Kazuki Miura

The first finding of a Rickettsia bacterium associated with parthenogenesis induction among insects

The larval endoparasitoid, Neochrysocharis formosa (Westwood), is an important natural enemy of the leafminer Liriomyza trifolii (Burgess) in Japan. The thelytokous strain of N. formosa mostly produces female progeny. Male progeny were produced by females treated with tetracycline, suggesting that microorganisms induce thelytoky in N. formosa. The result of nucleotide sequencing of the 16S rRNA gene indicated that the parasitoid is infected with a Rickettsia bacterium, which appears to be causative of the thelytoky. Although Rickettsia-bellii-like bacteria have been found to be associated with various reproductive disorders, this is the first finding of a parthenogenesis-inducing Rickettsia among insects.

Multiple infection with Wolbachia inducing different reproductive manipulations in the butterfly Eurema hecabe.

Wolbachia are rickettsial intracellular symbionts of arthropods and nematodes. In arthropods, they act as selfish genetic elements and manipulate host reproduction, including sex–ratio distortion and cytoplasmic incompatibility (CI). Previous studies showed that infection of feminizing Wolbachia and CI Wolbachia sympatrically occurred in the butterfly Eurema hecabe. We demonstrate that feminization–infecting individuals can rescue sperm modified by CI–infecting males. Phylogenetic analysis revealed that feminized individuals are infected with two distinct Wolbachia strains: one is shared with CI–inducing matrilines, and the other is only found in feminized matrilines. Therefore, the simultaneous double manipulation, CI rescue and feminization, is caused by different Wolbachia strains in feminized individuals, not by a single Wolbachia with two functions. This is the first finding of double infection of Wolbachia with different reproductive manipulations.

A new cytogenetic mechanism for bacterial endosymbiont-induced parthenogenesis in Hymenoptera

Vertically transmitted endosymbiotic bacteria, such as Wolbachia, Cardinium and Rickettsia, modify host reproduction in several ways to facilitate their own spread. One such modification results in parthenogenesis induction, where males, which are unable to transmit the bacteria, are not produced. In Hymenoptera, the mechanism of diploidization due to Wolbachia infection, known as gamete duplication, is a post-meiotic modification. During gamete duplication, the meiotic mechanism is normal, but in the first mitosis the anaphase is aborted. The two haploid sets of chromosomes do not separate and thus result in a single nucleus containing two identical sets of haploid chromosomes. Here, we outline an alternative cytogenetic mechanism for bacterial endosymbiont-induced parthenogenesis in Hymenoptera. During female gamete formation in Rickettsia-infected Neochrysocharis formosa (Westwood) parasitoids, meiotic cells undergo only a single equational division followed by the expulsion of a single polar body. This absence of meiotic recombination and reduction corresponds well with a non-segregation pattern in the offspring of heterozygous females. We conclude that diploidy in N. formosa is maintained through a functionally apomictic cloning mechanism that differs entirely from the mechanism induced by Wolbachia.

Comparison of Life History Characters of Arrhenotokous and Wolbachia-Associated Thelytokous Trichogramma kaykai Pinto and Stouthamer (Hymenoptera: Trichogrammatidae)

Abstract We analyzed the life history characters related to rate of potential population growth for two genetically identical lines of Trichogramma kaykai Pinto and Stouthamer (Hymenoptera: Trichogrammatidae). These lines differed in that one was infected with parthenogenesis-inducing Wolbachia (W+), whereas the other was free of Wolbachia (W−) through antibiotic treatment. For both lines, the number of females emerging per day was the highest from eggs laid during the first 2 d of oviposition. Although adult longevity in W− was shorter than that in W+, number of eggs reaching the prepupal stage, adult emergence, and female emergence were significantly higher for W− than for W+. There were no significant differences in the number of mature ovarian eggs at emergence between W− and W+. The values of mean length of generation (T) in W+ was similar to that in W−, but the values of net reproduction rate (R0) and intrinsic rate of natural increase (rm) in W+ were lower than those in W−. In most field populations, the W+ individuals form a small percentage (<5%) of the total population. The relative low growth rate caused by the Wolbachia infection may contribute to this low prevalence of the infection. Because differences in the relative population growth rates would soon result in the extinction of the infected population in the field, other factors must play a role in allowing the infection to maintain itself in the field.

Gene flow between sexual and asexual strains of parasitic wasps: a possible case of sympatric speciation caused by a parthenogenesis-inducing bacterium.

Sympatric speciation is strictly defined as the emergence of two species from a population in which mating has been random with respect to the place of birth of the mating partners. Mathematical models have shown that sympatric speciation is possible, but very few examples have been documented in nature. In this article, we demonstrate that arrhenotokous and thelytokous strains of a parasitic wasp, Neochrysocharis formosa, speciated sympatrically through infection by a symbiotic bacterium Rickettsia for the following reasons: First, Rickettsia infection was detected in all of the thelytokous strains collected throughout Japan. Second, the arrhenotokous and thelytokous strains have been collected sympatrically. Third, crossing experiments between the two strains did not result in fertilized offspring. In addition, the two strains were genetically isolated at the nuclear and mitochondrial genes. Fourth, the two strains showed a sister relationship in nuclear 28S rRNA gene. Finally, thelytokous females treated with antibiotics produced Rickettsia‐free male offspring of the same reproductive form as arrhenotokous females indicating that the thelytokous strain could have speciated sympatrically from an individual of the arrhenotokous strain.

