Orcokinin: a novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus.

A myotropic peptide, named orcokinin, was isolated from approximately 1200 abdominal nerve cords of the crayfish, Orconectes limosus. Its amino acid sequence was determined as follows: Asn-Phe-Asp-Glu-Ile-Asp-Arg-Ser-Gly-Phe-Gly-Phe-Asn. This structure was confirmed by synthesis. There is no sequence similarity to any known neuropeptide. Orcokinin exhibits high potency on the crayfish hindgut, enhancing both frequency and amplitude of spontaneous contractions. The threshold of biological activity in vitro was determined to be approximately 5 x 10(-11) M.     

Insect peptide hormones, an overview of the present literature.

A comprehensive overview of the recent state of the art of insect peptide hormones with chemical structures is presented. An increased interest in insect neuropeptides and dynamic development of that research area has been influenced by a rapid improvement of instrumentation necessary for isolation and structural characterization. Several research teams have studied the relationships between biological properties of insect and vertebrate peptide hormones. Thus hormones from the AKH family can be considered glucagon counterparts, whereas the myotropic hormones such as proctolin and Lem-PK (LPK) are a substance P equivalent. Insect melanization hormones Bom-MRCH in their structural characteristics and properties resemble those of mammal MSH, and leucosulfakinins Lem-SK-I and -II show some similarities with gastrin II and cholecystokinin. Bombyxin-II (Bom-PTTH-II) reveals a structural homology with human insulin and similar biological properties to adenocorticotropic mammal hormone. Allatostatin (Dip-JHS-I) may be compared to somatostatin as it can be inferred from the observations that this peptide modulates JH secretion in cockroach, Blattella germanica. Determination of the primary structure of eclosion hormones Mas-EH and Bom-EH-II as well as the amino acid sequence of allatotropin and allatostatin is a significant contribution to the understanding of the molecular mechanisms of metamorphosis and insect development.     

Orcokinin immunoreactivity in the accessory medulla of the cockroach Leucophaea maderae

The accessory medulla is the master circadian clock in the brain of the cockroach Leucophaea maderae and controls circadian locomotor activity. Previous studies have demonstrated that a variety of neuropeptides are prominent neuromediators in this brain area. Recently, members of the orcokinin family of crustacean neuropeptides have been identified in several insect species and shown to be widely distributed in the brain, including the accessory medulla. To investigate the possible involvement of orcokinins in circadian clock function, we have analyzed the distribution of orcokinin immunostaining in the accessory medulla of L. maderae in detail. The accessory medulla is densely innervated by approximately 30 orcokinin-immunoreactive neurons with cell bodies distributed in five of six established cell groups in the accessory medulla. Immunostaining is particularly prominent in three ventromedian neurons. These neurons have processes in a median layer of the medulla and in the internodular neuropil of the accessory medulla and send axonal fibers via the posterior optic commissure to their contralateral counterparts. Double-labeling experiments have revealed the colocalization of orcokinin immunostaining with immunoreactivity for pigment-dispersing hormone, FMRFamide, Mas-allatotropin, and γ-aminobutyric acid in two cell groups of the accessory medulla, but not in the ventromedian neurons or in the anterior and posterior optic commissure. Immunostaining in the ventromedian neurons suggests that orcokinin-related peptides play a role in the heterolateral transmission of photic input to the pacemaker and/or in the coupling of the bilateral pacemakers of the cockroach.This study was supported by the Deutsche Forschungsgemeinschaft, grant HO 950/9.     

Myotropic effect of helicokinins,tachykinin-related peptides and Manduca sexta allatotropin on the gut of Heliothis virescens (Lepidoptera: Noctuidae)

