A novel insect-infecting positive sense single-stranded RNA virus, Riptortus pedestris virus-1 (RiPV-1), was found in the Riptortus pedestris transcriptome data by de novo assembly and further confirmed by RACE method. The genome of RiPV-1 consists of 10,554 nucleotides (nt) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 10,371 nt encoding a 3,456 aa polyprotein and flanked by 71 and 112 nt 5' and 3' noncoding regions, respectively. RiPV-1 genome contains the consensus genome organization of picorna-like RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) array in that order from the 5' to the 3' end. From the phylogenetic analysis, RiPV-1 was clustered with unassigned insect RNA viruses, APV and KFV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses. Tissue tropism analysis revealed that RiPV-1 was relatively abundant in the thorax, abdomen, midgut and fat body. Interestingly, RiPV-1 replication was enhanced by Beauveria bassiana JEF-007 infection that was quantified using qRT-PCR. This study identified a novel insect-infecting virus and provided further insight into the relationship between virus, fungus and host.

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.

Various techniques have been developed to monitor insect behavior in the field. Harmonic radar tagging is a promising method of tracking target insects because it can allow efficient detection of the insects with negligible effects on their behavior or physiology. However, availability of the light limits the effectiveness of the method as it is almost impossible to spot an insect in the dark. On the other hand, fluorescent-marking, when combined with strong light source, makes the organisms distinct at night in a non-destructive manner. Therefore, we conducted a field experiment to determine the effectiveness of the combination of the two methods. Riptortus pedestris (Fabricius) adults were marked with both harmonic radars and fluorescent paint. The marked insects were haphazardly arranged and pinned onto trees or the ground in the forest at night. Then, a group of paired researchers, one equipped with a harmonic radar and the other with a hand-held laser, attempted to find R. pedestris within 20 minutes. The detection rate showed a high variance ranging from 40% to 100%. Thus, with a proper training, harmonic radar system combined with fluorescent-marking can be a powerful technique to detect insects in the field. With these equipments, tracking insect behavior in the field will be more efficient in the dark.

Riptortus pedestris is a major agricultural pest in Korea. To manage this pest, it is necessary to understand the complete life history of R. pedestris. However, most studies have focused on the summer period when the pest attacks crops. Therefore, we conducted laboratory and field experiment to identify overwintering behavior of adult R. pedestris. First, we conducted two laboratory experiments to examine overwintering habitat preferences and spatial distribution of R. pedestris in the habitat. Second, we conducted field survey to characterize overwintering sites of R. pedestris in diverse landscapes. In the laboratory conditions, R. pedestris almost exclusively showed overwintering behavior in the leaf litter arena (63%) among the structures tested (rocks: 3%; rotten wood: 0%). And spatial distribution of R. pedestris showed solitary behavior in leaf litters. In the field, we located 12 individuals of overwintering adults from the urban areas, whereas no individuals were found from the high-elevation mountains and agricultural landscapes. Our results suggest that R. pedestris mainly use leaf litter as an overwintering structure, with solitary behavior, and low-elevation forested areas near urban landscapes seem to be more preferred overwintering sites.

A mycoinsecticide, Beauveria bassiana (strain GHA) marketed as BotaniGard® ES, was evaluated as a biological control agent against Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) a major soybean pest. It was tested on commercial soybean fields with one time spray of both high (1×109) and medium (1×108spore/ml) concentration and double spray of the medium concentration. Nymph population decreased in all treated fields regardless of dose and frequency of spray. But, adult and egg populations were not affected. Alive nymphs collected from the fields and kept in laboratory showed infection rate ranging 35-68% in treated plots while 0% in control plots. It also reduced damage on soybean pods and seeds by 17.7-21.2 and 18.0-22.5% compare to control, respectively. Additionally, B. bassiana reduced other pests found in soybean fields including brown marmorated stink bug Halyomorpha halys (Stål) and two striped leaf beetle Medythia nigrobilineata Motschulsky. Also there was no negative effect on the parasitism of released non-viable eggs of R. pedestris. These results suggest that, in field condition, one time spraying of B. bassiana in the concentration of 1×108spore/ml can help reducing crop damage as well as R. pedestris nymph population.

Insect antennae play important roles in finding mates and in locating food source and oviposition sites. Riptortus pedestris is an important pest of soybean and sweet persimmon in Korea. The male R. pedestris adult produce the aggregation pheromone attracting the conspecific nymphs and both sexes of adults. The pheromone was known as a cue for food finding, but the 1st instar nymph can develop to the 2nd instar without food. This phenomenon may suggest that the 1st instar nymph may have different sensilla system from other instars. Thus, we investigated the morphology and distribution of antennal sensilla and antennal response to the aggregation pheromone (AG) of each nymphal stage using Scanning Electron Microscope (SEM) and Electroantennography (EAG). As expected, first instar nymph did not have sensilla trichodea 3 (T3) and chaetica 3 (Ch3) which existed in other instar nymphs. The antennae of the 1st instar nymph did not responded to AG, with no difference from control. For further elucidation of the functions of sensilla T3 and CH3, single sensillum recording to AG will be done.

