William G. Bendena

Allatostatins: Identification, Primary Structures, Functions and Distribution

Publisher Summary This chapter focuses on the neuropeptides of known amino acid sequence that inhibit juvenile hormone (JH) production by the corpora allata (CA). During insect development, juvenile hormone (JH) maintains the juvenile form and metamorphosis to the adult form appears to require a reduced titre of JH. The regulation of JH production involves many factors, both extrinsic and intrinsic. With the discovery of at least 30 different Diploptera punctata allatostatin-like peptides in several species of insect, most showing the characteristic pentapeptide Tyr-Xaa-Phe-Gly-Leu-NH2 carboxyl (C)-terminus, it is now clear that these allatostatins represent a unique family of neuropeptides that probably serve several different functions in insects and related arthropods. The occurrence of multiple allatostatin peptides has prompted the search for receptors of important peptides. The neurosecretory cells producing allatostatins are identified immunohistochemically with antibodies produced against synthetic allatostatins. The immunocytochemical localization of allatostatin-like peptides in interneurons of the central nervous system; in neurons that innervate visceral muscle and glands other than the corpora allata, neurohaernal organs; and in midgut cells suggests that allatostatins are multifunctional neuropeptides even though inhibition of JH synthesis by corpora allata in vitro was the only bioassay utilized in their isolations. The coding region that specifies an allatostatin gene in Diploptera punctata was identified and isolated by polymerase chain reaction (PCR). Study of the regulation of allatostatin release is possible now, because the peptides have been identified and sensitive methods have been developed to quantitate them. The search for neuropeptide regulators of JH synthesis by the CA has led to the identification of a peptide from the moth Munducu sexfa and quite a different family of peptides from the cockroaches, Diploptera punctata and Periplaneta americana.

Allatostatins: A Growing Family of Neuropeptides with Structural and Functional Diversity

Abstract: The high degree of conservation of the core sequence of the “cockroach‐types” of AST and their widespread distribution suggest that they should be considered a ubiquitous family of peptides within the invertebrates, regulating a range of important physiological processes. These functional processes, by either neural or humoral routes of action, include the inhibition of endocrine function, interneuronal functions, neuromodulatory roles, myotropic and myoendocrine roles, and direct action on biosynthetic pathways. The myomodulatory function appears to be conserved through evolutionary time, whereas the JH inhibitory activity appears to be confined to specific orders. This suggests that the myomodulatory role was the more ancestral of these two particular functions. Certainly, further purification and gene cloning as a means to precursor identification and functional analysis will be a prerequisite to understanding the diverse functions of this peptide family.

The effect of the thirteen Dip-allatostatins on myogenic and induced contractions of the cockroach (Diploptera punctata) hindgut

Abstract The Dip-allatostatins are a group of 13 neuropeptides, ranging in size from 6 to 18 amino acids, identified from a cloned cDNA sequence of Diploptera punctata . All 13 Dip-allatostatins inhibited both myogenic and proctolin-induced contractions of the hindgut of D. punctata . Dip-allatostatins were capable of reducing a 5 × 10 −6 M proctolin-induced contraction in a dose-dependent manner, with ED 50 values ranging from 1.7 × 10 −7 M for Dip-allatostatin 7 to greater than 1.3 × 10 −5 M for Dip-allatostatin 12. The effects of the Dip-allatostatins were reversible upon washing with saline. The free carboxylic acid (non-amidated) form of Dip-allatostatin 11, callatostatin 3 and callatostatin 5 showed a loss of inhibitory activity. In contrast to the inhibitory actions of the Dip-allatostatins on hindgut contractions, selected Dip-allatostatins (Dip-allatostatins 4, 6, 7, 10, 12), at concentrations ranging from 10 −7 M to 2 × 10 −5 M, did not alter the frequency or amplitude of myogenic contractions or inhibit proctolin-induced contractions of the cockroach oviduct muscle. Dip-allatostatins (Dip-allatostatins 1, 2, 4, 5, 6, 7) also had no effect on either myogenic or proctolin-induced contractions of the oviduct of Locusta migratoria .

Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.

FMRFamide-related peptides: a multifunctional family of structurally related neuropeptides in insects

Publisher Summary This chapter reviews the information available on the extensive FMRFamide (Phe-Met-Arg-Pheamide)-related peptides (FaRPs) family in insects. The chapter examines the principles behind peptide discovery and the way information can be used as a tool for further investigation. In addition, it puts into perspective the FaRP family from gene to behavior, examining the molecular biology, distribution, receptor activation through to biological activity, and integrative actions on behavior. FMRFamide is regarded as the primary member of an extensive family of peptides with diverse biological activities. The FaRP family may itself be divided into subfamilies, including the extended FMRFamides, FLRFamides, HMRFamides and related peptides. Several molecular biological techniques have been applied to isolate either new members of the FaRP gene family or the homologous gene from a different species using available FaRP-encoding genomic DNA or cDNA as a probe. The most common method is screening of libraries of DNA sequences under conditions of reduced stringency. However, more research is needed on this family of neuropeptides before a clear picture emerges as to their true physiological relevance.

