Bean Bug Research Articles

Multiple Precursor Proteins of Thanatin Isoforms, an Antimicrobial Peptide Associated With the Gut Symbiont of Riptortus pedestris.

Thanatin is an antimicrobial peptide (AMP) generated by insects for defense against bacterial infections. In the present study, we performed cDNA cloning of thanatin and found the presence of multiple precursor proteins from the bean bug, Riptortus pedestris. The cDNA sequences encoded 38 precursor proteins, generating 13 thanatin isoforms. In the phylogenetic analysis, thanatin isoforms were categorized into two groups based on the presence of the membrane attack complex/perforin (MACPF) domain. In insect-bacterial symbiosis, specific substances are produced by the immune system of the host insect and are known to modulate the symbiont’s population. Therefore, to determine the biological function of thanatin isoforms in symbiosis, the expression levels of three AMP genes were compared between aposymbiotic insects and symbiotic R. pedestris. The expression levels of the thanatin genes were significantly increased in the M4 crypt, a symbiotic organ, of symbiotic insects upon systemic bacterial injection. Further, synthetic thanatin isoforms exhibited antibacterial activity against gut-colonized Burkholderia symbionts rather than in vitro-cultured Burkholderia cells. Interestingly, the suppression of thanatin genes significantly increased the population of Burkholderia gut symbionts in the M4 crypt under systemic Escherichia coli K12 injection. Overgrown Burkholderia gut symbionts were observed in the hemolymph of host insects and exhibited insecticidal activity. Taken together, these results suggest that thanatin of R. pedestris is a host-derived symbiotic factor and an AMP that controls the population of gut-colonized Burkholderia symbionts.

Circadian clock outputs regulating insect photoperiodism: A potential role for glutamate transporter

Many temperate ectotherms survive winter by entering diapause – a state of developmental (or reproductive) suppression or arrest – in response to short autumnal day lengths. Day lengths are assessed by the circadian clock, the biological time-keeping system that governs biological rhythms with a period of approximately 24 h. However, clock output molecules controlling this photoperiodic response are largely unknown for many insects. To identify these molecules in Hemiptera, we performed RNAi knockdowns of several candidate genes in the bean bug Riptortus pedestris to determine whether their silencing affects photoperiodic regulation of ovarian development (reproductive diapause). Knockdown of diuretic hormone 31, short neuropeptide F, neuropeptide F, ion transport peptide, neuropeptide-like precursor 1, and choline acetyltransferase had no effect on ovarian development and were therefore ruled out as regulators of the photoperiodic response. However, knockdown of vesicular glutamate transporter promoted ovarian development under diapause-inducing short days, and this is the first report of the functional involvement of glutamate signalling in insect photoperiodism. Improved knockdown of this transporter (or receptor) and RNAi of other genes involved in glutamate signal transduction is required to verify its role as an output of the circadian clock.

Insecticide resistance by a host-symbiont reciprocal detoxification

Insecticide resistance is one of the most serious problems in contemporary agriculture and public health. Although recent studies revealed that insect gut symbionts contribute to resistance, the symbiont-mediated detoxification process remains unclear. Here we report the in vivo detoxification process of an organophosphorus insecticide, fenitrothion, in the bean bug Riptortus pedestris. Using transcriptomics and reverse genetics, we reveal that gut symbiotic bacteria degrade this insecticide through a horizontally acquired insecticide-degrading enzyme into the non-insecticidal but bactericidal compound 3-methyl-4-nitrophenol, which is subsequently excreted by the host insect. This integrated “host-symbiont reciprocal detoxification relay” enables the simultaneous maintenance of symbiosis and efficient insecticide degradation. We also find that the symbiont-mediated detoxification process is analogous to the insect genome-encoded fenitrothion detoxification system present in other insects. Our findings highlight the capacity of symbiosis, combined with horizontal gene transfer in the environment, as a powerful strategy for an insect to instantly eliminate a toxic chemical compound, which could play a critical role in the human-pest arms race.