Different insect neuropeptides (helicokinins, tachykinin-related and allatoregulating peptides) were investigated with regard to their myostimulatory effects using whole-gut preparations isolated from fifth instar Heliothis virescens larvae. The experiments demonstrated that representatives of all three peptide families are able to induce and amplify gut contractions in this species in a dose-dependent manner. Structure-activity studies (alanine scan, D-amino acid scan and truncated analogues) with the helicokinin Hez-K1 supported the finding, that the core sequence for biological activity of kinins is the amidated C-terminal pentapeptide (FSPWG-amide). Similar investigations with insect tachykinin isolated from Leucophaea madera (Lem-TRP1) revealed that the minimum sequence evoking a physiological gut response in H. virescens is the amidated hexapeptide (GFLGVR-amide), which represents the conserved amino acid sequence for Leucophaea TRPs in general. The peptide concentration causing a half-maximal gut contraction (EC(50)) for Lem-TRP1 was about 26 nM. Although the potency of Lem-TRP1 was 9-fold lower compared with Hez-KI (EC(50): 3 nM), the maximal tension of the gut obtained with Lem-TRP1 was 1.7-fold higher compared with Hez-KI. The EC(50) of Manduca sexta allatotropin (Mas-AT; 79 nM) was of lowest potency among all three peptides tested. In a pharmacological study, co-incubation experiments with Lem-TRP1, Hez-KI or Mas-AT and compounds interfering with signal transduction pathways were employed to investigate the mode of action of the myotropic effects of these peptides. Cadmium and the protein kinase C (PKC) inhibitor tamoxifen attenuated the contractile effects of all three peptides tested. The data suggest that in the gut muscle of H. virescens the myotropic peptides bind to G-protein-coupled receptors that cause contraction by promoting the entry of extracellular calcium mediated by a PKC involved pathway.     

Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab,Cancer borealis

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab (Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750-3000Da. Comparison of the molecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala(13)]-orcokinin, and [Val(13)]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic analysis of the crustacean nervous system.     

Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure-function studies

Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2-8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK.     

HOM/Hox genes of Artemia: implications for the origin of insect and crustacean body plans

BACKGROUND: Insects and crustaceans are generally assumed to derive from a segmented common ancestor that had a distinct head but uniform, undifferentiated trunk segments. The subdivision of the body into functionally distinct regions (e.g. thorax and abdomen) is thought to have evolved independently in these two lineages. In insects, the differences between segments in the trunk are controlled by the Antennapedia-like genes of the homeotic gene clusters. Study of these genes in crustaceans should provide a basis for comparing body plans and assessing their evolutionary origin. RESULTS: Using a polymerase chain reaction (PCR) / inverse PCR strategy, we have isolated six genes of the HOM/Hox family from the crustacean Artemia franciscana. Five of these are clearly identifiable as specific homologues of the insect homeotic genes Dfd, Scr, Antp, Ubx and abdA. The sixth appears to have no close counterpart in insects. CONCLUSION: All the homeotic genes that specify middle body regions in insects originated before the divergence of the insect and crustacean lineages, probably not later than the Cambrian (about 500 million years ago). A commonly derived groundplan may underlie segment diversity in these two groups.     

Imaging mass spectrometry of neuropeptides in decapod crustacean neuronal tissues

Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.     

fushi tarazu: a Hox gene changes its role

The Hox genes play a role in anteroposterior axis specification of bilaterian animals that has been conserved for more than 600 million years. However, some of these genes have occasionally changed their roles in evolution. For example, the insect gene fushi tarazu (ftz), although localised in the Hox cluster, no longer acts as a Hox gene, but is involved in segmentation and nervous system development. Recent data of Mouchel-Vielh et al., and Hughes and Kaufman on ftz homologues in a crustacean and a myriapod, respectively, shed new light onto the evolution of this gene.     

Increasing parasitism by the German yellow jacket wasp, Paravespula germanica, on dairy cattle in Israel

During the past two decades, parasitism by the German yellow jacket wasp, Paravespula germanica , on lactating dairy cattle has occurred in Israel during August to October annually, affecting up to 65% of cows in certain herds. The nibbled and exposed tissues of teats and sometimes udders become infested by bacteria, especially Streptococcus dysgalactiae and Actinomyces pyogenes, causing clinical and subclinical mastitis. Normally, German wasps are primarily insect predators, but the urbanization around many dairy farms has reduced open space and associated standard food sources, i.e. insects, plants and carcasses. This has resulted in P. germanica nesting more often on dairy farms. In some instances, when high densities of P. germanica correspond with scarcity of prey, a segment of the wasp population preys primarily on the older and heavier cows with weak defensive behaviour. The teat feeding colonies of P. germanica may have an advantage, in that they are less dependent on fluctuations in the number of prey insects.     

