Environmental effects on cytoplasmic incompatibility and bacterial load in Wolbachia-infected Drosophila simulans

The effects of high temperatures, antibiotics, nutrition and larval density on cytoplasmic incompatibility caused by a Wolbachia infection were investigated in Drosophila simulans. Exposure of larvae from an infected stock to moderate doses of tetracycline led to complete incompatibility when treated females were crossed to infected males; the same doses only caused a partial restoration of compatibility when treated males were crossed to uninfected females. In crosses with treated females, there was a strong correlation between dose effects on hatch rates and infection levels in embryos produced by these females. Ageing and rearing males at a high temperature led to increased compatibility. However, exposing infected females to a high temperature did not influence their compatibility with infected males. Male temperature effects depended on conditions experienced at the larval stage but not the pupal stage. Exposure to 25 °C reduced the density of Wolbachia in embryos compared with a 19 °C treatment. Low levels of nutrition led to increased compatibility, but no effect of larval crowding was detected. These findings show the ways environmental factors can influence the expression of cytoplasmic incompatibility and suggest that environmental effects may be mediated by bacterial density.     

The impact of Wolbachia,male age and mating history on cytoplasmic incompatibility and sperm transfer in Drosophila simulans

Most insects harbour a variety of maternally inherited endosymbionts, the most widespread being Wolbachia pipientis that commonly induce cytoplasmic incompatibility (CI) and reduced hatching success in crosses between infected males and uninfected females. High temperature and increasing male age are known to reduce the level of CI in a variety of insects. In Drosophila simulans, infected males have been shown to mate at a higher rate than uninfected males. By examining the impact of mating rate independent of age, this study investigates whether a high mating rate confers an advantage to infected males through restoring their compatibility with uninfected females over and above the effect of age. The impact of Wolbachia infection, male mating rate and age on the number of sperm transferred to females during copulation and how it relates to CI expression was also assessed. As predicted, we found that reproductive compatibility was restored faster in males that mate at higher rate than that of low mating and virgin males, and that the effect of mating history was over and above the effect of male age. Nonvirgin infected males transferred fewer sperm than uninfected males during copulation, and mating at a high rate resulted in the transfer of fewer sperm per mating irrespective of infection status. These results indicate that the advantage to infected males of mating at a high rate is through restoration of reproductive compatibility with uninfected females, whereas uninfected males appear to trade off the number of sperm transferred per mating with female encounter rate and success in sperm competition. This study highlights the importance Wolbachia may play in sexual selection by affecting male reproductive strategies.     

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.     

The effects of unidirectional incompatibility on cytonuclear disequilibria in a hybrid zone

Unidirectional incompatibility selection is examined as an alternate mechanism of natural selection to cytoplasmic male sterility (CMS) for generating cytonuclear disequilibria. Differences in the dynamics and equilibrium behavior of cytonuclear disequilibria between these two cytonuclear selection models may allow for statistical tests of CMS vs. unidirectional incompatibility between mating cytotypes. Unlike CMS without migration, unidirectional incompatibility causes the cytoplasmic allele frequency to change over time rather than remain constant, and the nuclear allele frequencies hitchhike on the cytoplasmic frequencies. The decay of disequilibria is also distinctive in the absence of migration. Furthermore, in comparing both models with migration it is seen that the opportunity for internal equilibrium can be two or three times higher in a unidirectional incompatibility vs. CMS model. An example is presented that shows how unidirectional incompatibility can be statistically eliminated as a possible mechanism of cytonuclear selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytoplasmic Incompatibility and Mating Preference in Colombian Drosophila Pseudoobscura

MACRAE and ANDERSON observed a large frequency change of mitochondrial DNA (mtDNA) haplotypes in a population initiated with two allopatric strains of Drosophila pseudoobscura, BogER from Colombia and AH162 from California. They concluded that mtDNA haplotypes in D. pseudoobscura are not always selectively neutral. NIGRO and PROUT suggested, however, that a maternally transmitted incompatibility system, similar to the one they observed in two strains of D. simulans from Italy, could account for the observed mtDNA frequency changes. SINGH and HALE postulated that a mating preference between the strains BogER and AH162 in MACRAE and ANDERSON's experiment, in the form of negative assortative mating, could also account for the mtDNA frequency changes. We report two experiments designed to test the hypotheses: that a maternally transmitted cytoplasmic incompatibility system exists between D. pseudoobscura strains BogER and AH162; and, that BogER females mate preferentially with AH162 males. Our results do not support either hypothesis.     

