Dale B. Gelman

Critical periods for the brain and prothoracic glands of 5th instars of the European corn borer, Ostrinia nubilalis hubner☆

1. 1. Classical ligation techniques were used to determine the critical periods for the head and the prothoracic glands in the larval-pupal molt of the European corn borer. 2. 2. Most larvae reared under LD 16:8 and 30°C released sufficient PTTH between lights on + 13 hr and lights on — 3 hr to stimulate the prothoracic glands to produce ecdysone. 3. 3. Ecdysone was first released just before lights on, with almost all larvae having produced sufficient ecdysone to elicit a molt by lights on + 6 hr. 4. 4. Critical periods for the brain and prothoracic glands of Ostrinia are compared with those of other lepidopterans.

Effect of azadirachtin on development, juvenile hormone and ecdysteroid titres in chilled Galleria mellonella larvae

Azadirachtin inhibited in a dose-dependent manner cold-induced supernumerary moults of last-instar Galleria mellonella larvae. Inhibition of supernumerary moults was greatest immediately after injection of azadirachtin into cold-treated, 1-day-old, last instars. Azadirachtin also induced disturbances in larval and pupal ecdysis and in metamorphic processes resulting in the formation of various intermediates and finally death. Azadirachtin decreased cold-induced elevation of juvenile hormone titres in the larval body but it had no effect on the allatotropic activity of the brain. However, it might have an effect on the prothoracicotropic function of the brain since, in chilled and azadirachtin-treated larvae, the ecdysteroid peak was higher and was delayed by 24h as compared with the first ecdysteroid peak in controls.

Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro.

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.

Haemolymph ecdysteroid titers of diapause- and nondiapause-bound fifth instars and pupae of the European corn borer, Ostrinia nubilalis (Hübner)☆

Abstract 1. 1. Haemolymph ecdysteroid titers of nondiapause-bound European corn borers (LD 16: 8, 30°C) were low at the beginning of the fifth (last) instar, increased gradually between days 2 and 4 and peaked sharply (4400 pg/μl) just before pharate pupal formation. 2. 2. Time of day influenced prepupal ecdysteroid titers in that levels rose sharply between 14:00 and 24:00 hr AZT (arbitrary Zeitgeber time) in nondiapause-bound animals. 3. 3. Haemolymph ecdysteroid titers decreased in pharate and new pupae, peaked in 2-day pupae (26,000 pg/μl) just before pharate adult formation and then fell more gradually in 3–5-day pupae. 4. 4. In diapause-bound and diapausing 5th instars haemolymph ecdysteroid titers were relatively low, 11–25 pg/μl 5. 5. HPLC followed by RIA of 5th stage larval and pupal haemolymph revealed the presence of a polar peak, 2 small peaks following the polar peak, material that comigrated with 20-hydroxyecdysone and ecdysone, and in 2- and 3-day pupae, material that comigrated with 26-hydroxyecdysone. 6. 6. Before formation of the pharate pupa and pharate adult, ecdysone levels were relatively low as compared to 20-hydroxyecdysone levels.

Ecdysteroid synthesis by testes of 5th instars and pupae of the European corn borer, Ostrinia nubilalis (Hubner)

Summary Testes from young fifth instar Ostrinia nubilalis produced very small amounts of ecdysteroids while those from larvae that had purged their gut produced considerably more immunodetectable ecdysteroid in vitro. Larval testes that had fused produced 2.2 times more ecdysteroid than those that remained separate. It was the sheath of the testes rather than the contents that was physiologically active. Synthesis was questionable in testes from day-1 pupae and was not observed in testes from pharate pupae, from day-2 pupae or from pharate adults. Thus, synthesis only occurred at specific times in the life cycle. Ecdysteroid profiles for testes from wandering larvae whose testes had fused showed a net increase in all normally observed ecdysteroids, with the greatest increase being in 20-hydroxyecdysone. For testes from day-1 pupae, the nature of the ecdysteroid profile changed after 24 h of incubation, with some ecdysteroids showing increases and other decreases. There appear to be considerable difference...

