Yoshiomi Kato

Feminization of genetic males by a symbiotic bacterium in a butterfly, Eurema hecabe (Lepidoptera: Pieridae)

Abstract.Wolbachia are symbiotic bacteria found in many arthropods and filarian nematodes. They often manipulate the reproduction of host arthropods. In the present study, female-biased sex-ratio distortion in the butterfly Euremahecabe was investigated. Breeding experiments showed that this distorted sex ratio is maternally inherited. When treated with tetracycline, adult females of the thelygenic line produced male progeny only. After PCR using Wolbachia-specific primers for the ftsZ gene a positive result was seen in the thelygenic females, but not in male progeny from tetracycline-treated females, or individuals from a Tokyo population with normal sex ratio and reproduction. Cytological observations showed that thelygenic females lack the sex chromatin body (W chromosome). The results strongly suggest that the sex-ratio distortion in E. hecabe is due to feminization of genetic males by Wolbachia.

Genetic structure of sibling butterfly species affected by Wolbachia infection sweep: evolutionary and biogeographical implications

It was recently recognized that in Japan, the common yellow butterfly, Eurema hecabe, consists of two sibling species, which have been unnamed yet and tentatively called yellow (Y) type and brown (B) type. We investigated the diversity of nuclear and mitochondrial genes in Japanese populations of Y type and B type of E. hecabe. The phylogeny based on nuclear genes agreed with the distinction between Y type and B type, which had been also supported by a wide array of biological data. However, the phylogeny based on mitochondrial genes did not reflect the distinction. PCR survey of Wolbachia revealed that B‐type populations were all infected while Y‐type populations contained both infected and uninfected individuals. A single genotype of Wolbachia, which was inferred to be a CI‐inducing strain from their wsp gene sequence, was prevalent in these populations. Notably, the mitochondrial phylogeny was in perfect agreement with the pattern of Wolbachia infection, suggesting that the Wolbachia infection had affected the mitochondrial genetic structure of the host insects. Probably, the Wolbachia strain and the associated mitochondrial genomes have been occasionally introduced from B‐type populations to Y‐type populations through migration and subsequent interspecific hybridization, and CI‐driven population sweep has been spreading the Wolbachia strain and the particular mitochondrial haplotypes, which originated from B‐type populations, into Y‐type populations. On the basis of these results together with the geological and biogeographical knowledge of the Japanese Archipelago, we proposed an evolutionary hypothesis on the invasion and spread of Wolbachia infection in B‐type and Y‐type of E. hecabe.

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.

Biogeography of the subspecies of Parides (Byasa) alcinous (Lepidoptera: Papilionidae) based on a phylogenetic analysis of mitochondrial ND5 sequences

Abstract.  The phylogeny of the butterflies Parides (Byasa) alcinous caught at various localities in Japan and the Ryukyu Islands and in the eastern part of the Eurasian Continent was analysed using mitochondrial DNA sequences coding for NADH dehydrogenase subunit 5 (778 bp). The same phylogenetic relationship among P. (B.) alcinous subspecies was obtained with all analytical methods used, and was supported by high bootstrap values. The female butterfly wing pattern that characterizes each subspecies was unrelated to the phylogenetic relationship among the subspecies. The phylogenetic trees show that one group composed of ssp. alcinous and yakushimanus, which are distributed in the main area of the Japan Archipelago, the Korean Peninsula and southern Primorski of Russia, and the other group comprised of ssp. loochooanus, bradanus and miyakoensis, all of which are distributed in the Ryukyu Islands, diverged from a common ancestor. The ssp. loochooanus distributed in Amami and Okinawa Islands then diverged, and ssp. bradanus and miyakoensis distributed in Yaeyama and the Miyako Islands, respectively, finally diverged. This divergence order nearly agrees with the palaeogeography of the Ryukyu Islands that has been established in Pliocene and Pleistocene (0.2–2 MYA), suggesting that P. (B.) alcinous has been isolated in the Ryukyu Islands since the establishment of the islands.

Molecular phylogeography of two sibling species of Eurema butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.

An FXPRLamide Neuropeptide Induces Seasonal Reproductive Polyphenism Underlying a Life-History Tradeoff in the Tussock Moth

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH2 (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.

Influence of visual stimuli on host location in the butterfly, Eurema hecabe

The influence of visual stimuli on female host location was investigated in Eurema hecabe (Lepidoptera: Pieridae). Mated females with no oviposition experience were presented with artificial plant models treated with the methanol extract of the host plant, Lespedeza cuneata (Fabaceae). When models of different colours were presented to females, they landed and deposited eggs predominantly on the yellow‐green model. However, the size of the model was not important. When females were presented with simple square models and a model on which small squares were assembled, they predominantly chose the assembly model. This suggests that female discern the pattern which resembles the leaf of their main host plants, which consists of many small leaflets. However, the whole image of a leaf of the host plant was not an essential cue for host location, as females also chose the square with the central space more frequently than that without the space. We suggest that the long contour of a complicated form plays a role in female host location.

Sexual Isolation Between Two Sympatric Types of the Butterfly Eurema hecabe (L.)

In Japan, the butterfly Eurema hecabe (L.) consists of two separate types. These types differ in wing fringe color (brown or yellow), which is linked with expression of seasonal wing morphs and with ability for host-plant use. It was recently found that these two types occur together on the subtropical Okinawa-jima Island (one of the Ryukyu Islands). In the present work we studied whether sexual isolation may exist between these sympatric types of E. hecabe on Okinawa-jima, using caged conditions. When living females (days 0, 1, and 7) were presented to males of the same or different type, females of the brown type received males of the same type and responded with a refusal posture to those of the yellow type. Females of the yellow type received males of the same type at all ages tested. However, when they were exposed to males of the brown type, the proportion of females showing a refusal posture changed with female age. The proportion was high at day 7 but relatively low at day 0 or day 1. When model females of the brown and yellow types were simultaneously presented to males of each type, males courted not only the model females of the same type, but also those of the different type. The results suggest that (1) sexual isolation exists between these types of E. hecabe on Okinawa-jima, although it is not perfect; (2) this isolation is mediated by female recognition but not male recognition; and (3) the sexual isolation system between two types is asymmetrical, depending on the female age after adult eclosion.

Temporal pattern of changes in mitotic frequency in the epidermis and other larval tissues of Bombyx mori

Abstract Temporal changes in mitotic frequency were examined in various tissues through late larval life of Bombyx mori . From the second larval ecdysis to the third and from the third larval ecdysis to the fourth, there was a definite temporal change of mitotic pattern in each tissue. In the epidermis as well as in the tracheal epithelium, mitoses began to appear about 1 day after an ecdysis, and showed a maximum 1 to 2 days after an ecdysis. In the fat body, mitoses were observed continuously through the instars, and the mitotic frequency showed a maximum state just before an ecdysis. In the abdominal muscle the frequency was highest at about the middle of the period between two successive ecdyses. Furthermore, epidermal mitoses coincided with the time when the density of epidermal nuclei per unit area decreased to a half. This suggests that epidermal mitoses may be initiated by some process related to the increase in cell size.

Wolbachia-induced feminisation newly found in Eurema hecabe, a sibling species of Eurema mandarina (Lepidoptera: Pieridae)

1. Complete feminisation of genetic males into functional females, a unique case among insects, is known in Eurema mandarina (former Eurema hecabe Y type) that are infected with two strains of Wolbachia, wCIEm and wFemEm.