Male aggressive behavior and exaggerated hindlegs of the bean bug Riptortus pedestris

Males of the bean bug species Riptortus pedestris possess larger hindlegs than females. Observations of male-male interactions showed that the enlarged hindlegs are used as weapons in male fights, and that males with larger hindlegs win fights more frequently. Morphological analysis based on the positive allometry test showed that the femora of larger males are relatively bigger than those of smaller males, but femora of larger females are not relatively larger than those of smaller females. These results suggest that sexual selection in R. pedestris favors larger hindlegs for male fighting. In addition, the thorax and abdomen lengths were larger in the male than in the female. The males often lift their abdomen with their back to the opponent for displays against an opponent. As a result, abdominal size may be under stronger selection in the male than in the female, as for the exaggerated hindlegs.     

Male Courtship Behavior and Weapon Trait as Indicators of Indirect Benefit in the Bean Bug,Riptortus pedestris

Females prefer male traits that are associated with direct and/or indirect benefits to themselves. Male–male competition also drives evolution of male traits that represent competitive ability. Because female choice and male–male competition rarely act independently, exploring how these two mechanisms interact is necessary for integrative understanding of the evolution of sexually selected traits. Here, we focused on direct and indirect benefits to females from male attractiveness, courtship, and weapon characters in the armed bug Riptortus pedestris. The males use their hind legs to fight other males over territory and perform courtship displays for successful copulation. Females of R. pedestris receive no direct benefit from mating with attractive males. On the other hand, we found that male attractiveness, courtship rate, and weapon size were significantly heritable and that male attractiveness had positive genetic covariances with both courtship rate and weapon traits. Thus, females obtain indirect benefits from mating with attractive males by producing sons with high courtship success rates and high competitive ability. Moreover, it is evident that courtship rate and hind leg length act as evaluative cues of female choice. Therefore, female mate choice and male–male competition may facilitate each other in R. pedestris. This is consistent with current basic concepts of sexual selection.     

Olfactory attraction to aggregation pheromone is mediated by disti-flagellum of antennal segments in Riptortus pedestris

Riptortus pedestris (Hemiptera: Alydidae) is a serious pest of soybean and sweet persimmon and uses male produced aggregation pheromone, (E)-2-hexenyl (Z)-3-hexenoate, (E)-2-hexenyl (E)-2-hexenoate, and tetradecyl isobutyrate to facilitate food location and recognition by conspecifics. Using electroantennogram (EAG) and greenhouse bioassay, we determined which antennal segment is involved in the detection of their aggregation pheromone. In the first EAG test using individual antennal segments, significant EAG responses to 1:1:1 mixture of the aggregation pheromone were observed only from the disti-flagellum segments of both male and female antennae at both pheromone doses tested (1 µg and 100 µg). In the following EAG tests using gradually removed antennal segment(s), EAG response was still maintained when the distal half of a disti-flagellum was surgically removed, while EAG response was lost when whole segment of disti-flagellum or other whole segments were gradually removed from intact antenna of both sexes. In greenhouse experiment, removing one or both segment(s) of disti-flagellum from male or female antennae resulted in significant reduction in their attraction to the aggregation pheromone. Together, these findings support that the disti-flagellum of R. pedestris houses olfactory neurons associated with attraction to their aggregation pheromone.     

Evaluation of different leguminous seeds as food sources for the bean bug Riptortus pedestris

This study was conducted to determine the effects of six different leguminous seeds (cowpea, white kidney bean, soybean cultivars Cheongja and Daepung, mung bean, and azuki bean) on the life history traits of the bean bug Riptortus pedestris (F.) (Hemiptera: Alydidae). The total developmental time from the first instar nymph to adult ranged from 18 to 25 d; R. pedestris fed with white kidney beans were the slowest to develop. In addition, bugs fed with white kidney beans had the highest mortality (93%) and the shortest longevity (18 d). R. pedestris fed with cowpeas, soybeans, or azuki beans had high reproductive success, with the highest egg hatching success recorded in bugs fed cowpeas. The results suggest that cowpea may be a beneficial food source for the development and reproduction of bean bugs.     

