去除Wolbachia 对丽蝇蛹集金小蜂繁殖适合度和成蜂寿命的影响

【目的】明确内共生菌 Wolbachia 对丽蝇蛹集金小蜂 Nasonia vitripennis 繁殖适合度和成蜂寿命的影响。【方法】通过给自然感染 Wolbachia 的丽蝇蛹集金小蜂成蜂喂食不同浓度的利福平来消除其体内的 Wolbachia,然后进行10个世代的连续饲养,探究不同浓度利福平对丽蝇蛹集金小蜂体内 Wolbachia 的去除效果和去除 Wolbachia 后对丽蝇蛹集金小蜂繁殖力、性比（雌蜂占子代数量的比值）和成蜂寿命的影响。【结果】低浓度利福平（0.1~0.5 mg/mL）对丽蝇蛹集金小蜂的毒害作用较小,而高浓度利福平（0.7~10.0 mg/mL）对丽蝇蛹集金小蜂的毒害作用较大,但二者均能去除丽蝇蛹集金小蜂体内的 Wolbachia;去除 Wolbachia 后丽蝇蛹集金小蜂的出蜂量显著下降（P <0.01）,子代中性比显著下降（P <0.01）,但寿命无明显差异。【结论】不同浓度利福平均能去除丽蝇蛹集金小蜂体内Wolbachia,但效果不一致;Wolbachia 对丽蝇蛹集金小蜂的出蜂量和子代性比均有显著影响,对成蜂寿命无显著影响。     

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.     

Tetracycline‐exposed Drosophila melanogaster males produce fewer offspring but a relative excess of sons

A large diversity of species possesses endosymbionts; these endosymbionts can exhibit mutualistic, parasitic, and commensal relationships with their hosts. Previous work has consistently revealed that depleting endosymbiont titer with antibiotic treatment can significantly alter host fitness and function, particularly with respect to reproductive phenotypes. Although these findings are often interpreted as resulting from the breakdown of highly coevolved symbioses, it is possible that antibiotic treatment itself rather than endosymbiont removal contributes to the observed perturbations in reproductive phenotypes. Here, we investigate the effect of tetracycline treatment on sex ratio and male reproductive fitness using Drosophila melanogaster as a model system. Our results indicate that tetracycline‐treated males produce a relative excess of sons. We also find that tetracycline treatment reduces the number of progeny produced by treated males but not treated females. These findings are independent of the effects of tetracycline on Wolbachia titer and implicate the antibiotic itself as mediating these changes. It is yet unclear whether the sex ratio shift and reduction in male reproductive fitness are direct or indirect consequences of tetracycline exposure, and more work is needed to determine the molecular mechanisms by which these disturbances in reproductive phenotypes arise. Our data highlight the importance of considering the potentially confounding effects of antibiotic treatment when investigating the effects of endosymbiont depletion on host phenotypes.     

Removing endosymbiotic Wolbachia specifically decreases lifespan of females and competitiveness in a laboratory strain of Drosophila melanogaster

To understand specific symbiotic relationships ensuring stable existing of the bacterium Wolbachia in laboratory strains of Drosophila melanogaster, the imago lifespan and senescence rate, as well as competitiveness, have been evaluated as components of fitness in females from the following laboratory strains: (1) inbred strain 95 infected with Wolbachia; (2) two uninfected strains obtained by tetracycline treatment that were genetically similar to strain 95; and (3) two control, uninfected, wild-type laboratory strains that were used to assess the possible effects of the antibiotic on the studied characters in the absence of Wolbachia. The results have shown that infected females have longer lifespan and competitiveness than females with the same genotype uninfected with Wolbachia. The increase in the senescence and mortality rates with age was also slower in infected females. It is noteworthy that tetracycline does not affect the lifespan of females from the two control, uninfected, wild-type strains. Therefore, the antibiotic is not the cause of the positive changes in fitness that were observed in infected females. The obtained results are the first direct evidence that the relationship s in the Wolbachia-D melanogaster symbiotic system are mutualistic rather than parasitic, at least in micropopulations adapted to laboratory conditions.     

