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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.

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.

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., (Copyright 2004 Wiley-Liss, Inc.)

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., (Copyright 2004 Wiley-Liss, Inc.)

Allatotropin (AT) is a 13-residue amidated neuropeptide, first isolated from pharate adult heads of the tobacco hornworm, Manduca sexta (Manse-AT), which strongly stimulates the biosynthesis of juvenile hormones (JH) in the corpora allata (CA) of adult moths. In Spodoptera frugiperda, a cDNA that encodes 134 amino acids, including an AT peptide, has been cloned. The S. frugiperda allatotropin mature peptide (Spofr-AT) [GFKNVEMMTARGFa] is identical to that isolated from M. sexta. The basic organization of the Spofr-AT precursor is similar to that of Agrius convolvuli, M. sexta, Pseudaletia unipuncta, and Bombyx mori with 83-93% amino acid sequence identity. The Spofr-AT gene is expressed in at least three mRNA isoforms with 134, 171 and 200 amino acids, differing from each other by alternative splicing. All allatostatins (AS) have an inhibitory action on the JH biosynthesis in the CA. A cDNA that encodes 125 amino acid residues including one copy of the Manse-AS peptide has been cloned from S. frugiperda (Spofr-AS; QVRFRQCYFNPISCF). The basic organization of the Spofr-AS precursor is similar to that of P. unipuncta with 85% amino acid sequence identity. Using one step RT-PCR for semi-quantification of the gene expression, we showed that the three mRNAs of the Spofr-AT gene and the Spofr-AS gene are expressed in brains of last instar larvae, prepupae, pupae, and adults of both sexes of S. frugiperda with variable intensity.