Refrigerated eggs of Riptortus pedestris (Hemiptera: Alydidae) added to aggregation pheromone traps increase field parasitism in soybean

Riptortus pedestris (F.) (Hemiptera: Alydidae) is a key pest of soybean [Glycine max (L.) Merr.] in Korea and Japan. Aggregation pheromone traps have been used to attract R. pedestris. An aggregation pheromone, produced by males of R. pedestris, also attracts the egg parasitoid, Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae). As a new method of pest management, we propose the use of nonviable host eggs in the pheromone trap together to both trap R. pedestris and propagate parasitoids on host eggs attached to the trap. Approximately ten-thousand nonviable refrigerated host eggs were placed outside pheromone traps in three soybean fields (0.167-0.284 ha.). During the sampling period from 7 August to 8 October in 2008, statistical significance of treatment was not found in any life stage of R. pedestris population. However, significantly higher parasitism of both natural (78-91%) and refrigerated eggs (36-59%) occurred in the treatment sites, compared with control sites where parasitism was 0-62% and 16-34% on natural and refrigerated eggs, respectively. The proportion of bean pods with stink bug feeding damage was significantly reduced by 47% in the treatment sites. This study is the first trial of an integrated pest management (IPM) strategy using both nonviable host eggs and trap that simultaneously targets both the pest and its natural enemy.     

Refrigeration of Riptortus clavatus (Hemiptera: Alydidae) eggs for the parasitization by Gryon japonicum (Hymenoptera: Scelionidae)

Gryon japonicum (Ashmead) (Hymenoptera: Scelionidae) is a solitary egg parasitoid of Riptortus clavatus (Thunberg) (Hemiptera: Alydidae) one of the most important pests in soybean in Korean and Japan. Refrigeration of R. clavatus eggs for up to 120 days was evaluated as a method of mass production of G. japonicum. The refrigeration reduced the eclosion of eggs, thus no nymph emerged after 30 days of egg refrigeration. Increased duration of host acceptance behaviors by G. japonicum and some detrimental effects on parasitism rate, developmental time, longevity, and adult size of the parasitoid were found in refrigerated host eggs. However, all the biological parameters of G. japonicum were unaffected by the refrigeration of up to 30 days. Gryon japonicum parasitized 16 and 14 host eggs daily that were refrigerated for 15 and 30 days, respectively, which did not differ from parasitization of fresh host eggs. Furthermore, refrigeration of host eggs did not reduce the reproduction of the emerged adult parasitoids and emergence and sex ratio of their progeny. Gryon japonicum also parasitized 14 and 13 refrigerated host eggs per day kept at 26.3°C and 78.7% RH for 2 and 4 days of post-refrigeration without significant reduction, respectively. These results show that refrigeration of R. clavatus eggs can be a good method for mass rearing of the parasitoid, and the host eggs killed by cold storage can be supplemented in the field to boost field parasitism.     

Flight behavior of the bean bug,Riptortus clavatus (Thunberg) (Hemiptera: Alydidae), relative to adult age,sex and season

Studies related to the flight behavior of the bean bug, Riptortus clavatus, an insect pest of soybean, provide information, which can aid the development of management tactics. R. clavatus flight activities were determined using the tethered flight technique in the laboratory and a field study. We compared the flight parameters (flight distance, duration, speed and frequency) of laboratory-reared mated or unmated females and males of different adult age groups, and field-collected females and males from different seasons in a year. Mating effect was only significant for flight frequency, which was higher for unmated adults. Only the flight frequency was different between females and males. Among age groups, 25 d old individuals were shown to have higher flight parameters while the 35 or 45 d old groups showed the lower flight. Among the season, flight activities decreased as the season progressed. The flight distance of R. clavatus in a day was estimated to be 1.6–5.1 km with an average speed of 0.8 m/s from the laboratory data. However, from the field study, it was shown that bean bugs flew a 45–54 m distance with a flight speed of 3.0–3.6 m/s for the first single flight. The second flight was much shorter and slower. We discuss the possible difference of flight parameters between the laboratory and field studies with features of flight mill, variable field conditions and host plant finding behaviors. Further study on flight behavior may provide a better understanding of R. clavatus biology which may guide proper management.     

