Heather G. Marco

Structure, Function and Mode of Action of Select Arthropod Neuropeptides

ABSTRACT: This overview summarizes important features of the majority of neuropeptide families that occur in two species-rich and widely-radiated arthropod taxa, the crustaceans and the insects. The neuropeptides may act as true neurohormones, which are released into the circulation, or as local neurotransmitter and/or neuromodulator. By comparing the primary structures of members of peptide families, the biosynthesis (including preprohormone structure and the peptidergic control of release), the structures of the receptors and transduction of the message via second messenger systems, the inactivation and the multiple functions of selected neuropeptides, we want to draw the reader’s attention to the following main conclusions: 1) neuropeptides are important physiological regulators in arthropods; 2) neuropeptides can be structurally and functionally highly conserved in major arthropod groups (for example, proctolin, crustacean cardioactive peptide), or where peptide isoforms exist, there may be different scenarios: - (i) in the case of identical isoforms in insects and crustaceans, the peptide function may have changed in the two taxa (for example, red pigment-concentrating hormone affects pigmentation in crustaceans but mobilizes lipids in insects), - (ii) the isoforms are different in the two taxa and may have the same effect (for example, the ion-transporting peptide in insects and the crustacean hyperglycaemic hormone in crustaceans both play a role in osmoregulation), - (iii) the structurally different isoforms have different functions in the two taxa (for example, pigment-dispersing hormone affects pigmentation in crustaceans, whereas pigment-dispersing factor affects circadian rhythmicity in insects.

The adipokinetic hormones of Heteroptera: a comparative study.

The adipokinetic hormones (AKHs) from 15 species of heteropteran Hemiptera (encompassing eight families, six superfamilies and three infraorders) have been isolated and structurally identified using liquid chromatography coupled with mass spectrometry. None of the structures are novel and all are octapeptides. These peptide sequence data are used, together with the previously available AKH sequence data on Heteroptera, to create a larger dataset for comparative analyses. This results, in total, in AKH sequences from 30 species (spanning 13 families), which are used in a matrix confronted with the current hypotheses on the phylogeny of Heteroptera. The expanded dataset shows that all heteropterans have octapeptide AKHs; three species have two AKHs, whereas the overwhelming majority have only one AKH. From a total of 11 different AKH peptides known from Heteroptera to date, three AKHs occur frequently: Panbo‐red pigment‐concentrating hormone (RPCH) (×10), Schgr‐AKH‐II (×6) and Anaim‐AKH (×4). The heteropteran database also suggests that particular AKH variants are family‐specific. The AKHs of Heteroptera: Pentatomomorpha (all terrestrial) are not present in Nepomorpha (aquatic) and Gerromorpha: Gerridae (semiaquatic); AKHs with a Val in position 2 are absent in the Pentatomomorpha (only AKHs with Leu2 are present), whereas Val2 predominates in the nonterrestrial species. An unexpected diversity of AKH sequences is found in Nepomorpha, Nepoidea, Nepidae and Nepinae, whereas Panbo‐RPCH (which has been identified in all infraorders of decapod crustaceans) is present in all analysed species of Pentatomidae and also in the only species of Tessaratomidae investigated. The molecular evolution of Heteroptera with respect to other insect groups and to crustaceans is discussed

Neuropeptide Action in Insects and Crustaceans

Physiological processes are regulated by a diverse array of neuropeptides that coordinate organ systems. The neuropeptides, many of which act through G protein–coupled receptors, affect the levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues. In this perspective, their roles in molting, osmoregulation, metabolite utilization, and cardiovascular function are highlighted. In decapod crustaceans, inhibitory neuropeptides (molt‐inihibiting hormone and crustacean hyperglycemic hormone) suppress the molting gland through cAMP‐ and cGMP‐mediated signaling. In insects, the complex movements during ecdysis are controlled by ecdysis‐triggering hormone and a cascade of downstream neuropeptides. Adipokinetic/hypertrehalosemic/hyperprolinemic hormones mobilize energy stores in response to increased locomotory activity. Crustacean cardioacceleratory (cardioactive) peptide, proctolin, and FMRFamide‐related peptides act on the heart, accessory pulsatile organs, and excurrent ostia to control hemolymph distribution to tissues. The osmoregulatory challenge of blood gorging in Rhodnius prolixus requires the coordinated release of serotonin and diuretic and antidiuretic hormones acting on the midgut and Malpighian tubules. These studies illustrate how multiple neuropeptides allow for flexibility in response to physiological challenges.

