The genetic basis of male fertility in relation to haplodiploid reproduction in Leptopilina clavipes (Hymenoptera: Figitidae)

Traits under relaxed selection are expected to become reduced or disappear completely, a process called vestigialization. In parthenogenetic populations, traits historically involved in sexual reproduction are no longer under selection and potentially subject to such reduction. In Leptopilina clavipes, thelytokous (parthenogenetic) populations are infected by Wolbachia bacteria. Arrhenotokous populations do not harbor Wolbachia. When antibiotics are applied to infected females, they are cured from their infection and males arise. Such males are capable of producing offspring with uninfected females, but with lower fertilization success than sexual males. This can be attributed to the lack of selection on male fertility in thelytokous lines. In this study we used this variation in L. clavipes male fertility to determine the genetic basis of this trait. Males from cured thelytokous populations were crossed to females from uninfected populations. Using AFLP markers, a genetic linkage map was generated, consisting of five linkage groups and spanning a total distance of 219.9 cM. A single QTL of large effect (explaining 46.5% of the phenotypic variance) was identified for male fertility, which we call male fertility factor (mff). We discuss possible mechanisms underlying the effect of mff, as well as mechanisms involved in vestigialization of traits involved in sexual reproduction.     

Sperm chromatin remodelling and Wolbachia-induced cytoplasmic incompatibility in Drosophila.

Wolbachia pipientis is an obligate bacterial endosymbiont, which has successfully invaded approximately 20% of all insect species by manipulating their normal developmental patterns. Wolbachia-induced phenotypes include parthenogenesis, male killing, and, most notably, cytoplasmic incompatibility. In the future these phenotypes might be useful in controlling or modifying insect populations but this will depend on our understanding of the basic molecular processes underlying insect fertilization and development. Wolbachia-infected Drosophila simulans express high levels of cytoplasmic incompatibility in which the sperm nucleus is modified and does not form a normal male pronucleus when fertilizing eggs from uninfected females. The sperm modification is somehow rescued in eggs infected with the same strain of Wolbachia. Thus, D. simulans has become an excellent model organism for investigating the manner in which endosymbionts can alter reproductive programs in insect hosts. This paper reviews the current knowledge of Drosophila early development and particularly sperm function. Developmental mutations in Drosophila that are known to affect sperm function will also be discussed.incompatibility.     

Clonal genetic variation in a Wolbachia‐infected asexual wasp: horizontal transmission or historical sex?

Wolbachia are endocellular bacteria known for manipulating the reproductive systems of many of their invertebrate hosts. Wolbachia are transmitted vertically from mother to offspring. In addition, new infections result from horizontal transmission between different host species. However, to what extent horizontal transmission plays a role in the spread of a new infection through the host population is unknown. Here, we investigate whether horizontal transmission of Wolbachia can explain clonal genetic variation in natural populations of Leptopilina clavipes, a parasitoid wasp infected with a parthenogenesis‐inducing Wolbachia. We assessed variance of markers on the nuclear, mitochondrial and Wolbachia genomes. The nuclear and mitochondrial markers displayed significant and congruent variation among thelytokous wasp lineages, showing that multiple lineages have become infected with Wolbachia. The alternative hypothesis in which a single female became infected, the daughters of which mated with males (thus introducing nuclear genetic variance) cannot account for the presence of concordant variance in mtDNA. All Wolbachia markers, including the hypervariable wsp gene, were invariant, suggesting that only a single strain of Wolbachia is involved. These results show that Wolbachia has transferred horizontally to infect multiple female lineages during the early spread through L. clavipes. Remarkably, multiple thelytokous lineages have persisted side by side in the field for tens of thousands of generations.     

Sexual functionality of Leptopilina clavipes (Hymenoptera: Figitidae) after reversing Wolbachia-induced parthenogenesis

Females infected with parthenogenesis-inducing Wolbachia bacteria can be cured from their infection by antibiotic treatment, resulting in male production. In most cases, however, these males are either sexually not fully functional, or infected females have lost the ability to reproduce sexually. We studied the decay of sexual function in males and females of the parasitoid Leptopilina clavipes. In western Europe, infected and uninfected populations occur allopatrically, allowing for an investigation of both male and female sexual function. This was made by comparing females and males induced from different parthenogenetic populations with those from naturally occurring uninfected populations. Our results indicate that although males show a decay of sexual function, they are still able to fertilize uninfected females. Infected females, however, do not fertilize their eggs after mating with males from uninfected populations. The absence of genomic incompatibilities suggests that these effects are due to the difference in mode of reproduction.     

