Negative Effects of Low Temperatures on the Vertical Transmission and Infection Density of a Spiroplasma Endosymbiont in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

Maternally transmitted endosymbionts of the genus Spiroplasma infecting several species of Drosophila are known to cause selective death of male offspring (male killing). The male-killing trait is considered to be advantageous for maternally transmitted endosymbionts. However, a non-male-killing spiroplasma is present in Japanese populations of Drosophila hydei at high frequencies (23-66%). This spiroplasma is phylogenetically closely related to the male-killing spiroplasma infecting other Drosophila species. It is unknown why this spiroplasma is maintained in its host populations despite its inability to cause male killing. We examined the susceptibilities of the spiroplasma in D. hydei to four different temperatures (28, 25, 18, and 15 degrees C). Diagnostic PCR revealed that vertical transmission of the spiroplasma was nearly perfect at 28 and 25 degrees C, partially suppressed at 18 degrees C, and completely blocked at 15 degrees C. Furthermore, quantitative PCR demonstrated that offspring treated at 18 degrees C exhibited dramatically lower densities of spiroplasma (i.e., approximately one-tenth) compared to offspring treated at 28 and 25 degrees C. Considering the low temperatures during winter in Japan, some unknown advantageous effects of the spiroplasma that compensate for the failure of vertical transmission are suggested to act in natural populations of D. hydei.     

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.     

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.     

Tissue-specific infection dynamics of male-killing and nonmale-killing spiroplasmas in Drosophila melanogaster

The male-killing spiroplasma strain NSRO causes an extremely female-biased sex ratio of the host, Drosophila melanogaster, as a result of selective death of male offspring during embryogenesis. The spiroplasma strain NSRO-A, a variant of NSRO, does not cause such symptoms. In an attempt to gain insights into the mechanism underlying the symbiont-induced reproductive phenotype, infection densities of the spiroplasmas in different tissues were monitored during host aging using a quantitative PCR technique. The density dynamics in the hemolymph were reminiscent of those in the whole body, whereas the density dynamics in the fat body, intestine and ovary were not. These results suggest that the majority of the spiroplasmas colonize and proliferate in the hemolymph of the host. In the hemolymph and whole body, the infection densities of NSRO were generally higher than those of NSRO-A, which may be related to the different reproductive phenotypes caused by the spiroplasmas.     

Loss of Wolbachia infection during colonisation in the invasive Argentine ant Linepithema humile

WOLBACHIA are maternally inherited bacteria, which are very common in arthropods and nematodes. Wolbachia infection may affect host reproduction through feminisation, parthenogenesis, male-killing, cytoplasmic incompatibility and increased fecundity. Previous studies showing discrepancies between the phylogenies of Wolbachia and its arthropod hosts indicate that infection is frequently lost, but the causes of symbiont extinction have so far remained elusive. Here, we report data showing that colonisation of new habitats is a possible mechanism leading to the loss of infection. The presence and prevalence of Wolbachia were studied in three native and eight introduced populations of the Argentine ant Linepithema humile. The screening shows that the symbiont is common in the three native L. humile populations analysed. In contrast, Wolbachia was detected in only one of the introduced populations. The loss of infection associated with colonisation of new habitats may result from drift (founder effect) or altered selection pressures in the new habitat. Furthermore, a molecular phylogeny based on sequences of the Wolbachia wsp gene indicates that L. humile has been infected by a single strain. Horizontal transmission of the symbiont may be important in ants as suggested by the sequence similarity of strains in the three genera Linepithema, Acromyrmex, and Solenopsis native from South and Central America.     

Population dynamics of a maternally-transmitted <Emphasis Type="Italic">Spiroplasma</Emphasis> infection in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006–2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments.     

Low temperature cure of a male killing agent in Drosophila melanogaster

Environmental factors can affect transmission or phenotype expression of selfish cytoplasmic endosymbionts such as embryonic male killers. Temperature is one factor that usually affects the transmission rate of selfish cytoplasmic endosymbionts. Heat cures have been described for several host-parasite systems, cold cures, however, are rare. We report a temperature cure of the Drosophila melanogaster male-killing agent, which occurs when flies are raised at 16.5 degrees C. Flies grown at 20, 24, and 28 degrees C maintained an extremely female biased sexual proportion.     

