Yonggyun Kim

Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode Steinernema carpocapsae

Xenorhabdus nematophilus is a pathogenic bacterium causing insect haemolymph septicemia, which leads to host insect death. To address the fundamental mechanisms underlying this haemolymph septicemia, or the immunodepressive response of the host insects following bacterial infection, we tested a hypothesis that the insect immune-mediating eicosanoid pathway is blocked by inhibitory action of the bacterium. Haemocoelic injection of the bacteria into the fifth instar larvae of Spodoptera exigua reduced the total number of living haemocytes with postinjection time and resulted in host death in 16 h at 25 degrees C. The lethal efficacy, described by the median lethal bacterial dose (LD(50)), was estimated as 33 colony-forming units per fifth instar larva of S. exigua. The lethal effect of the bacteria on the infected larvae decreased significantly with the addition of exogenous arachidonic acid (10 µg), a precursor of eicosanoids. In comparison, injections of dexamethasone (10 µg), a specific inhibitor of phospholipase A(2), and other eicosanoid biosynthesis inhibitors elevated significantly the bacterial pathogenicity. Live X. nematophilus induced the infected larvae to form less nodules than did the heat-killed bacteria, but the addition of arachidonic acid increased the number of nodules formed significantly in response to live bacterial injection. The treatment with dexamethasone and other inhibitors, however, decreased the nodule formation after injection of heat-killed bacteria. These results indicate that eicosanoids play a role in the immune response of S. exigua, and suggest strongly that X. nematophilus inhibits its eicosanoid pathway, which then results in immunodepressive haemolymph septicemia.

Hemocyte Apoptosis Induced by Entomopathogenic Bacteria, Xenorhabdus and Photorhabdus, in Bombyx mori

Abstract Entomopathogenic bacteria of Xenorhabdus nematophila, Xenorhabdus sp., and Photorhabdus temperata subsp. temperata are symbionts of entomopathogenic nematodes including Steinernema and Heterorhabditis. These bacteria have potent insecticidal pathogenicity causing hemolymph septicemia. To explain a mechanism of the septicemia, this research raises a hypothesis that the hemolymph septicemia is due to the induction of the programmed cell death (=apoptosis) of the hemocytes by the entomopathogenic bacteria. Injection of the bacteria into the hemocoel of the fifth instar larvae of Bombyx mori led to septicemia (few viable hemocytes) at 12h in all three bacterial treatments. During the pathogenicity, the infected hemocytes exhibited the membrane blebbing, nuclear chromatin condensation and DNA fragmentation, which reflected typical morphological changes of cells undergoing apoptosis. The apoptosis began as early as 4h after bacterial injection and increased with post-injection time. Three pathogenic bacteria, however, differed in the degree of apoptosis-inducing effect on B. mori by the effective bacterial dose and time. These results support the hypothesis that hemolymph septicemia caused by the bacteria is due to their apoptosis-inducing effect.

Effect of thermoperiod and photoperiod on cold tolerance of Spodoptera exigua (Lepidoptera: Noctuidae).

Abstract Beet armyworm, Spodoptera exigua (Hübner), is a freeze-susceptible and migratory species, but can overwinter in temperate areas without diapause. Most developmental stages reared under constant environments are able to induce a cold-hardening process in response to a brief exposure to cool temperature. In this study, we analyzed the effect of daily temperature and photoperiod cycles on the cold-hardening process. Rearing temperatures significantly affected cold tolerance of larvae. In constant temperature regimes, as rearing temperature decreased, cold tolerance increased. Cyclic temperature regimes caused a significantly higher cold tolerance than did constant temperatures with the same daily average. In cyclic regimes with three combinations of a thermophase temperature and three different cryophase temperatures below a developmental threshold (13°C), cryophase temperature had a significant effect on cold tolerance. Cold-hardening induced by rearing temperatures was associated with decreases of supercooling points and increases of hemolymph osmolalities and glycerol contents. A cyclic photoperiod produced a higher cold tolerance than did constant dark or light. There was, however, no variation in cold tolerance among different cyclic daylength regimes. These results indicate that fluctuating temperature and photoperiod significantly affected cold tolerance of S. exigua.