Wolbachia infection lowers fertile sperm transfer in a moth

The endosymbiotic bacterium Wolbachia pipientis manipulates host reproduction by rendering infected males reproductively incompatible with uninfected females (cytoplasmic incompatibility; CI). CI is believed to occur as a result of Wolbachia-induced modifications to sperm during maturation, which prevent infected sperm from initiating successful zygote development when fertilizing uninfected females' eggs. However, the mechanism by which CI occurs has been little studied outside the genus Drosophila. Here, we show that in the sperm heteromorphic Mediterranean flour moth, Ephestia kuehniella, infected males transfer fewer fertile sperm at mating than uninfected males. In contrast, non-fertile apyrene sperm are not affected. This indicates that Wolbachia may only affect fertile sperm production and highlights the potential of the Lepidoptera as a model for examining the mechanism by which Wolbachia induces CI in insects.     

Cytology of Wolbachia-induced parthenogenesis in Leptopilina clavipes (Hymenoptera: Figitidae).

Parthenogenesis induced by cytoplasmatically inherited Wolbachia bacteria has been found in a number of arthropod species, mainly Hymenoptera. Previously, two different forms of diploidy restoration have been reported to underlie parthenogenesis induction in Hymenoptera by Wolbachia. Both are a form of gamete duplication, but each differs in their timing. We investigated the cytology of the early embryonic development of a Wolbachia-infected strain of the parasitoid wasp Leptopilina clavipes and compared it with that of an uninfected sexual strain. Both strains have a similar meiosis. In the infected parthenogenetic strain, diploidy is restored by anaphase restitution during the first somatic mitosis, similar to Trichogramma, but not to Muscidifurax. Our results confirm the occurrence of different cytological mechanisms of diploidy restoration associated with parthenogenesis-inducing Wolbachia in the order Hymenoptera.     

Opposite sex-specific effects of Wolbachia and interference with the sex determination of its host Ostrinia scapulalis

In the adzuki bean borer, Ostrinia scapulalis, the sex ratio in most progenies is 1 : 1. Females from Wolbachia-infected matrilines, however, give rise to all-female broods when infected and to all-male broods when cured of the infection. These observations had been interpreted as Wolbachia-induced feminization of genetic males into functional females. Here, we show that the interpretation is incorrect. Females from both lines have a female karyotype with a WZ sex-chromosome constitution while males are ZZ. At the time of hatching from eggs, WZ and ZZ individuals are present at a 1 : 1 ratio in broods from uninfected, infected and cured females. In broods from Wolbachia-infected females, ZZ individuals die during larval development, whereas in those from cured females, WZ individuals die. Hence, development of ZZ individuals is impaired by Wolbachia but development of WZ females may require the presence of Wolbachia in infected matrilines. Sexual mosaics generated (i) by transfection of uninfected eggs and (ii) by tetracycline treatment of Wolbachia-infected mothers prior to oviposition were ZZ in all tissues, including typically female organs. We conclude that: (i) Wolbachia acts by manipulating the sex determination of its host; and (ii) although sexual mosaics can survive, development of a normal female is incompatible with a ZZ genotype.     

Wolbachia-induced unidirectional cytoplasmic incompatibility and speciation: mainland-island model

Bacteria of the genus Wolbachia are among the most common endosymbionts in the world. In many insect species these bacteria induce a sperm-egg incompatibility between the gametes of infected males and uninfected females, commonly called unidirectional cytoplasmic incompatibility (CI). It is generally believed that unidirectional CI cannot promote speciation in hosts because infection differences between populations will be unstable and subsequent gene flow will eliminate genetic differences between diverging populations. In the present study we investigate this question theoretically in a mainland-island model with migration from mainland to island. Our analysis shows that (a) the infection polymorphism is stable below a critical migration rate, (b) an (initially) uninfected "island" can better maintain divergence at a selected locus (e.g. can adapt locally) in the presence of CI, and (c) unidirectional CI selects for premating isolation in (initially) uninfected island populations if they receive migration from a Wolbachia-infected mainland. Interestingly, premating isolation is most likely to evolve if levels of incompatibility are intermediate and if either the infection causes fecundity reductions or Wolbachia transmission is incomplete. This is because under these circumstances an infection pattern with an infected mainland and a mostly uninfected island can persist in the face of comparably high migration. We present analytical results for all three findings: (a) a lower estimation of the critical migration rate in the presence of local adaptation, (b) an analytical approximation for the gene flow reduction caused by unidirectional CI, and (c) a heuristic formula describing the invasion success of mutants at a mate preference locus. These findings generally suggest that Wolbachia-induced unidirectional CI can be a factor in divergence and speciation of hosts.     

