Cloning of an octopamine/tyramine receptor and plasticity of its expression as a function of adult sexual maturation in the male moth Agrotis ipsilon.

In the male moth Agrotis ipsilon behavioural response and antennal lobe (AL) neuron sensitivity to the female-produced sex pheromone increase with age and juvenile hormone (JH) level. We recently showed that the neuromodulator, octopamine (OA), interacts with JH in this age-dependent olfactory plasticity. To further elucidate its role, we cloned a full cDNA encoding a protein that presents biochemical features essential to OA/tyramine receptor (AipsOAR/TAR) function. The AipsOAR/TAR transcript was detected predominantly in the antennae, the brain and, more specifically, in ALs where its expression level varied concomitantly with age. This expression plasticity indicates that AipsOAR/TAR might be involved in central processing of the pheromone signal during maturation of sexual behaviour in A. ipsilon.

Molecular cloning and expression patterns of a putative olfactory diacylglycerol kinase from the noctuid moth Spodoptera littoralis.

In the aim of the characterization of the molecular actors of insect olfactory transduction, we have cloned the full cDNA encoding a Spodoptera littoralis diacylglycerol kinase (DGK) named SlDGK. In male adults, SlDGK transcript was detected predominantly in the brain and in the olfactory sensilla trichodea located on the antennae. SlDGK expression was first detected at day 3 of the pupal stage, then reached a maximum at the end of this stage and was maintained at this level during the adult period. These data provide the first molecular characterization of a DGK potentially involved in the regulation of signalling pathways responsible for the establishment and/or the functioning of the olfactory system in Lepidoptera.

A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster.

Drosophila melanogaster shows sexually dimorphic cuticular hydrocarbons, with monoenes produced in males and dienes produced in females. Here we describe a female-specific desaturase gene, desatF. RNAi knock-down led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males. The desatF gene was also expressed in females from D. sechellia, rich in dienes, but not D. simulans, which produce only monoenes. When hydrocarbons were feminized in D. melanogaster males by targeted expression of the transformer gene, the expression of desatF occurred. These results strongly suggest that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster.

Cloning and characterization of a gut-specific cathepsin L from the aphid Aphis gossypii.

We have characterized proteinase activities in gut extracts from the cotton-melon aphid (Aphis gossypii Glover), an insect feeding strictly on protein-poor phloem. The major, if not exclusive, intestinal proteinases of this aphid are of the cysteine type. A cDNA has been cloned from a gut library and codes for the cysteine proteinase AgCatL, a cathepsin L-like cysteine proteinase. The AgCatL protein shows high sequence similarity with mammalian and some arthropod cathepsin L-like proteinases, but can be reliably distinguished from the secreted (digestive) proteinases identified in other arthropods. AgCatL is widely expressed in aphid intestinal cells. Immunolocalization of AgCatL showed an intense signal at the level of the anterior 'stomach' part of the midgut, and especially at intracellular localization. Although the precise role of AgCatL in aphid midgut physiology is still unclear, this enzyme could be involved in the processing of exogenous ingested polypeptides.

Periodic expression of an ecdysteroid-induced nuclear receptor in a lepidopteran cell line (IAL-PID2).

A set of DNA primers was designed within the DNA-binding domain of the Manduca hormone receptor 3 (MHR3) cDNA. These primers were used in RT-PCR to isolate a 204 bp cDNA fragment from IAL-PID2 cells exposed to 10(-6) M 20-hydroxyecdysone (20E) for 12 h. The amino acid sequence deduced from the cDNA fragment presented 100% identity with the zinc finger domain of Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Choristoneura hormone receptor 3 (CHR3). This cDNA fragment was used as a probe on total RNA from IAL-PID2 cells exposed to 20E and hybridized to mRNA, the size of which was close to 4.5 kb and named Plodia hormone receptor 3 (PHR3). Kinetics of induction of PHR3 mRNA were similar to that of HR3 genes but varied according to the position of cells in their cell cycle. The non-steroidal ecdysone agonist, RH-5992 induced the expression of PHR3 at lower concentrations than 20E. From sequence similarity, mRNA size, 20E and RH-5992 inducibilities, we conclude that PHR3 transcript could encode a Plodia hormone receptor 3 involved in the genetic signalling cascade of 20E. Thanks to its periodic expression, this putative orphan nuclear receptor could serve as a suitable cellular marker for studying changes of epidermal cell sensitivity to 20E during the cell cycle.