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.     

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.     

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods.

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.     

Dual reproductive cost of aging in male Medflies: dramatic decrease in mating competitiveness and gradual reduction in mating performance

Although age-based effects on the reproductive success of males have been reported in several animal taxa the cost of aging on male mating success in lekking species has not been fully explored. We used the Mediterranean fruit fly, a lekking species, to investigate possible cost of aging on male reproductive success. We performed no choice and choice mating tests to test the hypothesis that aging does not affect the mating performance (mating success in conditions lacking competition) or the mating competitiveness (mating success against younger rivals) of males. The mating probability of older males decreased significantly when competing with younger males. Aging gradually reduced the mating performance of males but older males were still accepted as mating partners in conditions lacking competition. Therefore, older males are capable of performing the complete repertoire of sexual performance but fail to be chosen by females in the presence of young rivals. Older males achieved shorter copulations than younger ones, and female readiness to mate was negatively affected by male age. Older and younger males transferred similar amount of spermatozoids to female spermathecae. Females stored spermatozoids asymmetrically in the two spermathecae regardless the age of their mating partner. Aging positively affected the amount of spermatozoids in testes of both mated and nonmated males. No significant differences were observed on the amount of spermatozoids between mated and nonmated males.     

Wolbachia-Host Interactions: Host Mating Patterns Affect Wolbachia Density Dynamics

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and cause an array of effects on host reproduction, fitness and mating behavior. Although our understanding of the Wolbachia-associated effects on hosts is rapidly expanding, our knowledge of the host factors that mediate Wolbachia dynamics is rudimentary. Here, we explore the interactions between Wolbachia and its host, the two-spotted spider mite Tetranychus urticae Koch. Our results indicate that Wolbachia induces strong cytoplasmic incompatibility (CI), increases host fecundity, but has no effects on the longevity of females and the mating competitiveness of males in T. urticae. Most importantly, host mating pattern was found to affect Wolbachia density dynamics during host aging. Mating of an uninfected mite of either sex with an infected mite attenuates the Wolbachia density in the infected mite. According to the results of Wolbachia localization, this finding may be associated with the tropism of Wolbachia for the reproductive tissue in adult spider mites. Our findings describe a new interaction between Wolbachia and their hosts.     

Reproductive strategies of Aphidius ervi Haliday (Hymenoptera: Aphidiidae)

Hymenopteran parasitoids are usually arrhenotokous parthenogenetic, where females arise from fertilized and males from unfertilized eggs. Therefore, the reproductive fitness of females is a function of egg production and furthermore affected by mating, whereas that of males is mainly determined by the number of daughters they father. Aphidius ervi Haliday is a quasi-gregarious parasitoid of a number of aphid pests on economically important crops such as legumes and cereals. Females are monandrous whereas males are polygynous. Here, we tested how parental age at mating and male mating history affected mating success, fecundity and daughter production in this species. Once-mated males perform significantly better than naïve males with regard to mating success, suggesting that males learn from previous matings. The fecundity of virgin females is not significantly different from that of mated females regardless of parental age at mating and male mating history, indicating that mating does not stimulate egg production or contribute to female nutrient supply. Males can replenish sperm supply after mating, implying that they are at least moderately synspermatogenic. Preference for young over old mates for mating by both sexes may be explained by the fact that aging of both sexes contributes to the reduction of daughter production. Rather than sperm depletion, the reduced daughter production may be attributed to diminishing sperm viability and mobility in aging males and increasing constraints in fertilization process in aging females. Our results also show that female age has a stronger impact on the production of daughters, suggesting that fertilization process in females is more sensitive to aging than sperm vigor in males.     

