Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata (original) (raw)
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Molecular and Cellular Endocrinology, 1992
In an effort to identify the signal transduction mechanism associated with the inhibition of juvenile hormone (JH) biosynthesis by the neuropeptides allatostatins, levels of the cyclic nucleotides CAMP and cGMP were measured in corpora allata (CA) of virgin and mated Diplopteru punctuta females using radioimmunoassays. Treatment of isolated CA with varying concentrations of synthetic allatostatins 1, 2, 3 or 4 did not elicit significant changes in the levels of either CAMP or cGMP in any of the test glands, suggesting that these compounds do not act as second messengers for the four allatostatins tested. Simultaneous treatment of CA with allatostatin 4 and the adenylate cyclase activator forskolin did not increase the degree of inhibition of juvenile hormone biosynthesis relative to that obtained with forskolin (5 or 50 FM) alone. We interpret these results as lending further support to the suggestion that cyclic nucleotides do not play a role in the signal transduction of allatostatins 1-4 in cockroach CA.
Expression of the allatostatin gene in endocrine cells of the cockroach midgut
Proceedings of the National Academy of Sciences, 1994
Cockroach allatostatins are neuropeptides that have been isolated from the brain of Diploptera punctata and shown to inhibit juvenile hormone production by the corpora allata. Enzyme-linked immunoassay and Immunocytochemistry with antisera to two allatostatins, ASB2 (AYSYV-SEYKRLPVYNFGL-NH2) and ASAL (APSGAQRLYGFGL-NH2), revealed that allatostatins were located not only in the insect brain but also in several peripheral tissues Including the cockroach midgut and hindgut. Allatostatin-like immunoreactivity was found in nerve fibers of the stomatogastric nervous system as well as in intrinsic endocrine cells of the midgut. Midgut extracts were shown to be biologically active in an allatostatin bioassay and to contain several alatostatin-like peptides, including the octadecapeptide ASB2, which was identified by mass spectrometry following HPLC purification. Reverse transcription of brain mRNA followed by PCR with degenerate oligonucleotides for ASB2 and ASAL yielded a 338-bp fragment of the allatostatin gene that encoded six allatostatins. In situ hybridization with this probe confirmed that an allatostatin gene is expressed in intrinsic endocrine cells of the midgut. Reverse transcription of midgut mRNA followed by PCR and sequencing of the product revealed that the same gene is expressed in the midgut and in the brain. Allatostatins are thus an example of insect "brain-gut peptides" and we suggest that their function may not be restricted to the regulation of juvenile hormone production.
Allatostatins: Diversity in Structure and Function of an Insect Neuropeptide Family
Annals of The New York Academy of Sciences, 1997
The juvenile hormones (JHs) are a unique group of sesquiterpenoids, identified definitively only in insects, that are responsible for the maintenance of juvenile characteristics. A reduction in JH titer is generally believed to be required for metamorphosis to the adult form. Reproductive functions in most adult female insect species are also regulated by JH, and oocyte growth and maturation, including vitellogenesis, show an absolute dependency on JH-in the absence of the hormone, oocyte growth is arrested.' JH titer is regulated in part by the rate of biosynthesis within the endocrine glands known as the corpora allata (CA), which can he considered analogous to the adenohypophysis of vertebrates. Based on the now classic experiments involving severance of nerve tracts originating in the brain and innervating the CA, Scharrer demonstrated that CA function was under close-range neural control. Scharrer suggested that signals in the hemolymph might also act to regulate the CA.2 Consistent with Scharrer's original observations, the regulation of JH biosynthesis by CA is now known to be stimulated or inhibited by two groups of peptides, allatotropins and allatostatins (ASTs), respectively. This review will examine the ASTs which occur in multiple forms, are pleiotropic in function, and are widely distributed in both neural and nonneural tissue. The structural and functional features of the ASTs appear to parallel the vertebrate somatostatins and may provide one of the best examples of the parallel evolution of peptides.* MOLECULAR ISOLATION AND CHARACTERIZATION OF THE COCKROACH ALLATOSTATIN PRECURSOR The initial characterization of the first complete AST coding region was accomplished by polymerase chain reaction (PCR) amplifications3 of specific sequence from cDNA derived from mKNA isolated from the brains of virgin females of the cockroach Diploptera p~n c t a t a .~ Initially, a pair of highly degenerate primers was used to produce an internal DNA consensus sequence representing the peptide sequence of Dip-AST2
Journal of Insect Physiology, 2008
The cockroach allatostatin receptor (Dippu-AstR) is a 425 amino acid G-protein coupled receptor that is related to the mammalian galanin receptor. Using relative standard curve real-time PCR analysis, changes in Dippu-AstR mRNA expression levels were examined in tissues of adult mated and virgin female Diploptera punctata. Tissues were chosen that were either known targets of allatostatin (Dippu-AST) action or sites of Dippu-AST localization. Tissues examined included brain, corpora allata (CA), gut, ovaries, testes and abdominal ganglia. Dippu-AstR was expressed in all tissues examined for 7 days after adult emergence. Juvenile hormone (JH) biosynthesis is known to peak on day 5 postemergence in mated females. In mated females, Dippu-AstR mRNA was at the highest levels on day 6 post-emergence in brain and CA and day 2 post-emergence in midgut. Dippu-AstR expression was found to correlate with the decline in JH biosynthesis noted on day 5 post-emergence and early inhibition of feeding. Dippu-AstR mRNA expression in virgin female midgut and CA was dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR mRNA was found to be similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declined to low levels by day 4. Testes exhibited maximal Dippu-AstR mRNA expression on days 4 and 7 of adult life. A role for Dippu-AST in testes of Diploptera is unknown.
