Molecular characterisation of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sexta allatotropin and allatostatin preprohormone peptides

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.     

Triple co-localisation of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut

The triple co-localisation of peptidergic material immunoreactive to antisera raised against allatostatins of the Y/FXFGL-NH2 type, Manduca sexta allatostatin (Mas-AS), and allatotropin has been demonstrated in a single pair of anterodorsal neurones in the frontal ganglion of the tomato moth, Lacanobia oleracea (Noctuidae). Another pair of posterior neurones contain only Y/FXFGL-NH2-type allatostatin immunoreactivity. The neurites of all four cells trifurcate, and axons project to the brain in the frontal connectives and to the foregut in the recurrent nerve. Axons from the anterior neurones, within the recurrent nerve, have prominent lateral branches supplying muscles of the crop, and axons from both anterior and posterior cells show profuse branching and terminal arborisations in the region of the stomodeal valve. The brain contributes Y/FXFGL-NH2-immunoreactive material, but not allatotropin or Mas-AS, to the recurrent nerve via NCC 1+2 and NCC 3. All three peptides have a reversible effect on the spontaneous (peristaltic) contractions of the foregut (crop) in vitro. Thus, both types of allatostatin are inhibitory at 10(-12) to 10(-7) M, whereas allatotropin is strongly myostimulatory at 10(-14) M. This is the first demonstration of the gut myoinhibitory effects of Mas-AS and, taken together with the effects of Y/FXFGL-NH2-type allatostatins and allatotropin, reveals a different functional aspect to that normally attributed to these three peptides, i.e. control of juvenile hormone synthesis by the corpus allatum.     

Expression of allatostatins in the Mediterranean field cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae)

The allatostatin (AST) type A gene of the cricket Gryllus bimaculatus encodes a hormone precursor including at least 14 putative peptides with a common C-terminus Y/FXFGL/Iamide. By RT-PCR we have analyzed the expression of the allatostatin precursor in various tissues of 0-21 days old adult virgin and mated females. In 3-day-old virgin females, the gene is strongly expressed in the brain (oesophageal ganglion), the suboesophageal ganglion and the caecum, but to a lower extent in other parts of the digestive tract (ileum, midgut, colon), and in various other tissues such as the fat body, ovaries and female accessory reproductive glands. In the brain and ovaries of virgin females, the AST expression is rather constant throughout adult life, whereas in brains of mated animals, expression is low until day 7, but increases sharply from day 8 onwards to reach values triple those before day 7. In ovaries of mated animals AST gene expression is also age-dependent, with high expression rates during the first 4 days after imaginable moult, a second but smaller peak from day 15 to 21, and very low values in between. In the fat body of virgin crickets allatostatin expression is high during the first 9 days after ecdysis and declines thereafter, whereas in mated animals two peak values, day 1 and day 6, are observed, and a third peak in older animals.     

Biochemical and molecular characterization of allatotropin and allatostatin from the Eri silkworm, Samia cynthia ricini

In a previous study, allatotropic and allatostatic activities were observed in brain extract from the Eri silkworm, Samia cynthia ricini (Samcri) [Li, S., Jiang, R.-J., Cao, M.-X., 2002b. Allatotropic and allatostatic activities in brain extracts of the Eri silkworm, S. cynthia ricini, and the effects of Manduca sexta allatotropin and M. sexta allatostatin on juvenile hormone in vitro. Physiol. Entomol. 27, 322-329]. In the present study, the HPLC purified Samcri-allatotropin (AT) and -allatostatin (AST) factors were shown to have the same retention time as those of M. sexta (Manse)-AT and -AST, respectively. Moreover, the amino acid sequences of mature Samcri-AT and -AST deduced from their encoding cDNAs are identical to the Manse-AT and -AST amino acid sequences. Both Samcri-AT and -AST genes were expressed in brain, nerve cord, and midgut, with Samcri-AT also detected in gonads and epidermis, suggesting their pleiotropic physiological functions. The expression levels of Samcri-AT and -AST genes correlated well with the allatoregulatory activities during the period of adult emergence indicating the two peptides tightly control JH synthesis, in a contradictive and cooperative manner. Our biochemical and molecular data of Samcri-AT and -AST and other studies demonstrate that these two peptides regulate JH synthesis by corpora allata in Lepidoptera and have pleiotropic physiological effects.     

Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.     

昆虫神经肽allatostatin与allatotropin的研究新近展

昆虫神经肽ａｌｌａｔｏｓｔａｔｉｎ与ａｌｌａｔｏｔｒｏｐｉｎ的研究新近展关雪辰（中国科学院动物研究所北京１０００８０）昆虫神经激素是肽类激素,它控制昆虫许多关键的生理过程,例如生长、变态、生殖和行为等。保幼激素（ＪＨ）对昆虫的卵子发生成熟起着重要的调...     