Information on spatial distribution and peak occurrence is important for site and time specific pest management. So, we investigated seasonal occurrence and edge distribution of major soybean pests by visual count in six commercial fields located in Andong from July 16 to October 23 in 2015. We found a total of 19 species in 10 families. The major pest during vegetative stage was Medythia nigrobilineata (Motschulsky) while Riptortus pedestris (Fabricius) and Halyomorpha halys (Stal) showed peak occurrence during reproductive stage of soybean. Aphis glycines Matsumura was found throughout the season. Seasonal density of M. nigrobilineata was 0.20 and 0.38/plant in edge and interior without significance, respectively. The infestation of A. glycine was also found similar between edge and interior. Immatures of R. pedestris and H. halys distributed more in edge (0.21 and 0.36/plant) than interior (0.12 and 0.12/plant), respectively. The application of these informations on spatial distribution pattern of soybean pest was discussed.

톱다리개미허리노린재(Riptortus pedestris (Fabricius))는 콩과 작물을 가해하는 해충으로 국내 대부분의 지역에서 발견된다. 톱다리개미 허리노린재는 성충 수명이 길고 발육기간이 짧아 야외에서 여러 세대가 혼재되어 발생하고 있어 각 세대를 구분하기 어렵다. 특히, 제2세대 성충 의 발생시기는 콩의 착협기와 일치하여 큰 피해를 주고 있어, 제1세대 성충의 방제는 제2세대 밀도를 감소시켜 콩과 작물의 피해를 줄이는데 도움 이 될 것으로 예상된다. 따라서 본 연구는 약충기간의 광주기가 성충의 체색변이에 영향을 주는 특성을 이용하여 야외 포장에서 월동세대와 제1 세대의 발생시기를 구분하였다. 또한 톱다리개미허리노린재 개체군 모형을 수립하고 적용하여 이를 검증하였다. 여름형 성충의 비율이 20% 이 상 되는 시기를 제1세대 톱다리개미허리노린재 성충의 발생시기로 추정할 수 있으며, 이는 제2세대 성충의 밀도를 억제하기 위한 방제시기를 결 정하는데 활용될 것으로 기대된다.

Bean bug, Riptortus pedestris is an agriculturally serious pest in north eastern Asian countries, damaging to several legumes and fruit trees. Chemical pesticides have been largely used to control the pest but it encounters insecticide-resistance and environmental toxicity issues. Alternatively different mode of action and environmentally sound pest management system can be found in entomopathogenic fungal insecticides. Herein we developed a platform to optimize the fungal production to express their maximum virulence against bean bug, by focusing on solid culture system for thermotolerance, formulation to select effective surfactants to carry the fungal conidia on the cuticles, and relationship between environmental abiotic factors and fungal mortality. First to produce highly thermotolerance fungal conidia, Beauveria bassiana and Metarhizium anisopliae isolates were cultured on several granular cereal substrates, which could be subjected to formulation process. Among the tested media, four media (millet, non-glutinous italian foxtail millet, glutinous italian foxtail millet, brown rice) were superior to the other grains in the spore production and thermotolerance. Next to efficiently deliver the fungal conidia on the cuticles of bean bug, total of six surfactant (CO-2.5, CO-12, LE-7, PE-61, TED-3 and Siloxane) was used to experiment. CO-12 was superior to the other surfactant in mortality of 100 ppm consistence. This work suggests that solid culture system and formulation and application should be seriously considered to reach an optimal level of mortality by inducing their maximum virulence.