Control of Myzus persicae and Lipaphis erysimi (Hemiptera: Aphididae) by adults and larvae of a flightless strain of Harmonia axyridis (Coleoptera: Coccinellidae) on non-heading Brassica cultivars in the greenhouse

The green peach aphid [Myzus persicae (Sulzer)] and turnip aphid [Lipaphis erysimi (Kaltenbach)] are economically important pests with a worldwide distribution. We have evaluated the efficacy of releasing adults and larvae of a flightless strain of the multicolored Asian lady beetle (Harmonia axyridis Pallas) as a control measure against these aphids on plants of non-heading Brassica cultivars. Both adults and larvae of H. axyridis were observed to be effective biocontrol agents, markedly decreasing the numbers of aphids. The residence duration of adults was longer than that of larvae. The proportion of non-marketable plants damaged by aphids was lower in plots into which either adults or adults and larvae of H. axyridis had been released. These results suggest that both adults and larvae of this flightless strain of H. axyridis are effective in controlling aphids on plants of non-heading Brassica cultivars.

Application of Molecular Techniques to Distinguish Liriomyza trifolii from L. sativae (Diptera: Agromyzidae) on Tomato Cultivation in Japan

Abstract A molecular method is applied for differentiating the morphologically related leafminers Liriomyza trifolli and L. sativae on tomato cultivation. The method requires multiplex polymerase chain reaction (PCR) amplification of a region of mitochondrial cytochrome oxidase DNA using multiprimer sets. The method divides the mitochondrial fragment of L. trifolli into two fragments and L. sativae into three fragments. It is faster, less costly, and easier than random amplified polymorphic DNA-PCR, PCR-restriction fragment-length polymorphism, and DNA sequencing and more sensitive than the enzyme electrophoresis method, which are other ways to differentiate these two species. We applied the method to samples from populations of another place, sex, and stage and obtained the same results.

Transfer of a parthenogenesis-inducing Wolbachia endosymbiont derived from Trichogramma dendrolimi into Trichogramma evanescens.

Wolbachia, which are maternally transmitted endosymbionts, are considered to have moved horizontally between invertebrate hosts multiple times. However, it is not well understood how easily Wolbachia are transmitted horizontally between different hosts and how frequently horizontally-transmitted Wolbachia become established in their new hosts. We transferred a parthenogenesis-inducing Wolbachia endosymbiont derived from the parasitic wasp Trichogramma dendrolimi to Trichogramma evanescens. Specifically, Wolbachia was cultivated in a mosquito cell line and the Wolbachia-infected cells were microinjected into uninfected T. evanescens. Among 276 pupae inoculated with Wolbachia-infected cells, 65 adults emerged (G0). Diagnostic PCR demonstrated that 25 of 37 G0 females (68%) were Wolbachia-positive. Among isofemale lines established from G0 females, the proportions of infected lines were 80% (20 of 25) in G1 and 100% (18 of 18) in G2. In an isofemale line, infection was stably maintained for more than 10 generations. These results indicate invasion of Wolbachia into the germline of the recipient insect. Quantitative PCR demonstrated that the Wolbachia titer in the recipient host was significantly lower than that in the native host. The absence or very low number, if any, of parthenogenetically-reproducing individuals in the recipient host may be caused by the low Wolbachia titer. The Wolbachia titer in the recipients was lower in G11 than in G5, suggesting a decline in the density. Together with a previous report, our study may imply that Wolbachia in Trichogramma species are highly adapted to their hosts, which hinders robust expression of the Wolbachia phenotype in non-native host species.

Distribution Patterns of Wolbachia Endosymbionts in the Closely Related Flower Bugs of the Genus Orius: Implications for Coevolution and Horizontal Transfer

Vertical transmission is the primary route of the endosymbiont Wolbachia for its own spread among invertebrate hosts, but horizontal transmission between different hosts is believed to have occurred multiple times. However, it is not well known how Wolbachia commonly spread among closely related hosts. We focused on the closely related species of the minute pirate bugs belonging to the genus Orius, which are important biological control agents in agricultural crops because they are the most useful natural enemy of various tiny pests, such as thrips. Here, we examined five Orius species (Orius sauteri, Orius nagaii, Orius minutus, Orius strigicollis, and Orius tantillus) from eight geographic localities in Japan for Wolbachia infection. Two distinct strains, wOus1 and wOus2, were detected based on Wolbachia surface protein (wsp) gene sequencing. Furthermore, multilocus sequence typing revealed that each of the strains comprised two variants that differed in a single nucleotide. The overall distribution patterns of the two Wolbachia strains were found to differ among host species: prevalent double infection with wOus1 and wOus2 in O. strigicollis; fixation of single infection with wOus2 in O. nagaii; occurrence of single infection with wOus1 in O. sauteri; prevalence of single infection with wOus1 in O. minutus with an exception in a single population; and lack of Wolbachia infection in O. tantillus. Such differences in the distribution patterns of Wolbachia may reflect the evolutionary history of Wolbachia infection among Orius species and/or ecological and physiological differences among the Orius species that determine the invasiveness and maintenance of the two Wolbachia strains.

Control of Aphis gossypii and Myzus persicae (Hemiptera: Aphididae) by a flightless strain of Harmonia axyridis (Coleoptera: Coccinellidae) on green pepper plants in open fields

The cotton aphid Aphis gossypii Glover and the green peach aphid Myzus persicae (Sulzer) are economically important pests with a worldwide distribution. We evaluated the efficacy of releasing a flightless adult strain of the multicolored Asian lady beetle Harmonia axyridis (Pallas) as a control measure against these aphids on green pepper plants in open fields. Flightless H. axyridis adults were observed on the green pepper plants in the releasing plots throughout the experimental period and were found to be effective biocontrol agents, markedly decreasing the numbers of aphids. These results suggest that adults of this flightless strain of H. axyridis are effective in controlling aphids on green pepper plants in open fields.