Predicted versus expressed adipokinetic hormones, and other small peptides from the corpus cardiacum–corpus allatum: A case study with beetles and moths

This mass spectrometric study confines itself to peptide masses in the range of 500-1500Da. Adipokinetic hormones (AKHs) that are predicted from the genome of the red flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori, are shown to exist as expressed peptides in the corpora cardiaca (CC) of the respective species as evidenced by various mass spectrometric methods. Additionally, some related species were included in this study, such as the tenebrionid beetles Tribolium brevicornis and Tenebrio molitor, as well as the moths Spodoptera frugiperda, Spodoptera littoralis, Mamestra brassicae and Lacanobia oleracea, to investigate whether AKH peptides are structurally conserved in the same genus or family. Interestingly, the AKH peptide of T. brevicornis is identical to that of T. molitor but not to the ones of its close relative T. castaneum. Moreover, other peptides in T. brevicornis, such as various FXPRL amides (=pyrokinins), also match the complement in T. molitor but differ from those in T. castaneum. All the CC of beetles lacked the signal for the mass of the peptide corazonin. All moths have the nonapeptide Manse-AKH expressed in their CC. In addition, whereas the silk moth has the decapeptide Bommo-AKH as a second peptide, all other moths (all noctuids) express the decapeptide Helze-HrTH. In M. brassicae and L. oleracea a novel amidated Gly-extended Manse-AKH is found as a possible third AKH. The noctuid moth species also all express the same FLRF amide-I, corazonin, and a group-specific isoform of a gamma-PGN-(=gamma-SGNP) peptide. In L. oleracea, however, the latter peptide has a novel sequence which is reported for the first time, and the peptide is code-named Lacol-PK.

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.

Biological activity of the predicted red pigment-concentrating hormone of Daphnia pulex in a crustacean and an insect.

The elucidation of the genome of the waterflea Daphnia pulex made it possible to search for orthologue genes for the crustacean red pigment-concentrating hormone (named Panbo-RPCH after the species Pandalus borealis in which the red pigment-concentrating hormone was first identified); Panbo-RPCH is a member of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) peptide family. The information pointed to a putative mature RPCH octapeptide in D. pulex with the primary sequence of pGlu-Val-Asn-Phe-Ser-Thr-Ser-Trp amide (=Dappu-RPCH). Since Panbo-RPCH is endogenous in decapod crustaceans and in the green stink bug Nezara viridula, we assayed Dappu-RPCH in the shrimp Palaemon pacificus and in N. viridula. Here we show that this variant member of the AKH/RPCH family has no activity to concentrate the red, brown, yellow and blue pigments in the epithelium of the shrimp at physiological doses but is effective in mobilising lipids in the green stink bug N. viridula. Moreover, since Panbo-RPCH and Dappu-RPCH differ structurally at three positions, viz. Leu(2) to Val(2); Pro(6) to Thr(6); Gly(7) to Ser(7), we tested other members of the peptide family which have single or dual amino acid substitutions at the appropriate positions, for their chromatophorotropic action at physiological doses. These studies show unequivocally that a single change from Gly(7) to Ser(7) (as in the peptide Corpu-AKH) does not inflict any loss of biological activity, and the same is true for a single change from Pro(6) to Thr(6) (represented by the peptide Schgr-AKH-II). The change from Leu(2) to Val(2) (embodied in Manto-CC), however, is accompanied with a substantial loss of chromatophorotropic activity; combinations of Val(2) and Ser(7) (as in Anaim-AKH) or Val(2) and Thr(6) (as in Grybi-AKH) result in almost complete loss of biological activity. Dappu-RPCH with its three substitutions is not active at all in the shrimp at the tested concentration range of up to 30 pmol.

