fmrfamide and leucomyosuppressin

Insect myosuppressins and myosuppressin analogues were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Acyrthosiphon pisum myosuppressin (Acypi-MS) and leucomyosuppressin (LMS) had significant dose-dependent effects (0.1-0.5μg peptide/μl diet) on feeding suppression, mortality, reduced growth and fecundity compared with control insects, but Acypi-MS was more potent than LMS. One hundred percent of aphids had died after 10days of feeding on 0.5μg Acypi-MS/μl diet whereas 40% of aphids feeding on 0.5μg LMS/μl diet were still alive after 13days. Myosuppressins were degraded by aphid gut enzymes; degradation was most likely due to a carboxypeptidase-like protease, an aminopeptidase and a cathepsin L cysteine protease. The estimated half-life of Acypi-MS in a gut extract was 30min, whereas LMS was degraded more slowly (t½=54min). No toxicity was observed when the analogues δR(9) LMS and citrolline(9) Acypi-MS or FMRFamide were fed to the pea aphid. These findings not only help to better understand the biological effects of myosuppressins in aphids but also demonstrate the potential use of myosuppressins in a strategy to control aphid pests.

Topics: Animals; Aphids; Fertility; FMRFamide; Insect Control; Neuropeptides

The potency (muscle force-generated) of a number of long-chain RFamide neuropeptides was examined in mechanical experiments with the radular-retractor and radular-sac muscles of gastropods Buccinum undatum and Neptunea antiqua. Many of the heptapeptides, octapeptides and the decapeptide LMS were found to induce greater contraction than FMRFamide in both smooth muscles and in both species. RFamide neuropeptides interacted with the neurotransmitter acetylcholine in an additive way and RFamide-induced contractions were inhibited by the neuromodulator serotonin. Pre-treatment with a calcium-free saline completely abolished acetylcholine-induced responses but only partially inhibited RFamide responses in the muscles, suggesting that acetylcholine acts to cause influx of extracellular calcium for contraction. In contrast, RFamide neuropeptides may mobilise intracellular calcium to maintain sustained tonic force in calcium-free conditions. This suggests that an additional involvement of a fast calcium channel may be present in the RFamide responses, since loss of the usual superimposed twitch activity is observed. Force regulation in these muscles appears to result from a complex interaction of RFamide neuropeptides with the primary transmitter acetylcholine and the neuromodulator serotonin.

Topics: Acetylcholine; Animal Structures; Animals; Calcium; FMRFamide; Muscle Contraction; Muscle, Smooth; Neuropeptides; Oligopeptides; Serotonin; Snails; Sodium Chloride; United Kingdom

The feeding pattern of the adult female of Blattella germanica peaks in the middle of the vitellogenic cycle. Following the hypothesis that a factor inhibiting gut peristalsis also inhibits food intake and is involved in the regulation of feeding, we searched for the most powerful myoinhibitory peptide in brain extracts from B. germanica females collected after the peak within the feeding cycle. Through HPLC purification and sequence analysis, we obtained the peptide leucomyosuppressin (LMS): pQDVDHVFLRFamide. LMS elicited a powerful myoinhibitory effect on B. germanica foregut and hindgut, with ED(50) values around 10(-10) M. In addition, it inhibited food intake in vivo in a dose-dependent manner at doses between 5 and 50 microg. The study of the distribution of ingested food in the foregut, midgut and hindgut of B. germanica females treated with LMS showed that food accumulates in the foregut, which may be due to the myoinhibitory effects of the peptide. We propose that this accumulation inhibits food intake because of the persistence of the signals from gut stretch receptors.

Topics: Animals; Behavior, Animal; Biological Assay; Blattellidae; Brain; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Feeding Behavior; Female; FMRFamide; Mass Spectrometry; Neuropeptides; Peptides; Pyrrolidonecarboxylic Acid; Time Factors

