Quantitative RT-PCR analysis of pacifastin-related precursor transcripts during the reproductive cycle of solitarious and gregarious desert locusts.

In locusts, little is known about the physiological and biochemical mechanisms regulating complex processes, such as reproduction and phase transition. The pacifastin family constitutes a family of peptidic inhibitors of serine proteases that are considered to be important regulators of several physiological processes in arthropods. We have performed a detailed transcript profiling analysis of two pacifastin-related peptide precursors, SGPP-2 and SGPP-4, during the reproductive cycle of adult desert locusts (Schistocerca gregaria). This quantitative real-time (RT)-PCR analysis revealed a temporal regulation of both transcripts, which is paralleled by several events that occur during the reproductive cycle of adult locusts. The observed temporal transcript profiles display a strong tissue-, gender- and phase-dependence. In addition, a partial regregarization experiment suggests that both transcript levels are regulated during phase transition and can be employed as molecular markers of the gregarization process.

Quantitative real-time RT-PCR analysis in desert locusts reveals phase dependent differences in neuroparsin transcript levels.

In different parts of the world, locust swarms cause severe ecological and economic damage. However, the physiological mechanisms underlying this gregarization process remain elusive. In this study, we present a detailed quantitative analysis of two neuroparsin precursor (Scg-NPP1 and Scg-NPP2) transcripts in the brain, fat body, gut, gonads and accessory glands of male and female, gregarious and solitarious desert locusts (Schistocerca gregaria). These transcripts are generally more abundant in solitarious than in gregarious animals. In contrast to their gregarious congeners, solitarious locusts contain detectable Scg-NPP1 and Scg-NPP2 transcript levels in the fat body. Moreover, our data reveal temporal changes of neuroparsin mRNA levels in the brains and fat bodies of adult isolated-reared locusts. This paper provides the first scientific evidence for phase-dependent transcriptional regulation of neuropeptide hormone encoding genes.

Neuroendocrinological and molecular aspects of insect reproduction.

This review summarizes recent advances and novel concepts in the area of insect reproductive neuroendocrinology. The role of 'classic' hormones, such as ecdysteroids and juvenoids, to control reproduction is well documented in a large variety of insect species. In adult gonads, ecdysteroids appear to induce a cascade of transcription factors, many of which also occur during the larval molting response. Recent molecular and functional data have created opportunities to study an additional level of regulation, that of neuropeptides, growth factors and their respective receptors. As a result, many homologs of factors playing a role in vertebrate reproductive physiology have been discovered in insects. This review highlights several neuropeptides controlling the biosynthesis and release of the 'classic' insect hormones, as well as various peptides and biogenic amines that regulate behavioural aspects of the reproduction process. In addition, hormone metabolizing enzymes and second messenger pathways are discussed with respect to their role in reproductive tissues. Finally, we speculate on future prospects for insect neuroendocrinological research as a consequence of the recent 'Genomics Revolution'.

cDNA cloning and transcript distribution of two novel members of the neuroparsin family in the desert locust, Schistocerca gregaria.

This study describes the identification and distribution of two novel neuroparsin precursor transcripts (Scg-NPP3/Scg-NPP4) in the desert locust, Schistocerca gregaria. Unlike Scg-NPP1 and Scg-NPP2, both transcripts were not only detected in the brain, but also in various other tissues, such as fat body, ventral nerve cord, testis and male accessory glands. Northern analysis showed that the levels of these transcripts are regulated during larval development, as well as during moulting and reproductive cycles. A significant increase in both mRNAs was observed during the period that just precedes the initial sexual activity of adult females and males. In silico analysis of sequence databases revealed the existence of several other neuroparsin-like peptides in a variety of arthropod species, including crustaceans and chelicerates. Neuroparsins also display similarities with vertebrate IGFBP.

Cloning of two cDNAs encoding isoforms of a pacifastin-related precursor polypeptide in the desert locust, Schistocerca gregaria: analysis of stage- and tissue-dependent expression.

A novel serine protease inhibitor peptide family, designated as the 'pacifastin family', has recently been described in insects (locusts, lepidopterans) and crustaceans (crayfish). This study presents the cDNA cloning of two isoforms of SGPP-3, a novel pacifastin-related precursor in the desert locust, Schistocerca gregaria, which codes for three putative inhibitor peptides. The precursor isoforms differ at a single amino acid position in the third, C-terminal peptide. Northern blot analysis confirmed the presence of two different transcripts (0.75 and 0.90 kb). Both transcripts are most abundant in the fat body and appear to be strongly regulated during the moulting cycle. In addition, the amount of transcript proved to be strictly regulated in the ovaries during the female reproductive cycle.

cDNA cloning of two different serine protease inhibitor precursors in the migratory locust, Locusta migratoria.