Screening Candidate Effectors of the Bean Bug Riptortus pedestris by Proteomic and Transcriptomic Analyses

The damage of Riptortus pedestris is exceptional by leading soybean plants to keep green in late autumn. Identification of the salivary proteins is essential to understand how the pest-plant interaction occurs. Here, we have tried to identify them by a combination of proteomic and transcriptomic analyses. The transcriptomes of salivary glands from R. pedestris males, females and nymphs showed about 28,000 unigenes, in which about 40% had open reading frames (ORFs). Therefore, the predicted proteins in the transcriptomes with secretion signals were obtained. Many of the top 1,000 expressed transcripts were involved in protein biosynthesis and transport, suggesting that the salivary glands produce a rich repertoire of proteins. In addition, saliva of R. pedestris males, females and nymphs was collected and proteins inside were identified. In total, 155, 20, and 11 proteins were, respectively, found in their saliva. We have tested the tissue-specific expression of 68 genes that are likely to be effectors, either because they are homologs of reported effectors of other sap-feeding arthropods, or because they are within the top 1,000 expressed genes or found in the salivary proteomes. Their potential functions in regulating plant defenses were discussed. The datasets reported here represent the first step in identifying effectors of R. pedestris.

Effects of Different Legume Seeds on Individual Performance in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae).

The bean bug, Riptortus pedestris (Fabricius), is a serious pest of legume crops in East Asia. Here, we report how the consumption of different types of legume seeds affects various aspects of nymphal and adult traits related to fitness in R. pedestris. Two experiments were conducted. In the first experiment, we assessed how R. pedestris nymphs and adults performed on one of 10 different legume seeds: adzuki bean, chickpea, cowpea, two kidney bean varieties (red, white), mung bean, peanut, small black bean, and two soybean varieties (black, yellow). Riptortus pedestris fed on yellow soybean and chickpea performed well in terms of nymphal survivorship and fecundity. However, R. pedestris fed on two kidney bean varieties suffered 100% nymphal mortality and reduced fecundity. Small black bean and black soybean supported high fecundity, but were not suitable for nymphal development. Lipid content was 4- to 11-folds higher in R. pedestris raised on peanuts than those on the others. In the second experiment, we recorded the key parameters of adult performance from R. pedestris assigned to one of nine combinations of three nymphal and three adult diets (yellow soybean, adzuki bean, peanut). Riptortus pedestris raised on yellow soybean during development produced more eggs over their lifetime than those raised on the others. However, this beneficial effect of consuming yellow soybean during development occurred only when yellow soybean was consumed during adulthood. Our data have implications for predicting the occurrence and population dynamics of this pest.

Identification of Riptortuspedestris Salivary Proteins and Their Roles in Inducing Plant Defenses.

Simple SummaryThe bean bug, Riptortus pedestris (Fabricius) is a notorious pest of soybean crops in Asia. During the feeding process, the bug secretes a mixture of salivary components, which play critical roles in the insect–plant interactions. In the present study, a total of 136 salivary proteins were identified by transcriptomic and proteomic approaches. Among them, five proteins (RpSP10.3, RpSP13.4, RpSP13.8, RpSP17.8, and RpSP10.2) were capable of inducing cell death, reactive oxygen species (ROS) burst, and hormone signal changes, indicating the potential roles of these proteins in eliciting plant defenses. Our results provide a good resource for future functional studies of bug salivary effectors and might be useful in pest management.The bean bug, Riptortus pedestris (Fabricius), is one of the most important soybean pests. It damages soybean leaves and pods with its piercing-sucking mouthparts, causing staygreen-like syndromes in the infested crops. During the feeding process, R. pedestris secretes a mixture of salivary proteins, which play critical roles in the insect–plant interactions and may be responsible for staygreen-like syndromes. The present study aimed to identify the major salivary proteins in R. pedestris saliva by transcriptomic and proteomic approaches, and to screen the proteins that potentially induced plant defense responses. Altogether, 136 salivary proteins were identified, and a majority of them were involved in hydrolase and binding. Additionally, R. pedestris saliva contained abundant bug-specific proteins with unknown function. Transient expression of salivary proteins in Nicotiana benthamiana leaves identified that RpSP10.3, RpSP13.4, RpSP13.8, RpSP17.8, and RpSP10.2 were capable of inducing cell death, reactive oxygen species (ROS) burst, and hormone signal changes, indicating the potential roles of these proteins in eliciting plant defenses. Our results will shed more light on the molecular mechanisms underlying the plant–insect interactions and are useful for pest management.

