2 search hits
-
The Allatoregulatory Neuropeptides and their Genes in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)
(2004)
-
Mohatmed Abdel-latief
- The genes encoding the S. frugiperda allatotropin (Spofr-AT), allatostatin (Spofr-AST), allatostatin type-A (Spofr-AST A) peptide family and allatotropin 2 (Spofr-AT 2) peptides were isolated from S. frugiperda brain cDNA. The Spofr-AT gene is expressed in three mRNA isoforms with 134, 171, and 200 amino acids, respectively, differing from each other by alternative splicing. The Spofr-AST cDNA encodes 125 amino acid residues including one copy of the Manse-AST mature peptide (type-C allatostatin). The deduced precursor sequence of Spofr-AST A gene contains 231 amino acids and allowed unambiguous identification of nine (or ten) peptides of YFXFGL-a peptide family, which are tandemly arranged in three blocks. A cDNA that encodes 53 amino acids was cloned from S. frugiperda brain cDNA, including one copy of a non-amidated decapeptide (Arg-Val-Arg-Gly-Asn-Pro-Ile-Ser-Cys-Phe-OH). This peptide strongly stimulates the synthesis and release of juvenile hormone (JH) in vitro by the corpora allata (CA) of S. frugiperda adult females and was code-named Spofr-AT 2. The primary structure of Spofr-AT 2 is identical at its C-terminus (-NPISCF) with the M. sexta type-C allatostatin (Manse-AST). One-step RT-PCR for semi-quantification of the gene expression, it is demonstrated that both genes (Spofr-AT and Spofr-AST) are expressed in brain, digestive tract, and reproductive organs of larvae, pupae, and adults of S. frugiperda in a time-, tissue-, and sex specific manner. The tissue-specific localization of the prohormone expression, as demonstrated by whole-mount in situ hybridization, confirms the overall tissue distribution of the prohormones as shown by RT-PCR and supports the pleiotropic functions of the peptides. Spofr-AST type-A gene is expressed in the brain, midgut, and reproductive organs of S. frugiperda larvae and adults in a time- and tissue-specific manner. Data confirm the nature of the allatostatin type-A peptides as brain/gut myoregulatory hormones. Northern blotting and RT-PCR analyses revealed that the Spofr-AT 2 gene is expressed as one transcript in the brain, midgut, and ovary in a tissue- and developmental-specific manner. Treating the CA with the synthetic peptide caused an up to tenfold increase in the release of JH. The stimulation of JH release was dose-dependent with an apparent EC50 of ca. 10-7 M. CA that were activated with Spofr-AT 2 could be inhibited by the addition of synthetic Manse-AST. In conclusion, the presented date strengthen the hypothesis that “allatoregulating” neuropeptides are diverse in structure, widely distributed and exhibit multiple functions. The functions may be tissue-specific as well as specific to particular developmental stages of insects. Knowledge of the various peptide precursor sequences has opened the way for synthesis of these peptides for detailed physiological and functional studies. Further quantitative experiments formulated in context of the life history of the animals will certainly yield a more detailed understanding of the mode of action of these peptides in S. frugiperda. Other major challenges in the future will be to clone the receptors for these peptides and to study the receptor distribution in the fall armyworm.
-
Isolation and characterization of the B-type allatostatin gene of Gryllus bimaculatus de Geer (Ensifera, Gryllidae)
(2004)
-
Junling Wang
- 1. Cricket B type allatostatins, which belong to a neuropeptide family sharing the conserved W(X)6Wamide structure, exhibited inhibitory functions on the biosynthesis of juvenile hormones (JH) in vitro in the corpora allata (CA) as well as on ecdysteroid biosynthesis in the ovary of adult crickets (Gryllus bimaculatus). To understand the mechanisms of function of the pleiotropic cricket B type allatostatins, it is necessary to characterize their gene (preprohormone) and study the spatial and temporal expression patterns of the gene. 2. By PCR screening of a random primer cDNA library and by RACE (Rapid Amplification of cDNA Ends), a 535 bp 3´cDNA sequence of the cricket B type allatostatin gene was yielded. This 3´cDNA fragment encodes a putative translation product of 85 amino acids with potential dibasic endoproteolytic cleavage sites, which may allow processing into six peptides including three copies of Grybi-AS B1 (GWQDLNGGWGa) and single copies of Grybi-AS B2 (GWRDLNGGWGa), Grybi-AS B3 (AWRDLSGGWGa), and Grybi-AS B6 (AWNNLGSAWGa), respectively. Three of these deduced peptides were previously isolated from cricket brain extracts by conventional chromatographic techniques and were designated as Grybi-AS B1, Grybi-AS B2, and Grybi-AS B3. The Grybi-AS B6 neuropeptide represents a novel member of the B type allatostatins. 3. The nucleotide sequences encoding the type B allatostatins are high in GC-content and show strong homology. The highest GC-content was found for Grybi-AS B3 with 83.3%. The similarity of the nucleotide sequences encoding Grybi-AS B2 and Grybi-AS B1 is 93.3%, whereas Grybi-AS B2 and B3 share 90% nucleotide identity. 4. By Southern blot analyses, it was proven that the Grybi-AS type B gene is present as a single copy per haploid genome of G. bimaculatus. 5. By RT in situ PCR technique, it could be demonstrated that the Grybi-AS B gene is expressed in various tissues of 1 day old female adult crickets: In the central nervous system the Grybi-AS B gene expression was detected in the brain. In the protocerebrum, strong positive signals were found in the median neurosecretory cells, and to a lesser extent in lateral neurosecretory cells and in neurons. Gene expression was also found in the neurosecretory cells of the deuterocerebrum and the tritocerebrum. Furthermore, Grybi - AS B gene expression was localized in neurosecretory cells of the suboesophageal ganglion (SOG), the thoracic ganglia, and the abdominal ganglia. In the germarium and in primary oocytes of the ovary, Grybi-AS B gene expression was detected as condensed signals in the nuclei, but not in the prefollicular cells or the cytoplasm. With ongoing development of the oocytes, the signals in the nuclei (germinal vesicles) appeared as separated granules with weaker intensity, which finally disappeared, whereas in the follicular cells strong signals became apparent. Grybi-AS B gene expression was also detected in the epithelial cells of the accessory reproductive glands of female crickets. In the caecum and midgut, Grybi-AS B gene expression was found in endocrine secretory and epithelial cells, whereas in the hindgut, positive RT in situ PCR signals were detected in both longitudinal and circular muscles and in the gut epithelial cells. Grybi-AS B gene expression was also found in cells of the fat body and in thoracic (flight) muscles. 6. The results on Grybi-AS B gene expression as obtained by RT in situ PCR were confirmed by RT-PCR and RNA dot blot analyses. The expression of the Grybi-AS B gene in various tissues of adult females varied in an age-dependent manner. In brains of virgin females gene expression increased from the day of emergence until day 8 of adult life. In the ovary of virgin females gene expression showed a maximum at day 4 after ecdysis, whereas in mated females gene expression was high during the first two days and at days 6 to 7, but low inbetween. In caecum and midgut of virgin females gene expression was low during the first 5 days after ecdysis, but peaked at days 6 and 7, whereas in the hindgut gene expression was highest at day 3 of adult life. In the fat body, gene expression showed highest values on day 1 and days 6 to 7 after ecdysis. 7. Gene expression in brain, testes, and accessory reproductive glands of 0 to 3 days old male crickets was also demonstrated by RT-PCR and RNA dot blot analyses.