Juvenile Hormone Esterase of Diamondback Moth, Plutella xylostella, and Parasitism of Cotesia plutellae

Abstract An endoparasitic wasp, Cotesia plutellae, inhibits pupal metamorphosis of diamondback moth, Plutella xylostella. The parasitized larvae extended their larval stage about 2 days at 25°C and died after escape of wasp larvae for pupation. We set up a hypothesis that the wasp parasitization inhibits juvenile hormone (JH) degradation, which leads to extend larval stage and blocks metamorphosis. In this study, the activities of JH esterase (JHE), a major JH degrading enzyme in hemolymph, were analyzed by use of a surrogate substrate, HEPTAT (methyl 1-heptylthioacetothioate). Hemolymph JHE activities of P. xylostella showed a typical Michaelis-Menten enzyme kinetics in response to HEPTAT, where it exhibited KM at 19.7 μM and Vmax at 228.8 μM/min/μ 1. The parasitized C. plutellae did not exhibit decrease of the JHE activity, but rather kept high JHE activity during the extended larval period.

Identification and Characterization of a Symbiotic Bacterium Associated with Steinernema carpocapsae in Korea

Abstract A symbiotic bacterium was identified from the entomopathogenic nematode, Steinernema carpocapsae, collected in Korea and its biological significance was examined for insecticidal and antibiotic effects. The symbiotic bacterium was isolated from the nematode-infected hemolymph of the fifth instar larvae of Spodoptera exigua. The intra-hemocoelic injection of the bacterial isolates killed the insect hosts within 24h. Several biochemical and morphological characters of the bacterium were identical to those of Xenorhabdus nematophilus. The bacterial growth was not inhibited on the media containing 4,000 ppm of streptomycin sulfate or 2,000 ppm of penicillin. They showed antibacterial effects on Escherichia coli, Ralstonia solanacearum and Pseudomonas aeruginosa, but not on Pseudomonas putida and Pseudomonas fluorescens.

Relationship Between Cold Hardiness and Diapause in the Smaller Fruit Tortrix, Adoxophyes orana (Fischer von Roslerstamm)

Abstract The smaller fruit tortrix, Adoxophyes orana (Fischer von Roslerstamm), overwinters at larval developmental stage. Cold-hardening is presumed to be expressed during diapause development for successful overwintering in this species. To address this hypothesis, we compared cold tolerance between diapausing and non-diapausing larvae, following overall cold tolerance assays in all stages. Supercooling points (SCPs) varied among developmental stages. The SCPs of adults (−19.8°C) were lower than those of pupae (−17.8°C) and eggs (−15.2°C). The larval stage had the most variable SCPs (−8.5 to −16.8°C) among developmental stages, which increased with larval ages. There was, however, no significant difference in SCPs between non-diapausing and diapausing larvae. Field-collected overwintering larvae had rather higher SCPs than did the laboratory-diapausing larvae. Cold tolerance also varied with developmental stages. All the stages died after a few hour exposure to low temperatures above their SCPs. Pupae were the most susceptible to low temperatures. Diapausing larvae were much more tolerant to cold temperatures than the non-diapausing larvae. Non-diapausing larvae acclimated to 0°C for 2 h had higher survival in response to lethal low temperature than did the unacclimated larvae. These cold tolerance differences were related with trehalose contents analyzed by an ion-exchange HPLC. Field-collected overwintering larvae and the laboratory diapausing larvae had significantly higher trehalose contents than did non-diapausing larvae. These results indicate that A. orana is a freeze-susceptible species and that diapause development elevates cold tolerance in this species. These also suggest that trehalose can be a major polyol conferring cold tolerance to the diapausing larvae.

