Behavioral response of Riptortus pedestris (Hemiptera: Alydidae) 2nd instar nymphs to their gut symbiont,Burkholderia sp., in laboratory conditions

Many arthropods including insects have symbiotic relationships with diverse microorganisms that can confer fitness benefits to their hosts. Riptortus pedestris is a serious pest on leguminous crops in East Asia including South Korea and Japan. Recently, has been reported that R. pedestris form symbiosis with Gram‐negative bacteria in the genus Burkholderia where they acquire symbionts from soil environments without vertical transmission from each generation. In order to address how R. pedestris acquire their symbiont from environment, we hypothesized that R. pedestris would detect and exhibit preference toward the Burkholderia symbionts. In this study, we conducted a series of dual‐choice experiments and Y‐tube olfactometer experiments to monitor and evaluate behavioral responses by 2nd instar R. pedestris toward the Burkholderia treatment and untreated control under various experimental settings in the laboratory. In dual‐choice experiments, there was no significant preference by R. pedestris to the Burkholderia treatment compared with untreated control. Also, no significant difference was detected in the first choice frequency, visiting frequency, and residence time of the insects between the two treatments. Likewise, there was no detectable preference by R. pedestris to microbial odors from the symbionts in Y‐tube experiments. Therefore, our results indicate that there was no evidence supporting that 2nd instar R. pedestris exhibit preference to the Burkholderia symbiont in the laboratory conditions of this study. Further studies are required to evaluate behavioral responses of later immature stages (i.e., 3rd and 4th instar) to the symbionts and provide choice settings at larger semi‐field arenas.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Fruits of apple and sweet persimmon are not essential food sources for Riptortus pedestris (Hemiptera: Alydidae) which causes fruit-spotting

Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) has recently become an important fruit-spotting bug of apple and sweet persimmon trees in Korea. Although fruits of cultivated species are unsuitable food for some fruit-spotting bugs, the dietary importance of fruits of apple and sweet persimmon for R. pedestris is unknown. We evaluated harvested fruits of apple and sweet persimmon with or without soybean seeds as a food source for R. pedestris by investigating nymphal development time, immature mortality, preovipositional period, fecundity, and adult size in the laboratory. No first instars of R. pedestris that were fed only apple or sweet persimmon fruits became third instar nymphs. When supplemented with soybean seeds, both fruits supported nymphal development and reproduction of R. pedestris. However, fruits combined with soybean seeds were not a better diet than soybean seeds only. Therefore, apples and sweet persimmons are not essential food sources for the development and the reproduction of R. pedestris. These results suggest that R. pedestris may not reproduce on fruit trees, thus explaining the absence of eggs and nymphs of this bug on fruit trees in orchards. Management of nearby vegetation of fruit orchards would be important for reducing damage by adult R. pedestris.     

Ecotoxicological effect of insecticides on Ooencyrtus nezarae (Hymenoptera: Encyrtidae) reared from refrigerated and unrefrigerated Riptortus pedestris (Hemiptera: Alydidae) host

Due to increased field occurrence of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) on various crops including soybean, persimmon and apple in recent years in Korea, demand for insecticide applications to control the stink bug has increased. Acute toxicity of eight pesticides on Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae), a major egg parasitoid of R. pedestris, was compared in the laboratory. Fenitrothion, spinosad, cyfluthrin, etofenprox and carbosulfan caused 100% mortality of O. nezarae within 24 hours by topical application or exposure to residue. Fenitrothion was also highly toxic to the parasitoid when ingested orally. In a previous study, release of refrigerated inviable eggs of R. pedestris was found to increase field parasitism; therefore, we evaluated the sublethal effect of fenitrothion when O. nezarae parasitised refrigerated or unrefrigerated host eggs. Although parasitism rates on both kinds of eggs significantly decreased when O. nezarae were provided with host eggs sprayed with fenitrothion, no difference in parasitism rate, adult emergence, sex ratio, development time and longevity of O. nezarae was found between the refrigerated or unrefrigerated host eggs when the insecticide was treated either before or after oviposition. There was no significant sublethal effect when parasitised host eggs were treated with the insecticide. From these results, all the insecticides tested showed high, acute toxicity against O. nezarae with relatively lower sublethal effects. Refrigeration of host eggs did not affect the susceptibility of O. nezarae to insecticides.     

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.     

Diurnal rhythm of male–male combat behavior in the bean bug Riptortus pedestris (Heteroptera: Alydidae)

In many insects, mating is affected by the day–night cycle, i.e., diurnal rhythm. Although there are many reports that mating and other reproductive behaviors are controlled by daily rhythms in various taxonomic insect species, little attention has been paid to the effect of daily rhythms on male fighting behavior. Here, we investigate whether the frequency and escalation of male–male aggressive interaction exhibit diurnal rhythms under a long‐day condition in the bean bug Riptortus pedestris. Despite the fact that male aggressive behaviors were most often observed in the middle of the later half of light periods, no interaction was found between escalation of fighting and the time period. The results, at least, suggest that male aggressive behaviors are influenced by diurnal rhythms like other reproductive behaviors in R. pedestris.     

