The ant species, Vollenhovia emeryi Wheeler (Hymenoptera: Myrmicinae) is endemic in East Asia and has invaded into North America. In this species, the queen caste shows polymorphism in its wing morphology; long-winged queen and short-winged queen, and two morphs are thought not to coexist in nature. This research is conducted to 1) deduce the phylogeographical structure of the two wing morphs in South Korea and to trace the distribution pattern from East Asia to North America, and 2) investigate the Wolbachia and WO phage infection frequency of the species. Either individuals or colonies of V. emeryi were collected from 80 locations, encompassing 68 locations in South Korea, 11 in Japan, and one in USA. Among the collected samples in South Korea, the long-winged morph is dominant and considered as the ancestral type, while the short-winged morph is very rare and derived character. The origin of the US population is neither Korea nor Japan at least in this study. However, we do not exclude the possibility that its origin is the other parts of Japan or the other countries. All of the long-winged morph are infected with Wolbachia, while the short-winged seems to be geographically partially infected. It suggests the possibility that the short wing trait is linked with the evolution of resistance to Wolbachia infection. Bacteriophage WO infection status has no correlation with host insect lineage.

The invasive black soldier fly, Hermetia illucens, has been paid much attention as an excellent organic matter decomposer. We conducted the nationwide survey and the population genetic study using a mitochondrial cytochrome C oxidase 1 gene to understand its genetic diversity and distribution pattern in Korea. The results show that it has successfully settled down in South Korea and there are only 10 haplotypes and the populations of the insect are highly differentiated. The results indicate that only few maternal lineages were introduced and their dispersal was restrained due to their short distance flying tendency since their introduction.

The little sized ant species, Vollenhovia emeryi Wheeler (Hymenoptera: Myrmicinae) is the only species belonging to genus Vollenhovia in South Korea, and it is endemic in East Asia encompassing Korea, Japan, China, Taiwan and has recently invaded into North America. In this species, the queen caste shows polymorphism in its wing form, the queen with normal wings called the long-winged (L) queen and short-winged (S) queen with aberrant small wings, and the two morphs are thought not to coexist in nature, however the morphology of workers and males of the two wing morphs are indistinguishable. We obtained a L gyne from a S colony that had been maintained in the laboratory. In addition, we compared the genome size of entire castes of the two wing morphs using flowcytometry. Our results confirm that the two wing morphs are obviously the same species, and moreover, the wing morph may be determinated by the epigenetical process.

In Hymenoptera, thanks to its haplodiploid sex determination, arrhenotokous parasitoid females are able to control offspring sex ratio by determining whether to use sperm or not at the time of oviposition. However, the offspring sex ratio may also be influenced by the mating behavior of a male. When a male searches females, he will have more chances to encounter with other males in a high male density population than low male density population. If the male is competitive, the male can find females faster and may have longer copulation time, and eventually transfer more sperm than other males. In this study, we investigated mate searching time, copulation duration and offspring sex ratio as a function of the male density. Although the results show difference between searching time and copulation duration, they are not statistically significant. We will discuss the statistical consequence, male density dependent offspring sex ratio and its evolutionary implications.

In Korea, twelve species in genus Myrmica have been described. Some of them are morphologically similar and this makes their identification difficult. For this reason, we collected several Myrmica species in question and inferred their phylogenetic relationship using the 418bp partial COI (cytochrome C oxidase 1) region from a total of 33 individuals. We found that the CO1 haplotypes are effectively grouped into three clusters that match well to their external morphological characters. Although this three species could be distinguished by the only small part of the COI region, the two individual sample of the M. kotokuii and one sample of the M. carinata are included in the M. kurokii group. The results indicate that the morphological identification could be obscure in the three species and it requires a close examination for this phenomenon.

Wolbachia is one of the most common endosymbionts best known to induce several reproductive alterations in its insect hosts. In some cases, the insect hosts harbor more than two strains of the bacterium. The Vollenhovia emeryi ant lives in dead trees and is morphologically subdivided into the long-winged and the short-winged. Interestingly the short-winged morph is free of Wolbachia, but only the long-winged morph is multiple- infected with the Wolbachia bacterium. We sampled four populations of the long-winged morph in Korea and performed pyrosequencing in Multilocus Sequence Typing (MLST), to determine the bacterial strain diversity. Six different gene regions (coxA, fbpA, ftsZ, gatB, hcpA and wsp gene) were targeted and amplified. However, the result shows that diversity of haplotypes is very high. The pyrosequencing approach in MLST, a new method of discriminating Wolbachia strains, is promising to effectively detect multiple infections and rare haplotypes.

