Unexpected Mechanism of Symbiont-Induced Reversal of Insect Sex: Feminizing Wolbachia Continuously Acts on the Butterfly Eurema hecabe during Larval Development

When the butterfly Eurema hecabe is infected with two different strains (wHecCI2 and wHecFem2) of the bacterial endosymbiont Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia-mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex.     

Distribution and Evolutionary Impact of Wolbachia on Butterfly Hosts

Wolbachia are maternally inherited endosymbiotic alpha-proteobacteria found in terrestrial arthropods and filarial nematodes. They are transmitted vertically through host cytoplasm and alter host biology by inducing various reproductive alterations, like feminization, parthenogenesis, male killing (MK) and cytoplasmic incompatibility. In butterflies, some effects especially MK and sperm-egg incompatibility are well established. All these effects skew the sex ratio towards female and subsequently favor the vertical transmission of Wolbachia. Some of the insects are also infected with multiple Wolbachia strains which may results in some complex phenomenon. In the present review the potential of Wolbachia for promoting evolutionary changes in its hosts with emphasis on recent advances in interactions of butterfly–Wolbachia is discussed. In addition to this, strain diversity of Wolbachia and its effects on various butterfly hosts are also highlighted.     

Artificial selection on ant female caste ratio uncovers a link between female‐biased sex ratios and infection by Wolbachia endosymbionts

Social insect sex and caste ratios are well‐studied targets of evolutionary conflicts, but the heritable factors affecting these traits remain unknown. To elucidate these factors, we carried out a short‐term artificial selection study on female caste ratio in the ant Monomorium pharaonis. Across three generations of bidirectional selection, we observed no response for caste ratio, but sex ratios rapidly became more female‐biased in the two replicate high selection lines and less female‐biased in the two replicate low selection lines. We hypothesized that this rapid divergence for sex ratio was caused by changes in the frequency of infection by the heritable bacterial endosymbiont Wolbachia, because the initial breeding stock varied for Wolbachia infection, and Wolbachia is known to cause female‐biased sex ratios in other insects. Consistent with this hypothesis, the proportions of Wolbachia‐infected colonies in the selection lines changed rapidly, mirroring the sex ratio changes. Moreover, the estimated effect of Wolbachia on sex ratio (~13% female bias) was similar in colonies before and during artificial selection, indicating that this Wolbachia effect is likely independent of the effects of artificial selection on other heritable factors. Our study provides evidence for the first case of endosymbiont sex ratio manipulation in a social insect.     

No seasonal trend in infection of the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae), by Wolbachia

Wolbachia are Gram-negative bacteria that cause intracellular inherited infections in many invertebrates. They are extremely common, with 20–75 % of all insects being infected. Wolbachia belong to taxa of the Anaplasmataceae family, alpha proteobacteria. Because previous studies have shown that Wolbachia generally disappear from the host insect’s body under high-temperature conditions in laboratories, we investigated seasonal changes in infection of the pale grass blue butterfly, Zizeeria maha (Kollar, 1848) by Wolbachia, for 7 months of the year. Total annual infection was 86.7 % (n = 15) of females and 96.3 % (n = 81) of males. Statistical analysis showed that monthly infection was not significantly different among months. In addition, no significant difference was found between Wolbachia densities in cells of summer and winter butterfly samples by use of real-time PCR during the months examined. The results suggest that Wolbachia infection is not affected by seasonal factors, at least for Z. maha.     

Genetic diversity in lac resin-secreting insects belonging to Kerria spp., as revealed through ISSR markers

The Indian lac insect Kerria lacca is harnessed in India for commercial production of lac, which has diversified industrial applications. Many of the geographical races of this species are under threat of extinction due to increasingly drastic local deviations in climate patterns. Thus, there is need for documentation and conservation of the lac insect biodiversity adapted especially for local climatic conditions and host species. The genetic diversity among twenty lines of commercially important Kerria spp. was analyzed using Inter-Simple Sequence Repeat (ISSR) technique. Seventeen ISSR primers produced 96.1% genetic polymorphism in the lac insect lines under study. The clustering dendrogram segregated the twenty lines into four major clusters with similarity coefficients between 0.25 and 0.81. The first three principal coordinates revealed 43.1% of the total genetic variation. The above results reveal significant genetic variability in the lines, which could be used for genetic improvement of lac insects.     