Wolbachia-induced 'hybrid breakdown' in the two-spotted spider mite Tetranychus urticae Koch

The most common post-zygotic isolation mechanism between populations of the phytophagous mite Tetranychus urticae is 'hybrid breakdown', i.e. when individuals from two different populations are crossed, F1 hybrid females are produced, but F2 recombinant male offspring suffer increased mortality. Two-spotted spider mites collected from two populations, one on rose and the other on cucumber plants, were infected with Wolbachia bacteria. These bacteria may induce cytoplasmic incompatibility in their hosts: uninfected (U) females become reproductively incompatible with infected (W) males. We report on the effect of Wolbachia infections in intra- and interstrain crosses on (i) F1 mortality and sex ratios (a test for cytoplasmic incompatibility), and (ii) the number of haploid offspring and mortality in clutches of F1 virgins (a test for hybrid breakdown). U x W crosses within the rose strain exhibited partial cvtoplasmic incompatibility. More interestingly, F2 males suffered increased mortality, a result identical to the hybrid breakdown phenomenon. The experiments were repeated using females from the cucumber strain. In interstrain U x W and U x U crosses, hybrid breakdown was much stronger in the former (80 versus 26%). This is the first report of a Wolbachia infection causing a hybrid breakdown phenotype. Our results show that Wolbhachia infections can contribute to reproductive incompatibility between populations of T. urticae.     

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (Gmm(Apo)) females when mated with Wolbachia-infected (Gmm(Wt)) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.     

Wolbachia Infections and the Expression of Cytoplasmic Incompatibility in Drosophila Sechellia and D. Mauritiana

Various stocks of Drosophila mauritiana and D. sechellia were found to be infected with Wolbachia, a Rickettsia-like bacterium that is known to cause cytoplasmic incompatibility and other reproductive abnormalities in arthropods. Testing for the expression of cytoplasmic incompatibility in these two species showed partial incompatibility in D. sechellia but no expression of incompatibility in D. mauritiana. To determine whether absence of cytoplasmic incompatibility in D. mauritiana was due to either the bacterial or host genome, we transferred bacteria from D. mauritiana into an uninfected strain of D. simulans, a host species known to express high levels of incompatibility with endogenous Wolbachia. We also performed the reciprocal transfer of the natural D. simulans Riverside infection into a tetracycline-treated stock of D. mauritiana. In each case, the ability to express incompatibility was unaltered by the different host genetic background. These experiments indicate that in D. simulans and D. mauritiana expression of the cytoplasmic incompatibility phenotype is determined by the bacterial strain and that D. mauritiana harbors a neutral strain of Wolbachia.     

The etiological agent of cytoplasmic incompatibility in Culex pipiens

All individuals of Culex pipiens (wide sense) which have been examined were infected with Wolbachia pipientis. Larvae reared in tetracycline were freed of these symbiotes and remained free (aposymbiotic) in future generations. When males were freed of their symbiotes, they no longer displayed incompatibility. Aposymbiotic males were compatible with all females, whether infected or not. Aposymbiotic females, on the other hand, laid fertile eggs after mating with aposymbiotic males, but not after mating with normal males. Most eggs laid by aposymbiotic females after mating with normal males showed no development at all, even though the females had been inseminated.     

MICROSCOPICAL OBSERVATIONS OF INITIAL INTERACTIONS IN VARIOUS MATINGS OF SCHIZOPHYLLUM COMMUNE AND OF COPRINUS LAGOPUS

In Schizophyllum commune and Coprinus lagopus the frequency of hyaline hyphal tips adjacent to anastomoses reflects the degree of incompatibility between mated strains. The lowest percentage is associated with full compatibility, the highest with full incompatibility. The hyaline hyphal tips appear several hours after an anastomosis has been detected. The data suggest that the factor(s) responsible for a cytoplasmic incompatibility is not constitutive but is induced at the time of mating.     

UNIDIRECTIONAL INCOMPATIBILITY BETWEEN POPULATIONS OF DROSOPHILA SIMULANS

Drosophila simulans females from a strain collected at Watsonville, CA produce very few offspring when mated with males from a strain collected at Riverside 510 km away. This incompatibility does not exist in the reciprocal cross. In the incompatible cross, mating and oviposition are normal, but eggs fail to hatch. A survey of other California populations indicates that unidirectional incompatibility is widespread. The incompatibility is maternally inherited over one generation. Using older Riverside males or rearing the strains at 28°C suppresses incompatibility. Culturing the strains on medium with tetracycline restores compatibility, suggesting the involvement of a microorganism.     

