Beauveria bassiana (Bb) is an entomopathogenic fungus with a wide host range, and is commonly used as an environment-friendly biopesticide. However, the molecular mechanisms of Bb-host interactions are not well understood. Here, RNA isolated from a highly virulent strain of B. bassiana (Bb JEF-007) and Riptortus pedestris (Hemiptera: Alydidae) (bean bug) infected with this strain were subjected to high throughput next generation sequencing (NGS) to analyze and compare transcriptomes. Differentially expressed gene (DEG) analysis showed that 2,381 genes were up-regulated and 2,303 genes were down-regulated upon infection. Most DEGs were classified into the categories of single-organism, cellular and metabolism processes by gene ontology (GO) analysis. Carbon metabolism-related enzymes in the glyoxylate cycle were significantly up-regulated, suggesting a possible role for them in Bb growth in the host. This work provides insight into how entomopathogenic B. bassiana occupies agriculturally harmful bean bug at the late stage, which might be essential during fungal infection.

Bean bug, Riptortus pedestris (Hemiptera: Alydidae) is an agriculturally serious pest in East Asian countries. Chemical pesticides have been contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Herein entomopathogenic Beauveria bassiana JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by contact-exposure and spray methods. The Agrobacterium tumefaciens-mediated transformation of B. bassiana JEF-007 generated random transformants and some mutants showed reduced virulence against Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and R. pedestris nymph. Compared to the wild-type, the two transformants showed remarkably different morphology, conidial production, and thermotolerance. To figure out pathogenicity-related genes, thermal asymmetric interlaced (TAIL) PCR of the random transformants was performed and possibly some virulence-related genes were predicted. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Although expression of foreign genes in a crop species has been target techniques for powerful protection against insect pests, classical breeding programs using varietal resistance of a crop are still being processed. It is, however, frequently difficult to find key products expressed from resistance-related genes in the variety, and those action mechanism. Here, an unterminated story about seed of a mungbean (Vigna radiata) variety with high resistance against the adzuki bean weevil and the bean bug is introduced. The two insects cannot survive on seeds of the variety, although they well develop on susceptible ones. Molecular markers linked to the resistance were selected through BAC library screening and near isogenic lines, and finally a bruchid resistance gene was suggested after map-based cloning approaches. Starvation and chemicals were suggested for a resistant mechanism and a related factor through feeding and behavior experiments, respectively. The seed flour was extracted with organic solvents, and isolated into several fractions on chromatography. Several peaks on HPLC in a fraction were related with the high mortality of the bean bug. A partial structure in an isolated chemical was observed before full identification.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. Supernatant showed different level of enzyme activity including chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence. TAIL-PCR of the random transformants revealed virulence-related genes. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Sensory system of insect is important for their fitness in the environment. Riptortus pedestris (Hemiptera: Alydidae) is a major pest of bean plants and some fruit trees in Korea, Japan, China, and South Asian countries. This study was conducted to investigate morphology and distribution of antennal sensilla of R. pedestris, using scanning electron microscopy. Antennae of R. pedestris was 11 and 9.84 mm in length in male and female, respectively, and consists of four segments; scape, pedicel, flagellum 1 and 2 (F1 and F2). Five types of sensilla (s.) trichodea, four types of s. basiconica, two types of s. chaetica, and one type of s. coeloconica were preliminary identified in both sexes of adult R. pedestris, based on their size, shape, presence of socket and surface structure. Three types of trichoid sensilla, four types of basiconic sensilla, one type of chaetic sensilla and coeloconic sensilla had numerous pores along the surface of the sensilla, suggesting their olfactory function. Eight types of sensilla (3 trichoid, 2 basiconic, a chaetic and a coeloconic) showed a distinct socket structure at the base of each sensillum. Different types of sensilla showed different distribution along the antennal segments. Two types of trichoid sensilla and two types of basiconic sensilla were distributed on scape, pedicel and F1 flagella subsegment. However, the distribution of eight other types of sensilla was limited to one of two flagella subsegments.