“Phloem sap analysis of Schleichera oleosa (Lour) Oken, Butea monosperma (Lam) Taub. and Ziziphus mauritiana (Lam) and hemolymph of Kerria lacca (Kerr) using HPLC and tandem mass spectrometry”

Females of lac insects especially of Kerria lacca (Kerr) secret a resin known as lac for their own protection, which has tremendous applications. Lac insect completes its lifecycle on several host taxa where it exclusively feeds on phloem sap but Schleichera oleosa (Lour.) Oken, Butea monosperma (Lam.) and Ziziphus mauritiana (Lam.) are its major hosts. Analysis of phloem sap constituents as well as hemolymph of lac insect is important because it ultimately gets converted into lac by insect intervention. Main phloem sap constituent’s viz. sugars and free amino acids and hemolymph of lac insect were analyzed using HPLC and tandem mass spectrometry, respectively. The results were transformed to relative percentage of the total sugars and free amino acids analyzed in each sample for comparison among lac insect hemolymph and the phloem sap of the three different host taxa. Sucrose (58.9 ± 3.6–85.6 ± 0.9) and trehalose (62.3 ± 0.4) were the predominant sugars in phloem sap of three taxa and hemolymph of lac insect, respectively. Glutamic acid (33.1 ± 1.4–39.8 ± 1.4) was found to be main amino acid among the phloem sap of three taxa while tyrosine (61 ± 2.6) was the major amino acid in hemolymph of lac insect. The relative percentage of non-essential amino acids (60.8 %–69.9 %) was found to be more in all the three host taxa while essential amino acids (30.1 %–35.4 %) were present at a lower relative percentage. In contrast to this, the relative percentage of essential amino acids (81.9 %) was observed to be higher as compared to non-essential amino acids (17.7 %) in lac insect hemolymph. These results led to the detection of lac insect’s endosymbionts. Moreover, this study revealed a clue regarding the importance of development of a synthetic diet for this insect so that a precise pathway of lac biosynthesis could be investigated for thorough understanding.     

Wolbachia infection in the Korean endemic firefly,Luciola unmunsana (Coleoptera: Lampyridae)

Wolbachia is one of the most prevalent endosymbiontic bacteria of arthropods. The bacterium induces sex ratio distortions in various host insects through processes such as cytoplasmic incompatibility, feminization, male killing, and parthenogenesis. We investigated if the Korean endemic firefly, Luciola unmunsana was infected with the bacterium because the species had an abnormal sex ratio in the field. The results show that some individuals are infected with the bacterium. Phylogenetic analyses showed that the bacterial strain infecting the firefly is closely related to strains that infect phylogenetically distant hosts.     

High Levels of Multiple Infections,Recombination and Horizontal Transmission of Wolbachia in the Andricus mukaigawae (Hymenoptera; Cynipidae) Communities

Wolbachia are maternally inherited endosymbiotic bacteria of arthropods and nematodes. In arthropods, they manipulate the reproduction of their hosts to facilitate their own spread in host populations, causing cytoplasmic incompatibility, parthenogenesis induction, feminization of genetic males and male-killing. In this study, we investigated Wolbachia infection and studied wsp (Wolbachia surface protein) sequences in three wasp species associated with the unisexual galls of A. mukaigawae with the aim of determining the transmission mode and the reason for multiple infections of Wolbachia. Frequency of Wolbachia infected populations for A. mukaigawae, Synergus japonicus (inquiline), and Torymus sp. (parasitoid) was 75%, 100%, and 100%, respectively. Multiple Wolbachia infections were detected in A. mukaigawae and S. japonicus, with 5 and 8 Wolbachia strains, respectively. The two host species shared 5 Wolbachia strains and were infected by identical strains in several locations, indicating horizontal transmission of Wolbachia. The transmission potentially takes place through gall tissues, which the larvae of both wasps feed on. Furthermore, three recombination events of Wolbachia were observed: the strains W₈, W₂ and W₆ apparently have derived from W₃ and W_5a, W₆ and W₇, W₄ and W₉, respectively. W₈ and W₂ and their respective parental strains were detected in S. japonicus. W₆ was detected with only one parent (W₄) in S. japonicus; W₉ was detected in Torymus sp., suggesting horizontal transmission between hosts and parasitoids. In conclusion, our research supports earlier studies that horizontal transmission of Wolbachia, a symbiont of the Rickettsiales order, may be plant-mediated or take place between hosts and parasitoids. Our research provides novel molecular evidence for multiple recombination events of Wolbachia in gall wasp communities. We suggest that genomic recombination and potential plant-mediated horizontal transmission may be attributable to the high levels of multiple Wolbachia infections observed in A. mukaigawae and S. japonicus.     

Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes.     

Bidirectional Cytoplasmic Incompatibility Induced by Cross-Order Transfection of Wolbachia: Implications for Control of the Host Population

Wolbachia are widespread endosymbionts in arthropods and some nematodes. This genus of bacteria is known to manipulate host reproduction by inducing cytoplasmic incompatibility (CI). This important phenotype is implicated in the control of host populations since Wolbachia can suppress host populations through the induction of CI in a way similar to the sterile insect technique. Here, we identified a candidate CI-inducing Wolbachia strain from the parasitic wasp Scleroderma guani (wSguBJ) by sequencing and phylogenetic analysis. This Wolbachia strain was then isolated, purified, and artificially transfected into the new whitefly host Bemisia tabaci through nymphal microinjection. Infection frequency monitoring by molecular detection showed that 60–80 % of the offspring from transfected whitefly populations was infected with wSguBJ six generations after the transfer. Laboratory rearing experiments indicated that the artificial transfection caused no significant difference in the numbers of offspring between the transfected and naturally infected populations and had no significant detrimental effects on the development of transfected males, although the development of transfected females was delayed. Reciprocal crossings revealed that bidirectional CI was induced between the transfected and naturally infected whiteflies. These data indicated that the cross-order transfer of the heterologous Wolbachia strain by nymphal microinjection was successful. Mass release of the transfected males that could stably carry the heterologous Wolbachia without significant compromise of fecundity/development may provide an alternative approach to control of host populations.     

Characterization of Wolbachia Host Cell Range via the In Vitro Establishment of Infections

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories.     

The Toll and Imd Pathways Are Not Required for Wolbachia-Mediated Dengue Virus Interference

Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virus-blocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system.     

共生菌Wolbachia在宿主中诱导表达胞质不相容因子的研究进展

沃尔巴克氏体(Wolbachia)作为节肢动物的胞内共生菌,可以引起宿主产生雌性化、孤雌生殖、杀雄和胞质不相容性(cytoplasmic incompatibility, CI) 4种生殖表型。其中CI是最常见的现象,表现为受感染的雄性昆虫与未感染或感染不兼容Wolbachia的雌性昆虫交配时引起胚胎死亡;而雌性感染同种Wolbachia时胚胎能够正常发育。CI是由被称为CI因子(cifA和cifB)的Wolbachia基因对调控的。其中,CifB作为毒剂在雄性中表达诱导产生CI,而CifA作为解毒剂在雌性中表达拯救CI。本文综述了CI因子结构、功能和作用机制的研究,以期为未来利用Wolbachia和CI进行蚊媒疾病和农业虫害的防控奠定基础。     

Bacteriocyte-like cells harbour Wolbachia in the ovary of Drosophila melanogaster (Insecta, Diptera) and Zyginidia pullula (Insecta, Hemiptera)

Wolbachia is the most widespread bacterial endosymbiont in insects. It is responsible for a variety of reproductive alterations of the hosts. Wolbachia is transmitted through the germline from mother to offspring and, in rare cases, between individuals. This implies that acquired properties (through symbiosis with Wolbachia) can become heritable. We investigated the transovarial inheritance of Wolbachia in two phylogenetically distant insects, Drosophila melanogaster and Zyginidia pullula. We detected in both systems bacteriocyte-like cells, densely packed with Wolbachia endosymbionts, at the tip of the ovarioles. Bacteriocytes are cells specialized to harbour bacteria, typical of mutualistic insect symbiosis. Our observations of bacteriocyte-like cells harbouring Wolbachia in the ovary emphasize the plasticity of the female reproductive system of insects, which maintains its function while some cells are densely colonized by bacteria. In summary, there is evidence from different insects that bacteria which behave as parasites of reproduction are harboured by cells resembling bacteriocytes, which appear to mediate transmission of the bacteria to the progeny. It seems a valid hypothesis that the bacteriocyte-like cells that we observed are not the result of a co-evolution of host and symbiont, considering that Wolbachia is not an obligatory symbiont in Drosophila and Zyginidia.     

