Yasuo Chinzei

Vitellogenin synthesis, processing and hormonal regulation in the tick, Ornithodoros parkeri (Acari:Argasidae)

Abstract This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri . Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [ 35 S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.

Identification and cDNA cloning of novel juvenile hormone responsive genes from fat body of the bean bug, Riptortus clavatus by mRNA differential display

Juvenile hormone (JH) induces termination of diapause and initiation of reproductive maturation in the adult female bean bug, Riportus clavatus. Applying PCR-based differential display, we have identified four novel JH-responsive cDNAS, that is, three repressible (JR-1, 2 and 3) and one inducible (JI-1). These DNA fragments were partially sequenced and compared with sequences in the data base. JR-3 was shown to have similarity to the transferrins of other insects, which have been reported as JH-suppressed genes. JI-1 has similarity to vitellogenin of Aedes aegypti. On the other hand, JR-1-1 and 2 have no significant similarity to other known sequences. For JR-1, the full cDNA sequence was determined: it contained 913 bp, encoding 194 amino acid residues with a calculated M.W. of 21,531 Da in the mature protein. A total of six JH-responsive genes or cDNAs (four suppressible and two inducible or stimulated), including the already isolated JH-responsive cyanoprotein genes (CP-alpha and beta), have been isolated and are available for further comparative analysis of gene structure and regulatory mechanisms in the same tissue under the same hormonal conditions.

Vitellogenins and vitellins in the bean bug, Riptortus clavatus (hemiptera: alydidae): Purification, immunological identification, and induction by juvenile hormone

Vitellogenins (Vgs) and vitellins (Vns) were purified from reproductive female adult hemolymph (HL) and newly laid eggs of the bean bug, Riptortus clavatus. They were separated into two (Vg-1 and 2) and three (Vn-1′, Vn-1, and Vn-2) sub-components, respectively, by ion exchange chromatography. Fused rocket immunoelectrophoresis using anti-(Vn-1) serum showed that they are distinguished clearly into two immunologically distinct groups, i.e., Vg-1/Vn-1/Vn-1′ and Vg-2/Vn-2. SDS-PAGE analysis showed Vn-1 (Vn-1′) and Vn-2 have the same and/or smaller subunit component polypeptides as Vg-1 and Vg-2, respectively. Vn-1′ has some lower molecular weight peptides than Vn-1. Quantitative rocket immunoelectrophoretic analysis showed that Vg-1 and Vn-1 (plus V-1′) were detected first on day 3 in the HL and day 4 in the ovary, respectively, after adult emergence, and increased by day 10 in non-diapause female adults. Vg-1 synthesis is induced in diapause female adults in 1 day after the treatment with juvenile hormone (JH) and JH analog (JHA) or in a few days after transfer from short day to long day conditions.

Cyanoprotein: Quantitative changes and synthesis in diapause and juvenile hormone analog treated bean bug, Riptortus clavatus

Quantitative changes of cyanoproteins (CPs) in diapause and juvenile hormone (JH) analog treated bean bug, Riptortus clavatus, were analyzed by rocket immunoelectrophoresis (RIE). In diapause-oriented nymphal females and males, CP-A (CP-1, 2 and 3) and CP-B (CP-4) increased and reached a maximum level just before nymphal-adult ecdysis, which was the same in non-diapause female and male nymphs. Both CP-A and B disappeared immediately after adult emergence. After this initial decline CP-4 appeared again in the hemolymph, followed after a few days by CP-1, 2 and 3. CP-A and B then increased slowly but constantly in both diapause female and male adults. Both diapause females and males at day 30 after adult emergence had large amounts of CP-A (CP-1, 2 and 3) and CP-B (CP-4). Treatment of diapause females (day 30) with methoprene induced only CP-1 synthesis and increased CP-A content about twice in both the whole body and the hemolymph, but did not effect on CP-B content. Methoprene treated females developed ovaries which accumulated yolk containing CPegg and vitellin (Vn). In diapause males treated with methoprene CP-A and B were not induced and decreased gradully in concentration, eventually disappearing completely, similar to post-diapause males (30 days after transferred to long day condition) in which CPs were not detected. These results show that methoprene treatment of diapause females and males induced the same dynamical situations of CP-A and B seen in non-diapause adults, i.e. only CP-A was induced in females and CPs disappeared in males. This suggests that CP synthesis is regulated by juvenile hormone.

Juvenile hormone I is the principal juvenile hormone in a hemipteran insect,Riptortus clavatus

Juvenile hormone I (JH I) was identified by combined gas chromatography/mass spectrometry as the predominant JH in the hemolymph of female adults of the bean bug,Riptortus clavatus (Thunberg) (Hemiptera: Alydidae). Among JH I, II, and III, JH I was the most effective hormone for inducing the synthesis of yolk proteins in diapause adults.

