Tsuyoshi Hiraoka

Effects of lipophorin and 20-hydroxyecdysone on in vitro development of the larval endoparasitoid Venturia canescens (Hymenoptera: Ichneumonidae).

We previously reported that development of the embryos of the larval endoparasitoid Venturia canescens proceeded in the insect cell culture medium, MGM-450, and was promoted by the addition of a pupal extract from their host Galleria mellonella. The developmental promotion also was obtained by supplementing an equal amount of chicken egg yolk instead of the pupal extract to the medium. In this case, approximately 30% of the embryos developed into the second instar, but the value increased to more than 90% by treatment with 20-hydroxyecdysone. The medium supplemented with a G. mellonella pupal extract obtained by using Carlsons solution displayed growth-promoting ability, and in the extract, apolipophorin I was electrophoretically detected in large amounts. Both lipophorin purified from G. mellonella pupae and low density lipoprotein from chicken egg yolk acted as a growth-promoting substance for parasitoid development, although fetal bovine serum and 20-hydroxyecdysone were required as supplements to the medium for the expression of the ability. This indicated that lipophorin or lipophorin-transported lipids could act as a substance closely related to the growth-promoting factor(s) putatively involved in the host extract.

Isolation of a protein lethal to the endoparasitoid Cotesia kariyai from entomopoxvirus-infected larvae of Mythimna separata.

Virion-free plasma from entomopoxvirus (MyseEPV)-infected larvae of the armyworm, Mythimna separata, contains a factor that adversely affects the survival of the gregarious braconid endoparasitoid, Cotesia kariyai. Heating or proteinase K treatment eliminates the toxic effect of virion-free plasma on the parasitoid, suggesting that the lethal factor is a protein. We purified the Protein Lethal to C. kariyai larvae (PLCK) from the virion-free plasma of MyseEPV-infected M. separata larvae by a three-step procedure using gel filtration and cation-exchange chromatography. Toxic activity was measured using an in vitro-cultured parasitoid bioassay. Parasitoid larvae cultured in IPL-41 medium (Weiss et al., In vitro 17 (1981), 495) containing 4.7 microg/ml purified PLCK shrank and died within 3days. The molecular weight of PLCK was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 28,000, under both reducing and non-reducing conditions, indicating that in its native form the protein is a single 28-kDa polypeptide. Western blot analysis indicated that the lethal protein is not present in the hemolymph of uninfected host larvae, but is induced in the hemolymph by infection with MyseEPV. Western blot analysis also indicated that the proteins of virions and occlusion bodies of MyseEPV are not serologically related to PLCK.

Growth-promoting effects of ecdysteroids and juvenile hormone on in vitro development of the larval endoparasitoid, Venturia canescens (Hymenoptera: Ichneumonidae)

We previously reported that lipophorin, fetal bovine serum (FBS), and 20-hydroxyecdysone (20-HE) are essential for the development of the larval endoparasitoid Venturia canescens larvae in vitro. The present study was undertaken to determine the optimal concentrations of those three substances in the MGM-450 medium, and to examine the hormonal effects of ecdysteroids and juvenile hormone (JH) on the development of the parasitoid larvae in vitro. When the culture was started with embryos at the post-germband stage, concentrations of 3 mg/ml of lipophorin and 20% of FBS were most suitable for the development of the parasitoid. The growth-promoting effect of 20-HE increased in a concentration-dependent manner and peaked at a concentration of 1 &mgr;g/ml. Excess concentration led to malformations of the larvae. Three other ecdysteroids, ecdysone, 2-deoxy-20-hydroxyecdysone, and polypodine B had the same effect, although their activity was lower than that of 20-HE. Cholesterol had no effect; most larvae failed to develop. When the medium was supplemented with JH, the duration of the developmental period was significantly shortened, but this hormone was not found to be essential.

The carbohydrate ligands on the host embryo mediate intercellular migration of the parasitic wasp embryo

Invasive stage embryos of the parasitic wasp Copidosoma floridanum transmigrate through the epithelium of phylogenetically distant host embryos in a manner that is similar to mammalian leukocyte infiltration. Host embryonic cells appear to recognize the invading wasp embryo by components on the cell surface. We developed an in vitro wasp entry inhibition assay and found that C‐type lectins of the wasp embryo bound to N‐linked carbohydrate chains with fucose residues on the surface of host embryo. This is the first report showing a receptor–ligand interaction between heterologous multicellular organisms.

An ultrastructural study of polyembryonic parasitoid embryo and host embryo cell interactions.

