Nondevelopment ofCotesia kariyai(Hymenoptera: Braconidae) in Entomopoxvirus-Infected Larvae ofPseudaletia separata(Lepidoptera: Noctuidae)

Interaction between an entomopoxvirus (PsEPV) and a gregarious braconid endoparasitoid,Cotesia kariyai,inPseudaletia separatalarvae showed that infection of larvae with PsEPV was deleterious to the development and survival ofC. kariyai.The survival and development ofC. kariyaiin PsEPV-infectedP. separatalarvae depended on the length of time between parasitization and viral infection. No parasitoid larvae emerged from PsEPV-infected hosts when host larvae were exposed simultaneously to parasitization and PsEPV inoculation whereas more than 80% of the hosts produced parasitoids when PsEPV was administered 5 days postparasitization.C. kariyailarvae in PsEPV-infected hosts showed a retarded development, shrank, and died about 8 days after viral exposure. Virion-free plasma from PsEPV-infectedP. separatalarvae was toxic to the parasitoid larvae even up to a dilution level of 32 when it was injected intrahemocoelically into the host larvae. Development of parasitoids in hosts that were simultaneously parasitized and injected with the virion-free plasm never progressed beyond the egg stage. The parasitizedP. separatalarvae injected with the virion-free plasma did not pupate and died within 30 days after injection.     

Cotesia kariyai larvae need an anchor to emerge from the host Pseudaletia separata

Mature larvae of the gregarious endoparasitoid Cotesia kariyai construct cocoons for pupation approximately 10 days after parasitization and emerge from their host Pseudaletia separata under a long day photo-regime (16L8D) at 25 +/- 1 degrees C. The parasitoid larvae make capsules in the host hemocoel just prior to their emergence. These capsules function as "anchors," which enable them to press against the host integument from inside the host. It was predicted that this anchor might be composed of silk proteins secreted from the parasitoid larvae, because a previous study showed that the anchor was made up of a glycoprotein and that the silk gland of parasitoid larvae developed from 2nd larval stage. Fibroin-like proteins in C. kariyai larva mainly consist of two proteins with molecular masses of the 300.6 and 46.7 kDa estimated by SDS-PAGE. The fibroin-like proteins with the same molecular mass were detected from the anchor proteins just prior to parasitoid emergence. These results indicate that the anchor was assembled with fibroin-like proteins and was formed just before parasitoid emergence while in the host body cavity. Injection of bovine pancreatic trypsin inhibited the emergence of parasitoid larvae from the host because the anchor was decomposed by trypsin. Trypsin activity in the parasitized host hemolymph increased only after parasitoid emergence.     

Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae

A proteinaceous factor that represses plasma juvenile hormone esterase activity in parasitized insect larvae has been isolated and partially characterized from last instar larvae of the armyworm Pseudaletia separata parasitized with the wasp Apantales kariyai. Purification procedures consisted of extraction with 25% ethanol, gel filtration and reversed phase high performance liquid chromatography. Plasma juvenile hormone esterase activity in Day 3 last instar larvae was repressed by 50% when larvae were injected on Days 1 and 2 with 6.5 pmol of the purified peptide, which has a molecular weight of about 4,500 Da. The application of the factor also causes more than a 2-day delay in the onset of pupation. The sequence of 23 amino acid residues at the amino terminus of the factor was determined as follows: H-Glu-Asn-Phe-Ser-Gly-Gly-Xaa-Val-Ala-Gly-Tyr-Met- Arg-Thr-Pro-Asp-Gly-Arg-Xaa-Lys-Pro-Thr-Phe-Tyr-Gln-.     

A putative new juvenile peptide hormone in lepidopteran insects.

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.     

一种中红侧沟茧蜂畸形细胞分泌的寄生特异蛋白

SDS-PAGE电泳表明,黏虫Pseudaletia separa-ta、棉铃虫 Helicoverpa armigera、小地老虎 Agrotisypsilon的幼虫受中红侧沟茧蜂Microplitis mediator寄生后,血淋巴中都出现一个98.6 kDa的寄生特异蛋白(p98.6)。畸形细胞(teratocytes)的体外培养发现,p98.6是由来自中红侧沟茧蜂胚胎浆膜层的畸形细胞分泌的。这一结果将为研究寄生蜂的寄生生理和畸形细胞在协调寄生蜂和寄主关系中的作用打下基础。     

Structure of a growth-blocking peptide present in parasitized insect hemolymph

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.     