Wolbachia在山楂双叶螨中的感染及对寄主生殖的影响

【目的】共生菌Wolbachia在多种叶螨寄主中引起细胞质不亲和及适合度改变,影响寄主的生物学特性。山楂双叶螨Amphitetranychus viennensis是重要的果树害螨,常暴发成灾。本研究旨在明确Wolbachia在山楂双叶螨中的感染情况及对寄主生殖的影响。【方法】采集自然种群的山楂双叶螨,运用多位点序列分型技术（multilocus sequence typing, MLST）对其体内Wolbachia感染率及株系进行分析;通过杂交试验及生物学观察,分析感染Wolbachia对山楂双叶螨单雌产卵量、后代孵化率、性比及死亡率的影响。【结果】山楂双叶螨自然种群感染一种株系的Wolbachia （wVie）,该Wolbachia株系与小黑花椿象Orius strigicollis和丽蝇蛹集金小蜂Nasonia vitripennis中的Wolbachia株系亲缘关系较近,而与叶螨属Tetranychus叶螨感染的Wolbachia株系亲缘关系较远。Wolbachia与4种分化较小的线粒体单倍型相关联。Wolbachia感染雌虫与不感染雌虫产卵量没有显著差异（P>0.05）。不感染雌虫与感染雄虫交配,卵孵化率显著低于其他杂交组合 (P<0.05),但孵化率仍达近75%。各交配组合的后代性比及死亡率变化不明显（P>0.05）。【结论】Wolbachia在山楂双叶螨种群中的侵染历史较短,对山楂双叶螨的产卵力、后代性比、死亡率没有影响。Wolbachia在山楂双叶螨中诱导产生弱的CI表型。     

Influence of wolbachia infection on the fitness of the stored-product pest Liposcelis tricolor (Psocoptera: Liposcelididae)

Wolbachia are maternally inherited intracellular bacteria (Rickettsiaceae) that infect a wide range of arthropods and nematodes and that are associated with various reproductive abnormalities in their hosts. In this study, the effects of removal of Wolbachia infection on development, survival, and reproduction of Liposcelis tricolor Badonnel (Psocoptera: Liposcelididae) were investigated in laboratory. The Wolbachia-free strain was obtained by the removal of Wolbachia infection by using 1% rifampicin treatment on the Wolbachia-infected strain (control) for 4 wk, and no Wolbachia gene product was detected in this strain throughout the experiment. The results showed that the removal of Wolbachia infection had negative effects on the fitness of L. tricolor. Compared with the control strain, the Wolbachia-free strain (both in the first [F1] and second [F2] generation) had prolonged developmental times, reduced survivorship of immature stages, and reduced fecundity and longevity, resulting in much smaller rm values. Using rm values, the fitness for Wolbachia-free F1 and F2 relative to the control were calculated as 0.45 and 0.27, respectively. The results of this study further confirmed our previous conclusion that Wolbachia infection have positive effects on fecundity and fertility of L. tricolor, and for optimal reproduction of L. tricolor, Wolbachia must be present in psocids.     

Comparative multifactorial analysis of combined administration of injection and peroral forms of an antibiotic with a microbial immunomodulator in experimental anthrax

Comparative efficacy of the use of injection and oral dosage forms of rifampicin in the subtherapeutic doses in combination with peptidoglycan , an immunomodulator of microbial origin, was studied in respect to experimental anthracic infection with application of multifactorial analysis. It was shown that the antibiotic and immunomodulator had a pronounced synergistic effect. Polynomial statistic models were developed and nomograms or equal level curves defining the survival rate and average life-span (ALS) of the experimental animals within a wide range of the antibiotic and immunomodulator doses and the peptidoglycan dosing time were plotted. The combined use of the injection rifampicin in the subtherapeutic doses and the immunomodulator provided a significant increase in the survival rate and ALS, whereas the use of the oral antibiotic in combination with the immunomodulator increased only the ALS and not the survival rate. Multifactorial analysis proved to be an optimal methodical approach to comparative study of various antibiotic dosage forms used in combination with immunomodulators under experimental conditions.     

A natural population of the butterfly Eurema hecabe with Wolbachia-induced female-biased sex ratio not by feminization.

In butterflies, the adult sex ratio observed in the field is usually male-biased, although the sex ratio of their progeny is 1:1. This is due to the higher motility and larger behavioral range of males than females. As expected, the sex ratio of Eurema hecabe butterflies collected at 6 localities throughout Japan was male-biased. However, in Tsukuba, located in the central part of Japan, the sex ratio was found to be biased toward females. Their progeny reared in the laboratory also exhibited a female-biased sex ratio. A single strain of Wolbachia is considered to be the cause of the sex-ratio distortion, because antibiotic treatment reversed the sex ratio to 1:1, and only a single nucleotide sequence of wsp, a highly variable Wolbachia gene, was detected by molecular analysis. Cytogenetic analysis excluded the possibility of feminization as the underlying mechanism. In addition, when the wild-caught females that had already mated in nature were treated with antibiotics before oviposition, egg-hatch rates were extremely low, suggesting that the same Wolbachia strain also caused cytoplasmic incompatibility. Our findings suggest the possibility that a single strain of Wolbachia induces 2 distinct reproductive manipulations in the same host.     

