Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of <Emphasis Type="Italic">Penaeus monodon</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the -factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.     

The crayfish plague pathogen can infect freshwater‐inhabiting crabs

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Responses of the saucer scallop Amusium balloti to potential predators

A high natural mortality rate has been documented for the saucer scallop Amusium balloti, an Australian scallop found on sediment bottoms at 30–60 m deep, but little is known about the causes of mortality. We studied escape responses of A. balloti to five consumers common in the bycatch of the scallop fishery as a means of identifying potential predators. The scallop showed a negligible to weak response when touched with the sea star Pentaceraster regulus and the red portunid crab Portunus rubromarginatus. The response was similar to that observed when the scallop was touched with a plastic object. In contrast, A. balloti showed a consistent and vigorous swimming response to contact with the slipper lobster Thenus orientalis, the blue swimmer crab Portunus pelagicus and the coral crab Charybdis cruciata. This was not a generalized response to crustaceans, given the scallop's weak response to the red portunid crab. This is the first report of a scallop that has a strong swimming escape response to contact with decapod crustacean predators.     

A topographical and typological comparison of the rodlike setae of ambulatory dactylopodites in decapod crustaceans

The arrangement and external morphology of the rodlike setae and associated structures located on the dactylopodites of the walking legs of six species of decapod crustaceans are compared. The dactyls of littoral species, represented by the rock crab, Cancer antennarius, and the spiny lobster, Panulirus interruptus, have dense tufts and bands of rodlike setae, as is typical of many decapods, and additionally only a few small plumed setae. The arrangement of setae on the dactyls of the recently discovered Galapagos vent crab, Bythograea thermydron, closely resembles that of C. antennarius. Rodlike and long plumed setae occur in about equal numbers on the dactyls of the pelagic anomuran, Pleuroncodes planipes. The dactyls having the fewest rodlike setae are those of the terrestrial hermit crab, Coenobita perlatus, and those of the kelp crab, Pugettia producta, where flat setae typical of Majidae have replaced most rodlike setae. The presence and structures of the terminal pores in rodlike setae vary intra- and interspecifically, possibly as a function of molt stage. Variations in some features of rodlike setae, such as tip acuity and presence of microsetae and surface sculpting, appear to be related to development. Serrated setae occur on the dactyls of megalopal P. producta but not in later stages. The topography and typology of setae located on the ambulatory dactyls of decapod crustaceans are considered in light of recent interest in using setal characteristics to determine the sensory functions of sensilla and to clarify the phylogeny of arthropod groups.     

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.     

Atkinsiella dubia infection in the larvae of Japanese mitten crab,Eriocheir japonicus

Heavy mortalities reaching 100% among larvae of the Japanese mitten crab,Eriocheir japonicus, occurred in Yamaguchi Prefectural Naikai Fisheries Experimental Station, Yamaguchi, Japan. Under the microscope, infected zoeal larvae were seen to be filled with numerous aseptate hyphae. An investigation was carried out to identify the pathogenic fungus and determine its pathogenicity under laboratory conditions. The pathogenic fungus was identified asAtkinsiella dubia. Its optimum growth temperature was 25°C, and it grew only at 2.5% NaCl. Under laboratory conditions, it showed pathogenicity to the larvae of the swimming crab,Portunus trituberculatus. This is the first report of mass mortality in crustaceans due toA. dubia infection in Japan.     

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.     

Molecular and biological characterization of molt-inhibiting hormone of Penaeus monodon

The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.     

NO- and H<Subscript>2</Subscript>S-synthesizing neurons in the thoracic ganglion of crabs <Emphasis Type="Italic">Eriocheir japonicus</Emphasis> and <Emphasis Type="Italic">Pugettia quadridens</Emphasis> (Decapoda)

The presence and distribution of NO and H₂S-synthesizing elements in the thoracic ganglion in two crab species, Eriocheir japonicus and Pugettia quadridens, are shown using the staining methods of NADPH-diaphorase histochemistry and cystathionine-β-synthase (CBS) immunohistochemistry. NO and H₂S have been established to be synthesized by different neurons in the thoracic ganglion. The high CBS activity in efferent elements (segmental motoneurons, interneurons and nerve fibers in the limb neuropil) indicates the involvement of hydrogen sulfide in the organization of locomotor behavior in crustaceans. The presence of H₂S-synthesizing neurons in the wall of the sternal artery in E. japonicus allows suggesting the participation of H₂S in the regulation of vascular tone.     

