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Orcokinins (OKs) are neuropeptides that were first identified in crustacean through their myotropic activity. In insects, the OK gene gives rise to two mRNAs coding for two different families of conserved mature neuropeptides: OKA and OKB. Although OKs are conserved in many insect species, its physiological role in this animal class is not fully understood. Until now prothoracicotropic, regulatory of light entrainment to the circadian clock and "awakening" activities have been reported for these peptides in different insect species. Here we report the identification of OKA and OKB precursors in the cockroach Blattella germanica. OKA mRNA was detected in brain, whereas OKB mRNA was detected both in brain and midgut. In vivo silencing of OK precursors suggests the involvement of OK gene products in the regulation of vitellogenin expression in the fat body, an action that appears to be independent of juvenile hormone. This is the first time that a function of this kind has been reported for OKs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome., ((c) 2005 Wiley-Liss, Inc.)

We aimed to elucidate why cockroaches do not produce vitellogenin in immature stages, by studying the appearance of vitellogenin mRNA in larvae of Blattella germanica. Treatment of female larvae in any of the last three instars with 1 microg of juvenile hormone (JH) III induces vitellogenin gene transcription, which indicates that the fat body is competent to transcribe vitellogenin at least from the antepenultimate instar larvae. In untreated females, vitellogenin production starts on day 1 after the imaginal molt, when corpora allata begin to synthesize JH III at rates doubling the maximal of larval stages. This coincidence suggests that the female reaches the threshold of JH production necessary to induce vitellogenin synthesis on day 1 of adult life. These data lead to postulate that larvae do not synthesize vitellogenin simply because they do not produce enough JH, not because their fat body is incompetent.

The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.

In the cockroach Blattella germanica, the synthesis of vitellogenin is juvenile hormone III (JH III)-dependent. We have studied the effect of JH III upon vitellogenin gene expression in periovaric fat bodies incubated in vitro. Periovaric fat bodies were obtained from cardioallatectomized females. The response to JH III was measured in terms of vitellogenin and vitellogenin mRNA after 7 h of incubation. A hormonal concentration as low as 1 nM was enough to induce vitellogenin production and its release to the medium, whereas the concentration of 10 nM produced the maximal effects. Although the response of the vitellogenin gene to JH III is fast and efficient, it seems that the action is mediated by protein factors, given that cycloheximide treatment impairs the hormonal effect. The presence in the medium of brain extract (0.5 equivalents), corpora cardiaca (one pair) or hypertrehalosemic hormone (10(-7) or 10(-8) M), partially inhibited the response to JH III.

The cloning and sequencing of a cDNA of the vitellogenin gene from the cockroach Blattella germanica is reported. It is 5,749 nucleotides long and encodes an amino acid sequence of 1,862 residues (including a putative signal peptide of 17 residues). The vitellogenin sequence includes a long serine-rich stretch between amino acids 322 and 349, and two other stretches between amino acids 1691 and 1740. The vitellogenin of B. germanica shows a notable similarity (between 32 and 42%) to those described in other insects, and its alignment shows a high number of motifs conserved in all species, especially in the subdomains I-V. Non-parsimony methods (Neighbor Joining) of phylogenetic analysis of the insect vitellogenin sequences gave a tree showing a topology that is, in general, congruent with the currently accepted insect phylogenetic schemes. Arch., (Copyright 2000 Wiley-Liss, Inc.)

The present paper describes the effect of juvenile hormone III (JH III) upon vitellogenin (Vg) gene expression in cardioallatectomized females of Blattella germanica. Northern blot analyses of time course studies showed that Vg mRNA can be detected 2 h after the treatment with 1 microgram of JH III. Western blot analyses revealed that Vg protein is detectable 4 h after the same treatment. The study of the influence of the age showed that 48-h-old females seem more sensitive than 24-h-old females, whereas differences were less apparent between 48- and 72-h-old females. Dose-response studies indicated that 0.01 microgram of JH III is ineffective, whereas the doses of 0.1, 1 and 10 micrograms induced the synthesis of Vg in a dose-dependent fashion. Finally, the administration of three successive doses, of 0.01 microgram of JH III each, did not result in detectable Vg production, whereas two doses of 0.01 microgram followed by one of 1 microgram of JH III induced a greater response than that resulting from a sole dose of 1 microgram of JH III, which suggests that sub-effective doses of JH III elicit a priming effect on Vg production.

A partial cDNA clone of the vitellogenin gene from the cockroach Blattella germanica has been isolated from a cDNA expression library using an anti-vitellin-vitellogenin antiserum probe. The analysis of cDNA inserts gave a sequence of 2,645 nucleotides corresponding to the 3' region. The deduced amino acid sequence is 825 residues long and is similar to the homologous portion of the vitellogenin of other insect species, especially that of the mosquito Aedes aegypti. RNA hybridization studies indicated that the vitellogenin gene expression is limited to the fat body of adult females. The pattern of expression during the first vitellogenic cycle was approximately parallel to that of vitellogenin production by the fat body previously described. The availability of a cDNA probe for the B. germanica vitellogenin gene represents a useful tool to study the molecular action of hormones affecting vitellogenin synthesis in this species.

Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino psi [CH2NH] and ketomethylene psi [COCH2] peptide bond surrogates have been introduced at the position Leu3-Tyr4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu3-Phe4 of [Phe4]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica. The analogue was less active than BLAST-2 when tested in vitro, but more active than it when tested in vivo.

Allatostatins with a typical YXFGL-amide C-terminus constitute a neuropeptide family, which was discovered because of its inhibitory action on insect juvenile hormone synthesis. In the search for possible new functions for allatostatins we focused our attention on the fat body. Our previous studies on the cockroach Blattella germanica suggested the occurrence of factors terminating vitellogenesis, and the hypothesis here was that allatostatins might be one of these factors. Our experiments have shown that allatostatin impaired vitellogenin release in fat bodies incubated in vitro, and that this effect appears to be mediated by the inhibition of vitellogenin glycosylation. Fluvastatin also inhibited vitellogenin release, and mevalonolactone counteracted the inhibitory effects of allatostatin. These results suggest that allatostatin acts upon the mevalonate pathway and synthesis of dolichol, which would explain the inhibition of vitellogenin glycosylation. We finally conclude that allatostatins may effectively contribute to the termination of the vitellogenic cycle in B. germanica.