The morula‐stage embryo of the polyembryonic egg‐larval parasitoid Copidosoma floridanum forms outside the host embryo and secondarily invades the host body. Electron microscopic analyses of cellular interactions between the extraembryonic syncytium of the parasitic morula and the host embryonic epithelial cells showed that morula penetration into the host embryo did not cause obvious damage to the host cells, except for the abrasion of the embryonic cuticle. Epithelial cells of the host embryo extended microvilli toward the invading C. floridanum morula and also adjacent host cells in the same way. Shortly after settlement of the morula within the host body cavity, gap junctions and adherens junctions with host cells were formed. The morula was then surrounded by a cyst comprised of host cells into which host tracheoles were invaginated. J. Morphol., 2010.

IMMUNOCYTOCHEMICAL LOCALIZATION OF TREHALASE INHIBITOR IN SOME INSECT SPECIES

Antibody against cockroach trehalase inhibitor was prepared and tested against the plasma of adult locusts and larval silkworms to determine whether these species possess a similar protein. An immunopositive response was elicited in both species. Studies using immunogold labeling show that adult cockroaches have trehalase inhibitor protein in granules of plasmatocytes and in oenocytoid-like structures. Localization of the immunoreactive protein with trehalase inhibitor antibody in locust hemocytes indicated that the protein is also contained in the granules of plasmatocytes. However, in the hemolymph of silkworm larvae, the immunoreactive protein was found only in the spherules of spherulocytes. The results suggest that insect hemolymph commonly contains trehalase inhibitor both in plasma and in certain hemocytes.

The role of the effector caspases drICE and dcp-1 for cell death and corpse clearance in the developing optic lobe in Drosophila

In the developing Drosophila optic lobe, cell death occurs via apoptosis and in a distinctive spatio-temporal pattern of dying cell clusters. We analyzed the role of effector caspases drICE and dcp-1 in optic lobe cell death and subsequent corpse clearance using mutants. Neurons in many clusters required either drICE or dcp-1 and each one is sufficient. This suggests that drICE and dcp-1 function in cell death redundantly. However, dying neurons in a few clusters strictly required drICE but not dcp-1, but required drICE and dcp-1 when drICE activity was reduced via hypomorphic mutation. In addition, analysis of the mutants suggests an important role of effecter caspases in corpse clearance. In both null and hypomorphic drICE mutants, greater number of TUNEL-positive cells were observed than in wild type, and many TUNEL-positive cells remained until later stages. Lysotracker staining showed that there was a defect in corpse clearance in these mutants. All the results suggested that drICE plays an important role in activating corpse clearance in dying cells, and that an additional function of effector caspases is required for the activation of corpse clearance as well as that for carrying out cell death.

Compatible invasion of a phylogenetically distant host embryo by a hymenopteran parasitoid embryo.

Embryonic invasion into the tissue of genetically different organisms has been known only in mother-embryo interactions of viviparous organisms. Hence, embryonic invasions have been thought to occur only within the same or closely related species. For endoparasitic Hymenoptera, which are oviposited in their host egg but complete their development in the later stages, entry into the host embryo is essential. To date, the entry of these parasitoids is known to be accomplished by either egg deposition directly into the embryo or by the newly hatched larva boring into the embryo. However, Copidosoma floridanum is a polyembryonic parasitoid whose development is characterized by a prolonged embryonic stage, and which lacks a larval form during its host embryogenesis. We have analyzed the behavior and fate of C. floridanum embryos co-cultured with their host embryo in vitro. Here, we show that the morula-stage embryo of C. floridanum actively invades the host embryo. Histological analyses have demonstrated that C. floridanum embryonic invasion is associated with adherent junction to host cells rather than causing an obvious wound on the host cells. These findings provide a novel case of embryonic invasion into a phylogenetically distant host embryo, ensuring cellular compatibility with host tissues.

Hemolytic activity is mediated by the endogenous lectin in the mosquito hemolymph serum.

Although cytolysis of invading organisms is an innate form of immunity used by invertebrates, so far the underlying mechanism remains less explored. The pupal hemolymph of the mosquito Armigeres subalbatus induces an activity that causes hemolysis of human red blood cells (HRBC). This hemolytic activity was inhibited by sialic acid (N-acetylneuraminic acid) and serine protease inhibitors. We purified the sialic acid-specific lectin(s) from the pupal hemolymph using formaldehyde-fixed HRBC and determined the sequence of the amino-terminal 19 amino acid residues. A polyclonal antibody produced against this N-terminal peptide clearly inhibited the hemolytic activity of the hemolymph in vitro, thus suggesting that the hemolysis of HRBC is caused by the lectin present in the mosquito hemolymph. We suggest that mosquitoes possess a cytolysis system.