Male-killing <Emphasis Type="Italic">Wolbachia</Emphasis> do not protect <Emphasis Type="Italic">Drosophila bifasciata</Emphasis>against viral infection

BACKGROUND: Insect symbionts employ multiple strategies to enhance their spread through populations, and some play a dual role as both a mutualist and a reproductive manipulator. It has recently been found that this is the case for some strains of Wolbachia, which both cause cytoplasmic incompatibility and protect their hosts against viruses. Here, we carry out the first test as to whether a male-killing strain of Wolbachia also provides a direct benefit to its host by providing antiviral protection to its host Drosophila bifasciata. We infected flies with two positive sense RNA viruses known to replicate in a range of Drosophila species (Drosophila C virus and Flock House virus) and measure the rate of death in Wolbachia positive and negative host lines with the same genetic background. RESULTS: Both viruses caused considerable mortality to D. bifasciata flies, with Drosophila C virus killing 43% more flies than the uninfected controls and Flock House virus killing 78% more flies than the uninfected controls. However, viral induced mortality was unaffected by the presence of Wolbachia. CONCLUSION: In the first male-killing Wolbachia strain tested for antiviral effects, we found no evidence that it conferred protection against two RNA viruses. We show that although antiviral resistance is widespread across the Wolbachia phylogeny, the trait seems to have been lost or gained along some lineages. We discuss the potential mechanisms of this, and can seemingly discount protection against these viruses as a reason why this symbiont has spread through Drosophila populations.     

THE INTERACTION PHENOTYPE IN THE DROSOPHILA WILLISTONI–SPIROPLASMA SYMBIOSIS

Both the population and coevolutionary dynamics of hereditary male-lethal endosymbionts, found in a wide range of insect species, depend on host fitness and endosymbiont transmission rates. This paper reports on fitness effects and transmission rates in three lines of Drosophila willistoni infected with either male-lethal spiroplasmas or a spontaneous nonmale-lethal mutant. Overall fitness measures were reduced or unaffected by the infection; however, some infected females produced more offspring in early broods. Maternal transmission rates were high, but imperfect, and varied with a female's age, host line, and spiroplasma type. No evidence for paternal or horizontal transmission was found. If an altered temporal pattern of reproduction is not a factor in countering the loss of spiroplasma hosts through imperfect maternal transmission, persistence of this endoparasitism remains unexplained. Tolerance of the infection and ability to transmit bacteria varied with both host and spiroplasma line. Analysis of the interaction between the spontaneous nonmale-lethal mutant and its host suggests this symbiosis has undergone coevolution under laboratory culture.     

Intergenomic Arms Races: Detection of a Nuclear Rescue Gene of Male-Killing in a Ladybird

Many species of arthropod are infected by deleterious inherited micro-organisms. Typically these micro-organisms are inherited maternally. Consequently, some, particularly bacteria of the genus Wolbachia, employ a variety of strategies that favour female over male hosts. These strategies include feminisation, induction of parthenogenesis and male-killing. These strategies result in female biased sex ratios in host populations, which lead to selection for host factors that promote male production. In addition, the intra-genomic conflict produced by the difference in transmission of these cytoplasmic endosymbionts and nuclear factors will impose a pressure favouring nuclear factors that suppress the effects of the symbiont. During investigations of the diversity of male-killing bacteria in ladybirds (Coccinellidae), unexpected patterns of vertical transmission of a newly discovered male-killing taxon were observed in the ladybird Cheilomenes sexmaculata. Initial analysis suggested that the expression of the bacterial male-killing trait varies according to the male(s) a female has mated with. By swapping males between females, a male influence on the expression of the male-killing trait was confirmed. Experiments were then performed to determine the nature of the interaction. These studies showed that a single dominant allele, which rescues male progeny of infected females from the pathological effect of the male-killer, exists in this species. The gene shows typical Mendelian autosomal inheritance and is expressed irrespective of the parent from which it is inherited. Presence of the rescue gene in either parent does not significantly affect the inheritance of the symbiont. We conclude that C. sexmaculata is host to a male-killing γ-proteobacterium. Further, this beetle is polymorphic for a nuclear gene, the dominant allele of which rescues infected males from the pathogenic effects of the male-killing agent. These findings represent the first reported case of a nuclear suppressor of male-killing in a ladybird. They are considered in regard to sex ratio and intra-genomic conflict theories, and models of the evolutionary dynamics and distribution of inherited symbionts.     