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila.

Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met- and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.

Molecular characterization of a cDNA from Pseudaletia unipuncta encoding the Manduca sexta allatostatin peptide (Mas-AST)

A 15-residue neuropeptide, Manduca sexta allatostatin (Mas-AST), strongly inhibits juvenile hormone (JH) biosynthesis in vitro by corpora allata (CA) from Manduca fifth-stadium larvae and adult females as well as Helicoverpa zea adult females (Kramer et al., 1991 Proc. Natl. Acad. Sci (USA) 88, 9458-9462). In contrast, this study found that 1.0 microM Mas-AST has no JH biosynthesis inhibitory activity in Pseudaletia unipuncta sixth instar larvae or newly-emerged (day 0) adults but inhibited CA of 5-day-old adult females by 60%. From a P. unipuncta brain cDNA library, was isolated a cDNA that encodes a 125 amino acid polypeptide containing the Mas-AST sequence. Within the precursor, Mas-AST is situated at the carboxy terminus and is flanked by different dibasic proteolytic cleavage signals. The Pseudaletia gene specifying the Mas-AST peptide is present as a single copy per haploid genome. Expression of this gene was low in Pseudaletia sixth instar larvae, prepupae and early pupae but was relatively high in late pupae, and day 1 and 3 adults of both sexes. In day 5 adults, the relative transcript level appears to be maintained in females but declines in males. This pattern of Mas-AST expression does not correlate well with the profile of JH biosynthesis in Pseudaletia, which increases during the first 5 days of adult life, suggesting additional or alternative functions for this peptide.

Molecular cloning of the precursor cDNA for schistostatins, locust allatostatin-like peptides with myoinhibiting properties

The cDNA encoding the precursor polypeptide for schistostatins, allatostatin-like peptides which have been shown to inhibit peristaltic movements of the lateral oviducts of Schistocerca gregaria, has been cloned and sequenced. Translation of this sequence reveals the presence of a pre-proschistostatin consisting of 283 amino acids. It contains ten different peptide sequences which are flanked by dibasic cleavage sites and C-terminal amidation signals. Eight of these peptides were identical to the schistostatins (or Scg-ASTs) that were previously purified from Schistocerca gregaria brain extracts. Two novel peptide sequences were discovered. One of these is the first AST-like peptide which has a C-terminal valine residue. Two peptides contain within their sequence an internal dibasic site which suggests a possible role for alternative processing and/or degradation. The schistostatin precursor differs from cockroach pre-proallatostatins in size, in sequence and in organization. It contains a lower number of peptides (10 versus 13 or 14) which are interrupted only once by an acidic spacer region (versus four in Diploptera punctata and Periplaneta americana). Northern analysis showed the presence of a 2.4 kb mRNA band in the locust central nervous system and midgut. This indicates that schistostatins, like other ASTs, are a good example of insect brain/gut peptides.

IMMUNOCHEMICAL IDENTIFICATION AND EXPRESSION OF ALLATOSTATINS IN THE GUT OF DIPLOPTERA PUNCTATA

Abstract Dip-allatostatins are a family of related neuropeptides in the cockroach Diploptera punctata that were identified originally on the basis of their ability to inhibit juvenile hormone biosynthesis by corpora allata. These peptides were subsequently also found to inhibit myogenic and proctolin-induced contractions of the cockroach hindgut. Dip-allatostatin immunoreactive material is distributed in several sites within the brain, subesophageal and ventral ganglia and in nerves to the antennal pulsatile organ and hindgut muscles. We have demonstrated here that Dip-allatostatin immunoreactivity is also found in the proctodeal innervation and endocrine cells of the midgut. The endocrine cells which are distributed unequally in different regions of the midgut are also sites of Dip-allatostatin mRNA synthesis. The quantity of Dip-allatostatin immunoreactive material within and released from the gut was assessed by ELISA and RIA. The changes in Dip-allatostatin content in midgut and hindgut extracts during the first 8 days of the reproductive cycle, paralleled, to some degree, the changes noted previously in brain. Our data suggest that the midgut may be another source from which Dip-allatostatins are released into the hemolymph and this release may be regulated, in part, by nutritional status.

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta

Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10(-6)M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.