Occurrence of crustacean cardioactive peptide (CCAP) in the nervous system of an insect,Locusta migratoria

Summary Using a radioimmunoassay developed for the determination of crustacean cardioactive peptide (CCAP), immunoreactive material was detected in extracts of locust nervous tissue. Serial dilutions of a brain extract gave a displacement curve parallel to the CCAP standard curve. One locust nervous system was calculated to contain approximately 1.4 pmol CCAP-like material.In order to investigate whether the immunoreactive substance was similar or identical to the crustacean neuropeptide, isolation and complete characterization was carried out using 800 locust nervous systems. The isolation procedure consisted of pre-purification of the crude extract on a Sep-Pak cartridge, affinity chromatography on a column which was prepared by coupling of anti-CCAP antibody to CNBr-activated Sepharose, and reversed phase high performance liquid chromatography (HPLC). In the HPLC-profile immunoreactivity was confined to a single peak which co-chromatographed with authentic CCAP. The peptide was carboxymethylated and analyzed in an automated gas-phase sequencer. Its amino acid sequence, is identical to that of CCAP fromCarcinus maenas.Synthetic CCAP was tested on the isolated locust hindgut in vitro. The peptide proved to be a potent enhancer of gut contractions, with a significant effect being observable at concentrations of 10^–10 M. It is concluded that in the locust CCAP may function as a myotropic peptide.     

Identification of leucomyosuppressin in the German cockroach, Blattella germanica, as an inhibitor of food intake

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.     

The multi-tasking gut epithelium of insects

The insect gut epithelium plays a vital role in multiple processes, including nutrition, immunity and osmoregulation. Recent research is revealing the molecular and biochemical basis of these functions. For example, the pattern of nutrient acquisition by the gut epithelium is integrated into the overall regulation of nutrient allocation, as illustrated by evidence for systemic controls over expression of key genes coding digestive enzymes and transporters in carbohydrate acquisition; and the abundance and diversity of microorganisms in the gut lumen is regulated by multiple molecular properties of the gut epithelial cells, including the synthesis of enzymes that produce reactive oxygen species and anti-microbial peptides. These traits are underpinned by the function of the gut epithelium as a selective barrier which mediates the controlled movement of water, ions, metabolites and macromolecules between the gut lumen and insect tissues. Breakdown of the gut epithelial barrier has been implicated in muscle paralysis of insects at low temperatures (chill coma) and in aging. The key challenge for future research is to understand how the multiple functions of the insect gut epithelium are integrated by signaling interactions among epithelial cells, the gut microbiota and other insect organs.     

CLARITY组织透明化技术在蜜蜂肠道组织上的运用

【目的】以西方蜜蜂Apis mellifera工蜂肠道为例探究组织透明化技术--丙烯酰胺交联替换脂质透明硬化成像/免疫染色/原位杂交兼容组织水凝胶(clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue-hYdrogel, CLARITY)在昆虫组织上的应用,确定CLARITY与荧光原位杂交(FISH)相结合在昆虫肠道组织透明化中的适用性。【方法】依照CLARITY技术操作程序,用水凝胶固定西方蜜蜂肠道,并以被动方式透明化,再用靶向东方蜜蜂微孢子虫Nosema ceranae 16S rRNA带异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记和靶向真核细胞18S rRNA带Texas RED标记的寡核苷酸荧光探针进行肠道组织的荧光原位杂交,然后用DAPI(蓝色)进行细胞核复染,通过激光共聚焦显微镜观察透明化的染色组织。【结果】首次成功将西方蜜蜂肠道组织透明化。在激光共聚焦显微镜下,观察到马氏管的原始分布形态,以及东方蜜蜂微孢子虫在中肠末端分布更密集的空间分布特征,并实现了对肠道组织的3D重构。【结论】CLARITY能应用于蜜蜂肠道组织透明化,透明化组织能进行原位杂交和激光共聚焦观察。CLARITY和FISH相结合免除抗体制备和石蜡切片的麻烦,直观展示肠道内部的真实状态,为昆虫生理病理研究提供了一种可靠特异的标记方法。     

Food habits of fishes in the surf zone of a sandy beach at Sanrimatsubara, Fukuoka Prefecture, Japan

To clarify the feeding habits of fishes in surf zones, the gut contents of 19 fish species collected in the surf zone of a sandy beach at Sanrimatsubara, western Japan, were examined. Ontogenetic changes in food preference were recognized in seven species (Mugil cephalus cephalus, Lateolabrax latus, Sillago japonica, Paralichthys olivaceus, Paraplagusia japonica, Takifugu poecilonotus, and Takifugu niphobles). A cluster analysis based on dietary overlaps showed that the surf zone fish assemblage comprised six trophic groups (zooplankton, benthic and epiphytic crustacean, detritus, polychaete, fish, and insect feeders). Of these, the most abundant trophic group was zooplankton feeders, along with benthic and epiphytic crustacean feeders.     