从白符虫兆(弹尾纲)活性卵的形状变化探讨沃尔巴克氏体的共生作用(英文)

Wolbachia属共生菌的侵染是引起跳虫——白符虫兆孤雌生殖的原因。对带有正常沃尔巴克氏体菌群的白符虫兆卵和通过利福平处理剔除沃尔巴克氏体菌群的白符虫兆卵的胚胎发育进行实验观察。白符虫兆的活性卵产出3到4天后,卵体大小显著性地增大,并伴随卵体形状从球形到圆饼形的变化。这些变化在利福平处理的或者是7%自然失活的非活性卵中都没有出现。推测沃尔巴克氏体在白符虫兆卵产出后的3天之内或者3天之前的受精卵发育或胚胎发育中发挥着重要作用;同时根据目前已有的研究结果推断沃尔巴克氏体对白符虫兆卵发育可能的影响机制。     

Operational sex ratio in terrestrial isopods: interaction between potential rate of reproduction and Wolbachia-induced sex ratio distortion

Selfish genetic elements distorting sex ratio are known in several arthropods. By inducing a deficit of males, these sex ratio distorters may modify sexual selection by reversing the sex that competes for a mate. They also have potential to reduce the male proportion to values limiting mating possibilities and therefore limiting population size. Wolbachia endosymbionts are intracytoplasmic, vertically transmitted bacteria that convert genotypic males of terrestrial isopods (woodlice) into functional females. We have tested the impact of these feminizing symbionts on the operational sex ratio (OSR) in three woodlice species. Preliminary experiments consisted in estimating the potential rate of reproduction in males and females, and measuring the dynamics of the onset of reproduction in the wild. These parameters were then combined with population sex ratio to discriminate key factors influencing the OSR. The results suggest that the high potential rate of reproduction of males and the asynchrony in female receptivity both counterbalance female-biased sex ratios. The result is an overall balanced or slightly female-biased OSR. Male deficit can therefore not be considered as a factor strongly limiting reproduction in woodlice. Some females were nevertheless found not mated in the wild at the beginning of the reproductive season, most of them being infected by Wolbachia . This suggests that uninfected females may have an advantage as the first mate. Consequences of these findings on woodlice population dynamics are discussed.     

A cost of Wolbachia-induced sex reversal and female-biased sex ratios: decrease in female fertility after sperm depletion in a terrestrial isopod

A number of parasites are vertically transmitted to new host generations via female eggs. In such cases, host reproduction is an intimate component of parasite fitness and no cost of the infection on host reproduction is expected to evolve. A number of these parasites distort host sex ratios towards females, thereby increasing either parasite fitness or the proportion of the host that transmit the parasite. In terrestrial isopods (woodlice), Wolbachia bacteria are responsible for sex reversion and female-biased sex ratios, changing genetic males into functional neo-females. Although sex ratio distortion is a powerful means for parasites to increase in frequency in host populations, it also has potential consequences on host biology, which may, in turn, have consequences for parasite prevalence. We used the woodlouse Armadillidium vulgare to test whether the interaction between Wolbachia infection and the resulting excess of females would limit female fertility through the reduction in sperm number that they receive from males. We showed that multiple male mating induces sperm depletion, and that this sperm depletion affects fertility only in infected females. This decrease in fertility, associated with male mate choice, may limit the spread of Wolbachia infections in host populations.     

What maintains noncytoplasmic incompatibility inducing Wolbachia in their hosts: a case study from a natural Drosophila yakuba population

Cytoplasmic incompatibility (CI) allows Wolbachia to invade hosts populations by specifically inducing sterility in crosses between infected males and uninfected females. In some species, non-CI inducing Wolbachia, that are thought to derive from CI-inducing ancestors, are common. In theory, the maintenance of such infections is not possible unless the bacterium is perfectly transmitted to offspring--and/or provides a fitness benefit to infected females. The present study aims to test this view by investigating a population of Drosophila yakuba from Gabon, West Africa. We did not find any evidence for CI using wild caught females. Infected females from the field transmitted the infection to 100% of their offspring. A positive effect on female fecundity was observed one generation after collecting, but this was not retrieved five generations later, using additional lines. Similarly, the presence of Wolbachia was found to affect mating behaviour, but the results of two experiments realized five generations apart were not consistent. Finally, Wolbachia was not found to affect sex ratio. Overall, our results would suggest that Wolbachia behaves like a neutral or nearly neutral trait in this species, and is maintained in the host by perfect maternal transmission.     

