Cytoplasmic Incompatibility in Drosophila Simulans: Dynamics and Parameter Estimates from Natural Populations

In Drosophila simulans, cytoplasmically transmitted Wolbachia microbes cause reduced egg hatch when infected males mate with uninfected females. A Wolbachia infection and an associated mtDNA variant have spread northward through California since 1986. PCR assays show that Wolbachia infection is prevalent throughout the continental US and Central and South America, but some lines from Florida and Ecuador that are PCR-positive for Wolbachia do not cause incompatibility. We estimate from natural populations infection frequencies and the transmission and incompatibility parameter values that affect the spread of the infection. On average, infected females from nature produce 3-4% uninfected ova. Infected females with relatively low fidelity of maternal transmission show partial incompatibility with very young infected laboratory males. Nevertheless, crosses between infected flies in nature produce egg-hatch rates indistinguishable from those produced by crosses between uninfected individuals. Incompatible crosses in nature produce hatch rates 30-70% as high as those from compatible crosses. Wild-caught infected and uninfected females are equally fecund in the laboratory. Incompatibility decreases with male age, and age-specific incompatibility levels suggest that males mating in nature may often be 2 or 3 weeks old. Our parameter estimates accurately predict the frequency of Wolbachia infection in California populations.     

Factors Affecting the Distribution of Cytoplasmic Incompatibility in Drosophila Simulans

In Drosophila simulans a Wolbachia-like microorganism is responsible for reduced egg-hatch when infected males mate with uninfected females. Both incompatibility types have previously been found in North America, Europe and Africa. Some California populations have remained polymorphic for over two years, and the infection is apparently spreading in central California. Egg hatch proportions for wild-caught females from polymorphic populations show that the incompatibility system acts in nature, but egg mortality rates are apparently lower than observed in laboratory populations. Although infected females maintained under various laboratory conditions never produce uninfected offspring, some wild-caught infected females produce both infected and uninfected progeny. This helps explain the persistence of a low frequency of uninfected flies in predominantly infected populations and may also explain the other polymorphisms observed. Fitness comparisons of infected and uninfected stocks, including both larval and adult fitness components, indicate that fecundity may be the component most affected. Infected females suffer a fecundity reduction of 10-20% in the laboratory, but the reduction seems to be smaller in nature. A theoretical analysis provides some insight into the population biology of the infection.     

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.     

Cytoplasmic Incompatibility in Australian Populations of Drosophila Melanogaster

In Drosophila melanogaster, weak incompatibility in crosses between infected and uninfected strains is associated with a Wolbachia microorganism. Crosses between infected males and uninfected females show a reduction (15-30%) in egg hatch. Progeny tests indicated that the infection is widespread in Australian D. melanogaster populations and that populations are polymorphic for the presence of the infection. The infection status of 266 lines from 12 populations along the eastern coast of Australia was determined by 4',6-diamidino-2-phenylindole (DAPI) staining of embryos. All populations contained both infected and uninfected flies. Infection frequencies varied between populations but there was no discernible geographical pattern. Laboratory experiments indicated that the infection was not associated with a reduction in fecundity as in Drosophila simulans. Incompatibility levels could not be increased by laboratory selection on isofemale lines. Factors contributing to the persistence of the infection in D. melanogaster populations are discussed.     

Wolbachia and cytoplasmic incompatibility in the California Culex pipiens mosquito species complex: parameter estimates and infection dynamics in natural populations

Before maternally inherited bacterial symbionts like Wolbachia, which cause cytoplasmic incompatibility (CI; reduced hatch rate) when infected males mate with uninfected females, can be used in a program to control vector-borne diseases it is essential to understand their dynamics of infection in natural arthropod vector populations. Our study had four goals: (1) quantify the number of Wolbachia strains circulating in the California Culex pipiens species complex, (2) investigate Wolbachia infection frequencies and distribution in natural California populations, (3) estimate the parameters that govern Wolbachia spread among Cx. pipiens under laboratory and field conditions, and (4) use these values to estimate equilibrium levels and compare predicted infection prevalence levels to those observed in nature. Strain-specific PCR, wsp gene sequencing, and crossing experiments indicated that a single Wolbachia strain infects Californian Cx. pipiens. Infection frequency was near or at fixation in all populations sampled for 2 years along a >1000-km north-south transect. The combined statewide infection frequency was 99.4%. Incompatible crosses were 100% sterile under laboratory and field conditions. Sterility decreased negligibly with male age in the laboratory. Infection had no significant effect on female fecundity under laboratory or field conditions. Vertical transmission was >99% in the laboratory and approximately 98.6% in the field. Using field data, models predicted that Wolbachia will spread to fixation if infection exceeds an unstable equilibrium point above 1.4%. Our estimates accurately predicted infection frequencies in natural populations. If certain technical hurdles can be overcome, our data indicate that Wolbachia can invade vector populations as part of an applied transgenic strategy for vector-borne disease reduction.     