Reproductive aging and pace-of-life syndromes: more active females age faster

Abstract A common pattern of reproductive aging is that reproductive performance increases during early life and reaches a peak, followed by a decline with age. Such quadratic reproductive aging patterns can differ among individuals. Moreover, if individual differences in reproductive aging patterns reflect individual-specific life-history trade-off strategies, they are also predicted to be associated with behavior according to the pace-of-life syndrome. For example, more active, aggressive, or bolder individuals may invest more in early reproduction, resulting in more rapid reproductive aging. In this study, we estimated individual differences in quadratic reproductive aging patterns and the relationship between reproductive aging and the activity of the virgin female bean bug (Riptortus pedestris) in the absence of mating costs. We found that the egg production of virgin females followed a parabolic trajectory with age and that individuals varied significantly in their quadratic reproductive aging patterns. In addition, we found that females that were relatively more active during early life invested in egg production more heavily at a young age and suffered from a sharper decline in egg production later in life. Thus, our results indicate that individual reproductive aging patterns may be a key component in the study of pace-of-life syndromes. We suggest that within-individual plastic characteristics of life-history traits such as reproductive aging patterns may explain the mixed results from multiple studies on pace-of-life syndromes.

Chromosome-level genome assembly of the bean bug Riptortus pedestris.

The bean bug (Riptortus pedestris) causes great economic losses of soybeans by piercing and sucking pods and seeds. Although R. pedestris has become the focus of numerous studies associated with insect-microbe interactions, plant-insect interactions, and pesticide resistance, a lack of genomic resources has limited deeper insights. Here, we report the first R. pedestris genome at the chromosomal level using PacBio, Illumina, and Hi-C technologies. The assembled genome was 1.080Gb in size with a contig N50 of 2.882Mb. More than 96.3% of the total genome bases were successfully anchored to six unique chromosomes. Genome resequencing of male and female individuals and chromosomic staining demonstrated that the sex chromosome system of R. pedestris is XO, and the shortest chromosome is the X chromosome. In total, 19,026 protein-coding genes were predicted, 18,745 of which were validated as being expressed. Temporospatial expression of R. pedestris genes in six tissues and 37 development stages revealed 4,657 and 7,793 genes mainly expressed in gonads and egg periods, respectively. Evolutionary analysis demonstrated that R. pedestris and Oncopeltus fasciatus formed a sister group and split ∼80million years ago (Mya). Additionally, a 5.04Mb complete genome of symbiotic Serratia marcescens Rip1 was assembled, and the virulence factors that account for successful colonization in the host midgut were identified. The high-quality R. pedestris genome provides a valuable resource for further research, as well as for the pest management of bug pests.

Mapping PERIOD-immunoreactive cells with neurons relevant to photoperiodic response in the bean bug Riptortus pedestris.

Circadian clock genes are involved in photoperiodic responses in many insects; however, there is a lack of understanding in the neural pathways that process photoperiodic information involving circadian clock cells. PERIOD-immunohistochemistry was conducted in the bean bug Riptortus pedestris to localise clock cells and their anatomical relationship with other brain neurons necessary for the photoperiodic response. PERIOD-immunoreactive cells were found in the six brain regions. In the optic lobe, two cell groups called lateral neuron lateral (LNl) and lateral neuron medial (LNm), were labelled anterior medial to the medulla and lobula, respectively. In the protocerebrum of the central brain, dorsal neuron (Prd), posterior neuron (Prp), and antennal lobe posterior neuron (pAL) were found. In the deutocerebrum, antennal lobe local neurons (ALln) were detected. Double immunohistochemistry revealed that PERIOD and serotonin were not co-localised. Furthermore, pigment-dispersing factor-immunoreactive neurons and anterior lobula neurons essential for R. pedestris photoperiodic response were not PERIOD immunopositive. LNl cells were located in the vicinity of the pigment-dispersing factor immunoreactive cells at the anterior base of the medulla. LNm cells were located close to the somata of the anterior lobula neurons. Fibres from the anterior lobula neurons and pigment-dispersing factor-immunoreactive neurons had contacts at the anterior base of the medulla. It is suggested that LNl cells work as clock cells involved in the photoperiodic response and the region at the medulla anterior base serves as a hub to receive photic and clock information relevant to the photoperiodic clock in R. pedestris.

Field Cage Assessment of Feeding Damage by Riptortus pedestris on Soybeans in China.