Computational analysis of molt-inhibiting hormone from selected crustaceans

Molt-inhibiting hormone (MIH) is a principal endocrine hormone regulating the growth in crustaceans. In total, nine MIH peptide sequences representing members of the family Penaeidae (Penaeus monodon, Litopenaeus vannamei, Marsupenaeus japonicus), Portunidae (Portunus trituberculatus, Charybdis japonica, Charybdis feriata), Cambaridae (Procambarus bouvieri), Parastacidae (Cherax quadricarinatus) and Varunidae (Eriocheir sinensis) were selected for our study. In order to develop a structure based phylogeny, predict functionally important regions and to define stability changes upon single site mutations, the 3D structure of MIH for the crustaceans were built by using homology modeling based on the known structure of MIH from M. japonicus (1J0T). Structure based phylogeny showed a close relationship between P. bouvieri and C. japonica. ConSurf server analysis showed that the residues Cys⁸, Arg¹⁵, Cys²⁵, Asp²⁷, Cys²⁸, Asn³⁰, Arg³³, Cys⁴¹, Cys⁴⁵, Phe⁵¹, and Cys⁵⁴ may be functionally significant among the MIH of crustaceans. Single amino acid substitutions ‘Y’ and ‘G’ at the positions 71 and 72 of the MIH C-terminal region showed an alteration in the stability indicating that a change in this region may alter the function of MIH. In conclusion, we proposed a computational approach to analyze the structure, phylogeny and stability of MIH from crustaceans.     

Lagenidium infection in eggs and larvae of mangrove crab (Scylla serrata) produced in Indonesia

A fungal infection occurred in the eggs and larvae of mangrove crab (Scylla serrata) in seed production in Bali, Indonesia. The causative fungus was classified as a member of the genusLagenidium (Oomycetes, Lagenidiales). After comparison of its biological and physiological characteristics with those ofL. callinectes ATCC 24973, a known parasite of various crustaceans, was concluded that the isolate is a new species ofLagenidium, L. thermophilum, because of its rapid and thermotolerant growth and unique discharge process. Fungal growth was observed on PYG agar containing 0–5.0% (w/v) NaCl and 0–2.5% (w/v) KCI. Similar pathogenicity toward the zoeae of swimming crab (Portunus trituberculatus) was demonstrated.     

Differential distribution of β-pigment-dispersing hormone (β-PDH)-like immunoreactivity in the stomatogastric nervous system of five species of decapod crustaceans

Summary Pigment-dispersing hormone (PDH) acts to disperse pigments within the chromatophores of crustaceans. Using an antibody raised against -PDH from the fiddler crab Uca pugilator, we characterized the distribution of -PDH-like immunoreactivity in the stomatogastric nervous system of five decapod crustaceans: the crabs, Cancer borealis and Cancer antennarius, the lobsters, Panulirus interruptus and Homarus americanus, and the crayfish, Procambarus clarkii. No somata were stained in the stomatogastric ganglion (STG) or the esophageal ganglion in any of these species. Intense PDH-like staining was seen in the neuropil of the STG in P. interruptus only. In all 5 species, cell bodies, processes, and neuropil within the paired circumesophageal ganglia (CGs) showed PDH-like staining; the pattern of this staining was unique for each species. In each CG, the -PDH antibody stained: 1 large cell in C. borealis; 3 small to large cells in C. antennarius; 3–8 medium cells in P. clarkii; 1–4 small cells in H. americanus; and 13–17 small cells in P. interruptus. The smallest cell in each CG in C. antennarius sends its axon, via the inferior esophageal nerves, into the opposite CG; this pair of cells, not labeled in the other species studied, may act as bilateral coordinators of sensory or motor function. These diverse staining patterns imply some degree of evolutionary diversity among these crustaceans. A -PDH-like peptide may act as a neuromodulator of the rhythms produced by the stomatogastric nervous system of decapod crustaceans.     

IgE Reactivity of Blue Swimmer Crab (Portunus pelagicus) Tropomyosin,Por p 1, and Other Allergens; Cross-Reactivity with Black Tiger Prawn and Effects of Heating

Shellfish allergy is a major cause of food-induced anaphylaxis, but the allergens are not well characterized. This study examined the effects of heating on blue swimmer crab (Portunus pelagicus) allergens in comparison with those of black tiger prawn (Penaeus monodon) by testing reactivity with shellfish-allergic subjects'' serum IgE. Cooked extracts of both species showed markedly increased IgE reactivity by ELISA and immunoblotting, and clinical relevance of IgE reactivity was confirmed by basophil activation tests. Inhibition IgE ELISA and immunoblotting demonstrated cross-reactivity between the crab and prawn extracts, predominantly due to tropomyosin, but crab-specific IgE-reactivity was also observed. The major blue swimmer crab allergen tropomyosin, Por p 1, was cloned and sequenced, showing strong homology with tropomyosin of other crustacean species but also sequence variation within known and predicted linear IgE epitopes. These findings will advance more reliable diagnosis and management of potentially severe food allergy due to crustaceans.     