Substrate usage and its regulation during flight and swimming in the backswimmer, Notonecta glauca

Abstract.  The metabolites that are generally used by insects during exercise are present in quite different concentrations in the haemolymph of the backswimmer Notonecta glauca L. Lipids are most abundant (between 10 and 20 mg/mL), whereas carbohydrates (2–3 mg/mL) and proline (approximately 1 mg/mL) are at very low concentrations. Injection of an extract of conspecific corpora cardiaca causes pronounced hyperlipaemia in the backswimmer. A neuropeptide with the same effect was isolated from the corpora cardiaca in a single high‐performance liquid chromatography (HPLC) step; the primary sequence was deduced from mass spectrometric measurements (matrix‐assisted laser desorption/ionization‐time of flight and electrospray quadrupol time‐of‐flight mass spectrometry) of whole corpora cardiaca, and the mass was confirmed in the HPLC fraction that had adipokinetic activity. The biologically active octapeptide has the sequence pGlu‐Val‐Asn‐Phe‐Ser‐Pro‐Ser‐Trp amide, which was characterized previously from the corpora cardiaca of the Emperor dragonfly, Anax imperator, and denoted Anaim‐adipokinetic hormone (AKH). The synthetic Anaim‐AKH peptide causes lipid mobilization when injected at a dose of 1 pmol into N. glauca. When other synthetic AKH members that occur in Hemiptera are injected into N. glauca at the same dose, the hyperlipaemic responses are significantly lower than after injection of Anaim‐AKH. Because only lipids increase upon activity, such as continuous swimming for 1 h or during a 1‐h rest period after a 3‐min flight episode in the laboratory, it is assumed that Anaim‐AKH serves as a true adipokinetic hormone in the backswimmer during bouts of natural swimming and flight.

Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii.

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.

A comparative immunocytochemical study of the hyperglycaemic, moult-inhibiting and vitellogenesis-inhibiting neurohormone family in three species of decapod Crustacea

Abstract Eyestalks of the palinuran species Jasus lalandii and Panulirus homarus, and the brachyuran species Carcinus maenas, were examined with antisera raised against purified crustacean hyperglycaemic hormone (cHH) of the astacidean species Homarus americanus and Procambarus bouvieri, as well as the brachyuran species Cancer pagurus. Other antisera used in this investigation were raised against purified moult-inhibiting hormone (MIH) of C. pagurus and vitellogenesis-inhibiting hormone (VIH) of H. americanus. Positive immunoreactions to all the antisera were localised in perikarya of the X-organ and the axon terminals in the sinus gland of all the crustaceans investigated. These results illustrate the existence of an immunological similarity, detectable at the immunocytochemical level, between the cHH/MIH/VIH neurohormones of the Astacidae, Palinura and Brachyura infraorders. Furthermore, results from consecutive tissue sections indicate that cHH, MIH and VIH are co-localised in a subpopulation of X-organ neurons.

A novel adipokinetic peptide in a water boatman (Heteroptera, Corixidae) and its bioanalogue in a saucer bug (Heteroptera, Naucoridae).

The corpora cardiaca (CC) of two water bug species, the water boatman Corixa punctata and the saucer bug Ilyocoris cimicoides, contain a substance that cause hyperlipemia in the migratory locust. The primary sequence of one octapeptide belonging to the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family was deduced from the multiple MS(N) electrospray mass data of CC material from each species. Whereas the saucer bug contains the known octapeptide pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Anaim-AKH, the water boatman has a novel peptide identified as pGlu-Leu/Ile-Asn-Phe-Ser-Pro-Ser-Trp amide, code-named Corpu-AKH. The ambiguity about the amino acid at position 2, i.e. Leu or Ile, in Corpu-AKH was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with the synthetic peptide containing Leu at position 2. Functionally, the peptides regulate lipid mobilization, as evidenced by an adipokinetic effect after injecting synthetic Anaim-AKH and Corpu-AKH into the respective acceptor species. Swimming activity of I. cimicoides also causes hyperlipemia.