The hologenome theory of evolution proposes that an organism is simply not an individual but the totality of numerous microbial symbionts to the host. In fact, the evidence of biochemical and physiological associations between the symbionts and the host has been growing fast in all major taxa. The Vollenhovia emeryi ant is tiny and found nationwide in Korea. The ant can be further categorized by its wing morphology, eg. long-winged and short-winged. Our initial screening process showed that the microbial reproductive manipulator, the Wolbachia bacterium, only infected the long-winged morph. This gave us a good opportunity to investigate the effect of the Wolbachia infection on the bacterial community diversity using the next generation sequencing technique. We find that there are about 180 bacterial symbionts in the short-winged morph. On the other hand, the long-winged morph has only about 20 bacterial symbionts. This implies that the bacterial community diversity may be subject to the existence of Wolbachia. Furthermore, the Wolbachia strain diversity is unexpectedly high. In addition, the bacterial structure difference among castes indicates that there may be labour division even between queens. The results and future research direction will be discussed from the hologenome theory perspective.

In haplodiploid sex determination, females are sexually reproduced from fertilized diploid eggs, and males from unfertilized haploid eggs. Haplodiploid sex determination seems simple in that sex depends simply on the ploid level. However, the underlying genetic mechanisms are thought to be much more complicated than expected. Among them, a powerful proposed mechanism is genomic imprinting. All epigenetic on-off systems require target genes, unless the systems target histone proteins on chromosomes. For Hymenoptera, a good candidate target gene in terms of sex determination is known either as feminizer (fem) or transformer (tra) in many insects. These two genes are essential for expressing femaleness. In most Hymenopteran insects, the maternal tra seems to be methylated and consequently not expressed, while the paternally derived tra gene is not methylated. Therefore, a fertilized egg with the paternally derived active tra gene will develop into a functional female. Like all Hymenoptera, ants (Formicidae) have haplodiploid sex determination. In Vollenhovia emeryi, however, queens are produced clonally while workers derive from fertilized eggs. Males are haploid, likewise deriving from fertilized eggs, but only after selective elimination of their maternal genome. Under the conventional genomic imprinting model, we would have expected that the opposite pattern of what is observed in others. Here we present extraordinary sex determination and suggest our hypothesis about genomic imprinting pattern in V. emeryi

Vollenhovia emeryi ant is distinguished by its wing morphology; short winged (SW) and long winged (LW). Its reproduction shows a bizarre genetic caste system distinct from other social hymenopteran insects. Unfertilized eggs undergo genome duplication and develop into clonal gynes. Fertilized eggs develop either into workers or males. The fate of the fertilized eggs is determined whether maternal genome loss (MGL) takes place after fertilization. Eggs with MGL become haploid males with only paternal half of the genome. Without MGL, the eggs become workers with maternal and paternal half of the genome. In this research, we analysed 5 nuclear genes of SW and LW individual ants. Among them, two genes from an SW male are identical to those of LW, and one gene from the SW male seems a variant of LW. The result indicates that SW males are derived from LW colonies. From the genetic relatedness point of view individuals in the same castes are genetically identical. On the other hand, between workers and two reproductives, the relatedness is asymmetrical and there is even no gene sharing between gynes and males. The conventional genetic relatedness by Hamilton is revised under this condition.

The ant species, Vollenhovia emeryi, is distributed in Far East. The species can be divided into two major groups by their wing morphology of reproductives: short-winged and long-winged. A nationwide survey of the species was conducted for analyzing the mitochondrial haplotype diversity and genetic population structure. We collected 91 samples from 40 locations. A total of the 1239 bp partial COI (cytochrome C oxidase 1) region was used for the analyses. We found the total of 21 haplotypes. The mitochondrial haplotypes may correspond to the wing morphology. The genetic population structure examined potential geographic barriers of gene flow such as distance, mountains, rivers and plains which are non-mountain areas to prevent dispersal through mountain range. The result implied that no barriers considered in this study affected differently gene flow. Therefore, the behavioral characteristics of the ant may be the causal constraint of its genetic exchange.