Circadian clock regulates photoperiodic responses governed by distinct output pathways in the bean bug,Riptortus pedestris

Effects of RNA interference (RNAi) targeted against circadian clock genes on two distinct types of photoperiodic responses – ovarian development and lipid accumulation – were investigated in a bean bug Riptortus pedestris, to explore which physiological process in the photoperiodic response involved the circadian clock. Ovarian development and lipid accumulation are known to be regulated by distinct output pathways. Control insects showed clear photoperiodic responses; i.e. induction of ovarian development and suppression of lipid accumulation under long-day conditions, whereas opposite characteristics under short-day conditions. We found that RNAi directed against period, a negative element of the circadian clock, produced a long-day effect for both the ovarian development and lipid accumulation, while RNAi directed against Clock, a positive element of the circadian clock, produced a short-day effect for both, irrespective of photoperiod. These results indicate that the circadian clock comprised of these genes regulates a process governing both distinct photoperiodic responses.     

In vitro rearing of Ooencyrtus nezarae (Hymenoptera: Encyrtidae), an egg parasitoid of Riptortus pedestris (Hemiptera: Alydidae)

Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) is a polyphagous egg parasitoid of various true bugs, including Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), a major pest of soybean and fruit trees in northeastern Asia. This study was conducted to develop artificial host eggs containing insect haemolymph for mass rearing of O. nezarae. The haemolymph of Chinese oak silkworm (Antheraea pernyi Guérin-Méneville) pupae was found to be most suitable for artificial eggs for the complete development of O. nezarae. Among 764 parasitoid eggs laid in artificial eggs, 49.2% successfully developed to adult parasitoids. The developmental time in artificial eggs was delayed by 3–5 days relative to that observed in natural host eggs; however there was no significant difference in the length of hind tibia between adults that emerged from artificial and natural host eggs. Moreover, new generations that emerged from artificial eggs reproduced on all egg types offered (either natural or artificial eggs). We propose that the artificial eggs developed herein can therefore be an effective means for mass rearing O. nezarae.     

Changes in cuticular hydrocarbons in different developmental stages of the bean bug,Riptortus pedestris (Hemiptera: Alydidae)

The function of “cuticular hydrocarbons” (CHCs) in insects is to protect the body as well as chemical communication and recognition between species. Gas chromatography and mass spectrometry were used to compare the composition of CHCs in eggs, all nymph instars, and male and female adults of the bean bug, Riptortus pedestris (Hemiptera: Alydidae). CHC carbon chain numbers of each developmental stage ranged from 14 to 19 in eggs to the fourth nymph and were 24 in the last nymph. CHCs' carbon chain numbers in male and female adults increased from 16 to 23 and 16 to 27 until 6 days of age, respectively. Major components consisted of n-nonacosane in the larval stages, which changed to n-hentriacontane, n-heptacosane and 13,17-;15,19-demethyltritriacontane in adults. The major component from eggs was not identified. The major component from newly emerged adults was n-heptacosane. CHCs in different developmental stages of the bean bug consisted of n-alkanes with saturated hydrocarbons (27–63%), followed by mono or di-methyl alkanes. However, the CHCs of newly emerged female and male adults consisted mostly of methyl-alkanes. The major constituents of CHCs in the developmental stages of R. pedestris were proportionally different but showed only slight differences in composition.     