Identification and characterization of an ecdysiotropic peptide from brain extracts of the gypsy moth, Lymantria dispar

A peptide (Lymantria TE) was isolated from brains of the gypsy moth, Lymantria dispar, which stimulates synthesis of ecdysteroid in the testes of larval and pupal insects. This ecdysiotropic peptide was purified and its structure determined to be NH2-IIe-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH using protein sequence analysis and electrospray mass spectrometry. The peptide was biphasic in activity, with maximal activity in the pupal testes at 10−13 M and 10−9 M, with a minimum at 10−10 M, and with maxima at 10−15 M and 10−10 M and minimum at 10−13 M for larval testes. Arch. Insect Biochem. Physiol. 34:175–189, 1997.

Ecdysteroid conjugates in pupal and pharate adult haemolymph of the European corn borer, Ostrinia nubilalis (Hubner)

Abstract Hydrolysis of a polar ecdysteroid HPLC fraction by treatment with glucuronidase revealed the presence of at least six ecdysteroid conjugates in haemolymph from pupal and pharate adult European corn borers. Conjugates of ecdysone, an ecdysteroid more polar than ecdysone, 26-hydroxyecdysone, 20-hydroxyecdysone and two ecdysteroids more polar than 20-hydroxyecdysone were detected. Titres of conjugates were maximal on day 2, 3 or 4 post-pupation and fell to lower levels on day 5. This pattern is similar to that for the corresponding free haemolymph ecdysteroids.

Improved techniques for the rapid radioimmunoassay of ecdysteroids and other metabolites

Summary An RIA procedure for assaying a large number of samples in a relatively short time has been developed. It utilizes multi-channel repeater pipettors for the addition of reagents, specially designed racks which accommodate 48 6 x 50-mm tubes and which fit directly into the centrifuge, multitube vortexers, and a specially designed aspiration device. The racks allow the tubes to remain in the same support throughout most of the assay. The aspirator permits the rapid aspiration of eight tubes at a time, 192 in 2 min. One hundred ninety-two samples can be accurately assayed in as little as 2.5 h.

Effects of ecdysone and 20-hydroxyecdysone on apyrene spermiogenesis in the European corn borer, Ostrinia nubilalis

European corn borer testes were cultured in vitro to determine the effects of physiological doses of ecdysteroids on apyrene spermiogenesis. Ecdysone and 20-hydroxyecdysone stimulated spermiogenesis in larval testes excised 4–5 days after ecdysis to the last instar. The stimulatory effect on the testes was direct. Both the degree of larval maturation and the concentration of ecdysteroid in the incubation medium affected the amount of stimulation. Promotional capability of 20-hydroxyecdysone was greatest in wandering larvae that had not yet completed gut purge. Spermatocysts from larval testes taken 3 days after ecdysis were refractory to stimulation. Determination of 20-hydroxyecdysone levels in larval, pharate pupal and day-1 pupal haemolymph confirmed that the observed stimulation occurred at physiological doses.

Action cascade of an insect gonadotropin, testis ecdysiotropin, in male Lepidoptera

Summary Testis sheaths from late last instar larvae and mid-developing pupae of Heliothis virescens and Lymantria dispar synthesize ecdysteroid in vitro. Gonadal ecdysteroid can stimulate the production of growth factors from the sheaths which, in turn, promote the growth and development of the genital tract. Ongoing basal synthesis is controlled by positive feedback to exogenous ecdysteroid; titers of this hormone approaching those of molting last instar larvae and developing pupae effect maximum synthesis. These findings suggest that circulating titers of ecdysteroid hormone promote gonadal ecdysteroidogenesis, and thus coordinate the actions of the gonads with metamorphic events in the whole animal. Synthesis of ecdysteroid by testes is initiated, however, by a brain neuropeptide, testis ecdysiotropin (TE). TE is a 21 amino acid peptide of molecular weight 2472 Da. TE boosts basal steroid synthesis by pupal testis sheaths as well. It acts primarily via Gi protein and second messengers diacyl glycerol a...