Mechanisms promoting the long-term persistence of a Wolbachia infection in a laboratory-adapted population of Drosophila melanogaster

Intracellular bacteria of the genus Wolbachia are widespread endosymbionts across diverse insect taxa. Despite this prevalence, our understanding of how Wolbachia persists within populations is not well understood. Cytoplasmic incompatibility (CI) appears to be an important phenotype maintaining Wolbachia in many insects, but it is believed to be too weak to maintain Wolbachia in Drosophila melanogaster, suggesting that Wolbachia must also have other effects on this species. Here we estimate the net selective effect of Wolbachia on its host in a laboratory-adapted population of D. melanogaster, to determine the mechanisms leading to its persistence in the laboratory environment. We found i) no significant effects of Wolbachia infection on female egg-to-adult survival or adult fitness, ii) no reduced juvenile survival in males, iii) substantial levels of CI, and iv) a vertical transmission rate of Wolbachia higher than 99%. The fitness of cured females was, however, severely reduced (a decline of 37%) due to CI in offspring. Taken together these findings indicate that Wolbachia is maintained in our laboratory environment due to a combination of a nearly perfect transmission rate and substantial CI. Our results show that there would be strong selection against females losing their infection and producing progeny free from Wolbachia.     

Wolbachia对赤眼蜂的性别调控机制及生理影响

沃尔巴克氏体(Wolbachia)是广泛分布于节肢动物生殖组织内的一类共生细菌。它们通过共生诱导宿主赤眼蜂的产雌孤雌生殖,在生物防治上具有重要意义。文章概述了Wolbachia在赤眼蜂中的分布状况,对赤眼蜂的性别调控机制,在赤眼蜂种群中的传播及限制因素,以及对赤眼蜂适合度的影响。     

Effects on male fitness of removing Wolbachia infections from the mosquito Aedes albopictus

Abstract.  Cytoplasmic incompatibility (CI) induced by maternally inherited Wolbachia bacteria is a potential tool for the suppression of insect pest species with appropriate patterns of infection. The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) is known to be infected by two strains of Wolbachia pipientis Hertig (Rickettsiales: Rickettsiaceae), w Alb A and w Alb B, throughout its geographical distribution. This infection pattern theoretically restricts the application of CI-based control strategies. However, Wolbachia can be horizontally transferred using embryonic microinjection to generate incompatible transfected lines harbouring a single new strain of Wolbachia. In order to assess the feasibility of this approach, the effects of Wolbachia removal on mosquito fitness need to be clearly evaluated as the removal of natural superinfection is an inescapable step of this approach. Previous research has shown that uninfected females, produced by antibiotic treatment, showed a decrease in fitness compared with those infected with Wolbachia. In this study, the effect of Wolbachia removal on male fitness was investigated. Longevity and reproductive potential (mating competitiveness and sperm capacity) were assessed in both laboratory cages and greenhouses. No differences were observed between uninfected and infected males with respect to longevity, mating rate, sperm capacity and mating competitiveness in either laboratory conditions or greenhouses. The preservation of fitness in males of Ae. albopictus deprived of natural Wolbachia infection is discussed in relation to the development of incompatible insect technique suppression strategies. Finally, the potential application of aposymbiotic males in mark–release–recapture studies is suggested.     

Interspecific transfer of Wolbachia between two lepidopteran insects expressing cytoplasmic incompatibility: a Wolbachia variant naturally infecting Cadra cautella causes male killing in Ephestia kuehniella

Wolbachia is known as the causative agent of various reproductive alterations in arthropods. The almond moth Cadra cautella is doubly infected with A- and B-group Wolbachia and expresses complete cytoplasmic incompatibility (CI). The Mediterranean flour moth Ephestia kuehniella carries A-group Wolbachia and expresses partial CI. In the present study, the Wolbachia in C. cautella was transferred to E. kuehniella from which the original Wolbachia had been removed. We obtained transfected lines of three different infection states: single infection with A, single infection with B, and double infection with A and B. The doubly transfected lines and those transfected with only A produced exclusively female progeny. Two lines of evidence suggested that the sex ratio distortion was due to male killing. First, reduced egg hatch rate was observed. Second, removal of the Wolbachia from the transfected lines resulted in the recovery of a normal sex ratio of approximately 1:1. The occurrence of male killing following transfection showed that host factors influence the determination of the reproductive phenotype caused by Wolbachia. The transfected E. kuehniella males carrying exclusively B-group Wolbachia expressed partial incompatibility when crossed with the uninfected females. In addition, the transfected lines were bidirectionally incompatible with the naturally infected strain, which was the first demonstration of bidirectional CI in a lepidopteran.     