Simple SummaryRiptortus pedestris (Fabricius) is a serious pest of soybeans. The nymphs and adults of R. pedestris cause feeding injury to soybean pods, resulting in significant field damage losses. In this paper, we tested the damage to soybeans at different growth stages caused by different densities of R. pedestris, through the field cage test. Our results showed that R4 was the most sensitive stage to R. pedestris injury. The damage intensity of the soybeans increased with increased pest density, and soybean nutrition factors changed after suffering R. pedestris injury. These results will be beneficial to future management programs of R. pedestris in soybean fields.The bean bug, Riptortus pedestris, is a major pest of soybeans. In order to assess the critical stages of soybean damage by R. pedestris, we tested the damage to soybeans at different growth stages (R2, R4, and R6) caused by five densities of R. pedestris (1, 2, 3, 4, and 5) through a field cage experiment. The results show that the R4 stage was the most sensitive stage in terms of suffering R. pedestris injury damage, followed by the R6 stage and then the R2 stage. The number of stay green leaves was 7.04 per plant, the abortive pod rate of the soybeans was 56.36%, and the abortive seed rate of the soybeans was 46.69%. The dry weight of the soybeans was 14.20 g at the R4 stage; these values of R4 were significantly higher than at the R2 and R6 stages. However, the dry weight of soybean seed was 4.27 g and the nutrient transfer rate was 27.01% in the R4 stage; these values were significantly lower than in the R2 and R6 stages. The number of stay green leaves, abortive pod rates, and abortive seed rates were all increased significantly with increasing pest density at each stage of soybean growth. However, the nutrient transfer rate was significantly decreased with the increase in the pest density. Soybean nutrition factors changed after they suffered R. pedestris injury; the lipid content of the soybean seed decreased and the lipid content of the soybean plant increased compared to controls, when tested with a density of five R. pedestris in the R4 stage. These results will be beneficial to the future management of R. pedestris in soybean fields.

Dual oxidase enables insect gut symbiosis by mediating respiratory network formation

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.

Oviposition-promoting pars intercerebralis neurons show period-dependent photoperiodic changes in their firing activity in the bean bug

Animals show photoperiodic responses in physiology and behavior to adapt to seasonal changes. Recent genetic analyses have demonstrated the significance of circadian clock genes in these responses. However, the importance of clock genes in photoperiodic responses at the cellular level and the physiological roles of the cellular responses are poorly understood. The bean bug Riptortus pedestris shows a clear photoperiodic response in its reproduction. In the bug, the pars intercerebralis (PI) is an important brain region for promoting oviposition. Here, we analyzed the role of the photoperiodic neuronal response and its relationship with clock genes, focusing on PI neurons. Large PI neurons exhibited photoperiodic firing changes, and high firing activities were primarily found under photoperiodic conditions suitable for oviposition. RNA interference-mediated knockdown of the clock gene period abolished the photoperiodic response in PI neurons, as well as the response in ovarian development. To clarify whether the photoperiodic response in the PI was dependent on ovarian development, we performed an ovariectomy experiment. Ovariectomy did not have significant effects on the firing activity of PI neurons. Finally, we identified the output molecules of the PI neurons and analyzed the relevance of the output signals in oviposition. PI neurons express multiple neuropeptides-insulin-like peptides and diuretic hormone 44-and RNA interference of these neuropeptides reduced oviposition. Our results suggest that oviposition-promoting peptidergic neurons in the PI exhibit a circadian clock-dependent photoperiodic firing response, which contributes to the photoperiodic promotion of oviposition.

Ovicidal activity of juvenile hormone mimics in the bean bug, Riptortus pedestris.

Insect juvenile hormone (JH) mimics (JHMs) are known to have ovicidal effects if applied to adult females or eggs. Here, we examined the effects of exogenous JHMs on embryonic development of the bean bug, Riptortus pedestris. The expression profiles of JH early response genes and JH biosynthetic enzymes indicated that JH titer was low for the first 3 days of the egg stage and increased thereafter. Application of JH III skipped bisepoxide (JHSB3) or JHM on Day 0 eggs when JH titer was low caused reduced hatchability, and the embryos mainly arrested in mid- or late embryonic stage. Application of JHMs on Day 5 eggs also resulted in an arrest, but this was less effective compared with Day 0 treatment. Interestingly, ovicidal activity of synthetic JHMs was much lower than that of JHSB3. This study will contribute to developing novel insecticides that are selective among insect species.

Population parameters and growth ofRiptortus pedestris(Fabricius) (Hemiptera: Alydidae) under elevated CO2concentrations

AbstractWe evaluated the direct effects of three different CO2concentrations (400, 600 and 1,000 ppm) on the population parameters and growth of the bean bug,Riptortus pedestris,while being fed on soybean. The raw life history data fromR. pedestriswas analyzed using an age‐stage, two‐sex life table to take the viable development rate among individuals into account. Based on the age‐stage, two‐sex life table analysis, the population projections ofR. pedestrisprovide the stage structure and variability of the population growth under different CO2treatments. Our results showed significantly shorter immature durations and higher pre‐adult survival rate under elevated CO2(1,000 ppm) than those under ambient CO2(400 ppm). The population ofR. pedestrisreared under elevated CO2conditions showed higher intrinsic and finite rates of increase but a lower mean generation time thanR. pedestrisreared under ambient CO2conditions. Our results show the population parameters and growth ofR. pedestrisare influenced by increased CO2relative to ambient CO2treatment. Further studies on the long‐term direct effects of different CO2levels onR. pedestrisare essential to understand their population dynamics and to establish appropriate management strategies.

