Phenotype and transmission efficiency of artificial and natural male-killing Spiroplasma infections in Drosophila melanogaster

Many insect species carry inherited Spiroplasma bacteria which act as important partners and antagonists. The nature of symbioses between Spiroplasma and insects has been most extensively studied in the interaction between male-killing Spiroplasma infection and Drosophila melanogaster. For historical reasons, these studies have largely focussed on the Spiroplasma strain known as NSRO, derived from Drosophila nebulosa and transinfected into D. melanogaster. More recently, D. melanogaster naturally infected with Spiroplasma were discovered. Whilst the well studied strain NSRO is closely related to that found natively in D. melanogaster, it is unclear whether strains from D. nebulosa reflect a natural interaction when placed in D. melanogaster. In this paper, we determine if NSRO has similar or different properties from strains of Spiroplasma naturally infecting D. melanogaster in terms of transmission efficiency and the strength and timing of male-killing. Native infections were observed to have higher transmission efficiency than introduced NSRO infections during the early phases of host reproduction, but not during late reproduction. The timing and intensity of male-killing did not differ between infection classes. As a precautionary measure, it is proposed that future work seeking to reveal the nature of coevolved Spiroplasma-Drosophila interactions use the native strain.     

Prevalence of a non-male-killing spiroplasma in natural populations of Drosophila hydei

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.     

Spiroplasma in Drosophila melanogaster Populations: Prevalence, Male-Killing, Molecular Identification, and No Association with Wolbachia

Spiroplasma endosymbionts are maternally transmitted bacteria that may kill infected sons resulting in the production of female-biased broods. The prevalence of male killers varies considerably both between and within species. Here, we evaluate the spatial and temporal status of male-killing and non-male-killing Spiroplasma infection in three Brazilian populations of Drosophila melanogaster, nearly a decade after the first occurrence report for this species. The incidence of the male-killing Spiroplasma ranged from close to 0 to 17.7?% (so far the highest estimate for a Drosophila species) with a suggestion of temporal decline in a population. We also found non-male-killing Spiroplasma coexisting in one population at lower prevalence (3-5?%), and we did not detect it in the other two. This may be taken as a suggestion of a spreading advantage conferred by the male-killing strategy. Sequencing two loci, we identified the phylogenetic position of Spiroplasma strains from the three localities, showing that all strains group closely in the poulsonii clade. Due to intensive sampling effort, we were able to test the association between Spiroplasma infections and another widespread endosymbiont, Wolbachia, whose prevalence ranged from 81.8 to 100?%. The prevalence of Wolbachia did not differ between Spiroplasma-infected and uninfected strains in our largest sample nor were the prevalences of the two endosymbionts associated across localities.     

Male genotype influences female reproductive investment in Drosophila melanogaster

In many species, males can influence the amount of resources their mates invest in reproduction. Two favoured hypotheses for this observation are that females assess male quality during courtship or copulation and alter their investment in offspring accordingly, or that males manipulate females to invest heavily in offspring produced soon after mating. Here, we examined whether there is genetic variation for males to influence female short-term reproductive investment in Drosophila melanogaster, a species with strong sexual selection and substantial sexual conflict. We measured the fecundity and egg size of females mated to males from multiple isofemale lines collected from populations around the globe. Although these traits were not strongly influenced by the male's population of origin, we found that 22 per cent of the variation in female short-term reproductive investment was attributable to the genotype of her mate. This is the first direct evidence that male D. melanogaster vary genetically in their proximate influence on female fecundity, egg size and overall reproductive investment.     

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.     

