basis of CI. Results highlight the central role of prophage WO in shaping Wolbachia phenotypes that are significant to arthropod evolution and vector control.

The gene family that cheats Mendel.

Some alleles of the wtf gene family can increase their chances of spreading by using poisons to kill other alleles, and antidotes to save themselves.

Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility.

The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research and relevance to symbiont-induced speciation, as well as control of arbovirus vectors and agricultural pests, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO from Wolbachia strain wMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.

Disentangling a Holobiont - Recent Advances and Perspectives in Nasonia Wasps.

The parasitoid wasp genus Nasonia (Hymenoptera: Chalcidoidea) is a well-established model organism for insect development, evolutionary genetics, speciation, and symbiosis. The host-microbiota assemblage which constitutes the Nasonia holobiont (a host together with all of its associated microbes) consists of viruses, two heritable bacterial symbionts and a bacterial community dominated in abundance by a few taxa in the gut. In the wild, all four Nasonia species are systematically infected with the obligate intracellular bacterium Wolbachia and can additionally be co-infected with Arsenophonus nasoniae. These two reproductive parasites have different transmission modes and host manipulations (cytoplasmic incompatibility vs. male-killing, respectively). Pioneering studies on Wolbachia in Nasonia demonstrated that closely related Nasonia species harbor multiple and mutually incompatible Wolbachia strains, resulting in strong symbiont-mediated reproductive barriers that evolved early in the speciation process. Moreover, research on host-symbiont interactions and speciation has recently broadened from its historical focus on heritable symbionts to the entire microbial community. In this context, each Nasonia species hosts a distinguishable community of gut bacteria that experiences a temporal succession during host development and members of this bacterial community cause strong hybrid lethality during larval development. In this review, we present the Nasonia species complex as a model system to experimentally investigate questions regarding: (i) the impact of different microbes, including (but not limited to) heritable endosymbionts, on the extended phenotype of the holobiont, (ii) the establishment and regulation of a species-specific microbiota, (iii) the role of the microbiota in speciation, and (iv) the resilience and adaptability of the microbiota in wild populations subjected to different environmental pressures. We discuss the potential for easy microbiota manipulations in Nasonia as a promising experimental approach to address these fundamental aspects.

An optimized approach to germ-free rearing in the jewel wasp Nasonia.

Development of a Nasonia in vitrogerm-free rearing system in 2012 enabled investigation of Nasonia-microbiota interactions and real-time visualization of parasitoid metamorphosis. However, the use of antibiotics, bleach, and fetal bovine serum introduced artifacts relative to conventional rearing of Nasonia. Here, we optimize the germ-free rearing procedure by using filter sterilization in lieu of antibiotics and by removing residual bleach and fetal bovine serum. Comparison of these methods reveals no influence on larval survival or growth, and a 52% improvement in adult production. Additionally, adult males produced in the new germ-free system are similar in size to conventionally reared males. Experimental implications of these changes are discussed.

Speciation by Symbiosis: the Microbiome and Behavior.

Species are fundamental units of comparison in biology. The newly discovered importance and ubiquity of host-associated microorganisms are now stimulating work on the roles that microbes can play in animal speciation. We previously synthesized the literature and advanced concepts of speciation by symbiosis with notable attention to hybrid sterility and lethality. Here, we review recent studies and relevant data on microbes as players in host behavior and behavioral isolation, emphasizing the patterns seen in these analyses and highlighting areas worthy of additional exploration. We conclude that the role of microbial symbionts in behavior and speciation is gaining exciting traction and that the holobiont and hologenome concepts afford an evolving intellectual framework to promote research and intellectual exchange between disciplines such as behavior, microbiology, genetics, symbiosis, and speciation. Given the increasing centrality of microbiology in macroscopic life, microbial symbiosis is arguably the most neglected aspect of animal and plant speciation, and studying it should yield a better understanding of the origin of species.

Extensive reorganization of behavior accompanies ontogeny of aggression in male flesh flies.

Aggression, costly in both time and energy, is often expressed by male animals in defense of valuable resources such as food or potential mates. Here we present a new insect model system for the study of aggression, the male flesh fly Sarcophaga crassipalpis, and ask whether there is an ontogeny of aggression that coincides with reproductive maturity. After establishing that reproductive maturity occurs by day 3 of age (post-eclosion), we examined the behavior of socially isolated males from different age cohorts (days 1, 2, 3, 4, and 6) upon introduction, in a test arena, with another male of the same age. The results show a pronounced development of aggression with age. The change from relative indifference to heightened aggression involves a profound increase in the frequency of high-intensity aggressive behaviors between days 1 and 3. Also noteworthy is an abrupt increase in the number of statistically significant transitions involving these full-contact agonistic behaviors on day 2. This elevated activity is trimmed back somewhat by day 3 and appears to maintain a stable plateau thereafter. No convincing evidence was found for escalation of aggression nor the establishment of a dominance relationship over the duration of the encounters. Despite the fact that aggressive interactions are brief, lasting only a few seconds, a major reorganization in the relative proportions of four major non-aggressive behaviors (accounting for at least 96% of the total observation time for each age cohort) accompanies the switch from low to high aggression. A series of control experiments, with single flies in the test arenas, indicates that these changes occur in the absence of the performance of aggressive behaviors. This parallel ontogeny of aggressive and non-aggressive behaviors has implications for understanding how the entire behavioral repertoire may be organized and reorganized to accommodate the needs of the organism.