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.     

How do insects react to novel inherited symbionts? A microarray analysis of Drosophila melanogaster response to the presence of natural and introduced Spiroplasma

Maternally inherited endosymbionts are found in numerous insect species and have various effects on host ecology. New symbioses are most commonly established following lateral transfer of an existing symbiont from one host species to another. Laboratory study has demonstrated that symbionts commonly perform poorly in novel hosts, with weak vertical transmission and maladaptive pathogenicity being observed in the generations following transfer. This poor performance probably limits symbiont occurrence. We here use microarray technology to test whether poor symbiont performance observed following 1 year of vertical transmission through a new host is associated with alteration in host gene expression or whether it occurs independently of this. We utilize the Drosophila melanogaster--Spiroplasma interaction and test the response of the host in the presence of both natural Spiroplasma infections and novel Spiroplasma infections transinfected previously from other host species. None of the Spiroplasma infections investigated produced upregulation in host haemolymph/fat body-based immune responses, and we therefore rejected the hypothesis that failure to thrive was associated with immune upregulation. One infection was associated with a downregulation of genes associated with egg production compared to uninfected controls, indicative of damage to the host. The Spiroplasma infection showed that the weakest vertical transmission showed no significant disturbance to host gene expression compared to uninfected controls. We conclude that the failure of Spiroplasma in novel host species is associated either with causing harm to their new hosts or through a failure to thrive in the new host that occurs independently of host responses to infection.     

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.     

Wolbachia distribution and cytoplasmic incompatibility during sperm development: the cyst as the basic cellular unit of CI expression

The growth and distribution of the intracellular microbe Wolbachia pipientis during spermatogenesis in several different host/symbiont genetic combinations in Drosophila melanogaster and Drosophila simulans is described. Considerable intra- and inter-strain variation in Wolbachia density and tissue distribution was observed. Wolbachia were found inside spermatocytes and spermatids or within the somatic cyst cells surrounding the germ cells. Some strains displayed both tissue distributions. High rates of cytoplasmic incompatibility (CI) are correlated with high levels of Wolbachia only when spermatocytes and/or spermatids harbor the microbe. Wolbachia infection of somatic cyst cells, although sometimes present at high levels, did not result in significant CI expression. CI-inducing Wolbachia strains within D. simulans showed no distinguishable differences in distribution or density within infected spermatids. To dissect the relative contribution of host and symbiont to the expression of CI, Wolbachia from various host strains known to exhibit varying levels of CI were introgressed into new uninfected host genetic backgrounds. These introgression experiments confirm that the mod(+)/mod(-) phenotype is an intrinsic Wolbachia trait and is not determined by host factors. The level of sperm modification in those lines harboring Wolbachia capable of modifying sperm, however, is influenced by host genetic background. These results form the basis of the Wolbachia Infected Spermatocyte/Spermatid Hypothesis (WISSH). According to WISSH, Wolbachia infection in spermatocytes and then spermatids during sperm development is required for CI expression.     