The α-proteobacterium Wolbachia is maternally inherited and is known to induce reproductive distortions in a wide range of insect taxa such as cytoplasmic incompatibility, feminization, male killing and parthenogenesis (PI). When a female is infected with PI-Wolbachia, she does not need a male to produce female offspring, because the female can produce female offspring via gamete duplication without the aid of sperm. However, in the parasitoid wasp Trichogramma kaykai species, Wolbachia infected parthenogenetic females still produce a fraction of male offspring. Offspring sex ratio for 10 days are different according to each T. kaykai isofemale line that is infected with PI- Wolbachia. This may be caused by complex interactions between Wolbachia and the host genetic backgrounds on converting sex of infected eggs.

Vollenhovia emeryi chosenica (Wheeler) (Hymenoptera: Myrmicinae) is an ant species frequently found in forests. In nature, two phenotypically distinct forms are found e.g. long winged and short winged. Unlike other hymenopteran insects, the ant is unique in its mode of reproduction. In this species, queens are clonally reproduced from unfertilized eggs. On the other hand, workers develop from fertilized eggs. Strikingly, haploid males are reproduced from fertilized eggs after destroying the maternal half of the genome e.g. maternal genome loss (MGL) consequently only with the paternal half of the genome. We collected the ant colonies nationwide in 2011. In this study, we demonstrate that the ant is infected with Wolbachia, the bacterial reproductive manipulator in various insects. Interestingly, only the long winged morphs seem to be infected. Furthermore, most colonies are mulitple-infected except two colonies collected from Chuncheon and Mt. Deogyu. We will discuss potential interactions among the Wolbachia infection polymorphism and wing morphology, and evolution of clonal reproduction and MGL.

Haplodiploid sex determination occurs in a wide range of animals, especially in Hymenoptera, where a fertilized egg develops into a diploid female and unfertilized into a haploid male. However, recent studies on diploid functional males in some wasps suggest that the simple addition of paternal gene by fertilization may not be enough to explain female offspring production in the sex determination system. Recently, activation of sex determination gene (tra) was found to have a pivotal role in determining the sex of Nasonia vitripennis. In N. vitripennis, tra is activated only on the paternal genome (i.e. sperm) not on the maternal counterpart (i.e. egg). Such parent specific activation of a gene is controlled by a epigenetic factor, DNA methylation. However, in Trichogramma kaykai, Wolbachia induces female offspring production without sperm. Therefore all female offspring are clonal to the maternal gene. This violates the role of activated sex determination gene (tra) from sperm in the wasp. We hypothesize that Wolbachia has an ability to activate the gene by demethylation. This hypothesis indicates that the target of sex ratio distorting endosymbionts may be an upstream gene. It will enhance our understanding of evolution of haplodiploid sex determination.

To understand the evolution and speciation of closely related species, a multiple approach encompassing morphological, behavioral, and genetic analyses is necessary. In Korea, three species of Loxoblemmus crickets occur widely. L. campestris and L. equestris are morphologically indistinguishable, whereas males of L. doenitzi are different from the other two species in head morphology. The genetic analyses using the partial mitochondrial COI sequences showed that L. doenitzi diverged off earlier than L. campestris and L. equestris. The analyses of laboratory recordings revealed that distributions of calling song characters generally overlapped among three cricket species. However, the number of pulses in a chirp was two in L. doenitzi and four in L. campestris, but it was greater than or equal to six in L. equestris. Provided that females make mate choice based on this calling song character, the differentiation in this character may lead to premating reproductive isolation and may have evolved during the speciation proccess in these closely related species.

The α-proteobacterium Wolbachia pipientis infects a wide range of arthropods and filarial nematodes. Wolbachia is maternally inherited and is known to induce reproductive anomalies such as cytoplasmic incompatibility, feminization, male killing and parthenogenesis induction (PI). Trichogramma kaykai is a tiny wasp that parasitizes on lepidopteran eggs. When a female of the wasp is infected with PI-Wolbachia, the female produces female offspring via gamete duplication without the aid of sperm. As she ages, however, the fraction of male offspring increases. In this study, we investigated the effect of host genetic background on the expression of sex ratio between isofemale lines. Virgin females of six isofemale lines were allowed to lay eggs individually for 10 days. There was the positive relationship between female age and the offspring sex ratio. Furthermore, the sex ratio was significantly different among isofemale lines, implying that the host genetic background had an effect on the sex ratio. Based on the results, evolution of symbiosis in terms of sex ratio and future experimental design are discussed.

