Robert A. Bell

Circadian rhythm of sperm movement in the male reproductive tract of the gypsy moth, Lymantria dispar

Abstract Several days before adult eclosion sperm starts being released from the testis of the gypsy moth in a daily rhythmic fashion. This rhythm has circadian endogenous nature since it free-runs in constant darkness, undergoes rapid phase-shifts and is temperature compensated. The rhythm can be initiated, entrained by the photoperiod and free-run in isolated pupal abdomens. Two rhythmic events, the release of sperm from the testis and its transfer to seminal vesicles, are independently modulated by temperature and photoperiod.

THE INSECT GUT : A NEW SOURCE OF ECDYSIOTROPIC PEPTIDES

Proctodaea of European corn borer (Ostrinia nubilalis) and gypsy moth (Lymantria dispar) last instars (larvae) contain prothoracicotropic factors that stimulate the prothoracic glands (PGs) of the gypsy moth to produce both ecdysone and 3-dehydroecdysone (precursors to the insect molting hormone) in a dose-dependent manner. In a separate in vivo assay, injections of proctodaeal extracts into gypsy moth larvae that were head-ligated before the release of the molt-stimulating brain hormone, PTTH, resulted in a pupal molt.

Prothoracicotropic hormone levels in brains of the European corn borer, Ostrinia nubilalis: Diapause vs the non-diapause state

Abstract Brains from non-diapause-bound, diapause-bound and diapausing European corn borers contain prothoracicotropic hormone (PTTH) which stimulates the prothoracic glands of both Lymantria dispar and Ostrinia nubilalis to produce ecdysone and 3-dehydroecdysone in a dose-dependent manner. At a dose of 0.75 brain equivalents, PTTH activity is highest in non-diapause-bound and diapausing prepupae. Levels are approx. 50% as high in younger 5th instars. In diapausing prepupae, PTTH activity again falls to approx. 50% after 5–8 weeks of refrigeration. Prothoracic glands from diapausing O. nubilalis prepupae were refractory to stimulation. In vivo experiments indicate that brains from diapausing prepupae have more moult-stimulating activity than those from non-diapause-bound prepupae. However, this may be due to the presence of factors other than PTTH. Based on gel filtration HPLC, the molecular weight range of the small form of O. nubilalis PTTH is 1500–3300 Da, somewhat less than the 5000–7000 Da peptide reported for other lepidopterans.

Manipulation of diapause in the gypsy moth, Lymantria dispar L., by application of KK-42 and precocious chilling of eggs

Abstract The gypsy moth enters diapause in late embryonic development as a fully differentiated pharate larva surrounded by an abundant supply of yolk. Although diapause is known to be regulated by the endocrine system in other insect species that have been intensively studied, the mechanism involved in regulation of diapause in the gypsy moth is unknown. The present study is part of a series designed to develop a fundamental understanding of the physiology of diapause in this species and to develop methods to manipulate it in the laboratory. In nature and in the laboratory, diapause is gradually terminated by prolonged chilling of the eggs for several months. Recently it was discovered that KK-42, a novel insect growth regulator with anti-hormonal properties, was effective in averting diapause in the gypsy moth. Results of the present study show that 14 day old eggs are highly sensitive to KK-42 and treatment at this stage prevents diapause in 85% of the test populations. Studies in which successive age groups, incubated at 25 °C then transferred to 5 °C, showed that eggs must be at least 12–13 days old to survive prolonged chilling and to hatch successfully after re-incubation at higher temperature. Eggs chilled at 14–15 days post-oviposition retained complete sensitivity to KK-42, whereas, 21–22 and 29–30 day old eggs were not responsive unless they were chilled for 4–6 weeks. These findings are important in developing methods for manipulating diapause in stockpiled eggs of the gypsy moth for use in biocontrol programs and also provides knowledge useful for further inquiry into the mechanism of diapause regulation in this species.

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth, Lymantria dispar (L.) following studies on identification, titers and synthesis of ecdysteroids in last-instar females

SummaryHemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Graces medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Graces medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.

Ecdysiotropic activity in the lepidopteran hindgut—An update

Abstract The production of ecdysteroid by the insect prothoracic glands (PTGs) is initiated by an ecdysiotropin, prothoracicotropic hormone, which is produced by the brain and released into the hemolymph from its neurohemal organ. Recently we reported the discovery of another ecdysiotropin which is localized in the lepidopteran hindgut or proctodaeum. It is a small, heat-stable peptide which is resistant to freeze-thaw and to extraction with organic solvents. Based on size-exclusion HPLC, we now estimate its molecular weight to be 500–1500 Da. The hindgut ecdysiotropin stimulates the PTGs of Lymantria dispar to produce both ecdysone and 3-dehydroecdysone in a dose-dependent manner. Ecdysteroid production was maximum in the presence of 0.125 and 0.1 hindgut equivalents for Ostrinia nubilalis and L. dispar , respectively. Activity was detected throughout the pylorus and anterior intestine of the O. nubilalis hindgut. When proctodaea from 5th instar O. nubilalis were analyzed daily for ecdysiotropic activity, those from wandering larvae which had undergone gut purge were found to have the greatest concentration of ecdysiotropin. Cyclic AMP appears to act as a second messenger for the proctodaeal ecdysiotropin as evidenced by the increased levels of cAMP present in PTGs incubated with hindgut extract. At doses which caused maximum stimulation, effects of brain and proctodaeal extracts were additive indicating that the two ecdysiotropins utilize separate receptors. Size exclusion HPLC of hemolymph obtained from prepupae that have experienced gut purge revealed the presence of an ecdysiotropin(s) whose molecular weight range is similar to that of the proctodaeal ecdysiotropin but not to that of the small form of brain PTTH. While the physiological function of the proctodaeal ecdysiotropin(s) is unknown, the discovery of such a peptide(s) is noteworthy in light of the reported production of ecdysteroids by isolated insect abdomens.