Comparison of sexual compatibility in crosses between the southern and northern populations of the cabbage beetle Colaphellus bowringi

It is widely accepted that the genetic divergence and reproductive incompat- ibility between closely related species and/or populations is often viewed as an important step toward speciation. In this study, sexual compatibility in crosses between the southern XS population and the northern TA population of the polyandrous cabbage beetle Co- laphellus bowringi was investigated by testing their mating preferences, mating latency, copulation duration, and reproductive performances of post-mating. In choice mating ex- periments, the percentages ofmatings were significantly higher in intra-population crosses than in inter-population crosses. Both isolation index （/） and index of pair sexual isolation （/PSi） indicated partial mating incompatibility or assortative mating in crosses between the two different geographical populations. In single pair mating experiments, XS females in inter-population crosses mated significantly later and copulated significantly shorter than those in intra-population crosses. However, TA females in inter-population crosses mated significantly earlier and copulated longer than those in intra-population crosses, suggesting that larger XS males may enhance heterotypic mating. The lifetime fecundity was highest in XS homotypic matings, lowest in TA homotypic matings, and intermedi- ate in heterotypic rnatings between their parents. The inter-population crosses resulted in significantly lower egg hatching rate and shorter female longevity than intra-population crosses. These results demonstrated that there exist some incompatibilities in premating, postmating-prezygotic, and postzygotic stages between the southern XS population and northern TA population of the cabbage beetle Colaphellus bowringi.     

Naturally Occurring Incompatibilities between Different Culex pipiens pallens Populations as the Basis of Potential Mosquito Control Measures

Background

Vector-borne diseases remain a threat to public health, especially in tropical countries. The incompatible insect technique has been explored as a potential control strategy for several important insect vectors. However, this strategy has not been tested in Culex pipiens pallens, the most prevalent mosquito species in China. Previous works used introgression to generate new strains that matched the genetic backgrounds of target populations while harboring a new Wolbachia endosymbiont, resulting in mating competitiveness and cytoplasmic incompatibility. The generation of these incompatible insects is often time-consuming, and the long-term stability of the newly created insect-Wolbachia symbiosis is uncertain. Considering the wide distribution of Cx. pipiens pallens and hence possible isolation of different populations, we sought to test for incompatibilities between natural populations and the possibility of exploiting these incompatibilities as a control strategy.

Methodology/Principal Findings

Three field populations were collected from three geographic locations in eastern China. Reciprocal cross results showed that bi-directional patterns of incompatibility existed between some populations. Mating competition experiments indicated that incompatible males could compete with cognate males in mating with females, leading to reduced overall fecundity. F1 offspring from incompatible crosses maintained their maternal crossing types. All three populations tested positive for Wolbachia. Removal of Wolbachia by tetracycline rendered matings between these populations fully compatible.

Conclusions/Significance

Our findings indicate that naturally occurring patterns of cytoplasmic incompatibility between Cx. pipiens pallens populations can be the basis of a control strategy for this important vector species. The observed incompatibilities are caused by Wolbachia. More tests including field trials are warranted to evaluate the feasibility of this strategy as a supplement to other control measures.     

Wolbachia Infection and Cytoplasmic Incompatibility in Drosophila Species

Forty-one stocks from 30 Drosophila species were surveyed for Wolbachia infection using PCR technology. D. sechellia and two strains of D. auraria were found to be infected and were tested for the expression of cytoplasmic incompatibility, along with D. ananassae and D. melanogaster strains, which are already known to be infected. D. ananassae and D. melanogaster show levels of incompatibility up to 25%, while D. auraria and D. sechellia exhibit levels of egg mortality ~60%. A dot-blot assay using the dnaA sequence as probe was developed to assess the infection levels in individual males that were used in incompatibility crosses. A positive correlation between bacterial density and cytoplasmic incompatibility was observed. The stocks examined can be clustered into at least two groups, depending on the levels of infection relative to the degree of cytoplasmic incompatibility exhibited. One group, containing D. simulans Hawaii, D. sechellia, and D. auraria, exhibits high levels of cytoplasmic incompatibility relative to levels of infection; all the other species and D. simulans Riverside exhibit significantly lower levels of cytoplasmic incompatibility relative to levels of infection. These data show that, in addition to bacterial density, bacterial and/or host factors also affect the expression of cytoplasmic incompatibility.