The Wolbachia bacterium, one of the most prevalent endosymbiotic bacteria, is known to induce reproductive anomalies in various invertebrate taxa. We investigated Wolbachia infection frequency in 203 Coleopteran insects collected from Korea. Among them 26 species (12.8%) across families proved to harbor Wolbachia. The phylogenetic trees of two Wolbachia specific genes imply that there have been complex horizontal gene transfers and recombination events within and between divergent Wolbachia subgroups. Interestingly, we also find that the infection frequency is noticeably low and incongruent at the family level when compared to other literature cases of Wolbachia infection in beetle species.

Wolbachia is intra-cellular bacteria and frustrate host biology, such as parthenogenesis, feminization, male killing andsperm-egg incompatibility. It has been reported that > 90% of Aedes albopictus are infected with Wolbachia in the Republicof Korea (ROK). However any roles of the Wolbachia strains have not been reported in ROK. In this study, we infectedWolbachia to Vero cell line to investigate the cell migration which is related with the development of multi-cellular organisms,immune response and inflammation. Changes of wound healing and viability in vero cell after Wolbachia infection wereassessed. Cell migration was induced by Wolbachia after 24hr. Cell viability was not affected by Wolbachia after 24hr.This study will be helpful to understand the role of Wolbachia strain and support various information in Dengue andZika vector management programs.

The intracellular endosymbiont bacterium Wolbachia is currently known to be the most common in arthropods. It mayleads to male-killing, cytoplasmic incompatibility, parthenogenesis and feminization of genetic males with large impacton host ecology and evolution in mosquitoes. Mosquitoes was collected in four areas with various ecological conditionsusing black light traps and Wolbachia detection from the collected mosquitoes was carried out by PCR using WSP andFtsZ genes primers. A total of 385 individual mosquitoes were screened. 213 mosquitoes (55.3%) belonging to four specieswere positive for Wolbachia infections. Aedes albopictus showed the highest infection rate (100%). Wolbachia infectionand type were firstly reported in Aedes in Korea.

Aedes albopictus (Skuse) has been involved as the potential vector of imported zika virus disease and dengue fever throughout Republic of Korea. It has been reported that the successful introduction of a life-shortening strain of Wolbachia into Ae. albopictus halves adult lifespan and also directly inhibits the ability of a range of pathogens to infect this mosquito species. In our previous studies, Ae. albopictus collected from field was naturally infected by two strains of Wolbachia (wAlbA and wAlbB). However, virus regulation of two Wolbachia strains in Ae. albopictus have not been assessed, yet. In our studies, the two Wolbachia strains in about 20% among Ae. albopictus were removed for the virus regulation test using tetracycline. In this presentation, the detailed results and methods are introduced.

Vollenhovia emeryi (Formicidae: Myrmicinae) is divided into two morphs based on the wing length of the queen caste: the long-winged with a normal wing and the short-winged with the aberrant short wing. The phylogenetic analysis shows that the short-winged is derived from the long-winged. In Korea, only the long-winged morph is infected with Wolbachia while the short-winged is devoid of the bacterium suggesting that the short-winged evolved the resistance to the bacterial infection. Intriguingly, some Japanese short-winged colonies proved to still be infected with the bacterium. We hypothesized that the infected Japanese short-winged is the intermediate form in the process to complete cure. However, the data of the MLST and the measurement of Wolbachia density did not support our hypothesis. Our further experiment using microsatellite markers shows that the infected Japanese short-winged shows the similar genetic background to the long-winged. In this presentation we will discuss the potential resistance evolution in the Korean short-winged and future research direction at the genome level.

알파 프로테박테리아(α-proteobacterium)인 볼바키아(Wolbachia) 세균은 절지동물 세포내의 중요한 공생균 중의 하나이다. 그람 음성 세 균인 이 공생균은 기주동물의 여러 생물적 과정에 관여하고 있으며, 현재 생물적 방제 수단으로 주목 받고 있다. 볼바키아는 기주 세포의 세포질에 서식하는 세균인데 암컷을 통하여 세대간 전염된다. 볼파키아의 감염 개체 밀도를 높이기 위해 기주의 생식방식을 조작하는 다양한 전략을 발달시 켰다. 볼바키아 유전자형 계통은 볼바키아 표면 단백질(WSP)의 고변이영역 아미노산 서열과 복합좌위 서열 타이핑(Multilocus sequence typing, MLST)으로 결정된다. 상이한 유전계통 판별은 wsp, 16S rRNA, ftsZ, gltA, groEL 등 유전자 분자표지를 이용하게 된다.. 이 계통 볼바키아 세균과 그들의 우월한 표현형이 농업해충과 인간의 질병매개 곤충에 대한 방제 프로그램에서 이용 가능성이 고려되고 있다. 볼바키아 표현형들은 세포질불일치(cytoplasmic incompatibility, CI), 단성생식 유도(parthenogenesis induction, PI), 여성화(feminization, F), 수컷치사(male killing, MK) 등을 유발하는 것으로 알려져 있다. 기타 볼바키아 세균의 농업과 위생곤충 방제 프로그램에서 응용 방안을 고찰하였다.

The Wolbachia bacterium is one of the best studied reproductive manipulators found in variuos invertebrate taxa. Theoretically it infects two thirds of invertebrates. It is thought to have a huge impact on its hosts’ evolution. The ant is a very successful organism in terrestrial ecosystems especially with its charicteristic sociality. However, the influence of Wolbachia on the animal remains to be paid more attention. This study is to examine Wolbachia infection frequency in some ant species in Korea. The result shows that 9 out of 12 ant species harbor the bacterium. Furthermore, at least two species show polymorphism in their infection status at the colony level. This will be used as a foundation of further study on the interactions between the bacterium and ant species.