Mutualistic Wolbachia infection in Aedes albopictus: accelerating cytoplasmic drive

Maternally inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods, often behaving as reproductive parasites by manipulating host reproduction to enhance the vertical transmission of infections. One manipulation is cytoplasmic incompatibility (CI), which causes a significant reduction in brood hatch and promotes the spread of the maternally inherited Wolbachia infection into the host population (i.e., cytoplasmic drive). Here, we have examined a Wolbachia superinfection in the mosquito Aedes albopictus and found the infection to be associated with both cytoplasmic incompatibility and increased host fecundity. Relative to uninfected females, infected females live longer, produce more eggs, and have higher hatching rates in compatible crosses. A model describing Wolbachia infection dynamics predicts that increased fecundity will accelerate cytoplasmic drive rates. To test this hypothesis, we used population cages to examine the rate at which Wolbachia invades an uninfected Ae. albopictus population. The observed cytoplasmic drive rates were consistent with model predictions for a CI-inducing Wolbachia infection that increases host fecundity. We discuss the relevance of these results to both the evolution of Wolbachia symbioses and proposed applied strategies for the use of Wolbachia infections to drive desired transgenes through natural populations (i.e., population replacement strategies).     

Cytoplasmic incompatibility in Drosophila populations: influence of assortative mating on symbiont distribution.

Cytoplasmic incompatibility is known to occur between strains of both Drosophila simulans and D. melanogaster. Incompatibility is associated with the infection of Drosophila with microorganismal endosymbionts. This paper reports survey work conducted on strains of D. simulans and D. melanogaster from diverse geographical locations finding that infected populations are relatively rare and scattered in their distribution. The distribution of infected populations of D. simulans appears to be at odds with deterministic models predicting the rapid spread of the infection through uninfected populations. Examination of isofemale lines from four localities in California where populations appear to be polymorphic for the infection failed to find evidence for consistent assortative mating preferences between infected and uninfected populations that may explain the basis for the observed polymorphism.     

Behavior of Wolbachia endosymbionts from Drosophila simulans in Drosophila serrata, a novel host

Many species harbor the incompatibility-inducing microbe Wolbachia, a maternally inherited endoparasite that causes reduced egg hatch in crosses between infected males and uninfected females. Infected females are immune to this effect, which gives them a relative fitness advantage that results in the spread of the infection. The strength of incompatibility, fitness deficits associated with the infection, and transmission rate from mother to offspring largely determine the rate and extent of spread of Wolbachia in a population. We transferred Wolbachia from Drosophila simulans to Drosophila serrata, a novel host, and compared parameter estimates with those from three naturally occurring Drosophila-Wolbachia associations believed to be of different ages. Transfected D. serrata showed strong incompatibility, low transmission efficiency, and an associated fitness deficit, and they would probably be unable to spread in nature. The comparisons generally supported the predicted evolution of a host-Wolbachia association. The parameters peculiar to any given host-Wolbachia association may determine whether the microbial strain can spread in that host.     

Dynamics of double and single Wolbachia infections in Drosophila simulans from New Caledonia

The bacterial symbiont Wolbachia can cause cytoplasmic incompatibility in Drosophila simulans flies: if an infected male mates with an uninfected female, or a female with a different strain of Wolbachia, there can be a dramatic reduction in the number of viable eggs produced. Here we explore the dynamics associated with double and single Wolbachia infections in New Caledonia. Doubly infected females were compatible with all males in the population, explaining the high proportion of doubly infected flies. In this study, males that carry only wHa or wNo infections showed reduced incompatibility when mated to uninfected females, compared with previous reports. These data suggest that either the DNA of these bacterial isolates have diverged from those previously collected, or the genetic background of the host has lead to a reduction in the phenotype of incompatibility. Mitochondrial sequence polymorphism at two sites within the host genome was assayed to investigate population structure related to infection types. There was no correlation between sequence polymorphism and infection type suggesting that double infections are the stable type, with singly infected and uninfected flies arising from stochastic segregation of bacterial strains. Finally, we discuss the nomenclature of Wolbachia strain designation.     