Compensatory regeneration of antennae after removal of the distal segment in Riptortus clavatus (Thunberg) (Heteroptera : Alydidae)

In Riptortus clavatus (Thunberg) (Heteroptera : Alydidae), growth and development of cuticular structures were compared between normal antennae and the antennae whose distal (4th) segment had been amputated during the 1st instar. The total length of the remaining 3 segments was 51% of the normal antenna. From the 2nd ecdysis onwards, the 2nd and 3rd segments grew excessively, and after adult emergence, the length of the operated antennae was 84% that of a normal antenna, although a typical 4th segment, separated from the 3rd segment by an intersegmental membrane, never developed. On the new distal (3rd) segment of operated antennae, long fine sensory hairs and grooved pegs, which characterize the normal distal (4th) segment, began to appear at the 2nd ecdysis, and successively increased in number between molts. Thus, when the distal segment was removed, the remaining segments tended to gradually compensate for the loss, both in terms of length and cuticular structures.     

Influences of food sources on reproduction and aggregation pheromone production of Riptortus pedestris (Hemiptera: Alydidae) in its adult stage

The dietary importance of sweet persimmon fruit (SPF) was determined in adult bean bug, Riptortus pedestris, by investigating the effect of feeding SPF and soybean seeds and peanut on longevity, weight change, fecundity and ability for pheromone production in the bean bug. Adult female R. pedestris fed on SPF had significantly shorter lifespans than those fed on soybean, while in males there was no significant difference. Males and females fed on SPF were significantly lighter than those fed on soybean seeds. Females fed on SPF did not lay eggs at all, but those fed on soybean laid 155.8 eggs, and those fed on soybean seed complemented with peanut seed laid 120.8 eggs in 50 days. The amount of pheromone components (E)‐2‐hexenyl (E)‐2‐hexenoate and (E)‐2‐hexenyl (Z)‐2‐hexenoate produced by male R. pedestris fed on SPF was significantly less than that produced by males fed on soybean. Other pheromone components, tetradecyl isobutyrate and octadecyl isobutyrate, were not produced by adult males fed on SPF. This difference was apparent at one week after emergence and was more at three weeks after emergence. In the present study, we demonstrated that SPF had adverse effect on female survivorship, reproduction of female adults and production of the aggregation pheromone by adult males, compared to soybean food. These results suggested that SPF is unsuitable for adult R. pedestris. Management of nearby vegetation of fruit orchards may play an important role in reducing damage by adult R. pedestris.     

Interspecific larval competition between two egg parasitoids in refrigerated host eggs of Riptortus pedestris (Hemiptera: Alydidae)

In a previous study, we found that soybean fields could be supplemented with refrigerated eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) to enhance parasitism. As a part of a study to evaluate the effect of host egg refrigeration on parasitism, host acceptance behavior and interspecific larval competition between Gryon japonicum (Ashmead) (Hymenoptera: Scelionidae) and Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) were studied in multiparasitized unrefrigerated and refrigerated eggs. O. nezarae showed complete host acceptance behavior when offered refrigerated host eggs that were preparasitized by G. japonicum. Adult emergence rate of O. nezarae was 43 and 74% when the interval between the first and second oviposition was 0 and 4 days, respectively, and was not different between refrigerated and unrefrigerated eggs. Refrigeration did not change host acceptance behavior of G. japonicum, but adult emergence declined from 80% in unrefrigerated eggs to 37% in refrigerated eggs that were pre-parasitized by O. nezarae on the same day. No negative effects of refrigeration on sex ratio, adult longevity, and adult size of the both parasitoids were found. Generally host egg refrigeration did not negatively affect host acceptance behavior of the both parasitoids on preparasitized eggs or larval competition between the two parasitoids in multiparasitized host eggs with exceptions in the development time and emergence rate of G. japonicum. Therefore, host egg refrigeration may not interrupt interactions between the parasitoid populations in the field.     