The ascomycete fungus Beauveria bassiana is a wide host range entomo- pathogenic fungus, which is commonly used as an environmental friendly biopesticide. However, the molecular mechanisms of host-pathogen interaction of B. bassiana are not well understood. Here, the high throughput next generation sequencing was performed to analyze the transcriptome of B. bassiana JEF-007 infected bean bug (Riptorus pedestris). Differentially expressed gene (DEG) analysis results showed that total 4,684 genes including 2,381 up and 2,303 down regulated genes were identified. Most of the DEGs were classified into single- organism, cellular and metabolism processes by gene ontology (GO) analysis. Metabolism pathway was the most abound category of DEGs via KEGG pathway mapping. Several possible candidates of virulence factors were dramatically expressed after infection, such as cytotoxic lectin, bacterial-like toxin, and proteins related to cell wall, hyphal growth, nutrient uptake and halogenated compounds synthesis. Furthermore, we also found the highest expression of a novel small RNA virus in the infected bean bug, but the relationship between fungal virulence and the RNA virus was under determination. The functional roles of these possible virulence factors are remained unclear, but this work provides a new insight for further fungal studies. Our results reflect systemic impacts of fungal pathogenesis and these findings represent a significant advance in the fungal functional genomics.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. The isolates produced high levels of pathogenesis-related enzymes, such as chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence against bean bugs, which provided some materials to figure out pathogenicity-related genes in the fungi. Now characterization of flanking region of the integrated fragment is underway and this work may reveal some important genes in the pathogenesis. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Symbiotic bacteria are common in insects. Because symbiotic bacteria are known to intimately affect the various aspects of insect host biology, ideally insects can be controlled by manipulating their symbiont. However, the attempts to control insects through their symbiont have been very limited. The paucity of the insect pest control using their symbiont is most likely due to the poor understanding of the symbiotic interactions between host insect and symbiont, which is attributed to the difficulty in cultivation of insect symbionts. However, the recently established bean bug, Riptortus pedestris, symbiotic system provides good opportunities to study insect’s symbiont in molecular level through their cultivable symbionts. Bean bugs acquire genus Burkholderia cells from environment and harbor them as their gut symbionts in the specialized posterior midgut. The genome of the Burkholderia symbiont was sequenced, and the genomic information has been used to generate the genetically manipulated Burkholderia symbiont strains. After orally administering the mutant Burkholderia symbionts into bean bugs for symbiotic association, the bacterial colonization levels in the host gut and host phenotypes were analyzed. As a result, we have identified novel symbiotic factors necessary for establishing successful association with host. Our recent understandings on the bacterial symbiotic factors demonstrate a great possibility to control the bean bug pest using genetically modified Burkholderia symbiont.

The legume family (Fabaceae) is the third largest family of flowering plants worldwide and often damaged by stink bugs, particularly Riptortus clavatus Thunberg (Heteroptera: Alydidae). This insect selectively feeds and oviposits on these plants, resulting in >50% of damage to soybeans harvested in autumn. The most common control methods for this insect are periodic application of chemical pesticides, pheromone traps, and bait crops, but still they encounters residual problem, insect resistance or low control efficacy. The current management system needs alternative control methods, which possibly combined with the present methods. Some entomopathogenic fungi have high virulence against the nymphs and adults of R. clavatus in a favorable conditions, reaching ∼80% mortality with mycosis in 3∼5 days. The genera of Beauveria and Metarhizium are possible control agents for effective and safe management of the serious pest. Fungal infection in R. clavatus was observed using an egfp-expressing fungal transformant. Thermotolerant entomopathogenic fungal conidia can be produced in cereal substrates, which enables conidia to be stable for long times. Our considerations need to be given to the combinations with other control methods, such as chemical pesticides or pheromone trap.

톱다리개미허리노린재[Riptortus pedestris (Fabricius) (Hemiptera: Alydidae)]는 콩에서 중요한 난방제 해충이다. 톱다리개미허리노린재와 두 종의 알기생벌의 계절적 발생 패턴을 기주 식물과 연관하여 고찰하였다. 또한 천적 상호간의 상대적 발생 패턴의 차이와 이유를 분석하고 톱다리개미허리노린재의 기주 식물에 대한 적합도 검증 결과를 바탕으로 톱다리개미허리노린재의 관리 전략을 제시하였다. 톱다리개미허리노린재의 효과적인 관리를 위해서 저항성품종의 사용, 과수의 경우 주변 작물에서 콩을 배제하는 것, 기주알 공급을 통한 보존적 생물적 방제, 살충제 사용 시기의 조절 등이 고려되어야 한다.

This study was examined for activity of aggregation pheromone trap with residual effect of insecticides against Riptortus pedestris. Time of escape was from aggregation pheromone trap researched 49.74 min (ET99) and 65.01 min (ET99) with 1st and 2nd instar nymph respectively. Six insecticides for the stink bugs were treated to 3 different materials (wood, metal, and plastic). Bifenthrin and fenitrothion were showed 100% insecticidal activity at 48 h to all developmental stage of R. pedestris. Residual effect of bifenthrin and fenitrothion on plastic material were showed 100% insecticidal activity over 15 day after treatment with 1st, 3rd instar nymphs, and adults except 5th instars nymph. In field test using plastic material trap with two pesticides, 1st instar nymphs and adults were showed 100% mortality until 10 day after treatment. These results indicate that R. pedestris might be managed using aggregation pheromone trap with insecticides.

