Walter G. Goodman

Ecdysteroid titer during larval-pupal-adult development of the tobacco hornworm, Manduca sexta

Abstract The ecdysteroid titer during the fourth (penultimate) and fifth larval instars of Manduca sexta was characterized by large increases lasting approximately 24 and 60 hr, respectively, and these peaks occurred just before ecdysis. During the last larval instar, an earlier, smaller increase in the titer was also observed lasting approsimately 20 hr and is the increase presumed responsible for commitment to pupal development. After pupation, the ecdysteroid titer increases again, reaching a maximum between Days 7 and 9. At Day 10 postpupation, the titer declined dramatically and stabilized at ∼day 14. During pupal-adult development, there was no sexual dimorphism in the ecdysteroid titer. However, a difference in the ecdysteroid titer between males and females was observed after adult eclosion. Qualitative analysis of the ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxyecdysone at varying ratios depending on developmental stage. For the large peaks preceding larval and pupal ecdysis, 20-hydroxyecdysone was the predominant hormone, while ecdysone was the major component during pupal-adult development. The fifth instar commitment peak was comprised of approximately equal quantities of the two ecdysteroids.

Juvenile hormone and hemolymph juvenile hormone binding protein titers and their interaction in the hemolymph of fourth stadium Manduca sexta

A driving force in the initiation of an endocrine response is the modulation of hormone titers surrounding the responsive tissue. For nonpolar hormones, titer fluctuations are, in part, regulated by specific circulatory transport proteins that bind, protect and convey the hormones to the responsive tissues. In the present study, juvenile hormone (JH), hemolymph juvenile hormone binding protein (hJHBP), and total hemolymph protein in the fourth larval stadium of Manduca sexta were quantified from similarly staged insects to assess whether JH is bound or unbound when it interacts with the target. JH levels, as determined by radioimmunoassay, were highest, 12.87 nM, 4 h after ecdysis to the fourth stadium, and declined 6-fold to the lowest titer, 2.28 nM, at 44 h after ecydsis, i.e. 8 h after head critical period. Conversely, hJHBP titers peaked at 11.95 μM 44 h after ecdysis and dropped 5-fold by the time of ecdysis to the fifth stadium. Total hemolymph protein also reached its highest level, 19.83 mg/ml, at 44 h; however, hJHBP titers did not follow fluctuations in the total hemolymph protein. Calculations of bound and unbound JH indicated that >99% of the hormone was bound by hJHBP. In contrast, <1% of the binding protein was hormone-loaded at any time during the stadium. Due to the rapid metabolism of JH in the hemol the hemolymph is improbable at certain times during development. Thus, the rate limiting step in JH uptake at these times may be the interaction of hJHBP with the target cell.

Development and application of a radioimmunoassay for the juvenile hormones.

Abstract A radioimmunochemical method for the quantification of juvenile hormones from hemolymph and whole body extracts is described. Rabbit polyclonal antiserum developed against a JH III-bovine thyroglobulin conjugate displayed minimal cross-reactivity with juvenile hormone metabolites including juvenile hormone acids, juvenile hormone diols and analogs but substantial cross-reactivity between juvenile hormone homologs. Minimum sensitivity of the assay toward racemic juvenile hormone III was 65 pg. The degree and relative order of cross-reactivities for juvenile hormones I, II and III varied according to the identity of the radioligand used. A method for isolating juvenile hormones from whole body and hemolymph for radioimmunoassay was developed utilizing organic solvent extraction followed by thin-layer chromatography. Noninterfering dyes were used to bracket the position of the hormone on thin-layer chromatography plates. Hemolymph extracts known to contain no JH did not interfere with the assay when this procedure was employed. Radioimmunoassay analysis of hemolymph samples containing known amounts of JH and corrected for recovery yielded the expected results. Quantification of total juvenile hormone in whole body and hemolymph extracts of Manduca sexta was in good agreement with total mass of JH determined by a GC/MS method.

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

Abstract The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V5) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V5 larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V5, V6 and V7 larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10−5M to 10−10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.