Biological attributes of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) reared on refrigerated eggs of Riptortus pedestris (= clavatus) Fabricius (Hemiptera: Alydidae)

Storing host eggs at low temperatures has been used to mass rear parasitoids of stink bugs, including Riptortus pedestris Fabricius (Hemiptera: Alydidae), a major soybean pest in Korea and Japan. However, no information on the effect of cold storage on parasitization by Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae), one of the major parasitoids of R. pedestris, has been published. In this study, we examined biological attributes, including parasitism rate, development time, sex ratio, adult size, and longevity, of O. nezarae when the adult parasitoids were provided with host eggs refrigerated for 0, 15, 30, 60, and 120 days at 2.0 °C. None of the attributes of the first or second generation of O. nezarae was negatively affected by host egg refrigeration up to 30 days. In addition, O. nezarae could parasitize refrigerated host eggs successfully for the first four days of post-refrigeration period when they were kept at 26.3 °C and 78.7% RH conditions. Therefore, refrigeration of R. pedestris eggs can be a good method to mass rear O. nezarae.     

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.     

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.     

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.     

Purification and characterization of biliverdin-associated cyanoprotein from eggs and hemolymph of the bean bug,Riptortus clavatus (Heteroptera: Alydidae)

Biliverdin binding protein, cyanoprotein (CP), was purified from egg extracts of the bean bug, Riptortus clavatus. The preparation was shown to be homogeneous by DEAE-chromatography and polyacrylamide gel electrophoresis (PAGE). The native molecular weight (MW) of egg CP (CP_egg) was estimated to be 320,000 by PAGE and 335,000 by gel filtration. The apoprotein consisted of identical subunits with MW of 76,000. CP_egg had a high content of aromatic amino acids (17% mol ratio) with absorbance peaks at 370 and 620 nm which are characteristic of biliverdins. Native CP_egg was associated non-covalently with 16 molecules of biliverdin. CP_egg contained about 13.0% neutral sugar (mainly mannose) associated covalently with the apoprotein, no lipid was detected. In the hemolymph of the bugs, four CP bands (CP1, 2, 3 and 4) were detected by native PAGE. These bands were immunologically related and showed properties similar to CP_egg in biochemical analysis. Only CP1 was identical to CP_egg. CPs specific to the hemolymph, CP2, 3 and 4, were different from CP_egg in mobility by PAGE, pI and peptide mapping profiles.     

Population parameters and growth of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) under elevated CO2 concentrations

We evaluated the direct effects of three different CO₂ concentrations (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 from R. pedestris was 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 of R. pedestris provide the stage structure and variability of the population growth under different CO₂ treatments. 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 of R. pedestris reared under elevated CO₂ conditions showed higher intrinsic and finite rates of increase but a lower mean generation time than R. pedestris reared under ambient CO₂ conditions. Our results show the population parameters and growth of R. pedestris are influenced by increased CO₂ relative to ambient CO₂ treatment. Further studies on the long‐term direct effects of different CO₂ levels on R. pedestris are essential to understand their population dynamics and to establish appropriate management strategies.     

Genetic diversity of Riptortus pedestris (Hemiptera: Alydidae) populations in the Republic of Korea based on COI sequence

Riptortus pedestris (Fabricius) has caused severe agricultural damage in Asian regions. To clarify genetic relationships among different populations of R. pedestris in Korea, 294 COI sequences were generated from 294 individual samples collected in 42 local regions. In total, 36 haplotypes were detected from the 294 COI sequences, and the genetic distances among the 42 local populations ranged from 0.00 % to 1.50 %. In the AMOVA results, a variability of more than 98 % was observed within populations, and the median joining (MJ) networks revealed that R. pedestris has expanded by crossing the nine geographical groups. Most of the samples shared one haplotype, H2; however, some samples obtained from the same regions displayed slight genetic differences. These results indicate that R. pedestris have undergone a series of genetic variations.     

Receptivity of female remating and sperm number in the sperm storage organ in the bean bug,Riptortus clavatus (Heteroptera: Alydidae)

This study examines the relationship between the number of sperm in the seminal receptacle (spermatheca) and the receptivity of female remating in the bean bugRiptortus clavatus Thunberg. On the 21 st day after the first mating when receptivity to remating was > 70%, females receptive to remating had significantly fewer sperm ( < 40 on average) in the spermathecae than females reluctant to do (about 150 on average). However, averages of the number of eggs laid by receptive and reluctant females within 21 days were almost same. The proportion of fertilized eggs for receptive females at 15–21 days after copulation was significantly lower than that for reluctant females. Spermatozoa transferred from a male to a female’s spermatheca were detected 5 min after copulation and then increased continuously to about 500 with the first hour. When copulation durations were manipulated artificially, the shorter the copulation period (=females had less sperm in their spermathecae), the higher the remating rate became. Females may perceive the number of sperm in their seminal receptacles and then determine whether they copulate or not. These results support the hypothesis that females mate multiply in order to replenish inadequate sperm supplies to fertilize all eggs produced.     