Effect of entomopoxvirus infection of Pseudaletia separata larvae on the oviposition behavior of Cotesia kariyai

Parasitoid behavior and oviposition were monitored to investigate the ability of Cotesia kariyai (Watanabe) (Hymenoptera: Braconidae) to distinguish between entomopoxvirus-infected and noninfected larvae of Pseudaletia separata Walker (Lepidoptera: Noctuidae). Comparison of individual means revealed that searching time was not significantly different between treatments. Ovipositional time, however, differed significantly after day 8 post virus inoculation. The parasitoid adopted ovipositional posture on infected and noninfected host irrespective of the stage of infection and did not distinguish infected from noninfected larvae up to 4 days post infection. Mean number of larvae stung by the parasitoid was not influenced by the length of time interval between exposure to the virus. However, duration of ovipositor insertion was influenced by the time interval between exposure to the virus and subsequent exposure to parasitoid females. Female parasitoids did not completely reject infected larvae as unsuitable for egg deposition. However, 5 days after virus inoculation they rejected significantly more infected than noninfected larvae. Furthermore, significantly more eggs were laid in noninfected than infected larvae from day 5 following virus administration. These observations suggest that probing by Cotesia kariyai may provide information concerning host suitability.     

The endoparasitoid Cotesia kariyai (Ck) regulates the growth and metabolic efficiency of Pseudaletia separata larvae by venom and Ck polydnavirus

It was previously demonstrated that parasitization by Cotesia kariyai caused a decrease in weight gain and food consumption in host larvae, resulting in a lower final weight for parasitized hosts. It is predicted that C. kariyai regulates the physiological condition of the host to obtain maximum food under restricted nutritional conditions. Approximate digestibility (AD) was higher following parasitization but the efficiency of conversion of digested food (ECD) of the parasitized hosts was lower. This suggests that resources available to the parasitoid larvae are enhanced in the parasitized hosts. We evaluated the physiological changes caused by injection of calyx fluid (polydnavirus) plus venom (C+V) in nonparasitized hosts. Injection of C+V into the nonparasitized hosts duplicated the effects of parasitism, namely it increased the AD and decreased the ECD. Furthermore, C+V injections elevated trehalose concentrations in nonparasitized host 7 to 10 d after injection (2nd stadium of the parasitoid larva). Protein content also increased on days 9 and 10 after C+V injection. These results suggest that the nutrients that parasitoid larvae require for their growth increase in the hemolymph of the host during the 2nd stadium of the parasitoid larva.     

Venom of ectoparasitoid,Euplectrus sp near plathypenae (Hymenoptera: Eulophidae) regulates the physiological state of Pseudaletia separata (Lepidoptera: Noctuidae) host as a food resource

Euplectrus sp. near plathypenae is an ectoparasitoid that can parasitize from 3rd to day 0-6th instar Pseudaletia separata. The developmental period of the parasitoid from the egg to the pupal stage is about 13 days. Parasitized hosts are developmentally arrested and never molt to the next stadium. The injection of venom fluid results in similar effects on P. separata larvae as does parasitization. The inhibitory effect of the venom on molting was dose dependent. Injection of 0.3 female equivalents of venom into day 0-5th host instar resulted in a similar developmental arrest as seen in parasitized hosts. The amount of total lipid in the hemolymph of the host increased as a function of the amount of venom injected, while the lipid content of the fat body was similar to lipid levels in the fat body of parasitized larvae. The amount of total protein in the hemolymph also increased when venom was injected, whereas the protein level of the fat body did not increase. The lipid concentration within the parasitoid larva was maintained at the same level throughout larval development, but increased before pupation. We conclude that the injected venom increased the hemolymph content of lipid and protein to support the growth and development of the ectoparasitoid larva.     

粘虫幼虫血淋巴中的凝集素

粘虫Mythlmna separata Walker幼虫血淋巴中含有凝集某些脊椎动物红细胞的凝集素,凝集活性可被乳糖、岩藻糖或神经氨酸抑制.用CNBr-sepharose 4B 进行亲和层析从血淋巴中分离的凝集素成分比较复杂,聚丙烯酰胺凝胶电泳显示三条区带,SDS聚丙烯酰胺凝胶电泳出现6个亚基,亚基分子量分别为71000、65000、56000、35000、33000及31000道尔顿.     