Molecular cloning and structural analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase of the moth Agrotis ipsilon.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which plays a key role in isoprenoid biosynthesis, catalyses the synthesis of mevalonate from HMG-CoA. Insects do not synthesize cholesterol de novo, rather mevalonate derivatives lead to non-sterol isoprenoids which are essential for development and reproduction. In this paper, we describe an HMG-CoA reductase of the moth Agrotis ipsilon and we report its expression in fat body, ovary, muscle, brain and corpora allata tissues of adult specimens. The analysis of the cDNA reveals that it encodes a polypeptide of 833 amino acids (Mr = 89785). Alignments of this HMG-CoA reductase from A. ipsilon with the homologous sequences of other eukaryotes shows a high degree of conservation in all species studied. Parsimony analysis based on these alignments produced dendrograms congruent with the current systematic schemes. This suggests that, during eukaryote evolution, HMG-CoA reductase diversified in parallel with taxonomic splitting.

A cDNA, from Agrotis ipsilon, that encodes the pheromone biosynthesis activating neuropeptide (PBAN) and other FXPRL peptides.

A cDNA encoding the prohormone of the pheromone biosynthesis activating neuropeptide (PBAN) in the moth Agrotis ipsilon was isolated. The cDNA contains 834 nucleotides, coding for a 193-amino acid protein that exhibits 89% identity with PBAN prohormones of other moths. The prohormone contains five potential peptides belonging to the FXPRL family. The peptide corresponding to the Bombyx mori diapause hormone exhibits an extra residue, and the C-terminal leucine is replaced by an isoleucine, introducing a new type of variability in this family of peptides. Northern blot analysis revealed expression in suboesophagal ganglion complexes. Constitutive heterologous expression of Agi-PBAN cDNA in yeast, using three different antibodies, did not produce PBAN-immunoreactive material.

The pheromone biosynthesis activating neuropeptide (PBAN) of the black cutworm moth, Agrotis ipsilon: immunohistochemistry, molecular characterization and bioassay of its peptide sequence.

PBAN-like immunoreactivity has been detected in the suboesophageal ganglion and the brain (Br-SOG) of larvae and adult males and females of Agrotis ipsilon, using an antiserum against Helicoverpa zea PBAN (Hez-PBAN). The amino acid sequence of A. ipsilon PBAN (Agi-PBAN) was deduced from the cDNA sequence, using both Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and 5' Rapid Amplification of cDNA Ends (RACE). The primers were degenerate sets of oligonucleotides derived from known amino acid sequences of the PBAN precursor. The final cloned fragment contained the complete DNA sequence coding for the putative Agi-PBAN. Based on a comparison with known PBAN processing from the polypeptide precursor, we propose that Agi-PBAN is a 33-amino acid peptide. Agi-PBAN exhibits high sequence homology with Hez-PBAN (88%), Lymantria dispar PBAN (Lyd-PBAN, 88%) and Bombyx mori PBAN (Bom-PBAN, 73%). Agi-PBAN shares the C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-LeuNH2) with all identified PBANs but has only one methionine residue instead of two in Hez-PBAN and Lyd-PBAN, and three in Bom-PBAN. Based on predicted a.a. sequence, Agi-PBAN, with Leu-NH2 as C-terminal motif, has been synthesized and assayed for its ability to promote pheromone production in decapitated females of A. ipsilon. Synthetic Agi-PBAN induced pheromone production in decapitated females as evaluated by the male responsiveness to the pheromonal blend in a wind tunnel.

Biosynthetic activity of corpora allata, growth of sex accessory glands and mating in the male moth agrotis ipsilon (Hufnagel)

The involvement of both juvenile hormone acid (JHA) and the sex accessory glands (SAGs) in the reproduction of the male moth Agrotis ipsilon was studied as a function of age and mating status. Total protein content analysis followed by gel electrophoresis of the SAGs, radiochemical assay for JHA biosynthesis and surgical and behavioural experiments were performed. Both the protein content of the SAGs and the biosynthetic activity of the corpora allata (CA) increased with age. Allatectomy and JHA/JH treatments showed that the protein content of the SAGs is linked with the activity of the CA. The protein content of the glands, but not the rate of JHA biosynthesis, decreased just after mating, and both increased sharply 24 h later. Injection of fluvastatin, an inhibitor of JH biosynthesis, in males immediately after mating prevented the increase in JHA synthesis and lowered the total protein content of the SAGs. Moreover, fluvastatin disrupted normal spermatophore transfer during the next mating of the injected males. Our results show that JHA controls the reproduction of A. ipsilon males by its separate actions on the sex accessory glands and on sexual behaviour.