Re-opening of the symbiont sorting organ with aging in Riptortus pedestris

Abstract Because environments are full of diverse microorganisms including parasites and pathogens, how to select and maintain a beneficial microbial partner is a critical issue for host organisms. The bean bug Riptortus pedestris (Heteroptera: Alydidae) acquires a specific gut symbiont, Burkholderia insecticola, from environmental soil in the second instar stage and houses it in a crypt-bearing midgut region called M4. To sort the Burkholderia symbiont from a wide variety of soil microbes, R. pedestris develops a specialized organ named “constricted region (CR)”. The CR, located in front of the crypt-bearing symbiotic region, is immediately closed after colonization of M4 by the Burkholderia symbiont to block any contamination of microbes ingested with food. By using a food coloring and a red fluorescent protein (RFP)-expressing Burkholderia symbiont, we here revealed that the closed CR is re-opened at a later developmental stage of R. pedestris. Although the CR was re-opened at the late phase of the fifth instar, oral administration of food coloring and green fluorescent protein (GFP)-expressing symbiont demonstrated that ingested food and bacteria were stopped at the M4B despite the opened CR. Observations using confocal microscopy revealed reverse flow of gut content from M4 to M3 through the opened CR, the flow pressure of which seemed to prevent any contamination of the symbiotic M4 region. The morphological change of the CR with aging may cause a risk of contamination, but another mechanism, the reverse flow, plausibly maintains the specificity of gut symbiont in R. pedestris.

Chemical polymorphism regulates the attractiveness to nymphs in the bean bug Riptortus pedestris

Riptortus pedestris (Fab.) is an important pest on soybean plants in East Asia. Females lay eggs scattered not only on host plants, but also on non-host plants, probably for avoiding detection by natural enemies. Newly hatched nymphs can develop to the second instar without the need of food except water. Second instar nymphs, the first-feeding stage with a strong locomotion, need to find suitable feeding sites on host plants. The mechanism is still not well understood. Here, we found second instars were attracted by both male and female adults, but generally repelled by second and fifth instar nymphs, wherein their responses were little affected by food volatiles (soybean seeds or seedlings). Nymphs distinctly differed from adults in chemical composition of the whole-body extracts. 4-oxo-(E)-2-Hexenal was the major component in nymph’s extracts, but it was not found in adults, whereas some ester compounds, such as (E)-2-hexenyl esters, were exclusively found in adults and accounted for the main part in their extracts. The three esters, (E)-2-hexenyl (Z)-3-hexenoate (E2-6:Z3Hx), (E)-2-hexenyl (E)-2-hexenoate (E2-6:E2Hx) and myristyl isobutyrate, were male specific and had been reported as an aggregation pheromone. Synthetic 4-oxo-(E)-2-hexenal strongly repelled second instars, while (E)-2-hexenyl hexanoate emitted by both males and females attracted them. The main component of the aggregation pheromone, myristyl isobutyrate, also strongly attracted second instars. How the findings may improve the efficiency of chemical traps in fields was discussed.

Complete Genome Sequence of a Novel Putative RNA Virus, RiPV-2, from the Bean Bug Riptortus pedestris.

A novel putative single-stranded RNA virus was discovered from the transcriptome of a bean bug, Riptortus pedestris, infected with the entomopathogenic fungus Beaveria bassiana JEF-007. The complete genome sequence was 9,915 nucleotides long and encoded a 2,916-amino-acid polyprotein. This virus belonged to Iflaviridae based on phylogenetic analysis and was named RiPV-2.

Burkholderia insecticola triggers midgut closure in the bean bug Riptortus pedestris to prevent secondary bacterial infections of midgut crypts.