FMRFamide-related peptides are common to a wide variety of invertebrate species, including helminths and arthropods. In arthropods, five distinct FMRFamide-related peptide subfamilies are recognised: the myosuppressins, extended-FLRFamides, -FMRFamides, -RFamides, and sulfakinins, members of which induce potent and diverse myotropic effects. Whilst >80 FMRFamide-related peptides have been identified in nematodes, only four FMRFamide-related peptides have been characterised from flatworms. The Ascaris suum ovijector/body wall bioassay and the Procerodes littoralis muscle fibre bioassay have proved both reliable and sensitive systems for assessing the functional activities of FMRFamide-related peptides in vitro, and data describing the effects of native FMRFamide-related peptides in these systems are rapidly accumulating. This is the first study to determine the cross-phyla activities of non-native FMRFamide-related peptides in both nematode and flatworm species. In the present study, the effects of 10 arthropod FMRFamide-related peptides (leucomyosuppressin [pQDVDHVFLRFamide], schistoFLRFamide [PDVDHVFLRFamide] and truncated analogues [HVFLRFamide and VFLRFamide], lobster peptide I [TNRNFLRFamide], lobster peptide II [SDRNFLRFamide], manducaFLRFamide II [GNSFLRFamide], manducaFLRFamide III [DPSFLRFamide], calliFMRFamide 4 [KPNQDFMRFamide] and perisulfakinin [EQFDDY(SO(3)H)GHMRFamide]), representing the five subfamilies, were examined on the body wall and ovijector of the parasitic porcine nematode, A. suum and dispersed muscle fibres from the free-living turbellarian, P. littoralis. The muscle activity of the ovijector was found to be modulated significantly by each of the arthropod FMRFamide-related peptides tested; the effects were concentration-dependent, reversible and repeatable. All but one (perisulfakinin) of the 10 arthropod FMRFamide-related peptides examined modulated significantly the activity of A. suum body wall muscle. In addition, all of the arthropod FMRFamide-related peptides examined induced potent concentration-dependent contractions of P. littoralis muscle fibres. These results reveal similarities in the ligand requirement(s) between FMRFamide-related peptide receptors within the Phyla Arthropoda, Nematoda and Platyhelminthes, and indicate significant receptor promiscuity, which highlights the potential of FMRFamide-related peptide receptors as legitimate targets for novel endectocidal agents.

Topics: Animals; Ascaris suum; FMRFamide; Invertebrate Hormones; Ligands; Muscle Contraction; Muscle Fibers, Skeletal; Neuropeptides; Oligopeptides; Peptide Fragments; Receptors, Invertebrate Peptide; Turbellaria

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.

Topics: Amino Acid Sequence; Amylases; Animals; Digestive System; Endopeptidases; FMRFamide; In Vitro Techniques; Insect Hormones; Larva; Lepidoptera; Leucine; Methionine; Molecular Sequence Data; Neuropeptides

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.

Topics: Animals; Chromatography, High Pressure Liquid; Cockroaches; Digestive System; Digoxigenin; Female; FMRFamide; Ganglia, Invertebrate; Immunohistochemistry; In Situ Hybridization; Insect Hormones; Nervous System; Neuropeptides; RNA, Messenger

We have previously shown differential expression of leucomyosuppressin (LMS) mRNA in apparent endocrine cells in the anterior region of midguts of the cockroach Diploptera punctata, using in situ hybridization. In contrast, other FMRFamide-related peptides, as revealed by immunohistochemistry, have been found most abundantly in the posterior region in both apparent endocrine cells and nerve tracts. Here, we partially purified extracts of anterior and posterior cockroach midguts, using HPLC coupled with radioimmunoassay, and found, among multiple FMRFamide-like immunoreactive fractions, one fraction co-eluting with LMS in both regions. The presence of a co-eluting fraction in the posterior region, in the absence of LMS mRNA positive endocrine cells suggests that LMS might therefore be present in nerve tracts running along the length of the midgut. Using a circular muscle contraction assay from different portions of midgut, we determined the effects of LMS, proctolin and a variety of other midgut peptides on contractions of the midgut of Diploptera. Proctolin caused a sustained tonic contraction in the anterior midgut, the amplitude of which was dose-dependent. In contrast, LMS, and its relative SchistoFLRFamide, reduced the amplitude of these contractions. LMS and SchistoFLRFamide also inhibited spontaneous phasic contractions, which were elicited by proctolin application in only a few preparations. Other postulated midgut peptides did not induce or inhibit contractions, nor augment the proctolin-induced contractions. The C-terminal truncated sequences of LMS, HVFLRFamide and VFLRFamide, were sufficient to reduce the amplitude of the proctolin-induced contractions. This work illustrates a possible physiological role for LMS in Diploptera midguts, in the passage of food along the alimentary canal.

Topics: Animals; Cockroaches; FMRFamide; Muscle Contraction; Nerve Tissue Proteins; Neuropeptides; Oligopeptides; Peptide Fragments; Peptides

The insect peptides leucomyosuppressin (pEDVDHVFLRFamide) and dromyosuppressin (TDVDHVFLRFamide) have identical chemical sequences with the exception of the N-terminal amino acid; both inhibit spontaneous contraction of insect visceral muscles. Neurons in the hypocerebral ganglion of horn fly, Hematobia irritans (L.), and stable fly, Stomoxys calcitrans (L.), were found to contain material immunoreactive to antiserum produced against the C-terminal of leucomyosuppressin, but not to the N-terminal of dromyossuppressin. Two large lateral clusters containing 8 cells, linked dorsally and ventrally by 2 chains of 6 cells, encircled the anterior surface of the proventriculus and were immunoreactive of leucomyosuppressin and FMRFamide antisera. Axons from these cells were traced to the wall of the aorta and over the surface of the proventriculus. Ultrastructural analysis revealed these cells contained a singular type of elementary secretory granule that contained material of relatively low electron density, both in the cell body and at the axon terminals.

Topics: Animals; Female; FMRFamide; Ganglia, Invertebrate; Immunoenzyme Techniques; Insect Hormones; Male; Muscidae; Neuropeptides