Functional activity of allatotropin and allatostatin in the pupal stage of a holometablous insect, Tribolium castaneum (Coleoptera, Tenebrionidae).

Allatotropin (AT) and allatostatin (AS) neuropeptides are known to regulate the biosynthesis of juvenile hormones (JH) in insects. Furthermore, they possess myoregulatory and other activities in a wide range of insect species. The genome of Tribolium castaneum encodes two AS and one AT precursors. Here we cloned the cDNAs of the precursors, followed their expression patterns during the pupal stage, and established their putative roles in adult development and oviposition of the females using RNA interference (RNAi). Cloning of the cDNA and gene structure analyses of the Tc-AT gene confirmed that the gene is expressed in three mRNA isoforms. Real-time PCR data demonstrate that the Tc-AT isoforms and the AS genes, Tc-AS C and Tc-AS B, are expressed in discerning developmental and tissue-specific patterns. Single injections of dsRNAi (targeted against the Tc-AT, Tc-AS C, and Tc-AS B, respectively), into young pupae resulted in abnormal adult phenotypes, whereby about half of the animals (P1 phenotype) looked relatively normal, but the females laid low numbers of eggs. The other halves (P2) exhibited strong developmental defects with abnormal duration of the pupal stage, abnormal head and body sizes, short elytra, and incomplete sclerotization. Moreover, these females deposited no eggs and died within one week after emergence. Individual silencing of the Tc-AT mRNA isoforms showed that Tc-AT3 had the most disruptive influence on adult development and fecundity of the females. Our findings clearly indicate a significant role of AT and AS neuropeptides in the pupa. The distinct mechanisms of action, however, remain to be determined.

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle, Tenebrio molitor (Coleoptera, Tenebrionidae).

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)(6)Wamide and AS type C; PISCF-OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949-955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca-corpora allata (CC-CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica-AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica-AS C peptide inhibited the JH release from CA of 3-day-old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7-day-old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica-AT) also activated the JH release from the CA of 7-day-old females in a dose-dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram-AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10(-8) to 10(-4) M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles.

An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site.

Epoxide hydrolases (EHs) are enzymes occurring in virtually any living organism. They catalyze the hydrolysis of epoxide containing lipids and are involved in crucial mechanisms, such as the detoxification of xenobiotics or the regulation of inflammation and blood pressure. Here, we describe a function of a putative EH gene in the biosynthesis of a sex attractant in the jewel wasp Nasonia vitripennis and use this gene to localize the site of pheromone production. Males of this parasitic wasp release a mixture of (4R,5R)-( threo-) and (4R,5S)-( erythro-)5-hydroxy-4-decanolide (HDL) to attract virgin females. Using a stable isotope labeled precursor, we demonstrated that vernolic acid ( erythro-12,13-epoxy-octadec-9Z-enoic acid) is converted by N. vitripennis males to threo-HDL. This suggested the involvement of an EH in hydrolyzing the fatty acid epoxide under inversion of the stereochemistry into the respective diol, which might be further processed by chain shortening and lactonization to HDL. We cloned a putative N. vitripennis EH gene (Nasvi-EH1) encoding 470 amino acids and localized its transcripts in the male rectal papillae by in situ RT-PCR. Chemical analyses and histological studies confirmed that males synthesize the sex attractant in the rectal vesicle and release it via the anal orifice. Involvement of Nasvi-EH1 in HDL biosynthesis was established by RNAi-mediated gene silencing. Injection of Nasvi-EH1 dsRNA into male abdomens inhibited pheromone biosynthesis by 55% and suppressed the targeted gene transcripts in the rectal vesicle by 95%.

A family of chemoreceptors in Tribolium castaneum (Tenebrionidae: Coleoptera).

Chemoperception in invertebrates is mediated by a family of G-protein-coupled receptors (GPCR). To date nothing is known about the molecular mechanisms of chemoperception in coleopteran species. Recently the genome of Tribolium castaneum was sequenced for use as a model species for the Coleoptera. Using blast searches analyses of the T. castaneum genome with previously predicted amino acid sequences of insect chemoreceptor genes, a putative chemoreceptor family consisting of 62 gustatory receptors (Grs) and 26 olfactory receptors (Ors) was identified. The receptors have seven transmembrane domains (7TMs) and all belong to the GPCR receptor family. The expression of the T. castaneum chemoreceptor genes was investigated using quantification real- time RT-PCR and in situ whole mount RT-PCR analysis in the antennae, mouth parts, and prolegs of the adults and larvae. All of the predicted TcasGrs were expressed in the labium, maxillae, and prolegs of the adults but TcasGr13, 19, 28, 47, 62, 98, and 61 were not expressed in the prolegs. The TcasOrs were localized only in the antennae and not in any of the beetles gustatory organs with one exception; the TcasOr16 (like DmelOr83b), which was localized in the antennae, labium, and prolegs of the beetles. A group of six TcasGrs that presents a lineage with the sugar receptors subfamily in Drosophila melanogaster were localized in the lacinia of the Tribolium larvae. TcasGr1, 3, and 39, presented an ortholog to CO(2) receptors in D. melanogaster and Anopheles gambiae was recorded. Low expression of almost all of the predicted chemoreceptor genes was observed in the head tissues that contain the brains and suboesophageal ganglion (SOG). These findings demonstrate the identification of a chemoreceptor family in Tribolium, which is evolutionarily related to other insect species.