Male killing in three species of the tripunctata radiation of Drosophila (Diptera: Drosophilidae)

An excess of females in progenies is commonly referred to as sex-ratio (SR). In this report, we describe three new occurrences of SR in species of the tripunctata radiation, Drosophila neocardini , Drosophila ornatifrons and Drosophila paraguayensis. Repeated backcrosses with males from normal strains were used to maintain the SR strains in the laboratory, always with all-female broods. The egg–larva viability of the SR strains was approximately half that of a normal strain, and antibiotic treatment restored the production of males in SR strains. PCR screening with Spiroplasma -specific primers showed that only SR strains tested positive for Spiroplasma . The results showed that in the three species, SR was maternally inherited and involved early male-killing bacteria, most likely spiroplasmas. These new occurrences increase the number of cytoplasmic male killers described in Drosophila from 11 to 14. In this particular collection of flies, one quarter (3 out of 12) of the species from the tripunctata radiation of Drosophila was infected with a male killer, suggesting that this group may be a hot spot for the presence of male-killing organisms.     

Evolutionarily stable infection by a male-killing endosymbiont in Drosophila innubila: molecular evidence from the host and parasite genomes

Maternally inherited microbes that spread via male-killing are common pathogens of insects, yet very little is known about the evolutionary duration of these associations. The few examples to date indicate very recent, and thus potentially transient, infections. A male-killing strain of Wolbachia has recently been discovered in natural populations of Drosophila innubila. The population-level effects of this infection are significant: approximately 35% of females are infected, infected females produce very strongly female-biased sex ratios, and the resulting population-level sex ratio is significantly female biased. Using data on infection prevalence and Wolbachia transmission rates, infected cytoplasmic lineages are estimated to experience a approximately 5% selective advantage relative to uninfected lineages. The evolutionary history of this infection was explored by surveying patterns of polymorphism in both the host and parasite genomes, comparing the Wolbachia wsp gene and the host mtDNA COI gene to five host nuclear genes. Molecular data suggest that this male-killing infection is evolutionarily old, a conclusion supported with a simple model of parasite and mtDNA transmission dynamics. Despite a large effective population size of the host species and strong selection to evolve resistance, the D. innubila-Wolbachia association is likely at a stable equilibrium that is maintained by imperfect maternal transmission of the bacteria rather than partial resistance in the host species.     

Do female Drosophila melanogaster adaptively bias offspring sex ratios in relation to the age of their mate?

Modification of offspring sex ratios in response to parental quality is predicted when the long-term fitness returns of sons and daughters differ. One factor that may influence a mother's sex allocation decision is the quality (or attractiveness) of her mate. We investigated whether the sex ratios of offspring produced by female Drosophila melanogaster are biased with respect to the age of the males to which they are mated, and whether there is an adaptive basis for this phenomenon. We found that females mated to old males (13 d post-eclosion) initially produced a greater proportion of daughters than did females mated to young males (1 d post-eclosion). This pattern does not appear to be due to a systematic difference in the numbers or mortality of the X- and Y-bearing sperm originating from old and young fathers, as the overall sex ratios of all offspring produced from a single copulation did not differ between broods fathered by the two types of males. The sons of older males fared worse in competitive mating assays than did the sons of younger males, while daughters of old and young males were of comparable fitness. These results suggest that there is an adaptive basis for the observed sex ratio modification.     

Sex ratio and male killing in Siberian populations of Harmonia axyridis (Pass.)]

In populations of Harmonia axyridis Pall. from Novosibirsk and Kyzyl, females (three out of 34 studied) that produce exclusively female progeny were found. In one of the families studied, the inheritance of the male-killing trait was monitored over five generations. The male-killing trait was maternally inherited. The beetles of this family were infected with the bacteria that, according to the sequence analysis of the gene fragment for 16S rRNA, belong to the genus Spiroplasma (VI group).     