Recently, a novel serine protease-inhibiting peptide family, designated as the 'pacifastin family', has been described in locusts and crayfish. All members of this family possess a characteristic cysteine-rich domain. The present study describes the cDNA cloning, sequencing and transcript distribution of two novel pacifastin-related peptide precursors in the migratory locust, Locusta migratoria. Only one of the encoded peptides (HI) was identified previously, whereas six others represent new members of the pacifastin family. Northern blot analysis showed that both precursor transcripts are present in adult locust fat body. These could not be detected in the midgut. Interestingly, an in silico data mining approach of the expressed sequence tags (EST) database revealed the existence of Manduca sexta and Bombyx mori cDNAs that display pronounced sequence similarities with these locust pacifastin-related transcripts.

cDNA cloning and transcript distribution of two different neuroparsin precursors in the desert locust, Schistocerca gregaria.

Neuroparsins were originally identified in locust corpus cardiacum extracts as folliculostatic or 'antigonadotropic' neuropeptides. This paper presents the cloning of two different neuroparsin precursor cDNAs from the brain of the desert locust, Schistocerca gregaria. The first transcript encodes the precursor (Scg-NPP1) of S. gregaria neuroparsin A and B, whereas the second codes for a novel neuroparsin-related peptide precursor (Scg-NPP2). Both precursors display significant sequence similarities with each other and with the Locusta migratoria neuroparsin (Lom-NPP) and Aedes aegypti ovary ecdysteroidogenic hormone (Aea-OEH1) precursors. Northern blot analysis revealed that these neuroparsin transcripts are present in larval and adult locust brains. Interestingly, the Scg-NPP2 mRNA content proved to be strongly regulated during the reproductive cycle in both adult males and females.

Cloning of the cDNA encoding Scg-SPRP, an unusual Ser-protease-related protein from vitellogenic female desert locusts (Schistocerca gregaria).

The cDNA coding for a Ser-protease-related protein (Scg-SPRP) was cloned from desert locust (Schistocerca gregaria) midgut. The derived amino acid sequence consists of 260 residues and shows strong sequence similarity to insect trypsin-like molecules. It is, however, likely that Scg-SPRP is not a proteolytically active enzyme and that it plays another physiologically relevant role, since two out of three residues which are indispensable for catalytic activity of Ser-proteases are replaced. Northern analysis revealed that the Scg-SPRP gene is expressed in midgut tissue and that this expression is strongly induced in adult female locusts. Moreover, the occurrence of the transcript (1.2 kb) fluctuates during the molting cycle and during the female reproductive cycle. Juvenile hormone (JH III) dependence of transcription was investigated by chemical allatectomy (precocene I) of adult females. This resulted in inhibition of vitellogenesis and in disappearance of the Scg-SPRP transcript. Expression of Scg-SPRP in precocene-treated locusts could be reinduced by additional treatment with JH III or with 20-OH-ecdysone.

Cloning of two cDNAs encoding three small serine protease inhibiting peptides from the desert locust Schistocerca gregaria and analysis of tissue-dependent and stage-dependent expression.

This study describes the cloning of two cDNAs encoding three serine-protease-inhibiting peptides, SGPI I, II and III, which were recently identified from ovarian extracts of the desert locust, Schistocerca gregaria. The first cDNA codes for the precursor polypeptides of SGPI I and SGPI II; the second encodes only a single inhibitor, SGPI III. Northern-blot analysis revealed an approximate length of 0.8 kb for SGPI-I/II mRNA and 0.6 kb for SGPI-III mRNA. The transcripts are present in several locust tissues, but they could not be detected in the midgut. The gene for SGPI-I/II is abundantly transcribed during all larval and adult stages, whereas SGPI-III mRNA is mainly present in adults. Northern-blot hybridization also revealed important changes in the SGPI-mRNA content during the molting cycle and during the adult reproductive cycle. Moreover, a differential hormonal control was observed in adult females which had been treated with precocene, juvenile hormone or ecdysone.