Alexej B. Borkovec

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.

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.

Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis

Abstract Monoclonal antibodies to a mixture of Aedes atropalpus and A. aegypti soluble yolk proteins were produced by hybridomas between the fusion of P3X63.653 myeloma cells and splenocytes of immunized BALB/c mice. Ascites fluid collected from mice innoculated with cloned hybridoma cells contained high specificity and affinity to the soluble yolk proteins of both Aedes species. Seven different hybridoma lines produced antibodies with specificity to both A. atropalpus and A. aegypti and one cell line produced antibodies monospecific to A. aegypti soluble yolk proteins. Monoclonal antibodies specific to A. atropalpus vitellin and vitellogenin were characterized by a combination of gel electrophoresis, western blotting and immunohistochemical staining. An indirect double antibody sandwich enzyme-linked immunosorbent assay was developed using a mixture of the seven hybridoma antibodies to A. atropalpus vitellin for monitoring vitellogenin levels in individual mosquito haemolymph samples. With this procedure, the peak period of vitellogenin synthesis in A. atropalpus was found to be 18 to 30 h after adult eclosion.

Thyroxine-induced haemolymph protein and ecdysteroid increases in the silkworm, Bombyx mori: Effect on larval growth and silk production

Abstract The effect of feeding mulberry leaves treated with thyroid powder on larval growth and cocoon formation was analysed following treatment of different larval instars. Larvae treated during the second instar only, showed maximal growth along with other enhanced cocoon characters. This suggests that thyroxine could be used effectively for commercial silkworm rearing by treatment at an early stage of silkworm development. The larval period was shortened by 30–40 h with increased cocoon and shell (silk) weight in all treated groups, reaching 150% in some cases with no loss in silk quality. An increase in haemolymph ecdysteroid level was observed during the fourth instar, suggesting that thyroxine in some way affects ecdysteroid metabolism. As compared to controls, where haemolymph ecdysteroid levels reached 1.4 ng/μl during the fourth instar, ecdysteroid levels showed an almost 2-fold increase during the fourth instar when animals were treated with thyroxine from the first through the fifth instar (2.3 ng/μl) or when treated in the fourth instar only (2.8 ng/μl), and a 3.5-fold increase (4.8 ng/μl) for treatment in the second instar only. A peak of haemolymph ecdysteroid was observed on day 10 of the fifth instar for all treated groups, but not until day 11 for the control group. Thyroxine treatment from the first through the fifth instar caused a 2-fold increase in haemolymph protein level during the first (15.07 μg/μl) and second (10.99 μg/μl) instars, as compared to controls (7.73 and 5.48 μg/μl, respectively). Thyroxine treatment of second or fourth instars alone resulted in a 2-fold increase in haemolymph protein levels to 12.83 and 13.49 μg/μl, respectively, during the fourth instar, when compared to that of the control group (6.20 μg/μl). This suggests that thyroxine, in addition to affecting ecdysteroid metabolism, also affects protein metabolism.

Monitoring Aedes aegypti vitellogenin production and uptake with hybridoma antibodies

Abstract Gel electrophoretic analysis of Aedes aegypti vitellogenin showed that there are two components, 200,000 and 66,000 Daltons. By a combination of western blotting and immunohistochemical staining, two monoclonal antibody cell lines were demonstrated to bind specifically to the larger molecular form of vitellogenin. They were used as primary antibodies in an enzyme-linked immunosorbent assay procedure developed for the measurement of vitellogenin in individual female samples. Vitellogenin levels in the haemolymph and its uptake in the ovaries were monitored with this immunochemical method which had distinct advantages over other techniques developed for measuring mosquito vitellogenin. The temporal pattern of vitellogenesis events described in this study was also compared to existing knowledge generated by other methodologies.

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.

ENHANCED PRODUCTION OF MOUSE HYBRIDOMAS TO PICOMOLES OF ANTIGEN USING EL-4 CONDITIONED MEDIA WITH AN IN VITRO IMMUNIZATION PROTOCOL

SummaryWe report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological fluids.

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...

Prothoracicotropic Hormone and Ecdysteroid Ketoreductase from Pre-Hatch Eggs of the Gypsy Moth, Lymantria Dispar

The identification of prothoracicotropic hormone (PTTH) activity in Lymantria (Kelly et al., 1986; Masler et al., 1986) verified the existence of a molt/metamorphosis-regulating brain secretory factor, first postulated by Kopec (1922). Since Kopec’s discovery, this factor has received considerable attention in post-embryonic stages of Bombyxmori (Ishizaki et al., 1987) and Manduca sexta (Flanagan et al., 1988). Recently, PTTH activity was demonstrated in embryonic stages of Bombyx and Manduca (Chen et al., 1987; Fugo et al., 1987; Dorn et al., 1987). We have demonstrated similar activity in embryonic eggs of Lymantria (see also Bell et al., Masler et al. and Thyagaraja et al., this volume).