Characterization of non-cytoplasmic incompatibility inducing Wolbachia in two continental African populations of Drosophila simulans

Wolbachia is an endocellular bacterium infecting arthropods and nematodes. In arthropods, it invades host populations through various mechanisms, affecting host reproduction, the most common of which being cytoplasmic incompatibility (CI). CI is an embryonic mortality occurring when infected males mate with uninfected females or females infected by a different Wolbachia strain. This phenomenon is observed in Drosophila simulans, an intensively studied Wolbachia host, harbouring at least five distinct bacterial strains. In this study, we investigate various aspects of the Wolbachia infections occurring in two continental African populations of D. simulans: CI phenotype, phylogenetic position based on the wsp gene and associated mitochondrial haplotype. From the East African population (Tanzania), we show that (i) the siIII mitochondrial haplotype occurs in continental populations, which was unexpected based on the current views of D. simulans biogeography, (ii) the wKi strain (that rescues from CI while being unable to induce it) is very closely related to the CI-inducing strain wNo, (iii) wKi and wNo might not derive from a unique infection event, and (iv) wKi is likely to represent the same entity as the previously described wMa variant. In the West African population (Cameroon), the Wolbachia infection was found identical to the previously described wAu, which does not induce CI. This finding supports the view that wAu might be an ancient infection in D. simulans.     

Offsetting effects of Wolbachia infection and heat shock on sperm production in Drosophila simulans: analyses of fecundity, fertility and accessory gland proteins

Infection in Drosophila simulans with the endocellular symbiont Wolbachia pipientis results in egg lethality caused by failure to properly initiate diploid development (cytoplasmic incompatibility, CI). The relationship between Wolbachia infection and reproductive factors influencing male fitness has not been well examined. Here we compare infected and uninfected strains of D. simulans for (1) sperm production, (2) male fertility, and (3) the transfer and processing of two accessory gland proteins, Acp26Aa or Acp36De. Infected males produced significantly fewer sperm cysts than uninfected males over the first 10 days of adult life, and infected males, under varied mating conditions, had lower fertility compared to uninfected males. This fertility effect was due to neither differences between infected and uninfected males in the transfer and subsequent processing of accessory gland proteins by females nor to the presence of Wolbachia in mature sperm. We found that heat shock, which is known to decrease CI expression, increases sperm production to a greater extent in infected compared to uninfected males, suggesting a possible link between sperm production and heat shock. Given these results, the roles Wolbachia and heat shock play in mediating male gamete production may be important parameters for understanding the dynamics of infection in natural populations.     

Population dynamics of the Wolbachia infection causing cytoplasmic incompatibility in Drosophila melanogaster.

Field populations of Drosophila melanogaster are often infected with Wolbachia, a vertically transmitted microorganism. Under laboratory conditions the infection causes partial incompatibility in crosses between infected males and uninfected females. Here we examine factors influencing the distribution of the infection in natural populations. We show that the level of incompatibility under field conditions was much weaker than in the laboratory. The infection was not transmitted with complete fidelity under field conditions, while field males did not transmit the infection to uninfected females and Wolbachia did not influence sperm competition. There was no association between field fitness as measured by fluctuating asymmetry and the infection status of adults. Infected field females were smaller than uninfecteds in some collections from a subtropical location, but not in other collections from the same location. Laboratory cage studies showed that the infection did not change in frequency when populations were maintained at a low larval density, but it decreased in frequency at a high larval density. Monitoring of infection frequencies in natural populations indicated stable frequencies in some populations but marked fluctuations in others. Simple models suggest that the infection probably provides a fitness benefit for the host in order to persist in populations. The exact nature of this benefit remains elusive.     