The effect of temperature on sex ratio in the isopod Porcellionides pruinosus: Environmental sex determination or a by-product of cytoplasmic sex determination?

The sex ratios of the progenies of woodlice Porcellionides pruinosus (Crustacea, Isopoda) raised at different temperatures were studied. Females from three French populations sampled in the wild produced highly female-biased broods at 20°C and male-biased broods above 30°C. The effect of high temperature was not due to selective mortality of females. Sex determination was thus sensitive to temperature in P. pruinosus. We also found an interpopulation variability of sex ratio thermosensitivity and a weak inheritance of male-biased sex ratios at high temperatures. Samples taken from a wild population throughout the year showed that while the thermal conditions required for changes in the sex ratio occurred, there was no significant variation in the sex ratio. On the other hand, almost all the females and many males in the four populations studied harboured intracytoplasmic bacteria. These maternally inherited symbionts belong to the genus Wolbachia and are known to possess a feminizing effect. While in other arthropods Wolbachia are destroyed at high temperatures, the symbionts of P. pruinosus were detected by a PCR procedure whatever the rearing temperatures. In light of these results, we propose that the thermosensitivity of sex determination in P. pruinosus could reflect the removal of the cytoplasmic effect on sex determination rather than environmental sex determination sensu stricto. The reduction in the amount of bacteria (but not their entire elimination), or the inhibition of bacterial metabolism, may be responsible for sex ratio variations relating to temperature. The incomplete inheritance of male-biased sex ratios at high temperatures might reflect a selection of thermo-tolerant bacterial strains.     

Influence of antibiotics on the offspring production of the Wolbachia-infected parthenogenetic parasitoid Encarsia formosa

Three different concentrations of the antibiotic tetracycline in honey were tested for their influence on the offspring production and longevity of the parasitoid wasp Encarsia formosa. Several earlier publications did not provide a conclusive answer on the effect that the Wolbachia have on these wasps. The results of our experiments show that at high tetracycline hydrochloride concentrations in honey (50mg/ml) the antibiotic is toxic to the females, all females died within three days after the antibiotic treatment. The concentration 5mg/ml was less toxic although the treated females also lived shorter and produced less offspring than the control females. At the lowest tested concentration of 1mg/ml there was no significant difference either in offspring production or in longevity between the control and the treated females. The antibiotic treatment at both 5 and 1mg/ml resulted in exclusively male progeny after the first two days of oviposition. These results are consistent with the theory that in species in which all individuals are infected the Wolbachia should not impose a large fitness cost.     

Recent changes in phenotype and patterns of host specialization in Wolbachia bacteria

Wolbachia are a genus of bacterial symbionts that are known to manipulate the reproduction of their arthropod hosts, both by distorting the host sex ratio and by inducing cytoplasmic incompatibility. Previous work has suggested that some Wolbachia clades specialize in particular host taxa, but others are diverse. Furthermore, the frequency with which related strains change in phenotype is unknown. We have examined these issues for Wolbachia bacteria from Acraea butterflies, where different interactions are known in different host species. We found that bacteria from Acraea butterflies mostly cluster together in several different clades on the bacterial phylogeny, implying specialization of particular strains on these host taxa. We also observed that bacterial strains with different phenotypic effects on their hosts commonly shared identical gene sequences at two different loci. This suggests both that the phenotypes of the strains have changed recently between sex ratio distortion and cytoplasmic incompatibility, and that host specialization is not related to the bacterial phenotype, as suggested from previous data. We also analysed published data from other arthropod taxa, and found that the Wolbachia infections of the majority of arthropod genera tend to cluster together on the bacterial phylogeny. Therefore, we conclude that Wolbachia is most likely to move horizontally between closely related hosts, perhaps because of a combination of shared vectors for transmission and physiological specialization of the bacteria on those hosts.