The Wolbachia bacterium is known to induce reproductive anomalies in various insect taxa such as cytoplasmic incompatibility, feminization, male killing and parthenogenesis. It is hypothesized that the degree of reproductive anomalies is dependent on the bacterial infection density. In this study, we attempted to test the hypothesis using the tiny egg wasp, Trichogramma kaykai that has served as the model system of parthenogenesis where an unfertilized egg develops into a female due to the bacterial infection. So far this is only found in haplodiploid organisms. The results show that 1) as mothers aged, they started producing male offspring, 2) the sex ratio was negatively correlated with the bacterial infection density, 3) female offspring were more than six times heavily infected with Wolbachia than male offspring in the species. In conclusion, female offspring production, parthenogenesis, is as a function of the Wolbachia bacterial density in this species.

evolution of multicellular organisms. Especially in insects, a large amount of bacterial symbionts are illustrated. Among the insect taxonomic group, sap feeding insects, mostly Heteroptera, that feeds on poor nutrient sources from plants, have developed special organs where symbiotic bacteria can reside. The bacteria mainly provide the host insects with some essential amino acids. This has built evolutionarily unbreakable tie between the symbionts and the their hosts. Here we present an obligate symbiont from Lycorma delicatula (Heteroptera: Fulgoridae) being thought to invade the Korean penninsula recently. Based on the partial 16s rRNA gene sequence, the symbiotic bacterium is identified as Cadidatus Sulcia mullleri. The Genbank data indicates that the bacterium is found in various heteropteran families. This may imply the acquisition of the bacterium precedes the hosts' divergence, though there is an alternative postulation. We discuss its distribution in the sap-feeding insects and potential role on survival of L. delicatula and paratransgenetic application of the bacterium in controlling L. delicatula.

The black soldier fly (Hermetia illucens) (Diptera: Stratiomyidae) is an invasive species that is thought to be endemic in Korea. The insect has been paid much attention thanks to its ability to decompose organic wastes. We collected the insect nation-wide in 2006 and 2010 to investigate its genetic structure based on the mitochondrial cytochrome oxidase 1 (mt CO1) haplotype. The results show that during the time period the insect seems to lose some rare haplotypes. We conclude that the insect may have experienced genetic bottleneck while its settlement in Korea. In our presentation, we show its updated haplotype network and genetic differentiation and propose potential explanations.

The black soldier fly (BSF), Hermetia illucens, is known as a beneficial insect and feeds on organic materials derived from animals and human, resulting in reduction of food waste and conversion of organic materials. Despite of a lot of study about the BSF, there is a less information about composition of digestive enzyme of the BSF larva. Experimentally, there is no evidence about characterization of digestive enzyme of the BSF. We investigated biochemical property of digestive enzyme released from the salivary and gut of the BSF. Through digestive enzyme assay, we found that the BSF has amylase, lipase and protease activity in gut extracts, resulting in that the BSF belong to polyphagous insect group. In the BSF gut, trypsin-like protease activity showed one peak at various temperature and pH condition. This result means the BSF has probably a similar form of trypsin-like enzymes. On study of comparison of enzyme activity between the BSF and the housefly using the apiZYM kit, the BSF had more strongly digestive enzyme activity than one of the housefly about leucine arylamidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase. This finding supports that the BSF can ingest raw waste far more efficiently than any other known species of fly as reported previously.

Taxonomic resolution of the Nosema/Vairimorpah clade has been augmented with DNA sequences of the small subunit (SSU) and large subunit (LSU) ribosomal RNA (rRNA) and the arrangement of SSU and LSU. Based on the two characteristics, the clade is largely divided into two, i.e. 'true' Nosema sub-group and 'non-true' Nosema sub-group within the clade. Our study shows that a novel Nosema species isolated from Pieris rapae has mixed characteristics of the 'true' and non 'true' Nosema sub-group based on the topology of SSU and LSU sequences, and rRNA of the isolate is normally organized. Additionally, the length of ITS can be a diagnostic tool to distinguish 'true' Nosema from non 'true' Nosema in the Nosema/Vairimorpha clade based on its nucleotide length as reported before. To our knowledge, this is the first report of recombination event in the Nosema/Vairimorpha clade.