Within- and between-population variation for Wolbachia-induced reproductive incompatibility in a haplodiploid mite

Wolbachia pipientis is a bacterium that induces cytoplasmic incompatibility (CI), the phenomenon in which infected males are reproductively incompatible with uninfected females. CI spreads in a population of hosts because it reduces the fitness of uninfected females relative to infected females. CI encompasses two steps: modification (mod) of sperm of infected males and rescuing (resc) of these chromosomes by Wolbachia in the egg. Infections associated with CI have mod+ resa+ phenotypes. However, mod- resc+ phenotypes also exist; these do not result in CI. Assuming mod/resc phenotypes are properties of the symbiont, theory predicts that mod- resc+ infections can only spread in a host population where a mod+ resc+ infection already occurs. A mod- resc+ infection spreads if the cost it imposes on the infected females is lower than the cost inflicted by the resident (mod+ resc+) infection. Furthermore, introduction of a mod- Wolbachia eventually drives infection to extinction. The uninfected population that results can be recolonized by a CI-causing Wolbachia. Here, we investigated whether variability for induction of CI was present in two Tetranychus urticae populations. In one population all isofemale lines tested were mod-. In the other, mod+ resc+ and mod- resc+ isofemale lines coexisted. We found no evidence for a cost difference to females expressing either type (mod-/-). Infections in the two populations could not be distinguished based on sequences of two Wolbachia genes. We consider the possibility that mod- is a host effect through a population dynamics model. A mod- host allele leads to infection extinction in the absence of fecundity differences. Furthermore, the uninfected population that results is immune to reestablishment of the (same) CI-causing Wolbachia.     

Fitness advantage and cytoplasmic incompatibility in Wolbachia single- and superinfected Aedes albopictus

Wolbachia are obligate, maternally inherited, intracellular bacteria that infect numerous insects and other invertebrates. Wolbachia infections have evolved multiple mechanisms to manipulate host reproduction and facilitate invasion of naive host populations. One such mechanism is cytoplasmic incompatibility (CI) that occurs in many insect species, including Aedes albopictus (Asian tiger mosquito). The multiple Wolbachia infections that occur naturally in A. albopictus make this mosquito a useful system in which to study CI. Here, experiments employ mosquito strains that have been introgressed to provide genetically similar strains that harbor differing Wolbachia infection types. Cytoplasmic incompatibility levels, host longevity, egg hatch rates, and fecundity are examined. Crossing results demonstrate a pattern of additive unidirectional cytoplasmic incompatibility. Furthermore, relative to uninfected females, infected females are at a reproductive advantage due to both cytoplasmic incompatibility and a fitness increase associated with Wolbachia infection. In contrast, no fitness difference was observed in comparisons of single- and superinfected females. We discuss the observed results in regard to the evolution of the Wolbachia/A. albopictus symbiosis and the observed pattern of Wolbachia infection in natural populations.     

Increased male mating rate in Drosophila is associated with Wolbachia infection

The maternally inherited bacterium Wolbachia pipientis infects 25-75% of arthropods and manipulates host reproduction to improve its transmission. One way Wolbachia achieves this is by inducing cytoplasmic incompatibility (CI), where crosses between infected males and uninfected females are inviable. Infected males suffer reduced fertility through CI and reduced sperm production. However, Wolbachia induce lower levels of CI in nonvirgin males. We examined the impact of Wolbachia on mating behaviour in male Drosophila melanogaster and D. simulans, which display varying levels of CI, and show that infected males mate at a higher rate than uninfected males in both species. This may serve to increase the spread of Wolbachia, or alternatively, may be a behavioural adaptation employed by males to reduce the level of CI. Mating at high rate restores reproductive compatibility with uninfected females resulting in higher male reproductive success thus promoting male promiscuity. Increased male mating rates also have implications for the transmission of Wolbachia.     

EVOLUTION OF INCOMPATIBILITY-INDUCING MICROBES AND THEIR HOSTS

In many insect species, males infected with microbes related to Wolbachia pipientis are “incompatible” with uninfected females. Crosses between infected males and uninfected females produce significantly fewer adult progeny than the other three possible crosses. The incompatibility-inducing microbes are usually maternally transmitted. Thus, incompatibility tends to confer a reproductive advantage on infected females in polymorphic populations, allowing these infections to spread. This paper analyzes selection on parasite and host genes that affect such incompatibility systems. Selection among parasite variants does not act directly on the level of incompatibility with uninfected females. In fact, selection favors rare parasite variants that increase the production of infected progeny by infected mothers, even if these variants reduce incompatibility with uninfected females. However, productivity-reducing parasites that cause partial incompatibility with hosts harboring alternative variants can be favored once they become sufficiently abundant locally. Thus, they may spread spatially by a process analogous to the spread of underdominant chromosome rearrangements. The dynamics of modifier alleles in the host are more difficult to predict, because such alleles will occur in both infected and uninfected individuals. Nevertheless, the relative fecundity of infected females compared to uninfected females, the efficiency of maternal transmission and the mutual compatibility of infected individuals all tend to increase under within-population selection on both host and parasite genes. In addition, selection on host genes favors increased compatibility between infected males and uninfected females. Although vertical transmission tends to harmonize host and parasite evolution, competition among parasite variants will tend to maintain incompatibility.     