Origin and evolution of arthropod hemocyanins and related proteins

Arthropod hemocyanins are large, multimeric, (n x 6) copper-containing proteins that deliver oxygen in the haemolymph of many chelicerate, crustacean, myriapod, and also possibly some insect species. The arthropod hemocyanins belong to a large protein superfamily that also includes the arthropod phenoloxidases, certain crustacean and insect storage proteins (pseudo-hemocyanins and hexamerins), and the insect hexamerin receptors. Here I summarise the present knowledge of the origin, functional adaptations, and evolution of these proteins. Arthropod and mollusc hemocyanins are, if at all, only distantly related. As early as in the arthropod stem line, the hemocyanins emerged from a phenoloxidase-like enzyme. The evolution of distinct hemocyanin subunits, as well as the formation of multi-hexamers occurred independently within the arthropod subphyla. Hemocyanin subunit evolution is strikingly different in the Chelicerata, Myriapoda and Crustacea. Hemocyanins individually gave rise to two distinct copper-less storage proteins, the insect hexamerins and the crustacean pseudo-hemocyanins (cryptocyanins). The receptor responsible for the uptake of hexamerin by the larval fat body of the insects emerged from a hexamerin-precursor. Molecular phylogenetic analyses show a close relationship of the crustacean and insect proteins, providing strong support for a pancrustacean taxon, while structural data suggest a myriapod-chelicerate clade.     

Discovery and characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques

The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules-neuropeptides. Via a multifaceted mass spectrometry (MS) approach, 70 neuropeptides were identified including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH) precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), pigment dispersing hormone (PDH), proctolin, RFamides, RYamides, and HL/IGSL/IYRamide. Among them, 15 novel orcokinins, 9 novel CPRPs, 1 novel orcomyotropin, 1 novel Ork/Orcomyotropin-related peptide, and 1 novel PDH were de novo sequenced via collision induced dissociation (CID) from the SG of a model organism Callinectes sapidus. Electron transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and higher charge state. Capillary isoelectric focusing (CIEF) was employed for separation of members of the orcokinin family, which is one of the most abundant neuropeptide families observed in the SG. Collectively, our study represents the most complete characterization of neuropeptides in the SG and provides a foundation for future investigation of the physiological function of neuropeptides in the SG of C. sapidus.     

The Leucophaea maderae hindgut preparation: A rapid and sensitive bioassay tool for the isolation of insect myotropins of other insect species

The isolated hindgut preparation of the cockroach, Leucophaea maderae has provided an effective bioassay tool for the isolation of certain structural types of insect myotropic peptides. Initially, the preparation was used to monitor excitatory and inhibitory activities of numerous HPLC fractions in a study that resulted in the structural characterization of 12 Leucophaea neuropeptides. Subsequently, the preparation was used as the bioassay for the isolation and structural characterization of myotropic neuropeptides of the house cricket, Acheta domesticus, and the locust, Locusta migratoria. Five novel myotropic peptides from the cricket were structurally characterized, and 32 separate myotropic compounds were isolated from nervous tissue of the locust. At present, 8 of the locust peptides have been structurally characterized. Isolation studies using this bioassay have been responsible for the discovery of 25 unique neuropeptides, 4 new peptide families, and the initial demonstration of the natural analog phenomenon in insects.     

Identification of plasmalogen in the gut of silkworm (Bombyx mori)

Herbivorous insect species are constantly challenged with endogenous and exogenous oxidative stress. Consequently, they possess an array of antioxidant enzymes and small molecular weight antioxidants. Lipid-soluble small molecular antioxidants, such as tocopherols, have not been well studied in insects but may play important antioxidant roles. In this study, we identified plasmalogen phosphatidylethanolamines (pPEs) as well as α-, β/γ-, δ-tocopherol in the larvae of the silkworm Bombyx mori by LCMS analyses and examined their distribution. Plasmalogen are reported to inhibit the metal ion induced oxidation. The composition of tocopherols was the same among gut contents, gut tissues, and the other tissues. However, plasmalogens, a unique class of glycerophospholipids rich in polyunsaturated fatty acids and containing a vinyl ether bond at the sn-1 position, were mainly distributed in gut tissues. Plasmalogens might protect gut tissues from oxidation stress.