Male-killing Wolbachia in a flour beetle

The bacteria in the genus Wolbachia are cytoplasmically inherited symbionts of arthropods. Infection often causes profound changes in host reproduction, enhancing bacterial transmission and spread in a population. The reproductive alterations known to result from Wolbachia infection include cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males, fecundity enhancement, male killing and, perhaps, lethality Here, we report male killing in a third insect, the black flour beetle Tribolium madens, based on highly female-biased sex ratios of progeny from females infected with Wolbachia. The bias is cytoplasmic in nature as shown by repeated backcrossing of infected females with males of a naturally uninfected strain. Infection also lowers the egg hatch rates significantly to approximately half of those observed for uninfected females. Treatment of the host with antibiotics eliminated infection, reverted the sex ratio to unbiased levels and increased the percentage hatch. Typically Wolbachia infection is transmitted from mother to progeny, regardless of the sex of the progeny; however, infected T. madens males are never found. Virgin females are sterile, suggesting that the sex-ratio distortion in T. madens results from embryonic male killing rather than parthenogenesis. Based on DNA sequence data, the male-killing strain of Wolbachia in T. madens was indistinguishable from the CI-inducing Wolbachia in Tribolium confusum, a closely related beetle. Our findings suggest that host symbiont interaction effects may play an important role in the induction of Wolbachia reproductive phenotypes.     

Which way to manipulate host reproduction? Wolbachia that cause cytoplasmic incompatibility are easily invaded by sex ratio-distorting mutants

The bacterium Wolbachia manipulates its hosts by inducing cytoplasmic incompatibility (CI), where zygotes formed from crosses between uninfected mothers and infected fathers die. In addition, it distorts the host's sex ratio via male killing, parthenogenesis induction, or feminization. Here, we model transitions between these states, examining the evolution of mutants of CI strains that retain both the ability to induce and resist CI but, in addition, cause sex ratio distortion. The model shows that CI strains are highly susceptible to invasion and subsequent elimination by these mutants. For all three types of sex ratio distortion, there is some parameter space in which the strain showing sex ratio distortion becomes extinct following exclusion of the progenitor CI strain, leaving the population uninfected. Extinction of the new Wolbachia strain is common for the case of male killing but rarer for parthenogenesis induction and feminization. Our models predict that CI strains of Wolbachia will occur most commonly in hosts that are male heterogametic, where there is little interaction between siblings because these hosts are unlikely to favor the spread of male killing, feminization, or parthenogenesis induction. The models raise the question of why CI strains apparently predominate in nature, and it is suggested that this is a result of either fewer restrictions on CI strains spreading through novel host populations or restrictions to the mutability of Wolbachia strains.     

Molecular evidence for multiple infections of a new subgroup of Wolbachia in the European raspberry beetle Byturus tomentosus

Wolbachia, a group of maternally inherited intracellular parasitic bacteria, alter host reproduction, including the induction of thelytokous parthenogenesis, feminization of genetic males, son killing and, most commonly, the induction of cytoplasmic incompatibility (CI), in a diverse array of arthropods. CI can result in infertility and has attracted attention because of its potential in biological control and as an agent in speciation. Although there has been some analysis of overall infection rates in arthropods and within individual insect orders, there has been little exploration of within-species variation. In this study, primers specific for the ftsZ gene of Wolbachia were used to amplify it from different geographical samples of the European raspberry beetle (Byturus tomentosus), confirming the presence of Wolbachia. More than 99% of UK individuals were found to be infected with Wolbachia and 97% of these B. tomentosus beetles harboured multiple infections. Preliminary analysis of B. tomentosus beetles from continental European populations revealed a lower level of infection (24%) than those from the UK. Phylogenetic analysis using the ftsZ DNA sequences places Wolbachia from B. tomentosus into a new clade (Abt) within the A division, with some revisions to the existing Wolbachia phylogeny.     