The bean bug, Riptortus pedestris (F.) (Hemiptera: Alydidae) is one of the most important polyphagous pests in soybean in Korea and Japan. With the review of previous studies on the suitability of different diets, we additionally tested two leguminous plants as food sources of R. pedestris by assessing development and reproduction of the bugs. The tested diets include seeds of acacia, two mung bean varieties (var. Eoul and Dahyeon), and soybean (var. Daewon). Riptortus pedestris fed on mung bean var. Eoul showed higher nymphal mortality than those fed on mung bean var. Dahyeon, acacia, and soybean. On acacia, development time and preoviposition period of R. pedestris was shortest among the tested diets Also, five day fecundity the highest in bugs fed on acacia seeds. These results showed that acacia seed can be a good quality diet for R. pedestris, and both acacia and mung bean could be a suitable host resources for R. pedestris during the period when soybean, a major host crop, is unavailable.

톱다리개미허리노린재 (Riptortus pedestris) 상용 집합페로몬을 이용하여 포획할 때 유인되어지는 거리를 규명하고 이를 이용한 대량포획으로 콩 피해 경감 효과를 조사하였다. 집합페로몬 트랩의 재포획 비율은 최초 방사 지점에서 40 m 이내의 트랩에서는 전체 재포획 개체의 76.1%, 80 m 이내에서는 89.0%가 포획 되었으며, 트랩 설치 방위 간에 재포획 개체수는 큰 차이가 없었다. 대량 포획을 통한 피해 경감 효과를 알아보기 위해 집합페로몬 트랩을 농가 포장에 설치한 결과, 트랩수가 증가 할수록 톱다리개미허리노린재 총 포획수는 직선적으로 증가하였다. 페로몬 트랩 당 포획 할당 면적이 770 m2인 포장에서 10주당 수량이 261.8 g 으로 가장 높았으나, 트랩 당 할당 면적과 수량과의 관계에는 일정한 경향이 없었다. 트랩 당 포획 할당 면적에 따른 종자 형태 비율은 트랩 당 포획 할당 면적이 385 m2와 770 m2 인 포장에서 건전한 A형 종자의 비율이 62% 근처로 높았고 약한 피해립인 B형 종자의 비율이 27% 근처로 가장 낮았다. 톱다리개미허리노린재 집합페로몬 트랩을 이용한 피해 경감 효과는 포장 면적 385∼770 m2 당 1개를 포장 주변에 적절히 설치 운용 한다면 건전립의 비율을 높이고 약한 피해립의 비율을 어느 정도 감소시킬 수 있는 수단으로 사용할 수 있을 것으로 판단된다.

Ooencyrtus nezarae Ishii and Gryon japonicum (Ashmead), egg parasitoids of Riptortus pedestris (Fabricius), coexist despite direct competition for host eggs. As asymmetrical pattern of seasonal occurrence, i.e., more G. japonicum during spring-summer and more O. nezarae during summer-fall, has been reported, host resource partitioning may occur in temporal scales. To test this hypothesis, we demonstrated the interspecific competition between the two species by measuring parasitism in nine combinations of host densities and exposure times. To reflect gregarious-solitary dichotomy, three O. nezarae and one G. japonicum mated females were used in each experiment. O. nezarae was better competitor when exposure time was longer than 1 day irrespective of host densities. Parasitism rate and progeny emergence of O. nezarae was 1.6-2.8 and 4.7-7.3 times higher than G. japonicum. O. nezarae has higher potential rate of increase than G. japonicum due to gregariousness, and be more successful in larval competition inside multiparasitized host egg as it acts as a facultative hyperparasitoid. Although G. japonicum was more effective in host finding (as they showed relatively higher per capita parasitism and progeny emergence), their progeny suffered high mortality from the larval competition with O. nezarae. These results may explain the asymmetrical occurrence pattern in the field.

Soybean seed injury was analyzed in the experiments in which the bean bug, Riptortus pedestris(Hemiptera: Alydidae), was released into screen-caged pots containing full seed stage(R6) of soybean. When the different stages of insects, from the 3rd instar nymphs to adults, were released into pots during 8 days in soybean R6 stage, soybean seeds with injury marks (B-type seeds) increased. The weight reduction ratio in B-type seeds was highest in the injury by the 5th instar nymphs, while the daily-produced number ratio of B-type seeds was small in the treatment. In the injury by the different number of adult released into pots, 4, 8, 16 adults caused a significant increase of B-type seeds, while 16 adults caused the significant increase of the deformed (C-type) seeds. In the injury by the different release period of adults, the total number of pods was not significantly different among treatments, while the total seed number harvested was significantly small in the release for 48 days. The release for 8 and 16 days caused a significant increase of B-type seeds, while the release for 48 days caused the increase of C-type seeds. The results indicated that injury of soybean R6 stage by the bean bug produced soybean seeds with distinct injury marks at relatively low density and during short term attack period, while it produced deformed seeds at high density and during long attack period.