Neuropepride Regulators of Insect Corpora Allata

SYNOPSIS. Neuropeptides of the insect brain that regulate juvenilehormone synthesis by the corpora allata include allatotropins,stimulatory modulators, and allatostatins, inhibitory modulators.A radiochemical assay for juvenile hormone synthesis by corporaallata in vitro was utilized in the high pressure liquid chromatographicisolation of brain neuropeptides leading to the determinationof their primary structure. Identified are an allatotropin andan allatostatin from a Lepidopteran, Manduca sexta, and a familyof five allatostatins from a Dictyopteran, Diploptera punctata.These neuropeptides are all unique, effective at low concentration(10^–10 to 10^–8 M), act quickly (within hrs) andappear to be effective only within the same order of insectsas that from which the peptides were isolated. The physiologicalstate of the corpora allata conditions the effectiveness ofthe allatostatins of D. punctata. These neuropeptide regulatorsof corpora allatal function may have multiple regulatory roles.This is indicated for D. punctata allatostatin I by specificreceptors in brain and fat body as well as in corpora allatalmembrane preparations, and also by immunocytochemical localizationof allatostatin I in medial nerve cells that terminate withinthe brain as well as in the lateral neurosecretory cells thatterminate on corpus allatum cells.     

Determination of allatostatin levels in relation to the gonadotropic cycle in the female of Blattella germanica (L.) (Dictyoptera, Blattellidae)

A polyclonal antibody against the allatostatin BLAST-3 (AGSDGRLYSFGL-NH₂) of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) has been raised and characterized, and an ELISA (enzyme-linked immunosorbent assay) for allatostatin quantification has been developed. Allatostatin contents in brain, midgut and haemolymph have been measured in females of B. germanica during the first gonadotropic cycle. Brain allatostatin content increases steadily from adult emergence to the formation of the first ootheca. The values range from 2 ng/brain on the day of adult emergence to 25 ng/brain when the insect forms the ootheca 8 days later. In the midgut, the pattern is similar but the values are about half those of the brain. Allatostatin concentrations in the haemolymph after HPLC separation are in the nanomolar range. The occurrence of allatostatins in the haemolymph suggests that these peptides can act through a humoral pathway, as well as via nerves. The allatostatin content of both brain and midgut are high while the female is transporting the ootheca, which suggests that these peptides could be related to the low metabolic status characterising the period of oothecal transport.     

初探E2F1 对基因组内跨膜信号转导基因的调控作用

目的:从基因组全局性角度研究E2F1对包括离子通道和G蛋白偶联受体在内的重要的跨膜信号转导基因的调控作用。方法:对从TRED和IUPHAR获取的E2F1靶基因数据和基因组离子通道及G蛋白偶联受体基因数据进行数据联配,获取E2F1调控的离子通道基因(ICG)和G蛋白偶联受体基因,并对调控基因进行家族富集性分析和组织特异性分析。结果:发现E2F1对7个ICG具有调控作用,且具有钾离子通道富集性,调控的离子通道基因具有心脏、脑、消化系统组织表达特异性。获得的11个受E2F1调控的G蛋白偶联受体基因家族富集性不明显,组织特异性表达不一致。结论:E2F1可能通过对钾离子通道基因的表达调控,实现对相应组织的作用机理影响,相应调控作用的紊乱也将导致心脏、脑或消化系统疾病,但难以确定E2F1对GPCR的调控作用效果。     

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae)

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.     

Allatostatins of the tiger prawn,Penaeus monodon (Crustacea: Penaeidea)

More than 40 peptides belonging to the -Y/FXFGL-NH(2) allatostatin superfamily have been isolated and identified from the central nervous system (CNS) of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea). The peptides can be arranged in seven sub-groups according to the variable post-tyrosyl residue represented by Ala, Gly, Ser, Thr, Asn, Asp, and Glu. Two of the residues (Thr and Glu) have not been observed in this position previously in either insects or crustaceans. Also reported for the first time for allatostatins, two of the peptides are N-terminally blocked by a pyroglutamic acid residue. The yields of certain peptides with similar amino acid sequences to each other were, in some instances, very different. As an example, the yield of ANQYTFGL-NH(2) was 2pmol, compared with ASQYTFGL-NH(2), with a yield of 156 pmol. There are several possibilities to account for this. If, as in all species so far investigated, there is a single allatostatin gene in P. monodon, then it would appear that different sub-populations have contributed mutant forms of particular peptides to the extract. Another, less likely possibility is that this species has more than one allatostatin gene, producing a variable array of peptides albeit in different molar ratios. Several peptides were present apparently as a result of the loss of one or more residues at the N-terminus of a larger form, either due to N-terminal degradation or specific post-translational processing. The number of peptides identified exceeds that for any other insect or crustacean species previously investigated. None is identical to any of the 60-70 insect allatostatins so far identified, and only three are common to other crustaceans. Immunohistochemical study of the CNS of P. monodon, with the same antisera as used to monitor the purification, confirms the widespread nature and complexity of allatostatinergic neural pathways in arthropods. Thus, all neuromeres of the brain, and all except one of the ventral cord ganglia, possess allatostatin neurons and extensive areas of allatostatin-innervated neuropile. In addition to the cytological evidence that the allatostatins act as neurotransmitters, associated with tissues as varied as eyes and legs, their presence in neurohemal areas such as the sinus gland and the perineural sheath of the thoracic ganglia suggests a neuroendocrine function. As well as posing a challenge to physiologists assigning specific functions to the allatostatins, their extensive intra-species multiplicity, linked to their inter-species variability, also presents a complex problem to geneticists and evolutionists.