The adipokinetic hormones in the fall armyworm, Spodoptera frugiperda: cDNA cloning, quantitative real time RT-PCR analysis, and gene specific localization.

Small neuropeptides of the adipokinetic/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Helicoverpa zea) seems to be restricted to moths. Here we report the identification of the Manse-AKH-like Spofr-AKH 1 and the Helze-HrTH-like Spofr-AKH 2 prohormone precursors from the fall armyworm, Spodoptera frugiperda. Moreover, by PCR screening of a random primer cDNA library and by RACE, three 668, 835 and 1008 bp cDNA sequences were obtained, which encode putative translation products of 67-74 amino acids, each containing one copy of a peptide sequence that in its processed form has the sequence of QLTFSSGW-amide (Spofr-AKH 3). Another cDNA sequence of 634 bp encodes a putative translation product of 40 amino acids, potentially leading to one copy of an elongated, non-amidated Helze-HrTH (pQLTFSSGWGNCTS-OH; Spofr-AKH 4). Q-RT-PCR analysis showed that the Spofr-AKH mRNAs are expressed in 2d-old female brain/corpora cardiaca complexes, but also in ovaries, midgut, fat body, accessory glands and muscle tissues. Expression was also found in the ovaries of 4d-old females. Whole-mount in situ RT-PCR analysis with ovaries from 2d-old females showed that the Spofr-AKH 2 and Spofr-AKH 4 were mainly localized in the germarium (phase 3), whereas the Spofr-AKH 1, and the three mRNA isoforms of Spofr-AKH 3 were localized at the end of the vitellarium and in the fully developed oocytes (phase 1 and 2). The results suggest that Spofr-AKH genes play a role in the regulation of oocyte maturation in S. frugiperda.

Characterization of a novel peptide with allatotropic activity in the fall armyworm Spodoptera frugiperda.

A cDNA that encodes 53 amino acids, including one copy of the RVRGNPISCF-OH peptide, was cloned from Spodoptera frugiperda. This peptide strongly stimulates the synthesis and release of juvenile hormone (JH) in vitro by the corpora allata (CA) of S. frugiperda and was code-named Spofr-AT 2. Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that the preprohormone is expressed as one transcript in the brain, midgut (Mg) and ovary (Ov) in a tissue- and developmental-specific manner. Whole-mount in situ hybridization confirmed the gene expression in the suboesophageal ganglion (SOG) and in the ovary of adult females. Treating the CA with the synthetic peptide caused an up to tenfold increase in the release of JH. The stimulation was dose-dependent with an apparent EC(50) of ca. 10(-7) M. CA that were activated with Spofr-AT 2 could be inhibited by the addition of synthetic allatostatin type-C from Manduca sexta (Manse-AS). This is the first report on the presence and function of two different peptides with allatotropic activity in an insect species.

Type-A allatostatins from the fall armyworm, Spodoptera frugiperda: molecular cloning, expression and tissue-specific localization.

The gene encoding the Spodoptera frugiperda allatostatin type-A peptide family (Y/FXFGL-amides) was isolated from S. frugiperda brain cDNA. The gene encodes a precursor of 231 amino acids containing nine (or ten) Y/FXFGL-a peptides that are tandemly arranged in three blocks. The comparison of the Spofr-AST A precursor with the respective precursor genes from two other lepidopteran species, Helicoverpa armigera and Bombyx mori, shows high homology in size, sequence (84 and 57%, respectively), and organisation of the allatostatins. One-step RT-PCR analysis using a Spofr-AST A-6 to A-9 probe shows that the gene is not only expressed as one transcript in the brain and midgut of larvae and adults in a time- and tissue-specific manner, but also in the reproductive tissues of adult S. frugiperda. Data confirm the nature of the allatostatin type-A peptides as brain/gut myoregulatory hormones, whereas their function(s) in ovaries, oviduct, and testes still have to be resolved. The cell-specific localization of the preprohormone expression, as demonstrated by whole mount in situ hybridization, confirms the overall distribution of the Spofr-AST A preprohormone as shown by RT-PCR and supports the pleiotropic functions of the peptides.

Expression and localization of the Spodoptera frugiperda allatotropin (Spofr-AT) and allatostatin (Spofr-AS) genes.

Manduca sexta allatotropin and allatostatin were the first corpora allata (CA) regulating neuropeptides identified from Lepidoptera. Recently, we cloned the allatotropin (Spofr-AT) and the allatostatin (Spofr-AS) genes from the fall armyworm Spodoptera frugiperda. Using one-step RT-PCR for semi-quantification of the gene expression, we now demonstrate that three mRNA isoforms of the Spofr-AT gene and the Spofr-AS gene are expressed in brain, digestive tract, and reproductive organs of larvae, pupae, and adults in a time- and tissue-specific manner. Expression rates in the brain and in various parts of the digestive tract prove the dual role of the peptides as brain/gut (neuro)peptides. The functional meaning of ovarian and testes expression of the genes is not yet clear, although myoregulatory properties of the peptides are probable. The tissue-specific localization of the prohormone expression, as demonstrated by whole mount in situ hybridization, confirms the overall distribution of the prohormones as shown by RT-PCR and supports the pleiotropic functions of the peptides.