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.     

Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D. sechellia

Abstract.— The intracellular bacterium Wolbachia invades arthropod host populations through various mechanisms, the most common of which being cytoplasmic incompatibility (CI). CI involves elevated embryo mortality when infected males mate with uninfected females or females infected with different, incompatible Wolbachia strains. The present study focuses on this phenomenon in two Drosophila species: D. simulans and D. sechellia . Drosophila simulans populations are infected by several Wolbachia strains, including w Ha and w No. Drosophila sechellia is infected by only two Wolbachia : w Sh and w Sn. In both Drosophila species, double infections with Wolbachia are found. As indicated by several molecular markers, w Ha is closely related to w Sh, and w No to w Sn. Furthermore, the double infections in the two host species are associated with closely related mitochondrial haplotypes, namely si I (associated with w Ha and w No in D. simulans ) and se (associated with w Sh and w Sn in D. sechellia ). To test the theoretical prediction that Wolbachia compatibility types can diverge rapidly, we injected w Sh and w Sn into D. simulans , to compare their CI properties to those of their sister strains w Ha and w No, respectively, in the same host genetic background. We found that within each pair of sister strains CI levels were similar and that sister strains were fully compatible. We conclude that the short period for which the Wolbachia sister strains have been evolving separated from each other was not sufficient for their CI properties to diverge significantly.     

Wolbachia transfer from Drosophila melanogaster into D. simulans: Host effect and cytoplasmic incompatibility relationships.

Wolbachia are maternally transmitted endocellular bacteria causing a reproductive incompatibility called cytoplasmic incompatibility (CI) in several arthropod species, including Drosophila. CI results in embryonic mortality in incompatible crosses. The only bacterial strain known to infect Drosophila melanogaster (wDm) was transferred from a D. melanogaster isofemale line into uninfected D. simulans isofemale lines by embryo microinjections. Males from the resulting transinfected lines induce >98% embryonic mortality when crossed with uninfected D. simulans females. In contrast, males from the donor D. melanogaster line induce only 18-32% CI on average when crossed with uninfected D. melanogaster females. Transinfected D. simulans lines do not differ from the D. melanogaster donor line in the Wolbachia load found in the embryo or in the total bacterial load of young males. However, >80% of cysts are infected by Wolbachia in the testes of young transinfected males, whereas only 8% of cysts are infected in young males from the D. melanogaster donor isofemale line. This difference might be caused by physiological differences between hosts, but it might also involve tissue-specific control of Wolbachia density by D. melanogaster. The wDm-transinfected D. simulans lines are unidirectionally incompatible with strains infected by the non-CI expressor Wolbachia strains wKi, wMau, or wAu, and they are bidirectionally incompatible with strains infected by the CI-expressor Wolbachia strains wHa or wNo. However, wDm-infected males do not induce CI toward females infected by the CI-expressor strain wRi, which is found in D. simulans continental populations, while wRi-infected males induce partial CI toward wDm-infected females. This peculiar asymmetrical pattern could reflect an ongoing divergence between the CI mechanisms of wRi and wDm. It would also confirm other results indicating that the factor responsible for CI induction in males is distinct from the factor responsible for CI rescue in females.     

Dynamics of Cytoplasmic Incompatibility and Mtdna Variation in Natural Drosophila Simulans Populations

In Drosophila simulans a cytoplasmically transmitted microorganism causes reduced egg hatch when infected males mate with uninfected females. The infection is rapidly spreading northward in California. Data on a specific mtDNA restriction site length polymorphism show that changes in the frequency of mtDNA variants are associated with this spread. All infected flies possess the same mtDNA allele, whereas the uninfected flies are polymorphic. Given that both paternal inheritance of the infection and imperfect maternal transmission have been demonstrated, one might expect instead that both infected and uninfected flies would possess both mtDNA variants. Our data suggest that imperfect female transmission of the infection (and/or the loss of the infection among progeny) is more common in nature than paternal transmission. A simple model of intrapopulation dynamics, with empirically supported parameter values, adequately describes the joint frequencies of the mtDNA variants and incompatibility types.