Belgaum S. Thyagaraja

Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors

Recombinant baculoviruses were constructed that express the cDNA encoding the prothoracicotropic hormone (PTTH) of Bombyx mori. This hormone stimulates the production of ecdysteroids by the insects prothoracic glands. Two groups of viruses were constructed, expressing either the entire cDNA encoding prepro-PTTH, or a synthetic chimeric gene encoding a signal peptide fused to the mature PTTH subunit. In both cases, the genes were expressed in wild-type Autographa californica nuclear polyhedrosis virus (AcMNPV) and in vEGTDEL, an ACMNPV mutant that lacks a functional egt gene. The egt gene is required for viral-mediated inactivation of host ecdysteroids. High levels of functional PTTH were produced only by viruses expressing the mature subunit cDNA. This recombinant PTTH resembled the native hormone by all criteria examined. The overproduction of B. mori PTTH induced higher than normal levels of haemolymph ecdysteroids but had no observable effects on the development of infected Spodoptera frugiperda larvae. However, expression of PTTH by AcMNPV was found to inhibit the pathogenicity of the virus. This effect was particularly marked in the case of viruses lacking a functional egt gene.

Hormonal control of vitellogenesis in the gypsy moth, Lymantria dispar (L.): suppression of haemolymph vitellogenin by the juvenile hormone analogue, methoprene.

Abstract In most insects, female-specific egg-protein precursors (vitellogenins) are synthesized during pupal or adult stages in response to 20-hydroxyecdysone and/or juvenile hormone. In the gypsy moth, two female-specific haemolymph polypeptides (apo-vitellogenins) having electrophoretic and immunologic identity with Lymantria dispar egg-vitellin apo-proteins begin to accumulate in the haemolymph after day 3 of the fifth larval instar. Head ligation, starvation or treatment with doses of a juvenile hormone analogue, methoprene, >10 μg prior to day 3 block the accumulation of apo-vitellogenin in the haemolymph. Unlike head-ligated or starved larvae which do not gain weight and do no accumulate normal levels of non-vitellogenin haemolymph proteins following treatment, methoprene-treated larvae grow normally and accumulate non-vitellogenin haemolymph proteins. 20-Hydroxyecdysone (2.5 μg) has no effect on the haemolymph accumulation of apo-vitellogenins in normal larvae, and does not restore haemolymph apo-vitellogenins in methoprene-treated, head-ligated or starved larvae. The above results suggest that a low (or declining) juvenile hormone titre during a critical stage early in the last larval instar (prior to day 3) is necessary for the accumulation of vitellogenin in the haemolymph to proceed normally in the gypsy moth. Other factors such as nutrient intake may also be involved. This role of juvenile hormone as an inhibitor of vitellogenin accumulation/synthesis is a novel one for insect species.

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.

Regulation of pheromone production in the gypsy moth, Lymantria dispar, and development of an in vivo bioassay

Abstract Production of sex pheromone in females of the gypsy moth, Lymantria dispar , is regulated primarily by a pheromonotropic factor originating in the head. Decapitation of a female caused depletion of pheromone in the pheromone gland; however, in contrast with several other species of moths, biosynthesis could not be reinitiated by injection of brain-suboesophageal ganglion homogenates or synthetic pheromone biosynthesis activating neuropeptide (PBAN). Severing the ventral nerve cord (VNC) in the abdominal region of females cuased termination of pheromone production. While PBAN was inactive in decapitated females, if the VNC was also transected, PBAN injection resulted in significant pheromone production. Moreover, delivery of PBAN directly to the posterior cut-end of VNC, injection of PBAN into the terminal abdominal ganglion (TAG), or electrical stimulation of TAG, resulted in significant pheromone production. Together, these results suggest that in this species, the pheromonotropic factor travelling via the VNC is an important regulator of pheromone production. However, the pheromonotropic response was very high only if VNC posterior to the thoracic ganglia was severed before injection of PBAN into the TAG or electrical stimulation of TAG. The latter result indicates that an inhibitory factor may be present in the thoracic ganglia. Administration of pheromonotropic factors to the posterior cut-end of VNC can be used as a bioassay for the gypsy moth and possibly for other species in which these factors are transported via the VNC.

Prothoracicotropic Hormone Stimulation of Ecdysone Synthesis by the Prothoracic Glands of the Gypsy Moth, Lymantria Dispar

Kopec (1922), using Lymantria, first demonstrated that a factor(s) from the head was necessary for insect metamorphosis. This factor was later termed prothoracicotropic hormone (PTTH) (Kobayashi et al., 1965). PTTH stimulates ecdysone synthesis by the paired prothoracic glands (PGs) in lepidopterans, and ecdysone is subsequently converted by other tissues to the active hormone, 20-hydroxyecdysone (see Gilbert et al., 1980). In vitro bioassays for PTTH have been established in both Bombyx mori (Nagasawa et al., 1984; Okuda et al., 1985) and Manduca sexta (Bollenbacher et al., 1979) and have been utilized to quantitate PTTH activity in various tissues and fractions (see Ishizaki and Suzuki, 1984; Bollenbacher and Granger, 1985).

Discovery and Partial Characterization of Prothoracicotropic Hormones of the Gypsy Moth, Lymantria Dispar

The brain neuropeptide prothoracicotropic hormone (PTTH) is an ecdysiotropin that drives post-embryonic growth and development through the stimulation of ecdysteroid production by the prothoracic glands (PG) (Gilbert et al., 1980). Research to isolate PTTH has utilized primarily two species, Bombyx mori (Ishizaki and Suzuki, 1984) and Manduca sexta (Bollenbacher and Granger, 1985). Characterization and isolation studies have used both in vivo (Bombyx, Ishizaki et al, 1983, Nagasawa et al, 1984a; Manduca, Kingan, 1981) and in vitro (Bombyx, Nagasawa et al, 1984a; Okuda et al, 1985; Manduca, Bollenbacher et al, 1979) bioassays. Recent work on Bombyx and Manduca PTTH demonstrates that biological activity is associated with two molecular-weight ranges of ca. 20–28 kD and ca. 4–7 kD (Ishizaki et al., 1983, Bollenbacher et al., 1984). A family of 4 kD PTTH’s has been isolated from Bombyx (Nagasawa et al., 1984b).

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.

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.

Complete base sequence for the mitochondrial large subunit ribosomal RNA of the gypsy moth Lymantha dispar(L.)

A 1355 bp sequence (accession number L32141) isolated from a gypsy moth (Lymantria dispar) cDNA library showed 68–74% sequence identity to mitochondrial large subunit ribosomal RNA (mt IrRNA) sequences of Locusta migratoria, Apis mellifera, Aedes albopictus, Anopheles gambiae and two Dros‐ophila species. A comparison of the primary sequences of the mt IrRNAs from the above insects in four orders and from Esherichia coli demonstrated regions of conservation which presumably correspond to regions of functional and/or structural homology. A secondary structure for the gypsy moth mt IrRNA sequence was derived based on the proposed secondary structures of Drosophila yakubaand Aedes albopictus mt IrRNAs (Gutell & Fox, Nucleic Acid Res 16 (Suppl.), r175‐r269, 1988). This sequence was found to hybridize to about 10–15% of the clones in several (eleven) gypsy moth cDNA libraries.