Wolbachia, sex ratio bias and apparent male killing in the harlequin beetle riding pseudoscorpion

Bacterial endosymbionts that manipulate host reproduction are now known to be widespread in insects and other arthropods. Since they inhabit the cytoplasm and are maternally inherited, these microorganisms can enhance their fitness by biasing host sex ratio in favour of females. At its most extreme, sex ratio manipulation may be achieved by killing male embryos, as occurs in a number of insect species. Here, we provide evidence for the first case of male killing by a tetracycline-sensitive microbe in pseudoscorpions. Using a combination of inheritance studies, antibiotic treatment and molecular assays, we show that a new strain of Wolbachia is associated with extreme female bias in the pseudoscorpion, Cordylochernes scorpioides. In a highly female-biased line, sex ratio distortion was maternally inherited, and occurred in conjunction with a high rate of spontaneous abortion and low reproductive success. Antibiotic treatment cured females of the Wolbachia infection, restored offspring sex ratio to 1:1, and significantly enhanced female reproductive success. The discovery of apparent male-killing in C. scorpioides is of interest because pseudoscorpions are viviparous. Theory predicts that male killing should be favoured, if male death enhances the fitness of infected female siblings. In a live-bearing host, reallocation of maternal resources from dead male embryos to their sisters provides a direct, physiological mechanism through which fitness compensation could favour male killing by cellular endosymbionts. Our results suggest, however, that fitness compensation and the spread of male-killing endosymbionts may be undermined by a high rate of spontaneous abortion in infected females of this viviparous arthropod.     

Reproductive costs of mating with a sibling male: sperm depletion and multiple mating in Cephalonomia hyalinipennis

Polygynous parasitoid males may be limited by the amount of sperm they can transmit to females, which in turn may become sperm limited. In this study, I tested the effect of male mating history on copula duration, female fecundity, and offspring sex ratio, and the likelihood that females will have multiple mates, in the gregarious parasitoid Cephalonomia hyalinipennis Ashmead (Hymenoptera: Bethylidae: Epyrinae), a likely candidate for sperm depletion due to its local mate competition system. Males were eager to mate with the seven females presented in rapid succession. Copula duration did not differ with male mating history, but latency before a first mating was significantly longer than before consecutive matings. Male mating history had no bearing on female fecundity (number of offspring), but significantly influenced offspring sex ratio. The last female to mate with a given male produced significantly more male offspring than the first one, and eventually became sperm depleted. In contrast, the offspring sex ratio of first‐mated females was female biased, denoting a high degree of sex allocation control. Once‐mated females, whether sperm‐depleted or not, accepted a second mating after a period of oviposition. Sperm‐depleted females resumed production of fertilized eggs after a second mating. Young, recently mated females also accepted a second mating, but extended in‐copula courtship was observed. Carrying out multiple matings in this species thus seems to reduce the cost of being constrained to produce only haploid males after accepting copulation with a sperm‐depleted male. I discuss the reproductive fitness costs that females experience when mating solely with their sibling males and the reproductive fitness gain of males that persist in mating, even when almost sperm‐depleted. Behavioural observations support the hypothesis that females monitor their sperm stock. It is concluded that C. hyalinipennis is a species with a partial local mating system.     

Sex determination in the androdioecious plant Datisca glomerata and its dioecious sister species D. cannabina.

Datisca glomerata is an androdioecious plant species containing male and hermaphroditic individuals. Molecular markers and crossing data suggest that, in both D. glomerata and its dioecious sister species D. cannabina, sex is determined by a single nuclear locus, at which maleness is dominant. Supporting this conclusion, an amplified fragment length polymorphism (AFLP) is heterozygous in males and homozygous recessive in hermaphrodites in three populations of the androdioecious species. Additionally, hermaphrodite x male crosses produced 1:1 sex ratios, while hermaphrodite x hermaphrodite crosses produced almost entirely hermaphroditic offspring. No perfectly sex-linked marker was found in the dioecious species, but all markers associated with sex mapped to a single linkage group and were heterozygous in the male parent. There was no sex-ratio heterogeneity among crosses within D. cannabina collections, but males from one collection produced highly biased sex ratios (94% females), suggesting that there may be sex-linked meiotic drive or a cytoplasmic sex-ratio factor. Interspecific crosses produced only male and female offspring, but no hermaphrodites, suggesting that hermaphroditism is recessive to femaleness. This comparative approach suggests that the hermaphrodite form arose in a dioecious population from a recessive mutation that allowed females to produce pollen.     