Symbiosis lost: imperfect vertical transmission of fungal endophytes in grasses

Vertically transmitted symbionts associate with some of the most ecologically dominant species on Earth, and their fixation has led to major evolutionary transitions (e.g., the development of mitochondria). Theory predicts that exclusive vertical transmission should favor mutualism and generate high frequencies of symbiosis in host populations. However, host populations often support lower-than-expected symbiont frequencies. Imperfect transmission (i.e., symbiont is not transmitted to all offspring) can reduce symbiont frequency, but for most beneficial symbionts it is unknown whether vertical transmission can be imperfect or during which life-history stage the symbiont is lost. Using quantitative natural history surveys of fungal endophytes in grasses, we show that transmission was imperfect in at least one stage for all seven host species examined. Endophytes were lost at all possible stages: within adult plants, from adult tillers to seeds, and from seeds to seedlings. Despite this loss, uninfected seeds failed to germinate in some species, resulting in perfect transmission to seedlings. The type and degree of loss differed among host populations and species and between endophyte genera. Populations with lower endophyte frequencies had higher rates of loss. Our results indicate new directions for understanding cooperation and conflict in symbioses and suggest mechanisms for host sanctions against costly symbionts.     

Male-Killing Spiroplasma Induces Sex-Specific Cell Death via Host Apoptotic Pathway

Some symbiotic bacteria cause remarkable reproductive phenotypes like cytoplasmic incompatibility and male-killing in their host insects. Molecular and cellular mechanisms underlying these symbiont-induced reproductive pathologies are of great interest but poorly understood. In this study, Drosophila melanogaster and its native Spiroplasma symbiont strain MSRO were investigated as to how the host''s molecular, cellular and morphogenetic pathways are involved in the symbiont-induced male-killing during embryogenesis. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, anti-cleaved-Caspase-3 antibody staining, and apoptosis-deficient mutant analysis unequivocally demonstrated that the host''s apoptotic pathway is involved in Spiroplasma-induced male-specific embryonic cell death. Double-staining with TUNEL and an antibody recognizing epidermal marker showed that embryonic epithelium is the main target of Spiroplasma-induced male-specific apoptosis. Immunostaining with antibodies against markers of differentiated and precursor neural cells visualized severe neural defects specifically in Spiroplasma-infected male embryos as reported in previous studies. However, few TUNEL signals were detected in the degenerate nervous tissues of male embryos, and the Spiroplasma-induced neural defects in male embryos were not suppressed in an apoptosis-deficient host mutant. These results suggest the possibility that the apoptosis-dependent epidermal cell death and the apoptosis-independent neural malformation may represent different mechanisms underlying the Spiroplasma-induced male-killing. Despite the male-specific progressive embryonic abnormality, Spiroplasma titers remained almost constant throughout the observed stages of embryonic development and across male and female embryos. Strikingly, a few Spiroplasma-infected embryos exhibited gynandromorphism, wherein apoptotic cell death was restricted to male cells. These observations suggest that neither quantity nor proliferation of Spiroplasma cells but some Spiroplasma-derived factor(s) may be responsible for the expression of the male-killing phenotype.     

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.     

The Spiroplasma heritable bacterial endosymbiont of Drosophila

Since the discovery of the small, gram-positive bacterium, Spiroplasma, as a sex-ratio distorting agent in Drosophila over 50 years ago, substantial progress has been made in understanding the relationship of this bacteria with its insect host. Thus far, spiroplasmas have been found as heritable endosymbionts in sixteen different species of Drosophila. In some species these bacteria cause a male-killing phenotype, where the males die during embryogenesis. In other species, however, Spiroplasma does not cause male-killing, and its fitness effects are unclear. Though recent research has identified multiple factors that affect the prevalence and transmission of spiroplasmas in Drosophila populations, much work remains to fully characterize this symbiosis. Spiroplasma is the only identified heritable bacterial endosymbiont of Drosophila, other than Wolbachia, and can serve as a useful as model for elucidating the nature of insect/bacterial interactions.     

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection,but not vertical transmission efficiency,evolve rapidly after horizontal transfer into D. melanogaster

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.     

Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster

The endosymbiotic bacteria Spiroplasma spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 10(8) dnaA copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 10(9) dnaA copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10(-5) for NSRO and 3 x 10(-4) for NSRO-A.