Douglas L. Dahlman

A teratocyte gene from a parasitic wasp that is associated with inhibition of insect growth and development inhibits host protein synthesis

After parasitization, some wasps induce hosts prematurely to initiate metamorphic development that is then suspended in a postwandering, prepupal state. Following egression of the parasite larva, the host remains in this developmentally arrested state until death. Teratocytes, cells released at egg hatch from extra‐embryonic serosal membranes of some wasp parasites, inhibit growth and development when injected into host larvae independent of other parasite factors (e.g. venom, polydnavirus). Synthesis of some developmentally regulated, abundantly expressed Heliothis virescens host proteins is inhibited in hosts parasitized by Microplitis croceipes and by teratocyte injection. A cDNA encoding a 13.9 kDa protein (TSP14) that inhibited protein synthesis, growth and development was isolated from a protein fraction secreted by teratocytes. TSP14 appears to be responsible, in part, for the teratocyte‐mediated inhibition of host growth and development. Interestingly, this cDNA encoded a cysteine‐rich amino acid motif similar to that described from Campoletis sonorensis polydnavirus, a mutualistic virus that enables wasp parasitization of lepidopteran larvae. Moreover, TSP14 inhibited protein synthesis in a dose‐dependent manner in rabbit reticulocyte lysate and wheat germ extract translation systems. We hypothesize that some wasp parasites inhibit translation as a general means to regulate and redirect lepidopteran host physiology to support endoparasite development.

Teratocytes and host/parasitoid interactions

Abstract Teratocytes, derived from extraembryonic tissues of parasitic Braconidae, Platygasteridae, and Scelionidae, play several important roles in parasitoid-host interactions. Teratocytes have trophic, immunosuppressive, and secretory functions but their specific activities depend upon the life stage of the host, the time spent by the parasitoid within the host, and the individual species of parasite and host. Teratocytes have immunosuppressive, trophic, and secretory functions. Teratocytes from the braconid, Microplitis croceipes, were obtained at various times from the hemolymph of its host, Heliothis virescens, or from preparations obtained from M. croceipes eggs hatched in vitro. The teratocytes were viewed using scanning electron microscopy and were found to possess a dense coat of microvilli. In vitro cultures of teratocytes secrete proteins and these teratocyte secretory products, when injected into H. virescens larvae, produce many of the same developmental abnormalities observed when larvae are injected with intact teratocytes or are truly parasitized. Finally, it was demonstrated that DNA from the M. croceipes polydnavirus hybridizes with DNA extracted from teratocytes and various other tissues of M. croceipes. Potential implications of the teratocyte/host/polydnavirus interactions are discussed with respect to biological control applications.

Trehalose and glucose levels in hemolymph of diet-reared, tobacco leaf-reared and parasitized tobacco hornworm larvae*

Abstract o 1. Tobacco hornworms, Manduca sexta , were reared on either tobacco leaves or synthetic diet. 2. Glucose and trehalose levels in the hemolymph were significantly lower in leaf-reared larvae. 3. Levels of trehalose were higher in the earlier part of each stadium. 4. Fourth stadium levels of trehalose were generally greater than fifth stadium levels. 5. Trehalose, but not glucose, levels were lower in diet-reared larvae parasitized by Apanteles congregatus than in unparasitized diet-reared larvae.

Non-protein amino acid-insect interactions—I. Growth effects and symptomology of l-canavanine consumption by tobacco hornworm, Manduca sexta (L.)

Abstract 1. 1. Incorporation of l -canavanine, a naturally occurring structural analogue of arginine, into the artificial diet of the tobacco hornworm larvae, Manduca sexta (L.), produced severely toxic effects. 2. 2. The toxic effects included enhanced larval mortality, decreased larval growth rates and malformed pupae and adults. 3. 3. Larval response to canavanine was concentration dependent at least over a range of 3–45 mM. 4. 4. Diets supplemented with arginine, ornithine, glycine and lysine were not toxic to fifth instar hornworms.

Down-regulation of juvenile hormone esterase and arylphorin production in Heliothis virescens larvae parasitized by Microplitis croceipes

Previous studies have shown that juvenile hormone esterase (JHE) activity and arylphorin titer were dramatically reduced in the hemolymph of Heliothis virescens larvae parasitized by Microplitis croceipes. Similar changes were observed in calyx fluid-injected or tetratocyte-injected larvae. In this study, we showed that the mRNA levels of both JHE and arylphorin transcripts were reduced significantly in fat body of parasitized larvae compared to those in nonparasitized larvae. The reduction of mRNA level appears to be fat body-specific, because no reduction of arylphorin mRNA level was observed in testes of the same parasitized larvae. Significantly, the mRNA levels of JHE and arylphorin in calyx fluid-injected or teratocyte-injected larvae were not different from those in control larvae, although the activity of JHE and the protein level of arylphorin were substantially lower in the treated larvae. Thus, our data suggest that both suppressed transcription of JHE- and arylphorin-encoding genes and post-transcriptional/translational modification(s) of JHE and arylphorin mRNAs or proteins may be involved in the reduction of JHE and arylphorin levels in parasitized H. virescens larvae. Calyx fluid and teratocytes may negatively regulate JHE and arylphorin levels via a transcriptional/translational mechanism. The factor(s) responsible for the reduction of JHE and arylphorin transcript levels by parasitization appears to be distinct from those induced by injection of calyx fluid or teratocytes.