Feminizing Wolbachia in an insect,Ostrinia furnacalis (Lepidoptera: Crambidae)

Wolbachia, which forms a group of maternally inherited bacteria in arthropods, often cause reproduction alterations in their hosts, such as cytoplasmic incompatibility, parthenogenesis, male-killing, hybrid breakdown and feminization. To date, Wolbachia-induced feminization has been reported only in isopods. Here we report that a Wolbachia strain feminizes an insect host, Ostrinia furnacalis. Among 79 wild females of O. furnacalis examined, Wolbachia infection was detected in 13 females. Twelve of the 13 infected females produced all-female progenies, and this trait was maternally inherited. Tetracycline treatment of thelygenic matrilines resulted in the production of all-male progenies. The present findings indicate that the Wolbachia infection induces feminization of genetic males in O. furnacalis. Differences in the Wolbachia-induced feminization in O. furnacalis and that in isopods are discussed along with the differences in sex determination mechanisms between insects and isopods. Phylogenetic analysis of the wsp gene sequence of Wolbachia suggests independent evolutionary origins for the Wolbachia-induced feminizations in O. furnacalis and in isopods. Our findings over 5 years suggest that the infection has been maintained at a low prevalence in the O. furnacalis population.     

Wolbachia plays no role in the one-way reproductive incompatibility between the hybridizing field crickets Gryllus firmus and G. pennsylvanicus

Wolbachia are cytoplasmically inherited alpha-proteobacteria that can cause cytoplasmic incompatibility (CI) in insects. This incompatibility between sperm and egg is evident when uninfected females mate with infected males. Wolbachia-driven reproductive incompatibilities are of special interest because they may play a role in speciation. However, the presence of Wolbachia does not always imply incompatibility. The field crickets Gryllus firmus and G. pennsylvanicus exhibit a very clear unidirectional incompatibility and have been cited as a possible example of Wolbachia-induced CI. Here, we conduct curing experiments, intra- and interspecific crosses, cytological examination of Wolbachia in testes and Wolbachia quantifications through real-time PCR. All of our data strongly suggest that Wolbachia are not involved in the reproductive incompatibility between G. firmus and G. pennsylvanicus.     

Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps

The intracellular bacterium Wolbachia is one of the most common symbionts in arthropods and, because of its manipulative effects on host reproduction, is assumed to be an important factor in several evolutionary processes. These bacteria are mainly vertically transmitted from mother to daughter through the egg cytoplasm, and horizontal transmission is generally assumed to be rare. Here, we show natural inter- and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia between parasitoid wasps of the genus Trichogramma. Horizontal transfer was observed when infected and uninfected larvae shared the same host egg. This is the first report, to our knowledge, on interspecific horizontal transfer of Wolbachia between closely related sympatric species. Some originally uninfected immature wasps acquired Wolbachia while inside the host egg, but not all of these newly infected females exhibited the parthenogenesis phenotype. In general, intraspecific horizontal transfer was more successful than interspecific transfer. Wolbachia underwent vertical transmission in the new species but the infection tended to be lost within several generations. Our results have important implications for understanding the evolution of Wolbachia-host associations.     

Infection polymorphism and cytoplasmic incompatibility in Hymenoptera-Wolbachia associations

Most cases of Wolbachia infection so far documented in haplodiploid Hymenoptera are associated with parthenogenesis induction. Only three examples of Wolbachia-mediated cytoplasmic incompatibility (CI) have been reported, resulting either in haploidisation of fertilised eggs, which develop into viable males, or in their death. To better document this variability, we studied two new Wolbachia-wasp associations involving Drosophila parasitoids. In Trichopria cf. drosophilae, individuals are infected by two different Wolbachia variants, populations are nearly totally infected, and Wolbachia induces incomplete CI resulting in death of the fertilised eggs. On the other hand, Pachycrepoideus dubius harbours only one bacterial variant, populations are polymorphic for infection, and Wolbachia has no detectable effect. These two cases show that the range of variation in Wolbachia's effects in Hymenoptera is as wide as in diploids, extending from complete CI to an undetectable effect. Cases so far studied show some parallel between the strength of incompatibility, the number of Wolbachia variants infecting each wasp, and the natural infection frequency. These empirical data support theoretical models predicting evolution of CI towards lower levels, resulting in the decline and ultimate loss of infection, and place multiple infections as being an important factor in the evolution of host-Wolbachia associations.     