Infection densities of three Spiroplasma strains in the host Drosophila melanogaster

Maternally transmitted endosymbiotic bacteria of the genus Spiroplasma associate with numerous insect species, including the genus Drosophila. Among the Spiroplasma strains associated with Drosophila, several manipulate their host??s reproduction by killing the male offspring of the infected females. Although the male-killing mechanism is not well understood, previous studies of non-native strains transferred to D. melanogaster (strain Oregon-R) indicate that the male-killing strain achieves higher densities than two non-male-killing strains. Whether this pattern of higher male-killing strain densities occurs in other host-symbiont strain combinations is not known. Herein, we used quantitative PCR to examine infection densities of one non-male-killing strain native to D. hydei (Hyd1), and two male-killing strains; one native to D. nebulosa (NSRO), and one native to D. melanogaster (MSRO; recently discovered), upon artificial transfer to D. melanogaster (strain Canton-S). Infection densities were examined at four weekly intervals in adult flies, across three consecutive generations following artificial transfer. Infection densities of the non-male-killing strain were significantly lower than those of the two male killers immediately after adult emergence. At later time points, however, the non-male-killing strain (Hyd1) is capable of proliferating to densities similar to those of the two male-killing strains (NSRO and MSRO) in D. melanogaster (Canton-S). We also examined the effect of co-infection by the heritable bacterium Wolbachia, on Spiroplasma densities and male-killing ability. Wolbachia had little to no effect of Spiroplasma densities, but the male-killing ability of MSRO was lower in the presence of Wolbachia. Generation post-infection had little effect on Spiroplasma densities, but affected the male-killing ability.     

Expression and modulation of embryonic male-killing in Drosophila innubila: opportunities for multilevel selection

Organisms and the symbionts they harbor may experience opposing forces of selection. In particular, the contrasting inheritance patterns of maternally transmitted symbionts and their host's nuclear genes can engender conflict among organizational levels over the optimal host offspring sex ratio. This study uses a male-killing Wolbachia endosymbiont and its host Drosophila innubila to experimentally address the potential for multilevel selection in a host-symbiont system. We show that bacterial density can vary among infected females, and that females with a higher density have a more female-biased offspring sex ratio. Furthermore, bacterial density is an epigenetic and heritable trait: females with a low bacterial load have daughters with a lower-than-average bacterial density, whose offspring then experience less severe male-killing. For infected sons, the probability of embryonic mortality increases with the bacterial density in their mothers. The frequency distribution of Wolbachia density among individual D. innubila females, and therefore the dynamics of infection within populations of these flies, results both from processes affecting the growth and regulation of bacterial populations within cytoplasmic lineages and from selection among cytoplasmic lineages that vary in bacterial density. Estimates of effective population size of Wolbachia within cytoplasmic lineages and of D. innubila at the host population level suggest that selection among cytoplasmic lineages is likely to overwhelm the results of selection within lineages.     

Female choice for good genes and sex-biased broods in guppies

In a population of guppies Poecilia reticulata females were found to prefer large males and the offspring of these males had a higher survival rate than those sired by smaller males, suggesting that females were selecting their mates on the basis of their good genes. The possibility that females differentially invested in male or female offspring depending on the size or attractiveness of their mate was also investigated, but no relationship was found between a male's attractiveness or body size and the sex ratio of the offspring he sired. However, a strong female-biased sex ratio was detected in the broods and the proportion of males produced decreased with the amount of time that a female was confined with a male. The possible reasons for this are discussed.     

Drosophila melanogaster females change mating behaviour and offspring production based on social context

In Drosophila melanogaster, biological rhythms, aggression and mating are modulated by group size and composition. However, the fitness significance of this group effect is unknown. By varying the composition of groups of males and females, we show that social context affects reproductive behaviour and offspring genetic diversity. Firstly, females mating with males from the same strain in the presence of males from a different strain are infecund, analogous to the Bruce effect in rodents, suggesting a social context-dependent inbreeding avoidance mechanism. Secondly, females mate more frequently in groups composed of males from more than one strain; this mitigates last male sperm precedence and increases offspring genetic diversity. However, smell-impaired Orco mutant females do not increase mating frequency according to group composition; this indicates that social context-dependent changes in reproductive behaviour depend on female olfaction, rather than direct male-male interactions. Further, variation in mating frequency in wild-type strains depends on females and not males. The data show that group composition can affect variance in the reproductive success of its members, and that females play a central role in this process. Social environment can thus influence the evolutionary process.     