Currently, the management of Riptortus pedestris (Hemiptera: Alydidae) relies mainly on insecticide application in most crop productions. However, there have been very few detailed studies concerning the potential behavioral effects of sublethal dose of insecticide. This study was conducted to address sublethal effects of five insecticides on the mobility of R. pedestris. Adult R. pedestris were individually exposed to dry residue of insecticides for 4 hours in laboratory. Tested insecticides were fenitrothion (organophosphate), etofenprox (pyrethroid), bifenthrin (pyrethroid), acetamiprid (neonicotinoid) and dinotefuran (neonicotinoid). After the exposure, vertical climbing ability and flight capacity of survived R. pedestris were evaluated and compared to water-treated control. First, the mortality of R. pedestris after the 4-h insecticide exposure was as follows: 10% for fenitrothion, 3.4% for dinotefuran, 3.4% for acetamiprid, 0% for etofenprox and 0% for bifenthrin. The vertical mobility of R. pedestris was affected differently by insecticides tested. Acetamiprid resulted in decrease (37%) in the vertical mobility, whereas dinotefuran caused increase (153%) in the ability compared to the control. There was no effect by three other insecticides on the vertical mobility. In general, tested insecticides did not affect the flight capacity of R. pedestris compared to untreated individuals. However, etofenprox resulted in increase (230%) in the flight capacity. The results of this study indicate that insecticide-treated but survived R. pedestris can show similar or even increased mobility compared to untreated individuals. This should be considered in designing insecticide application programs against of R. pedestris.

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.

온도에 따른 톱다리개미허리노린재 (Riptortus pedestris)의 개체군 밀도 변동 예측 모델을 상용 소프트웨어인 DYMEX로 구축하고 월동 성 충밀도를 바탕으로 한 연간 발생 밀도 변동 패턴과 살충제 처리 시기에 따른 밀도 억제 효과를 시뮬레이션하였다. 구축된 모델은 총 10개의 모듈을 사용하였으며, Lifecycle 모듈은 알, 1, 2, 3, 4, 5령, 성충의 7개 발육 단계로 구성하였다. 월동 성충 개체군의 포획시기를 이용하여 연중 밀도 변 동을 예측한 결과 연도에 따라 3~4번의 신 성충 발생이 가능하여 페로몬 트랩 포획밀도 조사와 유사하였다. 콩 포장으로 침입해 들어오는 두 번째 신 성충의 경우 개발된 모델을 이용하여 예측된 성충 발생 최성일이 페로몬 트랩으로 조사된 포획 밀도 최성기와 거의 일치 하였다. 그러나 예측된 첫 번째 신 성충 발생 최성일은 페로몬트랩 포획 최성기보다 연도에 따라 9~16일 늦었으며, 마지막 세대의 발생 최성일은 연도에 따라 페로몬 트랩 포 획 최성기보다 17~23일 빨랐다. 살충제 사용을 가정한 첫 번째 신성충 개체군 밀도 억제가 다음 세대들의 밀도 증가에 미치는 영향을 시뮬레이션한 결과, 신 성충 발생 초기일수록 밀도 억제효과가 커서 7월 1일 살충제 처리를 가정하였을 때 다음 세대에 형성된 성충은 무처리의 3% 정도로 현저 하게 낮았다. 또한 포장에 침입해 들어오는 두 번째 신성충 개체군을 대상으로 시기별 살충제 처리 효과를 시뮬레이션한 결과 8월 30일 살충제 처 리를 가정한 경우 다음세대 성충 최고 밀도는 무처리의 25% 정도로 줄었고, 최고 밀도에 도달한 시기도 무처리에 비해 2주 이상 늦었다. 이상의 연구 결과들은 톱다리개미허리노린재의 효율적인 종합적 방제 계획을 세우는데 유용하게 사용될 수 있을 것으로 기대된다.

Riptortus pedestris (F.) (Hemiptera: Alydidae) is an important pest of soybeans in Korea and Japan. A synthetic aggregation pheromone trap has been commercialized and used in soybean fields in Korea for both monitoring and mass-capture of this bug. As the trap’s efficacy in reducing the pest population or crop damage is unknown, in this study we evaluated the aggregation pheromone trap in experimental soybean fields located in Andong National University. Two treatments, one with traps deployed for the entire cultivation period and one with no traps, were tested in six small experimental fields. The total numbers of R. pedestris (in all life stages) in soybean field were not significantly different between the treatments until August. But, in presence of pheromone, the pest’s abundance increased significantly in September and October. Relative to the size of the bug population in the field, trap catch rate was low during the fall (when bug density was highest) and high in early summer when the field population was very low. Placement of aggregation traps in plots caused pod and seed damage from R. pedestris to increase 2.7 and 2.2 times compared to the control. In conclusion, R. pedestris populations increased significantly during the fall in the presence of the aggregation pheromone trap, which should therefore be used with great caution whether as a control measure or as a monitoring tool.

Lethal concentration of Beauveria bassiana (Strain GHA, Botanigard, Arysta LifeScience) against Riptortus pedestris (F.) (Hemiptera: Alydidae) was compared with that of its’ parasitoid Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae). Five concentrations of the mycoinsecticide, i.e., 5×108, 1×108, 2×107, 4×106, and 8×105 conidia/ml, were used. Glass scintillation vial was coated with 100 μl solution, and insects were released and assessed up to 12 hours. The LC50 and LC20 of second instar R. pedestris were 1.8×1010 and 1.5×108 conidia/ml, and those of O. nezarae were 1.9×107 and 2.8×106 conidia/ml, respectively. The mortality of O. nezarae was observed 3.9 times higher than R. pedestris in higher concentration. This result shows that O. nezarae is more susceptible to the mycoinsecticide than R. pedestris.