大豆荚发育程度对点蜂缘蝽成虫寿命及生殖力的影响

【目的】为明确大豆荚发育程度对点蜂缘蝽Riptortus pedestris成虫存活及生殖力的影响。【方法】在室内24℃±1℃恒温下,用不同日龄(5, 15, 25, 35和45日龄)离体鲜豆荚饲喂点蜂缘蝽,测定成虫寿命、产卵前期、产卵期、单雌产卵量等生物学参数和不同日龄豆荚籽粒中脂肪、总蛋白和总糖含量,分析点蜂缘蝽成虫的生物学参数与不同日龄豆荚籽粒中主要营养成分含量之间的相关性。【结果】随着所取食豆荚日龄的增加,点蜂缘蝽成虫的平均寿命、产卵期、单雌产卵量呈先增加然后下降的趋势,而产卵前期的变化趋势则相反。当饲喂35日龄豆荚时,点蜂缘蝽成虫寿命最长(56.90±9.47 d),产卵前期最短(6.00±0.58 d),产卵期最长(29.67±6.61 d),单雌产卵量最高(129.56±36.53粒),且与取食其他日龄豆荚的成虫之间存在显著差异。点蜂缘蝽成虫寿命、产卵期、单雌产卵量均与豆荚籽粒脂肪和总蛋白含量呈显著正相关,产卵前期与豆荚籽粒脂肪和总蛋白含量呈显著负相关。【结论】大豆荚发育程度对点蜂缘蝽成虫寿命及生殖力等生物学参数具有显著的影响,35日龄豆荚对点蜂缘蝽成虫存活和繁殖最为有利。     

New occurrence of the invasive alien leaf‐footed bug Leptoglossus gonagra (Hemiptera: Coreidae) in South Korea

The invasive alien leaf‐footed bug Leptoglossus gonagra (Fabricius 1775), native to the Americas, is herein recorded from South Korea for the first time. Forty nymphs and adults were collected on 22 September 2016 from arable lands of two separate sites. These individuals were identified based on the analysis of morphological characters and cytochrome oxidase c subunit 1 sequences. A neighbor‐joining tree indicated the existence of two clades: Group A from Brazil, and Group B from Asia and Oceania. Mean genetic distance between clades was 8.11%, range 7.60–8.57%. Within clades, mean genetic distance was 0.42% (0.17–0.83%) in Group A and, 0.45% (0.00–0.83%) in Group B. Colonies of 2nd‐final instar nymphs and adults were found, on top of leaves, flowers, fruits, and between the stems of pumpkins (Cucurbita moschata). Nymphs and adults damaged host plants by sucking their leaves, stems, fruits, and buds. After the first discovery in 2016, we were unable to find the bugs again (in 2017–2019). Therefore, the invasion of this species seems to have occurred only temporarily, during the warm season. We suggest that the species may have failed to overwinter as it is normally distributed in regions warmer than Korea. Nevertheless, possible appearances of this species must be continuously monitored as it is a potential pest of various plants cultivated in South Korea.     

Effect of Burkholderia (Pseudomonas) cepacia and soil type on the control of crown rot in wheat

A rifampicin-resistant isolate of Burkholderia (Pseudomonas) cepacia (A3R) reduced crown rot (Fusarium graminearum Group 1) symptoms significantly (P 0.05) in wheat in glasshouse and field experiments and increased grain yield significantly (P 0.05) in one of two field experiments. In glasshouse experiments, applying the bacteria as a soil drench (2.5 × 109 cfu/g soil) was more effective than coating the bacteria on wheat seed (3.4 × 107cfu/seed). In field experiments, the bacteria were applied as a soil drench at the rate of 1.8 x 10¹⁰ cfu/m row. In both the glasshouse and the field, disease severity in the bacteria-inoculated treatments was significantly less in a silt loam than in a sandy loam. The silt loam had a large proportion of fine clay and silt particles (51.7%), which may have favoured the biocontrol activity and survival of the introduced B. cepacia. In a glasshouse experiment, control by B. cepacia was significantly greater in the silt loam than in the sandy loam, which in turn was greater than in a loamy sand. The loamy sand appeared to favour crown rot development but not the activity or survival of the bacterial antagonist. The latter was reflected by the relative populations of the rifampicin-resistant bacteria re-isolated from the various soils during a 5-week period after application of the bacteria (silt loam > sandy loam > loamy sand). This study further confirms that soil type can influence the populations and the level of biocontrol activity of some bacterial antagonists.     

Occurrence and density of Halobates micans (Hemiptera: Gerridae) in the eastern South Indian Ocean

Two species of ocean skaters, Halobates germanus and Halobates micans, live in the tropical and subtropical waters of the Indian Ocean. From December 1992 to December 1993, Halobates was intensively sampled in the easternmost region of the South Indian Ocean (13–18.5°S, 114–121E°), from which there have been a small number of records of Halobates. No H. germanus was caught, but a total of 1190 H. micans were collected, with densities estimated at 13 900–28 100 individuals/km². This suggests that H. micans lives in the study area at high densities comparable to those in the Atlantic and the Pacific Oceans. We also discuss the possible effects of ocean currents and winds on the geographic distributions of the two Halobates species in the eastern South Indian Ocean.