Influence of nutrient deficiency caused by host developmental arrest on the growth and development of a koinobiont parasitoid

Koinobiont parasitoids utilize nutrients obtained from hosts that contine to feed and grow after parasitization. However, if the ecdysis of early host instars is prevented, parasitized larvae will fail to grow large enough to support the development of the parasitoid brood and both organisms will perish. When L5 instar larvae (the penultimate stage) of Pseudaletia separata were parasitized by Cotesia kariyai and injected with Euplectrus separatae venom (5PV), the development of these hosts was arrested before molting to the next stage and the caterpillars thus failed to gain weight. These hosts remained at approximately 300 mg until parasitoid emergence. In contrast, hosts parasitized as L5 but without the injection of venom (5P) exhibited an increase in weight after molting to the next stage and ultimately grew to approximately 700 mg. The inhibition of ecdysis reduced the amount of food resource (e.g. fat body) for the parasitoid larvae. On the other hand, when final (= L6) host instars were parasitized and injected with E. separatae venom (6PV), the maximum weight attained by these larvae was about 710 mg, although weight gain was depressed compared to hosts parasitized without the injection of E. separatae venom (6P). The adult weight of C. kariyai that emerged from 5PV hosts was less than conspecifics that emerged from 5P, 6P, and 6PV respectively, although the egg-pupal period of the parasitoid from 5PV hosts was extended. The offspring sex ratio (percentage males) of adult wasps did not vary significantly with treatment. Female parasitoids that eclosed from 5PV hosts laid almost the same number of eggs in day 0-6th host instars as those emerging from 5P, 6P, 6PV hosts. Their egg-pupal period was extended and the cocoon cluster mass and the parasitoid body mass on subsequent generations was lighter than those reared from 5P, 6P, 6PV hosts. The sex ratio of F2 C. kariyai wasps that eclosed from 5PV increased more than in wasps that eclosed from the other host treatments (5P, 6P, 6PV). Our results reveal that a reduction in host quality and offspring fitness in the first generation negatively impacted female fitness in the second generation. An early arrestment of host growth, mediated by the addition of E. separatae venom, has severe implications on parasitoid fitness by reducing host quality, especially in smaller hosts.     

Correlation between concentration of hemolymph nutrients and amount of fat body consumed in lightly and heavily parasitized hosts (Pseudaletia separata)

Two states of parasitization in the Pseudaletia separata-Cotesia kariyai system were examined: one that was lightly parasitized and one that was heavily parasitized. We predicted that the consumption of fat body and hemolymph nutrients depends on the number of parasitoid larvae in the host. Lightly parasitized hosts (average clutch size+/-S.E.: 42.5+/-16.2, N=15) and heavily parasitized hosts (average clutch size+/-S.E.: 230.2+/-8.8, N=15) were prepared artificially. Eight days after parasitization, perivisceral fat body was depleted in the heavily parasitized host, although peripheral fat body was not yet consumed, but by day 10 most of the peripheral fat body was consumed. In lightly parasitized hosts, perivisceral fat body was not consumed by day 10. The parasitoid larvae deplete the perivisceral fat body first and then consume the peripheral fat body in the heavily parasitized host. The amount of trehalose, the major carbohydrate in the hemolymph, was related to the number of parasitoid larvae developing in the host. In a heavily parasitized host, trehalose concentrations remained low. However, in lightly parasitized hosts, the amount of trehalose increased 8 days after parasitization and then decreased by day 10. Protein and total lipid concentrations in the hemolymph of the heavily parasitized host were significantly lower than in lightly parasitized host on day 10, suggesting that the large number of parasitoid larvae depleted the fat body and hemolymph nutrients by day 10. High concentrations of total lipid on day 8 and 10 in lightly parasitized hosts and on day 8 in heavily parasitized host are likely to be attributed to the teratocytes.     

Cause of mortality in insects under severe stress.

Mortality in the host armyworm larvae Pseudaletia separata parasitized by the parasitic wasp Cotesia kariyai was dramatically increased when they were simultaneously infected by the entomopathogen Serratia marcescens. Previous studies have shown that this strong insecticidal effect is due to a metalloprotease-like insecticide (MPLI) released from S. marcescens enterobacter. This study was conducted to elucidate the exact cause of the mortality resulting from MPLI. Injection of MPLI caused a sharp increase in hemolymph dopamine concentration followed by elevated levels of brain dopamine in armyworm larvae. [3H]Dopamine injected into the hemocoel, was incorporated into the brains of MPLI-injected larvae to a level eight times greater than in BSA-injected control larvae. Transmission electron microscopy showed an obvious decrease in thickness and density of the brain sheath in insects injected with MPLI. This was probably due to the MPLI-induced elevation of hemocyte metalloprotease activities. Further, electron microscopic and TUNEL staining analyses showed a significant increase in apoptotic cells in the brain 12 h after the injection. Injection of 3-iodotyrosine (a tyrosine hydroxylase inhibitor) before MPLI completely prevented the increase in hemolymph dopamine in test larvae and their following death. From these observations, we conclude that MPLI-injected larvae may have suffered mortal damage through increased apoptosis of brain cells caused by an influx of dopamine from the hemolymph.     