Locomotion ability variation among instars of the bean bug, Riptortus pedestris (Fabricius) (Heteroptera: Alydidae) nymphs

The nymphal locomotion ability (walking distance) of the stenophagous bean bug Riptortus pedestris (Fabricius) was studied in each instar. We measured the walking distance using two systems. The walking distance in photophase was measured for 6 h using a tracking system with a charge coupled device (CCD) camera and computer software. The daily activity of nymphs was measured by an actograph system counting the number of infrared beam intercepts. The actograph data were converted to distance using a linear regression against the data of the tracking system. The longevity of nymphs without food was also studied to estimate the potential walking distance. Using both the tracking and actograph systems, it was determined that first instars walked less than the other instars (only 10.7 m within 6 h). The second to fifth instars could move 20-25 m within 6 h, and this distance did not differ among instar. This indicates that first instars seldom move after hatching in the field. The walking distance for 24 h varied and was greatest for the third instars (80.8 m). The potential longevity of nymphs was found to increase with instar age. Potential locomotion ability (walking distance for 24 hxpotential longevity) was high in the third to fifth instars (approximately 340 m). The potential locomotion ability for the second instars was relatively low compared with the elder instars (approximately 180 m). From these results, nymphs of R. pedestris seem to adapt by identifying feeding site locations after hatching and elder instars may be able to find a novel feeding site after the degradation of previous habitat.     

Transmission of the Trypanosomatid Flagellate Phytomonas davidi,a Symbiont of the Euphorbiaceae,by the Hemipteran Bug Pachybrachius bilobata scutellatus1

ABSTRACT. Phytomonas davidi is found in the latex of species of the plant family Euphorbiaceae, including Chamaesyce hirta and C. hyssopifolia, to latitude 32°N in the southeastern United States. The hemipteran Pachybrachius bilobata scutellatus (family Lygaeidae) serves as an agent of transmission from plant to plant. Heavy infections of flagellates in the salivary glands were observed; trypanosomatids were less than one-half the size seen in the plants and in the gut of the bug.     

Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

Gut symbiotic bacteria of the genus Burkholderia in the broad-headed bugs Riptortus clavatus and Leptocorisa chinensis (Heteroptera: Alydidae)

The Japanese common broad-headed bugs Riptortus clavatus and Leptocorisa chinensis possess a number of crypts in the posterior region of the midgut, whose lumen contains a copious amount of bacterial cells. We characterized the gut symbiotic bacteria by using molecular phylogenetic analysis, light and electron microscopy, in situ hybridization, and PCR-based detection techniques. Restriction fragment length polymorphism analysis of 16S rRNA gene clones suggested that a single bacterium dominated the microbiota in the crypts of the both bug species. The predominant 16S rRNA gene sequences obtained from different individuals and species of the bugs were not identical but were very similar to each other. Homology searches in the DNA databases revealed that the sequences showed the highest levels of similarity (96% to 99%) to the sequences of Burkholderia spp. belonging to the beta subdivision of the class Proteobacteria. In situ hybridization with specific oligonucleotide probes confirmed the localization of the Burkholderia symbiont in the lumen of the midgut crypts. Electron microscopy showed that the lumen of the crypts was filled with rod-shaped bacteria of a single morphotype. Molecular phylogenetic analysis demonstrated that the Burkholderia symbionts of the bugs formed a well-defined monophyletic group, although the group also contained several environmental Burkholderia strains. The phylogenetic relationship of the Burkholderia symbionts did not reflect the relationship of the host bug species at all. The sequences from R. clavatus and the sequences from L. chinensis did not form clades but were intermingled in the phylogeny, suggesting that horizontal transmission of the symbiont might have occasionally occurred between populations and species of the bugs.     