Effects of selected physical and chemical treatments of Colorado potato beetle eggs on host acceptance and development of the parasitic wasp, Edovum puttleri.

Effects of various physical and chemical treatments of Colorado potato beetle [Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)] eggs on parasitization and development of the egg parasitoid Edovum puttleri (Hymenoptera: Eulophidae) were investigated. UV irradiation did not affect host acceptance but reduced host suitability for UV exposure times ≥90 min. Susceptibility of host eggs to UV irradiation varied with host age; eggs were most vulnerable to damage from irradiation at 12, 18, and 24 h post‐oviposition. The rate of parasitization also was influenced by host age. Percent parasitization was greatest in freshly laid eggs and 24–30 h old eggs. Seventy‐seven percent of host eggs frozen at −20 °C (5 min) were parasitized by E. puttleri, but extended exposure of eggs to −20 °C reduced both acceptance and suitability. Host eggs that had been washed with hexane (removal of kairomone and sticky layer) also were parasitized. After 5 min of washing, application of kairomone significantly increased the rate of parasitism (from 74.7% to 88.2%), but with longer periods of washing, kairomone application had no significant effect on percent parasitism. Thus, the sticky material(s) coating the egg did not appear to be essential for parasitization to occur. Our results provide effective methods and times for treating Colorado potato beetle eggs to maximize parasitization and development of E. puttleri.

Prothoracicotropic hormone — like activity in the embryonated eggs of gypsy moth, Lymantria dispar (L.)

SummaryProthoracicotropic hormone (PTTH)-like activity was obtained from embryonated eggs of the gypsy moth, Lymantria dispar. Activity was detected using an in vitro prothoracic gland stimulation bioassay. Doseresponse kinetics of crude extract revealed a 4-fold activation range with a maximum activation of 35-fold. Nearly 70% of the activity was sensitive to denaturation by heat or organic solvent extraction. Heat and organic solvent-stable activity is due to a protein. Dose-response kinetics suggest the presence of a small molecular weight PTTH with pre-hatch eggs providing a rich source of the hormone.

Vitellogenesis in the gypsy moth, Lymantria dispar (L.): Characterization of hemolymph vitellogenin, ovarian weight, follicle growth and vitellin content

Summary Gypsy moth vitellin (Vt, greater than 500, 000 daltons) was partially purified from pupal ovarian follicles by high-salt extraction and analyzed by non-SDS- and SDS-PAGE. A large-molecular-weight, female-specific, pupal hemolymph protein (vitellogenin, Vg) had electrophoretic and immuno-staining properties similar to Vt. A slightly larger female-specific hemolymph protein larval-Vt) was found in last-instar female gypsy moth larvae. Vt, pupal-Vg and larval-Vg were recognized on Western blots by a rabbit polyclonal antiserum made against L. dispar Vt. The apoprotein subunits of Vt were 180,000 (apo-Vt180), 165,000 (apo-Vt165), and 38,000 daltons (apo-Vt38). Apo-Vg of similar sizes as the apo-Vt (apo-Vg180, apo-Vg165, and apo-Vg38) were recognized in larval and pupal hemolymph by the anti-Vt antisera. Apo-Vg began to accumulate in the hemolymph of last-instar larvae after day 2 and were not detected by Coomassie-or immuno-staining prior to this time. This indicates that synthesis of Vg must be initi...

Kopec revisited—Neuroendocrine regulation of metamorphosis in the gypsy moth (Lymantria dispar): Development of a larval in vivo assay for prothoracicotropic hormone

Abstract The role of the brain as a neuroendocrine regulator of metamorphosis in the gypsy moth, Lymantria dispar L. was originally demonstrated by Kopec more than 70 years ago. We have reexamined this role by determining haemolymph ecdysteroid titres, activity and responsiveness of the prothoracic glands, and growth of female last (i.e. fifth)-instar larvae in relation to secretion of the brain prothoracicotropic hormone (PTTH), the factor known to trigger moulting and metamorphosis in insects. The head critical period for secretion of PTTH in Lymantria reared at 25°C in a 16 h light-8 h dark cycle was shown by neck ligation to occur on day 7–8 of the fifth instar. The critical period occurred shortly after a small peak of haemolymph ecdysteroid that reached 600 pg 20-hydroxyecdysone equivalents/μl, as determined by radioimmunoassay. Feeding was completed by day 7, thus neck ligation did not affect nutrient intake. Animals starved from day 5 onward completed pupation at the same time as non-starved controls and showed normal increases in haemolymph ecdysteroid titres. Animals neck-ligated on day 7 and injected 5 days later with extracts of brain, retrocerebral complex, or brain tissues containing lateral or median neurosecretory cells were stimulated to pupate within 5 days, and brain-extract-injected animals showed an increase in haemolymph ecdysteroid titre of over 4000 pg/μl. Animals neck-ligated earlier had lower haemolymph ecdysteroid titres than day 7, neck-ligated larvae and their prothoracic glands showed lower activity in vitro . Using the in vivo assay, PTTH activity was detected in pre-hatch eggs and in brains taken from day-5 last-instar larvae, day-1 pupae and day-1 adults. The assay appears specific to tissues from the brain and retrocerebral complex, since no activity was found with extracts of the subesophageal ganglion, fat body or muscle.