A bacterial colony was isolated from the gut of the bean bug, Riptortus clavatus. From morphological and biochemical tests, the bacterial isolate showed the highest similarity to Staphylococcus succinus. DNA sequence of 16S rRNA gene of the bacterium supported the identification. Oral administration of penicillin G to adults of R. clavatus gave a dose-dependent mortality of adults of R. clavatus to adults along with significant decrease of the bacterial population in the gut. Similarly, three metabolites (benzylideneacetone, proline-tyrosine, and acetylated phenylalanine-glycine-valine) derived from an entomopathogenic bacterium, Xenorhabdus nematophila, also inhibited growth of the gut bacterial population and gave significant mortalities to R. clavatus. These results suggest that a gut bacterial population classified as Staphylococcus sp. is required for survival of R. clavatus and that the three bacterial metabolites had toxic effects on the bugs due to their antibacterial properties.

Cuticular hydrocarbons (CHCs) were analyzed using GC and GC-MS, and compared with developmental stages of the bean bug, Riptortus pedestris. Carbon numbers on each developmental stages differed from 14-19 in eggs to 4th nymph, 25 in 5th nymph, and over 30 in last nymph that until adult emergence. Carbon numbers are increased to 16-17 carbons over time in newly emerged adults, 18-22 in 1 and 3 days after emergence, respectively; 23-27 in 6 days after emergence; approximately 30 in over 10 days after emergence. Carbon numbers increased as passed days after emergence. Riptortus pedestris consisted of n-nonacosane on almost all developmental stages, and followed by n-hentriacontane and 13,17-;15,19-demethyltritriacontane. Eggs, however, consisted of unknown compounds with high proportion and followed by n-heptacosane, and newly emerged adults also has a high proportion of n-heptacosane. From the above results, CHCs on developmental stages of the bean bug consist of n-alkane with saturated hydrocarbons (36-65 %), followed mono- or di- methylalkanes. However, newly emerged female and male adults mostly consisted of methyl-alkane. The major constituents of CHCs on the developmental stages of Riptortus pedestris is differently proportioned, but hardly showed the difference in their composition.

Thirteen plant essential oils were tested for their repellent activity against the bean bug Riptortus clavatus. Among the tested oils, caraway (100%) and clove bud oil (92%) significantly repelled the bean bugs at a dose of 0.142㎕/cm2 by using a Y-tube olfactometer. GC and GC-MS analyses revealed that the active components responsible for the effective repellency of caraway and clove bud oil were carvone (75%) and limonene (76.9%); eugenol (100%), isoeugenol (54.3%) and β-caryophyllene (60.0%), respectively. Of the different active fractions, eugenol was the most significant one than the other components with reference to repellent activity against the bean bugs. In the GC-EAD, limonene and carvone of caraway oil were responded to the antenna of Riptortus clavatus.

Male adults of bean bug, Riptortus clavatus Thunberg (Heteroptera: Alydidae), release aggregation pheromone (AP) which consists of (E)-2-hexenyl (Z)-3-hexenoate (E2HZ3H), (E)-2-hexenyl (E)-2-hexenoate (E2HE2H), and tetradecyl isobutyrate (TI). Variation in the pheromonal secretion were checked by the factors of age, mating status, seasons, body weight and time in a day. There were no significant differences in the amounts of AP secretion by mating, body weight, and time in a day. Higher amount of AP was detected from older males than younger ones. The amount of AP detected was higher in the males collected from April to September than in those collected during October. No AP was detected in the males of November and December. These phenomena in the AP secretion were discussed in relation to food exploitation and reproductive diapause of the bean bug.

Oviposition preference of the bean bug, Riptortus clavatus to sites on a plant and within a field, to plants at different developmental stages, and to different leguminous germplasms was observed. The insect layed its eggs mainly on the back surface of leaf in the upper half of plant in both observation from oviposition cage and soybean field. The egg number in fields were observed at a statistically-same rate in marginal and inside area, and at a median value of one egg per leaf in oviposited leaves. Full seed stage of soybean hardly affected oviposition preference of the insect. A statistically-significant difference in oviposition to different leguminous germplasms was observed.