New soybean variety,Agakong, as a host of Riptortus pedestris (Fabricius): Study on field occurrence and biological attributes in the laboratory

A new variety of soybean, Agakong, has good nutritious values, thus is expected to be grown widely in the near future. To evaluate Agakong as a new soybean host of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), we assessed the biological attributes of the stink bug fed on Agakong and its field (Fabricius) occurrence in comparison with other varieties, Cheongjakong and Daewonkong. In laboratory studies, the nymphal development time and fecundity of R. pedestris was unaffected by the soybean varieties. However, nymphal mortality of R. pedestris fed on Agakong was significantly lower than those fed on Daewonkong. Furthermore, adult females become significantly larger when they fed on Agakong. R. pedestris preferred Cheongjakong seeds to Daewonkong or Agakong seeds in Petri dish choice experiments. The population dynamics of eggs, nymphs, and adults on the three soybean varieties planted in a 3 × 3 Latin square design were assessed by direct counting from 13 July to 22 September, 2007 at 10 days intervals. R. pedestris was the most abundant stink bug species on all the tested soybean varieties. The occurrence of R. pedestris eggs and nymphs was not significantly different among the three varieties. However, the seasonal density of R. pedestris adults was lower on Agakong than on Cheongjakong. Although higher R. pedestris populations were not observed in the field, the possibility of R. pedestris becoming a pest on this newly developed soybean variety can't be ruled out since some of its biological attributes were positively affected by feeding on Agakong seeds.     

Pheromonal cross-attraction in true bugs (Heteroptera): attraction of Piezodorus hybneri (Pentatomidae) to its pheromone versus the pheromone of Riptortus pedestris (Alydidae)

We investigated the attractiveness of a synthetic form of the pheromone of the soybean stink bug, Piezodorus hybneri (Gmelin), under field conditions, and compared it with that of (E)-2-hexenyl (E)-2-hexenoate, a pheromone component of a competitor, Riptortus pedestris (Fabricius). Many adult stink bugs were attracted to traps baited with 100 mg of the synthetic pheromone (1: 1: one mixture of β-sesquiphellandrene, (R)-15-hexadecanolide, and methyl (Z)-8-hexadecenoate), but few were attracted to 1 or 10 mg. More than twice as many females as males were attracted to this male-produced pheromone. None of the individual pheromone components (30 mg) attracted conspecifics. In summer (June-July), when field P. hybneri were not in diapause, (E)-2-hexenyl (E)-2-hexenoate was more attractive to P. hybneri than the synthetic pheromone. The sex ratio of the adults attracted to the synthetic pheromone was highly female-biased, yet almost equal numbers of both sexes were attracted to (E)-2-hexenyl (E)-2-hexenoate. Most females attracted to both attractants were mated and had mature ovaries. However, adults attracted to (E)-2-hexenyl (E)-2-hexenoate were likely to have less food in their stomach than those attracted to the synthetic pheromone. In late autumn (October-November), when the bugs were in reproductive diapause, both attractants attracted many sexually immature female and male adults that had well-developed fat body. The synthetic pheromone also attracted a large number of conspecific nymphs. These results suggest that P. hybneri pheromone and R. pedestris pheromone component, respectively, have different functions for P. hybneri. The male-produced pheromone system of P. hybneri seems to be sex-related but to have other roles.     

Roles of Vibrio fischeri and Nonsymbiotic Bacteria in the Dynamics of Mucus Secretion during Symbiont Colonization of the Euprymna scolopes Light Organ

During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 μm in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.     

Colonization of an artificial stream by fishes and aquatic macro-invertebrates

We examined colonization by fishes and macro-invertebrates from permanent streams into an artificial freshwater stream simulating lotic temporary bodies of water that exist for only a limited period each year. After introducing water, invertebrates such as chironomid larvae in mud increased in numbers rapidly in the experimental stream, although they were rare in mud in the permanent streams. Eleven of 12 fish species present in the permanent streams colonized the experimental stream and preyed upon invertebrates, although fish composition differed significantly between the two streams. About 100 days after the initiation of the experiment, both species richness and diversity in the experimental stream reached almost the same level as that in the permanent streams. More diverse fishes colonized the complex section where habitat diversity was high compared to the simple section in the experimental stream. Our study strongly suggests that lotic temporary waters such as temporary streams around main rivers have unique ecological characteristics and serve as valuable foraging sites for fish.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.