Purification and characterization of a male-specific protein in the hemolymph of the wax moth,Galleria mellonella L.

A male-specific protein (MSP) present only in males was identified from the hemolymph of the wax moth, Galleria mellonella L., by polyacrylamide gel electrophoresis (PAGE) and purified by anion-exchange chromatography. MSP has a native molecular mass of 55 kDa and consists of two 27-kDa subunits. An isoelectric point of MSP was measured to be approximately 5.8. MSP is a glycoprotein that contains 1.7% carbohydrate. The compositional analysis of carbohydrate component indicated a predominance of fructose and glucose. MSP also contains large amounts of asparagine, aspartic acid, glutamine, glutamic acid, and lysine but small amounts of tyrosine, methionine, and tryptophan. Western blot analysis of the hemolymph of each developmental stage indicated that MSP is present in the hemolymph of 8-day-old pupa and adult. Also, results from Western blotting indicated that MSP is not present in the tissues of larvae and of female adults but appears in the fat body of male pupae and adult and testis of adult. The fat body and testis of male pupae and adult were cultured in vitro to trace the place and time of MSP synthesis. The fat body began to synthesize MSP in late pupae and showed active synthesis during the adult stage. The distribution of MSP in the testis was observed by electron microscopic immunogold labeling, using the antibody against MSP. MSP is present between the germinal cysts and is taken up through the basal surface of the seminiferous tubular epithelium. Arch. Insect Biochem. Physiol. 37:257–268, 1998. © 1998 Wiley-Liss, Inc.     

Uptake of juvenile hormone esterase by pericardial cells of Manduca sexta

Immunohistochemical studies were conducted to determine tissue(s) which might be involved in the uptake of juvenile hormone esterase (JHE) from larval hemolymph. Purified JHE expressed by a recombinant baculovirus carrying the JHE gene from Heliothis virescens was injected into the hemolymph of second stadium larvae of Manduca sexta. Immunoreactive material detected with specific antibodies against the natural JHE purified by affinity chromatography from the hemolymph of H. virescens was localized only in the dorsal regions of whole larval mounts. Further immunohistochemical studies of whole and dissected larvae at the light and electron microscopic level showed the specific localization of JHE in pericardial cells. Western blot analysis confirmed the localization of injected JHE in pericardial cells and also indicated some apparent degradation of the incorporated JHE. Similar results were obtained with the JHE from H. virescens injected into larvae of H. virescens. These results indicate that pericardial cells are involved in the uptake of injected JHE from insect hemolymph and its degradation.     

Growth and development of Cardiochiles nigriceps viereck (hymenoptera,braconidae) larvae and their synchronization with some changes of the hemolymph composition of their host,Heliothis virescens (F.) (Lepidoptera,Noctuidae)

Larval development of the parasitoid Cardiochiles nigriceps Viereck occurs in the last instar larva of its host, Heliothis virescens (F.). This allows the parasitoid to exploit the nutritional increase in the biosynthetic activity occurring in the host in preparation for metamorphosis. To understand the biochemical basis of this host parasitoid developmental synchrony, we undertook host ligation studies and analyzed host hemolymph for proteins and glycerol esters. Parasitization affected the biochemical profile of the host. The hemolymph protein concentration of parasitized last instar H. virescens larvae increased through time, whereas unparasitized (control) larvae were characterized by a decrease in the protein titer when they reached the prepupal stage. The effect of parasitism on glyceride titers of host hemolymph was not as pronounced as the effect on proteins. Ligation conducted on 5th instar hosts, which were parasitized as 4th instars, affected parasitoid development in a time-dependent way. The percentage of successfully developing C. nigriceps larvae increased with the increase of the time interval between parasitization and ligation. Ligation performed before day 2 of the 5th larval instar of H. virescens completely inhibited parasitoid development. Ligations that disrupted parasitoid developmentwere associated with a low host hernolymph protein concentration. Parasitoid development was successful when hernolymph protein titer was high, as occurred when ligations were performed after day 3 of the 5th host instar in both control and parasitized larvae. Ligations in both situations resulted in a slight increase in glyceride titers. The results suggest that host proteins and/or some factor(s) associated with them may play a role in parasitoid growth and development. © 1993 Wiley-Liss, Inc.     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。