J. Vanden Broeck

Characterization of a cloned locust tyramine receptor cDNA by functional expression in permanently transformed Drosophila S2 cells

Abstract: The cDNA for Tyr‐Loc, a G protein‐coupled receptor that clearly shows homology to a number of mammalian and fruit fly receptors for biogenic amines, was cloned from the nervous system of Locusta migratoria. Functional expression of the cloned cDNA was obtained in cultured insect cells, i.e., in Spodoptera SF9 cells using a baculoviral expression system and in stably transformed Drosophila Schneider 2 (S2) cells. Multiple copies of the receptor expression construct are inserted into the genome of these permanently transformed cells. The expression of the receptor cDNA was driven by the upstream sequences of a Bombyx mori baculoviral immediate early gene. Tyramine shows a much higher binding affinity to this receptor than other possible endogenous ligands. It also reduces forskolin‐induced cyclic AMP production in the permanently transformed S2 cells. The pharmacological profile of the Tyr‐Loc receptor is distinct from that of any locust receptor‐type described so far, but it is similar to that of the Drosophila tyramine/octopamine receptor. In the locust CNS, the Tyr‐Loc mRNA is not present in the distal part of the optic lobes but has a widespread distribution in the brain and the ventral nerve cord.

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 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.

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.

Scavenger receptor‐mediated endocytosis facilitates RNA interference in the desert locust, Schistocerca gregaria

RNA interference (RNAi) has become a widely used loss‐of‐function tool in eukaryotes; however, the delivery of double‐stranded (ds)RNA) to the target cells remains a major challenge when exploiting the RNAi‐technology. In insects, the efficiency of RNAi is highly species‐dependent. Yet, the mechanism of cell entry in insects has only been characterized in a cell line of the fruit fly, Drosophila melanogaster, a species that is well known to be poorly amenable to environmental RNAi. In the present paper, we demonstrate that silencing vacuolar H‐ATPase 16 (vha16) and clathrin heavy chain (clath), two components of the Clathrin‐dependent endocytosis pathway, together with pharmacological inhibition of scavenger receptors with polyinosine and dextran sulphate, can significantly attenuate the highly robust RNAi response in the desert locust, Schistocerca gregaria.

Functional expression of a locust tyramine receptor in murine erythroleukaemia cells

The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein‐coupled receptor. Functional agonist‐dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL‐TyrLoc). Tyramine elicited a dose‐dependent increase of cytosolic Ca2+‐ions and an attenuation of forskolin‐induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein‐coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.

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.

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.

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.

The molecular characterisation of chicken pituitary N-terminal pro-opiomelanocortin (POMC)

Monoclonal antibodies (Mabs) specifically recognizing the chicken pituitary corticotropes were used to isolate a population of closely related peptides from crude chicken pituitary extracts. A homogeneous N-terminal sequence homologous to the extreme N-terminus of mammalian and amphibian pro-opiomelanocortin (POMC) was revealed. Further physicochemical analysis proved the existence of a series of C-terminally truncated peptides including 3 major molecular species corresponding to Ser1-Gly64, Ser1-Arg73 and Ser1-Gly105 respectively. The two latter molecules were shown to be N-glycosylated at position Asn67, with mass spectrometric data indicating a carbohydrate structure of the oligomannose 5 type, in addition to two more complex structures. No evidence was found in favour of O-glycosylation on Ser47. Degenerated PCR primers were deduced from the above protein sequence and from the known chicken adrenocorticotropic hormone (ACTH) sequence. The nucleotide sequence obtained by reversed transcription PCR (RT-PCR) completely confirmed the new amino acid sequence data including pro-gamma-MSH, the joining peptide and ACTH.