A genetic test of the mechanism of Wolbachia-induced cytoplasmic incompatibility in Drosophila

Cytoplasmic bacteria of the genus Wolbachia are best known as the cause of cytoplasmic incompatibility (CI): many uninfected eggs fertilized by Wolbachia-modified sperm from infected males die as embryos. In contrast, eggs of infected females rescue modified sperm and develop normally. Although Wolbachia cause CI in at least five insect orders, the mechanism of CI remains poorly understood. Here I test whether the target of Wolbachia-induced sperm modification is the male pronucleus (e.g., DNA or pronuclear proteins) or some extranuclear factor from the sperm required for embryonic development (e.g., the paternal centrosome). I distinguish between these hypotheses by crossing gynogenetic Drosophila melanogaster females to infected males. Gynogenetic females produce diploid eggs whose normal development requires no male pronucleus but still depends on extranuclear paternal factors. I show that when gynogenetic females are crossed to infected males, uniparental progeny with maternally derived chromosomes result. This finding shows that Wolbachia impair the male pronucleus but no extranuclear component of the sperm.     

From parasite to mutualist: rapid evolution of Wolbachia in natural populations of Drosophila

Wolbachia are maternally inherited bacteria that commonly spread through host populations by causing cytoplasmic incompatibility, often expressed as reduced egg hatch when uninfected females mate with infected males. Infected females are frequently less fecund as a consequence of Wolbachia infection. However, theory predicts that because of maternal transmission, these "parasites" will tend to evolve towards a more mutualistic association with their hosts. Drosophila simulans in California provided the classic case of a Wolbachia infection spreading in nature. Cytoplasmic incompatibility allowed the infection to spread through individual populations within a few years and from southern to northern California (more than 700 km) within a decade, despite reducing the fecundity of infected females by 15%-20% under laboratory conditions. Here we show that the Wolbachia in California D. simulans have changed over the last 20 y so that infected females now exhibit an average 10% fecundity advantage over uninfected females in the laboratory. Our data suggest smaller but qualitatively similar changes in relative fecundity in nature and demonstrate that fecundity-increasing Wolbachia variants are currently polymorphic in natural populations.     

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host-symbiont co-evolution.     

Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations

Wolbachia are maternally inherited endosymbionts that can invade arthropod populations through manipulation of their reproduction. In mosquitoes, Wolbachia induce embryonic death, known as cytoplasmic incompatibility (CI), whenever infected males mate with females either uninfected or infected with an incompatible strain. Although genetic determinants of CI are unknown, a functional model involving the so-called mod and resc factors has been proposed. Natural populations of Culex pipiens mosquito display a complex CI relationship pattern associated with the highest Wolbachia (wPip) genetic polymorphism reported so far. We show here that C. pipiens populations from La Réunion, a geographically isolated island in the southwest of the Indian Ocean, are infected with genetically closely related wPip strains. Crossing experiments reveal that these Wolbachia are all mutually compatible. However, crosses with genetically more distant wPip strains indicate that Wolbachia strains from La Réunion belong to at least five distinct incompatibility groups (or crossing types). These incompatibility properties which are strictly independent from the nuclear background, formally establish that in C. pipiens, CI is controlled by several Wolbachia mod/resc factors.     

Cardinium symbionts cause cytoplasmic incompatibility in spider mites

Intracellular symbiotic bacteria belonging to the Cytophaga-Flavobacterium-Bacteroides lineage have recently been described and are widely distributed in arthropod species. The newly discovered bacteria, named Cardinium sp, cause the expression of various reproductive alterations in their arthropod hosts, including cytoplasmic incompatibility (CI), induction of parthenogenesis and feminization of genetic males. We detected 16S ribosomal DNA sequences similar to those of Cardinium from seven populations of five spider mite species, suggesting a broad distribution of infection of Cardinium in spider mites. To clarify the effect of Cardinium on the reproductive traits of the infected spider mites, infected mites were crossed with uninfected mites for each population. In one of the populations, Eotetranychus suginamensis, CI was induced when infected males were crossed with uninfected females. The other six populations of four species showed no reproductive abnormalities in the F(1) generation, but the possibility of CI effects in the F(2) generation remains to be tested. One species of spider mite, Tetranychus pueraricola, harbored both Cardinium and Wolbachia, but these symbionts seemed to have no effect on the reproduction of the host, even when the host was infected independently with each symbiont.