Archives of Insect Biochemistry and Physiology, 1996
Juvenile hormone (JH) biosynthesis by corpora allata (CAI from embryos of the cockroach Diploptera punctata was measured at four stages during the latter half of embryogenesis. Individual glands from 32-day-old embryos that had completed 49% of embryonic development synthesized 0.3 pmol JH Ill h-'. By day 46 (70% development) gland activity rose to 1.1 pmol J H h-', but on subsequent days JH synthetic rates declined, measuring only 0.8 pmol h-' on day 56 (86% development) and 0.5 pmol h-' on day 60 (92% development). Differences in JH biosynthesis by CA from different-aged embryos were more evident when gland activity was corrected for either corpus allatum cell number, which increased progressively from fewer than 200 cells per gland on day 32 to almost 700 cells per gland on day 60, or embryo mass, which increased from 1.6 mg per embryo on day 32 to 10.8 mg per embryo on day 60. J H biosynthetic rates were significantly inhibited in a medium containing M Dip-allatostatin 7 which suppressed CA activity by 68, 83, 76, and 51°% o n days 32, 46, 56, and 60, respectively. In all embryonic stages JH production was significantly stimulated by incubation of glands with 200 pM farnesol, a late precursor in the J H biosynthetic pathway. D 1~9 6
Juvenile hormone and allatostatins in the German cockroach embryo
Insect Biochemistry and Molecular Biology, 2010
Levels of juvenile hormone III (JH), FGLamide allatostatin peptides (ASTs), ASTs precursor (preproAST) mRNA and methyl farnesoate epoxidase (CYP15A1) mRNA were measured in embryos of the cockroach Blattella germanica. JH starts to rise just after dorsal closure, reaches maximal levels between 60% and 80% of embryogenesis, and decrease subsequently to undetectable levels. ASTs show low levels during the first two thirds of embryogenesis, increase thereafter and maintain high levels until hatching. Pre-proAST mRNA shows quite high levels during the two days following oviposition, thus behaving as a maternal transcript, the levels then become very low until mid embryogenesis, and increase afterwards, peaking towards the end of embryo development. CYP15A1 transcripts were detected around 25% embryogenesis and the levels tended to increase through embryogenesis, although differences amongst the days studied were not statistically significant. The opposite patterns of JH and AST towards the end of embryo development, along with the detection of AST immunoreactivity in corpora allata from late embryos, suggest that JH decline is caused by the increase of AST. Moreover, the uncorrelated patterns of JH concentration and CYP15A1 mRNA levels suggest that CYP15A1 expression does not modulate JH production.
Journal of Biological Chemistry, 2006
Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization timeof-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10 ؊9 M. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti. Juvenile hormone (JH) 3 titers must be modulated to permit the normal progress of development and reproduction in insects (1, 2). In adult female Aedes aegypti mosquitoes, JH levels are low at adult eclosion, elevated in sugar-fed females, and low again after a blood meal (3). JH titer is fundamentally controlled by the rate of biosynthesis in the corpora allata gland (CA) (4). The rate of CA activity is, in turn, regulated, in part, by allato-regulatory peptides that exert either allatostatic (inhibitory) or allatotropic (stimulatory) activity (5, 6). We previously reported that the biosynthetic activity of the A. aegypti CA in vitro was inhibited by mosquito brain extracts (5), and then we used confocal, laser-scanning microscopy studies to show specific patterns of immunostaining for AS-C in the cells of the brain of A. aegypti (7). Here we describe the purification, biochemical, molecular, and functional characterization of A. aegypti PISCF-allatostatin (or Ae-AS-C). It is the first comprehensive report since the original description of a member of this peptide family as an inhibitor of JH synthesis in the lepidopteran Manduca sexta (8). In addition, we describe the developmental stage-dependent Ae-AS-C inhibition of JH synthesis, as well as the changes in peptide levels in head samples. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.
Insect Biochemistry and Molecular Biology, 1997
Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino ⌿[CH 2 NH] and ketomethylene ⌿[COCH 2 ] peptide bond surrogates have been introduced at the position Leu 3-Tyr 4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu 3-Phe 4 of [Phe 4 ]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica. The analogue was less active than BLAST-2 when tested in vitro, but more active than it when tested in vivo.