The effect of interspecific mating on sex ratios in the twospotted spider mite and the Banks grass mite (Acarina: Tetranychidae)

Mixed populations of the twospotted spider mite (TSM),Tetranychus urticae (Koch), and the Banks grass mite (BGM),Oligonychus pratensis (Banks), occur on corn and sorghum plants in late summer in the Great Plains. Interspecific matings between these arrhenotokous species occur readily in the laboratory but yield no female offspring. The effect of interspecific mating on female: male sex ratios was measured by examining the F₁ progeny of females that mated with both heterospecific and conspecific males in no-choice situations. TSM females that mated first with BGM males and then with TSM males produced a smaller percentage of female offspring than TSM females that mated only with TSM males (43.1±5.8 and 78.9±2.8% females, respectively). Similarly, BGM females mated with heterospecific males and then with conspecific males produced fewer female offspring than females mated only with BGM males (55.7±5.2 and 77.5±2.5%, respectively). Lower female: male sex ratios were produced also by BGM females that mated with TSM males after first mating with conspecifics (62.4±3.4%). In mixed populations containing males of both species, females also produced lower female: male sex ratios, but these ratios were not as low as expected based on mating propensities and progeny sex ratios observed in no-choice tests. These data suggest that interspecific mating may substantially reduce female fitness in both mite species by reducing the output of female offspring, but in mixed populations this effect is mitigated by unidentified behavioral mechanisms.     

Incomplete sexual isolation in sympatry between subspecies of the butterfly Danaus chrysippus (L.) and the creation of a hybrid zone

Subspecies chrysippus, dorippus and alcippus of the butterfly Danaus chrysippus differ at three biallelic colour gene loci. They have partially vicariant distributions, but their ranges overlap over a substantial part of central and East Africa, where hybridism is commonplace. We now report that the West African subspecies alcippus differs from other subspecies, not only in nuclear genotype but also in mitochondrial haplotype in both allopatry and sympatry. The maintenance of concordant nuclear and cytoplasmic genetic differences in sympatry, and in the face of hybridisation, is prima facie evidence for sexual isolation. Other evidence that suggests alcippus may be isolated from chrysippus and dorippus include differences in sex ratio (SR), heterozygote deficiency at one site and deduced differences in patterns of migration. We suggest that, within the hybrid zone, differential infection of subspecies by a male-killing Spiroplasma bacterium causes SR differences that restrict female choice, triggering rounds of heterotypic mating and consequent heterozygote excess that is largely confined to females. The absence of these phenomena from hybrid populations that test negative for Spiroplasma supports the hypothesis. The incomplete sexual isolation and partial vicariance of alcippus suggests that it is a nascent species.     

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.     

Interspecific transmission of endosymbiotic Spiroplasma by mites

The occurrence of closely related strains of maternally transmitted endosymbionts in distantly related insect species indicates that these infections can colonize new host species by lateral transfer, although the mechanisms by which this occurs are unknown. We investigated whether ectoparasitic mites, which feed on insect haemolymph, can serve as interspecific vectors of Spiroplasma poulsonii, a male-killing endosymbiont of Drosophila. Using Spiroplasma-specific primers for PCR, we found that mites can pick up Spiroplasma from infected Drosophila nebulosa females and subsequently transfer the infection to Drosophila willistoni. Some of the progeny of the recipient D. willistoni were infected, indicating successful maternal transmission of the Spiroplasma within the new host species. However, the transmission rate of the infection from recipient flies to their offspring was low, perhaps due to low Spiroplasma density in the recipient flies.