Identification of Peptides Implicated in Antibacterial Activity of Snow Crab Hepatopancreas Hydrolysates by a Bioassay-Guided Fractionation Approach Combined with Mass Spectrometry

Snow crab (Chionoecetes opilio) by-products are a rich source of biomolecules, such as lipids, proteins, and chitin, which have not been extensively investigated. This study aims to identify antibacterial peptides to enhance the value of C. opilio by-products. After hydrolysis of different component parts using Protamex®, and concentration by solid-phase extraction, the resulting fractions were tested for antibacterial activity against Escherichia coli, Listeria innocua, and Vibrio parahaemolyticus. Hepatopancreas was the only tissue to display antibacterial activity detected using this protocol. Four fractions obtained with and without enzymatic hydrolysis of hepatopancreas followed by SPE C18 fractionation and elution with 50 and 80% acetonitrile demonstrated bacteriostatic activity against L. innocua HPB13, from concentrations of 0.30 to 43.05 mg/mL of peptides/proteins. Eleven peptides sharing at least 80% amino acid homology with four antimicrobial peptides were identified by mass spectrometry. Two peptides had homology to crustin-like and yellowfin tuna GAPDH antimicrobial peptides belonging to the marine organisms Penaeus monodon and Thunnus albacares, respectively. Other peptide sequence homologies were also identified: Odorranain-C7 from the frog Odorrana grahami and a predicted antibacterial peptide in the Asian ladybeetle Harmonia axyridis. These active peptides may represent a novel group of bioactive peptides deserving further investigation as food preservatives.

     

Taxonomy and distribution of the hydrozoan and protozoan epibionts on Pagurus bernhardus (Linnaeus, 1758) (Crustacea, Decapoda) from Scotland

Protozoan and hydrozoan epibionts on the hermit crab Pagurus bernhardus and its shell, collected near Cumbrae Island (Scotland) were studied. The epibionts found were the following: (1) protozoans: the suctorian ciliates Ephelota plana, Acineta compressa, Conchacineta constricta, Corynophrya anisostyla; the peritrich ciliates Cothurnia mobiusi and Zoothamnium plumula; the chonotrich ciliate Chilodochona quennerstedti; and (2) hydrozoans: the species Leuckartiara sp. and Clytia sp. The morphological characteristics of the epibionts were analysed, as well as their taxonomic position. The distribution of epibionts on the crab surface and its shell was studied, and the density and biomass of epibionts were calculated on each anatomic unit. There was a differential distribution according to the type of epibiont: hydrozoans dominated in biovolume and were present mainly on the shell, meanwhile protozoans represented the highest fraction of density and they were found exclusively on the crab (principally on eyes, antennulae, antennae, maxillipeds, pereopods and uropods). The anterior area of the cephalothorax was the most colonized. On this area, the maxillipeds and second pereopods showed the highest densities. The location of each epibiont species was described. There was a correlation between the length of the crab and the total number of hydrozoans. There was a significant correlation between the right and the left units of the crab, taking into consideration the mean densities of epibionts on each anatomical unit. The shell was colonized by the same species of hydrozoa that appeared on the crab, although in a much higher density (mean 3024.38 per shell; 6.9 per crab). There was a significant difference between both species of hydrozoan epibionts with respect to the mean densities on the different areas of the shell. The zone of the shell more occupied by Clytia sp. was the apical zone of the shell, while the highest densities of Leuckartiara sp. were registered near the aperture of the shell. The hydrozoan and protozoan epibiont species found on P. bernhardus in this study represent the first mention of their presence on this hermit crab.     

Phylogeography of the whelk genus Cominella (Gastropoda: Buccinidae) suggests long‐distance counter‐current dispersal of a direct developer

The gastropod genus Cominella Gray, 1850 consists of approximately 20 species that inhabit a wide range of marine environments in New Zealand and Australia, including its external territory, the geographically isolated Norfolk Island. This distribution is puzzling, however, with apparently closely‐related species occurring either side of the Tasman Sea, even though all species are considered to have limited dispersal abilities. To determine how Cominella attained its current distribution, we derived a dated molecular phylogeny, which revealed a clade comprising all the Australian and Norfolk Island species nested within four clades of solely New Zealand species. This Australian clade diverged well after the vicariant separation of New Zealand from Australia, and implies two long‐distance dispersal events: a counter‐current movement across the Tasman Sea from New Zealand to Australia, occurring at the origination of the clade, followed by the colonization of Norfolk Island. The biology of Cominella suggests that the most likely method of long‐distance dispersal is rafting as egg capsules. Our robust phylogeny also means that the current Cominella classification requires revision. We propose that our clades be recognized as subgenera: Cominella (s.s.), Cominista, Josepha, Cominula, and Eucominia, with each subgenus comprising only of New Zealand or Australian species. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 315–332.     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Involvement of the hyperglycemic neurohormone family in the control of reproduction in decapod crustaceans

Summary

In the last few years, (bio)chemical and molecular biological studies have shown that several members of the hyperglycemic hormone family are present in different molecular forms. In vivo and in vitro bioassays revealed that some of these isoforms also play a role in the control of reproduction in decapod crustaceans. This communication gives a review of the cytological aspects of the eyestalk X-organ sinus gland complex, responsible for the synthesis, storage and release of these neuropeptides, and the molecular and functional aspects of those members involved in the control of reproduction. Finally, the role of the hyperglycemic hormone family in the regulation of reproduction in the female lobster is described as an example of the (possible) interactions of the members of the hyperglycemic hormone family with other (neuro)endocrine factors in the reproductive process of crustaceans.