Indirect Chilling Injury of Spodoptera exigua in Response to Long-Term Exposure to Sublethal Low Temperature

Abstract We report indirect chilling injury, which is caused by the long-term exposure to the low temperature above freezing point, found in the beet armyworm, Spodoptera exigua. Short (minute or hour) exposure to 5°C was not lethal to all stages of S. exigua. However, several day-exposure to 5°C gave significant nonfreezing injuries such as low egg hatchability, retarded larval development, and low pupation rates. Even though the larvae did not develop at 5°C, they used up their major nutrient reserves significantly. The mechanism of the indirect chilling injury of the beet armyworm was discussed on the basis of the oxidative stress hypothesis.

Differential Parasitic Capacity of Cotesia plutellae and C. glomerata on Diamondback Moth, Plutella xylostella and Dichotomous Taxonomic Characters

Abstract Two closely-related endoparasitoids of Cotesia plutellae and C. glomerata parasitize the diamondback moth, Plutella xylostella. The parasitized hosts by either parasitoid species exhibited the extended larval period and died without further metamorphosis to pupal stage. However, two parasitoid species exhibited significantly different parasitic capacity and developmental rate, in which C. plutellae showed higher parasitism and faster development in the parasitized P. xylostella. To discriminate these two similar species, morphological and molecular differences were analyzed. Three dichotomous morphological characters including antennal flagellum, hind-leg femur, and terminal abdominal terga were determined. Based on the presumptive polydnaviral particles found in the ovarian calyx of C. glomerata , three genes similar to C. plutellae bracoviral genes were cloned in the C. glomerata genome and compared in their cDNA and the deduced amino acid sequences. Several polymorphic sites were detected to be applicable to design molecular markers to discriminate these two species.

Disturbance of adult eclosion by fenoxycarb, a juvenile hormone mimic, in the beet armyworm, Spodoptera exigua.

Abstract Effect of exogenous juvenile hormones (JHs) on pupal development was assayed in the beet armyworm, Spodoptera exigua. Fenoxycarb, a potent JH mimic, was applied topically to different ages of the pupae, and showed significant inhibition of normal adult eclosion even at 0.1 μg dose when it was applied at the early pupal stage (day 0). As the pupal development underwent, the susceptibility of the pupae fenoxycarb decreased. RH5992, a potent ecdysteroid mimic, did not, however, any similar inhibitory effect on the pupae. Natural JH types (JH I, JH II, and JH III) were applied on day 0 pupae to compare their inhibitory effects on adult eclosion. Both JH I and JH II significantly inhibited adult eclosion at 1.0 μg dose, but JH III did not even at 10.0 μg dose. It was noted that fenoxycarb-treated pupae showed little rectum development. Fenoxycarb did not, however, show any negative effect on the development of compound eye and wing imaginal discs, and on the pupal hemolymph protein pattern. These results suggest that there should be a commitment period requiring an absence of JH for a normal adult metamorphosis during early pupal development and that the endogenous type of JH in S. exigua is JH I or JH II or both JHs like other lepidopteran species.

Eicosanoid-mediated immunity in insects

Abstract Eicosanoid is a collective term for oxygenated metabolites of C20 polyunsaturated fatty acids. As seen in mammals, eicosanoids play crucial roles in mediating various physiological processes, including immune responses, in insects. Upon microbial pathogen infection, non‐self recognition signals are propagated to nearly immune effectors such as hemocytes and fat body using various immune mediators, in which eicosanoid signals act as the ultimate downstream mediator. The chemical diversity of eicosanoids may operate to mediate various immune responses. Some entomopathogenic bacteria suppress eicosanoid biosynthesis, which inhibits host insect immunity and promotes their pathogenicity. This review introduces immune responses mediated by various eicosanoids. Then it explains the cross‐talks of eicosanoids with other immune mediators including cytokines, biogenic monoamines, and nitric oxide to clarify the complexity of insect immune mediation. Finally, we highlight the biological significance of eicosanoids by demonstrating bacterial pathogenicity inhibiting a key enzyme – phospholipase A2 – in eicosanoid biosynthesis using their secondary metabolites to defend host insect immune attack. HighlightsEicosanoids play crucial roles in mediating insect immune responses.Eicosanoid signals act as the ultimate downstream mediator.Some insect pathogens suppress insect immunity by inhibiting eicosanoid biosynthesis.