In addition to abiotic triggers, biotic factors such as microbial symbionts can alter development of multicellular organisms. Symbiont-mediated morphogenesis is well-investigated in plants and marine invertebrates but rarely in insects despite the enormous diversity of insect-microbe symbioses. The bean bug Riptortus pedestris is associated with Burkholderia insecticola which are acquired from the environmental soil and housed in midgut crypts. To sort symbionts from soil microbiota, the bean bug develops a specific organ called the "constricted region" (CR), a narrow and symbiont-selective channel, located in the midgut immediately upstream of the crypt-bearing region. In this study, inoculation of fluorescent protein-labeled symbionts followed by spatiotemporal microscopic observations revealed that after the initial passage of symbionts through the CR, it closes within 12-18 h, blocking any potential subsequent infection events. The "midgut closure" developmental response was irreversible, even after symbiont removal from the crypts by antibiotics. It never occurred in aposymbiotic insects, nor in insects infected with nonsymbiotic bacteria or B. insecticola mutants unable to cross the CR. However, species of the genus Burkholderia and its outgroup Pandoraea that can pass the CR and partially colonize the midgut crypts induce the morphological alteration, suggesting that the molecular trigger signaling the midgut closure is conserved in this bacterial lineage. We propose that this drastic and quick alteration of the midgut morphology in response to symbiont infection is a mechanism for stabilizing the insect-microbe gut symbiosis and contributes to host-symbiont specificity in a symbiosis without vertical transmission.

Behavioral and Electrophysiological Effects of Ajowan (Trachyspermum ammi Sprague) (Apiales: Apiaceae) Essential Oil and Its Constituents on Nymphal and Adult Bean Bugs, Riptortus clavatus (Thunberg) (Hemiptera: Alydidae).

We investigated the repellent effect of 12 Apiaceae plant essential oils on nymphal and adult (male and female) forms of the bean bug, Riptortus clavatus (Thunberg) (Hemiptera: Alydidae), using a four-arm olfactometer. Among the essential oils tested, ajowan (Trachyspermum ammi Sprague) essential oil showed the strongest repellent activity against the nymphal and adult bean bugs. For female adults, the repellent activity was significantly different between an ajowan oil-treated chamber and an untreated chamber down to a concentration of 14.15 μg/cm2. We also investigated the repellent activity of individual ajowan essential oil constituents. Of the compounds examined, carvacrol and thymol showed the most potent repellent activity against the nymphal and adult bean bugs. Carvacrol and thymol exhibited 73.08% and 70.0% repellent activity for the bean bug nymph at 0.71 and 2.83 μg/cm2, respectively, and 82.6% and 80.7% at 5.66 and 11.32 μg/cm2, respectively, for male adults. Carvacrol and thymol exhibited strong repellent activity against female adult bean bugs down to a concentration of 2.83 μg/cm2. Ajowan essential oil, thymol and carvacrol elicited a negative electroantennogram (EAG) response from adult bean bugs. This could explain the repellent activity of ajowan essential oil and its constituents. Our results indicate that ajowan essential oil and its constituents carvacrol and thymol can be potential candidates as the ‘push’ component in a ‘push-pull’ strategy for bean bug control.

Juvenile hormone III skipped bisepoxide, not its stereoisomers, as a juvenile hormone of the bean bug Riptortus pedestris

Juvenile hormone (JH) plays a pivotal role in many aspects of insect physiology. Although its presence was first reported in a blood-sucking bug belonging to the suborder Heteroptera (true bugs), JH species in the group has long been controversial. Although some recent studies proposed a putative JH molecular species in several Heteropteran species, it is not conclusive because physicochemical analyses were insufficient in most cases. Here, we studied this issue with an ultraperformance liquid chromatography–tandem mass spectrometer (UPLC-MS/MS) equipped with C18 and chiral columns in the bean bug Riptortus pedestris (Heteroptera, Alydidae), in which the JH species has long been controversial. Although a recent study describes JHSB3 as the major JH of this species, that finding was not conclusive because its chirality has not been clarified. In the present study, we detected methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named juvenile hormone III skipped bisepoxide (JHSB3), in the culture media of the corpora cardiaca-corpus allatum (CC-CA) complex and in the hemolymph of this species by a chiral ultraperformance liquid chromatography- tandem mass spectrometer (UPLC-MS/MS). Other JHSB3 stereoisomers were not detected. Topical application of JHSB3 effectively averted diapause. These results indicate that JHSB3 is the major JH of R. pedestris. The present study further revealed that JHSB3 and its (2R,3S,10S) isomer are more potent than (2S,3R,10R) and (2S,3R,10S) isomers, which suggests that there is a significance to the configuration of the 2,3-epoxide moiety in JH action. We further found a supplemental significance to the configuration of the 10-position.