Trehalose and glucose levels in the hemolymph of Heliothis virescens parasitized by Microplitis croceipes or Cardiochiles nigriceps

Abstract 1. 1. Heliothis virescens larvae were parasitized by either Microplitis croceipes or Cardiochiles nigriceps. 2. 2. Hemolymph trehalose levels from host larvae parasitized by M. croceipes nearly tripled within 1 day following parasitization and remained elevated during parasitoid development. 3. 3. C. nigriceps did not affect trehalose levels. 4. 4. Neither parasitoid affected the level of glucose in the hemolymph of its host. 5. 5. M. crociepes prevented any significant increase in host hemolymph dry weight beyond day 0 values; whereas, C. nigriceps elevated the dry weight during the latter part of its development.

Microplitis croceipes teratocytes: in vitro culture and biological activity of teratocyte secreted protein

Teratocytes originate from the dissociation of the extraembryonic serosal membrane in some Braconidae and Scelionidae. Methods used to culture teratocytes in vitro are described and the yield of teratocyte secreted proteins (TSP) was measured. Although 90% are viable after 6 days, in vitro teratocytes reached only half the diameter (32&mgr;m) of the same age teratocytes obtained in vivo. Teratocytes cultured in vitro secrete as much as 0.7&mgr;g of protein per day per larval equivalent ( approximately 900 cells). Presence of parasitoid larvae enhanced teratocyte viability while periodic exchange of medium did not. However, medium exchange significantly increased the total amount of protein secreted. Size and viability were improved with the addition of 10% FBS to the Ex-cell 400 culture medium. Non-denaturing PAGE showed at least 15 proteins with molecular sizes estimated to be between 24 to 347kDa in medium containing teratocytes. An in vitro fat body assay was developed to measure the effect of TSP on protein synthesis and juvenile hormone esterase (JHE) activity. Crude TSP inhibited in vitro incorporation of [(35)S]-methionine into protein synthesized by the fat body. The amount of JHE released from in vitro fat body treated with crude TSP was significantly less than controls, most likely caused by the inhibition of general protein synthesis. The active fraction of TSP passed through a 30kDa molecular weight cutoff filter but was retained by a 3kDa filter. SDS-PAGE revealed four proteins with molecular weights between 8 and 20kDa not present in control medium incubated without teratocytes.

Non-protein amino acid-insect interactions-II. Effects of canaline-urea cycle amino acids on growth and development of the tobacco hornworm, Manduca sexta L. (Sphingidae).

Abstract 1. Larvae of the tobacco hornworm, Manduca sexta L. (Sphingidae), were fed diets containing 0·5, 1, 2·5, 5 and 10 mM levels of the canaline-urea cycle amino acids. 2. Consumption of the canaline-urea cycle amino acids caused increased larval mortality, decreased larval growth, prevention of pupation, pupal and adult malformations and reduction in ovarial tissue mass. 3. For a given concentration, the relative toxicity of the canaline-urea cycle amino acids was: l -canavanine > l -canaline > O-ureido- l -homoserine > l -canavaninosuccinate. 4. Diets supplemented with 10 mM levels of the ornithine-urea cycle amino acids had no effect on insect growth and development.

Further studies of the effect of l-canavanine on the tobacco hornworm, Manduca sexta☆

Fifth instar Manduca sexta growth response to injected doses of canavanine was concentration-dependent over a range of 0·5 to 2·0 mg/g body weight. Twenty-four hr after injection of 14C-guanidinooxy-d,l-canavanine, M. sexta larvae incorporated approximately 3·6% of the labelled l-canavanine into protein of non-gut tissue. Adult M. sexta mortality was related to the level of injected canavanine over a range of 2 to 8 mg/g body weight. Injection of as little as 2 mg canavanine/g body weight caused hyperactivity in adult M. sexta. Arginine, able to negate the toxic effects of canavanine during larval growth, was only marginally capable of overcoming canavanine effects on larval-pupal ecdysis.

Allelochemical induced stress: Effects of l-canavanine on the pathogenicity of Bacillus thuringiensis in Manduca sexta

Abstract Increased susceptibility of Manduca sexta to commercial formulations of the microbial insecticide, Bacillus thuringiensis , as evidenced by lower LD 50 and LT 50 values, was observed when M. sexta were reared on an artificial diet supplemented with a sublethal concentration (2.5 m m ) of l -canavanine. At several dosages of B. thuringiensis , which were administered either by diet contamination or by per os forced feeding, a greater than 70% reduction ( P 50 response with canavanine-treated larvae. The LD 50 values also were lowered by canavanine treatment. This constitutes the first report of a plant allelochemical enhancing the effect of B. thuringiensis in vivo. It is suggested that canavanine enhances the effect of B. thuringiensis on gut permeability and active transport.