Induction of anti-malarial transmission blocking immunity with a recombinant ookinete surface antigen of Plasmodium berghei produced in silkworm larvae using the baculovirus expression vector system

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA, B. mori larvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 could be purified from the hemolymph of each infected larva using affinity chromatography. Immunization of Balb/c mice with BmrPbs21 induced high anti-BmrPbs21 and anti--ookinete antibodies but low anti-EcrPbs21 antibody. In contrast, EcrPbs21 induced high anti--EcrPbs21 antibody but low anti-BmrPbs21 and anti-ookinete antibodies. This suggests that most B-cell epitopes on nPbs21 are conformational and that many of the linear epitopes in EcrPbs21 are not normally exposed in nPbs21. Oocyst formation in Anopheles stephensi mosquitoes, which fed on mice immunized with purified BmrPbs21 and infected with P. berghei, was blocked by 85.5-97.1%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.

Cypermethrin induction of vitellogenesis and ovarian development in unfed adult female Ornithodoros moubata (Acari: Argasidae)

Summary Vitellogenesis in ticks is known to be induced by engorgement and mating. In this paper, the synthetic pyrethroid cypermethrin (CyM) is shown to induce production of yolk protein precursor, vitellogenin (Vg), and ovarian development in unengorged mated adult female Ornithodoros moubata. The levels of Vg found in the hemolymph and ovarian development induced by CyM were dose-dependent. i.e., CyM doses of more than 0.2 and 1.0 μg/tick were needed for significant increase of Vg titer in the hemolymph and yolk deposition in oocytes, respectively. Immunological and electrophoretical analyses of Vg and Vitellin (Vn) induced by CyM were identical with those induced by engorgement. Vg titer induced by CyM in unengorged females followed approximately the same time course as that in the normal engorged females. However, Vg titer induced by CyM continued to increase after day 8 and reached a maximum (95 μg/μ1) on day 10 after treatment, while Vg titer induced by engorgement decreased again after reaching a m...

Vitellin is the nutrient reserve during starvation in the nymphal stage of a tick

In the tickOrnithodorus moubata, the major protein of egg yolk, vitellin, was conserved in the guts of larvae and nymphs in aggregated forms for over four months after hatching. Vitellin was the nutrient which supported tick survival until the nymph could obtain a blood meal. This adds to the known role of yolk protein as the nutrient reserved for embryos a new role as a reserve for post-embryonic development and during starvation in the nymphal stage.

Cloning of mRNA Sequences for Two Antibacterial Peptides in a Hemipteran Insect, Riptortus clavatus

Abstract Escherichia coli injection rapidly induced bactericidal activity in the hemolymph of a hemipteran insect, Riptortus clavatus. This activity reached its maximum at 9 hr after injection and thereafter declined slowly. Two types of cDNA clones involved in this response were isolated by differential screening. The predominant type encoded for an open reading frame of 678 amino acids, which consisted of fourteen tandem repeats. Each repeat was rich in charged residues and had a proline-rich region which had striking sequence similarities to proline-rich antibacterial peptides from other insect species, indicating these clones encode a multipeptide precursor of antibacterial peptides. The other type encoded for a glycine-rich peptide similar to a known antibacterial peptide as well. Northern blot analyses revealed rapid induction of mRNAs corresponding to these clones after the injection. To our knowledge, this is the first report on the mRNA sequences of antibacterial peptides of hemimetabolous insects, and the second report on the occurrence of multipeptide precursor structure in insect antibacterial peptides.

Effects of precocenes on vitellogenesis in the adult female tick,Ornithodoros moubata (Acari: Argasidae)

Vitellogenin (Vg) concentrations in the hemolymph and ovarian development were studied inOrnithodoros moubata after treatment with precocenes 1 (P1) and 2 (P2). Precocene was dissolved in acetone or DMSO and topically applied to the dorsal surface of ticks: (1) at adult ecdysis; (2) 24 h before engorgement; (3) immediately after engorgement; and (4) 24 h after engorgement. Subsequently, P1 and P2 were dissolved in olive oil and injected through the gonopore into the body cavity 24 h after engorgement. Vitellogenin concentration was measured on days 5 and 10 after engorgement and ovarian development was scored on day 10, 20 or 30. Oviposition was also recorded and the average weight of eggs laid by females was determined. No differences in concentration of Vg in the hemolymph occurred between the control ticks and ticks treated topically or by injection with P1 and P2. Precocene did not suppress the synthesis of Vg inO. moubata. However, oviposition was reduced in ticks that survived repeated treatment with high doses of P2 dissolved in acetone.