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.

The use of radar systems for entomological research offers new opportunities that allow continuous tracking of individual insects in natural settings. In particular, harmonic radar systems can be applied for small animals such as insects because the radar tag does not rely on battery power, allowing them to be light weight. To apply this technology, it is essential to develop procedures to securely attach radar tags on target insects and thereafter demonstrate no adverse effects of radar tag attachment on insect survivorship and behaviors. In this study, we developed procedures to securely attach harmonic radar tags on adult Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) and verified there were no detectable adverse effects of radar tagging on R. pedestris survivorship and walking or flight capacity. With the radar technique, future studies will focus on understanding dispersal capacity and pattern of R. pedestris in diverse landscapes. This will help enhance monitoring and management programs for this pest.

숙주와 장내 공생균 간의 상호작용을 연구하기 위한 다양한 모델 중 우리는 중장 의 특이적인 구간인 M4 내에 오직 한 종류의 공생균(Burkholderia)를 가지는 톱다 리개미허리노린재(Riptortus pedestris)를 연구 모델로써 사용하였다. 본 모델은 노 린재의 공생균을 대량으로 쉽게 분리할 수 있다는 큰 장점을 가지고 있다. 우리는 배양한 Burkholderia가 숙주안에 공생균으로 적응하면서 많은 변화가 일어날 것이 라 예상하고, 먼저 세포벽의 형태학적 차이를 전자현미경을 통해 알아보았다. 그 결과 장내 공생균의 세포벽이 시험관 내에서 배양한 균보다 안정성이 떨어져 보이 는 현상을 볼 수 있었다. 이러한 차이는 계면활성제인 SDS에 대한 감수성이 공생균 이 배양균보다 높은 것으로도 확인할 수 있었다. 우리는 M4 구간에 공생균의 세포 벽을 변화시키는 물질이 있다고 가정하고, M4구간의 추출물이 배양한 Burkholderia의 SDS에 대한 감수성을 높이는지 알아보았다. 그 결과 M4구간 추출 물을 처리한 배양균이 M4 추출물 양에 비례하여 SDS에 대한 감수성이 증가되는 것을 관찰하였다. 다음으로, 곤충 장에서 공생균의 수를 조절하는 것으로 추정되어 지는 항균펩타이드인 riptosin에 대한 감수성도 M4 추출물에 의해 증가되는지 알 아보았다. 배양균에 M4구간 추출물을 처리하면 riptocin에 더 민감하게 반응하는 것을 관찰하였다. 이러한 결과로 M4구간에 공생균의 세포벽을 변화시키는 어떤 물질을 있다는 것을 사실을 증명하였다. 더 나아가, 공생균이 M4 구간의 세포벽을 변화시키는 물질에 의해 항균펩타이드에 더 민감해져 숙주가 장내 공생균의 수를 쉽게 조절할 수 있게 된다는 것을 추정해 볼 수 있었다.

Biological properties of antimicrobial peptides (AMPs) of hemimetabolous insect are poorly characterized in innate immunity field. To investigate the biochemical properties of hemimetabolous insect’s AMPs, we purified the pyrrhocoricin-like AMP from the hemolymph of Riptortus pedestris and then named as riptocin. We successfully determined the primary protein structure and its cDNA sequence. Interestingly, the determined cDNA revealed that riptocin precursor is composed of 12 repeating units of active riptocins, which implied that riptocin precursor might require to be processed to generate active riptocins by several unidentified processing enzymes. In order to characterize the bio-processing mechanisms of riptocin precursor, we generated the antibody against active riptocin. Using quantitative PCR and Western blot analyses, we showed that gene of riptocin was started to express from the fatbody after three hours post bacterial infection. To address our hypothesis that active riptocin is generated from riptocin precursor by several processing enzymes, we need to obtain the riptocin precursor. Currently, we are expressing the recombinant riptocin precursor using in vitro translation system. Meanwhile, we investigated whether naive hemolymph (naive HL), which may contain precursor riptocin, can generate active riptocin when riptocin precursor was co-incubation with bacteria-challenged hemolymph (active HL), which may contain all processing enzymes. Actually, when naive HL was incubated with active HL, antimicrobial activity was dramatically increased, suggesting that processing enzymes in active HL may induce processing of riptocin precursor to generate active riptocins.