The intracellular endosymbiont bacterium Wolbachia is currently considered the most abundant in arthropods and has also been isolated from nematodes, amphipods, isopods, mites and spiders. Recently, Wolbachia-based research was focused on the control of disease vector-population, such as several mosquitoes such as genus Aedes and Anopheles which cause dengue fever and malaria, respectively. For the analysis of regional difference between vector mosquito Aedes albopictus and Wolbachia, we selected different regions and collected Ae. albopictus which were distinguished with mountain chain and waterway. Whole genomic DNA were extracted from collected specimens with 9 regions. PCR analysis and sequencing were accomplished in each specimen for Wolbachia detection and identification using WSP gene. As a results, almost mosquitoes were infected with two strain of Wolbachia both wAlbA and wAlbB. However, regional separation of vector mosquitoes, wAlbA strain of Wolbachia were showed more than 98% sequence similarity. In this study, we first reported that Wolbachia infection and type of Wolbachia in Korea and endosymbiont Wolbachia was showed highly sequence homologies.

The phylogenetic analysis the queen polymorphic Vollenhovia emeryi ant reveals derivation of the Wolbachia- free short-winged from the Wolbachia-infected long-winged. However, intriguingly, some Japanese short-winged colonies harbor Wolbachia. Wolbachia specific bacteriophage (WO) is also detected in more than half of the infected colonies with no clear distribution pattern across the host insect lineage. We hypothesized that 1) the infected Japanese short-winged is in the intermediate stage to complete loss of Wolbachia and 2) the phage invaded the host after the host insect diverged. To test the hypotheses, we studied the strain diversity using the multi-locus sequence typing (MLST) of five ant colonies; three longwinged colonies from Korea and one long-winged colony and one short-winged colony from Japan. Both Korean and Japanese V. emeryi colonies show unexpectedly high level of Wolbachia strain diversity. However, the diversity is not significantly different between the long-winged and the short-winged against our first hypothesis. Phylogenies of Wolbachia show Korean strains and Japanese strains are largely monophyletic indicating prior infection before the host divergence. The strain diversity of the phage is also surprisingly high. Phylogenies of orf2 and orf7 genes are incongruent to that of Wolbachia and geographically distinct. This indicates that the phage is spatially static and the current infection pattern may be the consequence of local repeated gain and loss of the phage.

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 intracellular endosymbiont bacterium Wolbachia is currently considered the most abundant in arthropods. Many Wolbachia manipulate host reproductive systems, it lead to male-killing, cytoplasmic incompatibility, parthenogenesis and feminization of genetic males with large impact on host ecology and evolution in arthropods. In this study, we investigated the distribution of Wolbachia infection in Ae. albopictus according to geographical distribution. We selected 9 areas and collected Ae. albopictus, these areas were distinguished with mountain chain and waterway. Whole genomic DNA were extracted from collected specimens and PCR analysis were accomplished in each specimen using Wolbachia specific primers such as 16S and WSP genes. As a results, we collected 730 Ae. albopictus at least 30 specimens in each area. Wolbachia infection rate showed different patterns between geographical region. Cheonbook, Cheonnam and Yeongdong showed 100% infection rate followed by Jeju (97%), Chungchung (88%), and Gyungnam (85%) with two Wolbachia marker genes. Our results indicated that Ae. albopictus are commonly infected with Wolbachia, it is possible that Wolbachia may act as endosymbiont in Ae. albopictus regardless of geographical region. Although, low infection rate of Wolbachia, we need continuous survey for the evaluation of Wolbachia strain within Ae. albopictus as a vector of dengue fever.

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.

Whiteflies harbor several secondary endosymbionts, which are maternally inherited from mother to offspring, that have major effects on host preferences, biology, and evolution. Here, we identified Wolbachia bacteria in sweetpotato whitefly (Bemisia tabaci) as well as whitefly popluations from other countries by comparison of 16S rDNA sequences. Wolbachia were detected in all tested indigenous B. tabaci populations (Bangladesh, Myanmar, Nepal, and the Philippines) as well as Q1 biotype of Korea, whereas they were absent from B biotype of Korea and Q biotype of China. Wolbachia were also detected in all five tested Aleurodicus dispersus populations as well as Tetraleurodes acaciae, whereas they were not detected in all seven Trialeurodes vaporariorum populatuions. In addiiton, Wolbachia were detected in parasitic wasp (Encarsia formosa) of B. tabaci as well as honeybee (Apis mellifera). Among the 19 whitefly populations from different countries, our analysis identified four phylogenetic groups of Wolbachia, thereby demonstrating the high diversity of this genus. Wolbachia phylogeny suggests a correlation of geographical range with ecological variation at the species level.

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.

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.

The Wolbachia bacterium, one of the most prevalent endosymbiotic bacteria, is known to induce reproductive anomalies such as cytoplasmic incompatibility, feminization, male killing and parthenogenesis in various arthropod species. The bacterium is considered to have had huge impacts on hosts' reproductive biology, immunity, evolution, and molecular machineries. Infection surveys on the bacterium have rather been limited to specific taxa that are mainly of economical importance or conducted with randomly collected organisms. Here we investigated infection frequency of Wolbachia in 206 Coleopteran insects collected from Korea. Among them 28 species (13.59%) across families proved to harbor Wolbachia. The phylogenetic trees based on the partial 16s rRNA gene and the partial Wolbachia surface protein (wsp) gene of Wolbachia show that all the Wolbachia strains belong to either Supergroup A or B and Wolbachia evolved independently from its hosts. In addition, the cophylogenetic analysis of the 16s rRNA gene and wsp gene implies that there have been horizontal DNA transfers and recombination events within and between divergent Wolbachia supergroups.