Infection density,diversity, and distribution of Wolbachia bacteria in moths (Order Lepidoptera): First systematic report from Thailand

Members of the genus Wolbachia are a group of Rickettsia-like, intracellular, maternally inherited bacterial endosymbionts that infect a diverse range of insects and cause reproductive changes in their hosts. Although Wolbachia’s role in many insects has been extensively studied, only a little of their effects on host’s reproduction and their infection frequencies were reported in Lepidopteran which is one of the most diverse insects. Here, we present the first systematic survey of the Wolbachia infection status in different species of moths from three different geographic regions of Thailand, which was carried out during January to December in 2019 with the screening of 1,235 specimens in total of 58 moth species from 13 families. Specimens were collected from Khao Yai National Park (the Central and the Northeast regions), and Kaeng Krachan National Park (the West region). Infections of Wolbachia were screened by using polymerase chain reaction with 16S rRNA, ftsZ and wsp gene primers which the results indicated high rates of Wolbachia infection in moth populations from Thailand. Wolbachia was found in all different geographically populations in total of 625 individuals in total of 28 moth species from 9 families, including 144 individuals (46 males and 98 females) from the Central, 156 individuals (49 males and 107 females) from the Northeast, and 325 individuals (114 males and 211 females) from the West. The highest infection rate was 90.47% in the West populations and the average infection rate was 61.90%. The detection of Wolbachia in different moth populations from all regions was identical when all primers were used to screen for Wolbachia. The relative densities of Wolbachia within each individual were determined using quantitative real-time PCR and the result showed that there was a low Wolbachia infection density in these moth populations. These findings indicated that Wolbachia are distributed throughout the moth populations from Thailand.     

Microbial Symbionts in Insects Influence Down-Regulation of Defense Genes in Maize

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect.     

Reproductive compensation favours male-killing Wolbachia in a live-bearing host

Wolbachia are maternally inherited, cellular endosymbionts that can enhance their fitness by biasing host sex ratio in favour of females. Male killing (MK) is an extreme form of sex-ratio manipulation that is selectively advantageous if the self-sacrifice of Wolbachia in males increases transmission through females. In live-bearing hosts, females typically produce more embryos than can be carried to term, and reproductive compensation through maternal resource reallocation from dead males to female embryos could increase the number of daughters born to infected females. Here, we report a new strain of MK Wolbachia (wCsc2) in the pseudoscorpion, Cordylochernes scorpioides, and present the first empirical evidence that reproductive compensation favours the killing of males in a viviparous host. Females infected with the wCsc2 strain produced 26 per cent more and significantly larger daughters than tetracycline-cured females. In contrast to the previously described wCsc1 MK Wolbachia strain in C. scorpioides, wCsc2 infection was not accompanied by an increase in the rate of spontaneous brood abortion. Characterization of the wCsc1 and wCsc2 strains by multi-locus sequence typing and by Wolbachia surface protein (wsp) gene sequencing indicates that the marked divergence between these two MK strains in their impact on host reproductive success, and hence in their potential to spread, has occurred in association with homologous recombination in the wsp gene.     

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.     

Epidemiology of asexuality induced by the endosymbiotic Wolbachia across phytophagous wasp species: host plant specialization matters

Among eukaryotes, sexual reproduction is by far the most predominant mode of reproduction. However, some systems maintaining sexuality appear particularly labile and raise intriguing questions on the evolutionary routes to asexuality. Thelytokous parthenogenesis is a form of spontaneous loss of sexuality leading to strong distortion of sex ratio towards females and resulting from mutation, hybridization or infection by bacterial endosymbionts. We investigated whether ecological specialization is a likely mechanism of spread of thelytoky within insect communities. Focusing on the highly specialized genus Megastigmus (Hymenoptera: Torymidae), we first performed a large literature survey to examine the distribution of thelytoky in these wasps across their respective obligate host plant families. Second, we tested for thelytoky caused by endosymbionts by screening in 15 arrhenotokous and 10 thelytokous species for Wolbachia, Cardinium, Arsenophonus and Rickettsia endosymbionts and by performing antibiotic treatments. Finally, we performed phylogenetic reconstructions using multilocus sequence typing (MLST) to examine the evolution of endosymbiont‐mediated thelytoky in Megastigmus and its possible connections to host plant specialization. We demonstrate that thelytoky evolved from ancestral arrhenotoky through the horizontal transmission and the fixation of the parthenogenesis‐inducing Wolbachia. We find that ecological specialization in Wolbachia's hosts was probably a critical driving force for Wolbachia infection and spread of thelytoky, but also a constraint. Our work further reinforces the hypothesis that community structure of insects is a major driver of the epidemiology of endosymbionts and that competitive interactions among closely related species may facilitate their horizontal transmission.