Characterization of Burkholderia bacteria clade compositions in soil and Riptortus pedestris (Hemiptera: Alydidae) in South Korea

Riptortus pedestris (Hemiptera: Alydidae) is known to acquire the genus Burkholderia, symbiotic bacteria, from soil. Therefore, symbiont acquisition of R. pedestris would be directly affected by bacterial diversity in soil. Soil typically harbors diverse microbes including different Burkholderia clades such as SBE (stinkbug-associated beneficial and environmental), PBE (plant-associated beneficial and environmental), and BCC (Burkholderia cepacia and complex). Nevertheless, little is known about Burkholderia acquisition patterns of R. pedestris in nature, especially in the context of bacteria clade compositions in soil. Therefore, based on diagnostic PCR analysis, we investigated Burkholderia clade compositions in field-collected soil itself and R. pedestris when the insects were provided with the soil. Also, wild R. pedestris were surveyed to characterize their Burkholderia compositions. First, 88.44% of soil samples were detected with the genus Burkholderia, and triple clade (SBE + PBE + BCC) was most frequently detected. Second, R. pedestris nymphs readily acquired Burkholderia bacteria from field-collected soil where 91.25% of the reared insects harbored the bacteria in their midguts. In contrast to soil, the detection of single BCC clade was the most dominant among the three identified Burkholderia clades. Third, from wild R. pedestris, 80.62% of the insects were found to harbor the genus Burkholderia, and single BCC clade was most frequently detected. Finally, 29.13% and 47.06% of the reared and wild R. pedestris were detected with unidentified Burkholderia clade, which does not belong to any of the three identified clades. Our findings provide baseline information to better understand ecological associations between R. pedestris and Burkholderia bacteria in different clades.     

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.     

Quality assessment of Riptortus pedestris (Hemiptera: Alydidae) eggs cold-stored at different temperature and relative humidity regime

Supplementation of host resource can be more economical method for the biological control of insect pest compared to direct release of adult parasitoids. Periodical release of non-viable cold-stored eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) has been found to enhance parasitism of this pest in soybean fields. To find the optimum environmental conditions for cold storage of these host eggs, we evaluated nine different combinations of temperature (2, 6, and 10 °C) and relative humidity (high 90–95%, medium 70–75%, and low 30–35%). After 30 d of cold-storage, eggs were weighed and held at 26.6 °C and 75% relative humidity for 8 d before testing. To test the eggs’ suitability as hosts following cold storage, females of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) were released individually onto batches of eggs, and parasitization rates and the development, emergence, sex ratio, adult longevity, and size of parasitoid progeny were examined. Eggs stored at high relative humidity showed less weight loss than those stored at low relative humidity. The number of eggs parasitized was highest (5.9/15) on eggs stored at 6 °C and high relative humidity. Developmental times and adult emergence were optimal on host eggs stored at 2 °C and high relative humidity. A significantly lower proportion of eggs produced male parasitoids when eggs were stored at 2 or 6 °C. Adult longevity was not affected by egg storage conditions, but adult size of progeny decreased in eggs stored at 10 °C. In conclusion, eggs of R. pedestris stored below 6 °C and with a high relative humidity maintained the best quality for parasitization by O. nezarae.     

A revision of Neomegalotomus (Hemiptera: Alydidae)

We recognize two species in Neomegalotomus, N. parvus (Westwood), the type species; and N. rufipes (Westwood). The two species are redescribed from type specimens, and are keyed. We synonymize the following species with Neomegalotomus parvus: N. simplex (Westwood), N. latifascia (Berg), and N. pallescens (St?l) (all new synonymies). We synonymize N. jamaicensis (Westwood) with N. rufipes (new synonymy). The type specimens of all species hitherto placed in the genus are also redescribed, except those of N. debilis (Walker) and N. vicinus (Westwood). N. parvus occurs from México through Central America into northern Argentina and east into Venezuela and adjacent Caribbean islands. N. rufipes is widespread in the Caribbean south towards Venezuela. The two species overlap in distribution: N. parvus is found on St. Vincent and Tobago, whereas N. rufipes occurs on Grenada, which lies between those two islands.