Parasitism by Microplitis demolitor induces alterations in the juvenile hormone titers and juvenile hormone esterase activity of its host, Pseudoplusia includens

Microplitis demolitor is a polydnavirus-carrying parasitoid that attacks the larval stage of Pseudoplusia includens and other noctuids. Parasitism or injection of wasp components like polydnavirus and teratocytes has a juvenilizing effect on P. includens development. Here we measured hemolymph juvenile hormone (JH) titers and juvenile hormone esterase activity in P. includens larvae after parasitism or injection of wasp components. Results were compared to nonparasitized larvae. During the fifth stadium, JH titers of nonparasitized larvae fluctuated between 0.08 and 0.50 ng/ml, whereas titers in parasitized larvae never fell below 2 ng/ml. P. includens larvae injected with calyx fluid plus venom or calyx fluid plus venom and teratocytes exhibited JH titers intermediate between parasitized and nonparasitized larvae. Nonparasitized larvae exhibited two peaks of JH esterase activity. The first occurred between 12 and 48 h in association with cessation of larval feeding, while the second occurred immediately before pupation. In contrast, levels of JH metabolism remained at low levels in parasitized larvae and larvae injected with calyx fluid plus venom or calyx fluid, venom and teratocytes. Only trace amounts of JH were detected in in vitro assays of teratocytes or M. demolitor larvae suggesting neither released JH into the hemolymph of parasitized larvae. Although calyx fluid plus venom and teratocytes did not elevate JH titers to the levels measured in parasitized larvae, their effects on formation of larval-pupal intermediates by P. includens were similar to those generated by exogenous application of methoprene or JH.

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Abstract Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis . Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.

The juvenile hormone titer of Trichoplusia ni and its potential role in embryogenesis of the polyembryonic wasp CopidosomaFloridanum

Abstract The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum , was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of ( RS )-methoprene or JH II had a juvenilizing effect on parasitized T. ni , and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.

Hemolymph titers of the biliprotein, insecticyanin, during development of Manduca sexta

Abstract The levels of a biliverdin-associated protein in the hemolymph of larvae, pupae and adults, and in egg homogenates, of Manduca sexta were determined by radial immunodiffusion. The concentration of the protein fluctuates dramatically during development and displays an ontogenetic pattern different from that of the total hemolymph protein concentration. During the larval stages studied, the very early fourth instar displayed the highest concentration of insecticyanin (0.6 mg/ml), which dropped precipitously afterwards (0.3 mg/ml). During fifth instar development, the levels decreased after ecdysis (0.15 mg/ml), began to rise at wandering, and nearly doubled (0.3 mg/ml) by the time of pupation. Pupal titers of the protein remained fairly constant until the day before adult eclosion, when titers increased. The highest levels of any stage were recorded for adults 12 hr post eclosion (0.80 mg/ml).

Relative hemolymph juvenile hormone binding capacity during larval, pupal and adult development in Manduca sexta

Abstract Hemolymph from larval, pupal and adult Manduca sexta was analyzed for juvenile hormone binding protein activity by a hydroxylapatite adsorption assay. During each of the larval instars studied, the relative binding capacity followed a cyclical fluctuation with the early part of each instar displaying relatively lower capacities than the midpart of the instar. Binding levels declined in the period preceding ecdysis to the next instar or stage. Analysis of total hemolymph protein indicated that the binding titers generally followed the increase in hemolymph protein concentration. During the pupal and adult stages, sexual dimorphism was observed, with females displaying significantly more binding per unit hemolymph than males. The highest levels of binding at any time during development were observed in the adult female hemolymph 36 hr post eclosion. Adult male hemolymph showed no corresponding increase during the same period.

Ligand regulation of juvenile hormone binding protein mRNA in mutant Manduca sexta.

Insect hemolymph juvenile hormone binding protein (hJHBP) regulates peripheral titers of its ligands, the juvenile hormones. In larvae of the black (bl) strain of the tobacco hornworm, Manduca sexta, treatment with small doses of juvenile hormone I (JH I) can also regulate titers of hJHBP. To further investigate this regulation, responsiveness of hJHBP mRNA expression to JH I was characterized in vivo. RNA analyzes revealed that transcript levels in fat body, the site of hJHBP synthesis, increased fivefold within several hours of treatment with physiological doses of hormone and remained elevated for approximately 16 h. Sensitivity to JH treatment was found to vary temporally. To ensure transcript identity, a wild-type cDNA clone and a bl RT-PCR fragment were sequenced and found to be 99% homologous. Together, these results suggest that JH participates in regulating expression of its transport protein in bl larvae by modifying the in vivo abundance of hJHBPs mRNA transcript.