DNA metabarcoding reveals the importance of gelatinous zooplankton in the diet of Pandalus borealis, a keystone species in the Arctic.

Information about the dietary composition of a species is crucial to understanding their position and role in the food web. Increasingly, molecular approaches such as DNA metabarcoding are used in studying trophic relationships, not least because they may alleviate problems such as low taxonomic resolution or underestimation of digestible taxa in the diet. Here, we used DNA metabarcoding with universal primers for cytochrome c oxidase I (COI) to study the diet composition of the northern shrimp (Pandalus borealis), an Arctic keystone species with large socio-economic importance. Across locations, jellyfish and chaetognaths were the most important components in the diet of P. borealis, jointly accounting for 40%-60% of the total read abundance. This dietary importance of gelatinous zooplankton contrasts sharply with published results based on stomach content analysis. At the same time, diet composition differed between fjord and shelf locations, pointing to different food webs supporting P. borealis in these two systems. Our study underlines the potential of molecular approaches to provide new insights into the diet of marine invertebrates that are difficult to obtain with traditional methods, and calls for a revision of the role of gelatinous zooplankton in the diet of the key Arctic species P. borealis, and in extension, Arctic food webs.

The protandric life history of the Northern spot shrimp Pandalus platyceros: molecular insights and implications for fishery management.

The Northern spot shrimp, Pandalus platyceros, a protandric hermaphrodite of commercial importance in North America, is the primary target species for shrimp fisheries within Southeast Alaska. Fishery data obtained from the Alaska Department of Fish and Game indicate that spot shrimp populations have been declining significantly over the past 25 years. We collected spot shrimps in Southeast Alaska and measured reproductive-related morphological, gonadal and molecular changes during the entire life history. The appendix masculina, a major sexual morphological indicator, is indicative of the reproductive phase of the animal, lengthening during maturation from juvenile to the male phase and then gradually shortening throughout the transitional stages until its complete disappearance upon transformation to a female. This morphological change occurs in parallel with the degeneration of testicular tissue in the ovotestis and enhanced ovarian vitellogenesis. Moreover, we obtained the entire mRNA sequence of the yolk protein precursor, vitellogenin, and monitored its transcript levels throughout the entire shrimp life-cycle. Vitellogenin transcript levels in the hepatopancreas increased in the early transitional stage until reaching a peak prior to extruding eggs. Such transcriptomic analyses, coupled with a comprehensive description of the gonad, external sex characters and timing of the reproductive life history of spot shrimps contribute to a better understanding of the hermaphroditic reproduction process in the cold Southeast Alaskan waters. This knowledge can contribute to a revision of current conservation efforts to maintain wild populations sustainable for both commercial and ecological considerations.

Boreal marine fauna from the Barents Sea disperse to Arctic Northeast Greenland.

As a result of ocean warming, the species composition of the Arctic seas has begun to shift in a boreal direction. One ecosystem prone to fauna shifts is the Northeast Greenland shelf. The dispersal route taken by boreal fauna to this area is, however, not known. This knowledge is essential to predict to what extent boreal biota will colonise Arctic habitats. Using population genetics, we show that Atlantic cod (Gadus morhua), beaked redfish (Sebastes mentella), and deep-sea shrimp (Pandalus borealis) recently found on the Northeast Greenland shelf originate from the Barents Sea, and suggest that pelagic offspring were dispersed via advection across the Fram Strait. Our results indicate that boreal invasions of Arctic habitats can be driven by advection, and that the fauna of the Barents Sea can project into adjacent habitats with the potential to colonise putatively isolated Arctic ecosystems such as Northeast Greenland.

Three new species of the pandalid shrimp genus Pandalopsis Spence Bate, 1888 (Crustacea: Decapoda: Caridea) from the southwestern Sea of Okhotsk, with supplemental note on P. glabra Kobjakova, 1936.

The present study deals with a collection of deep-sea shrimp of the pandalid genus Pandalopsis Spence Bate, 1888 from the Nemuro Strait, Hokkaido, southwestern part of the Sea of Okhotsk. Three new species are described and illustrated: Pandalopsis capillus n. sp., P. houyuu n. sp. and P. princeps n. sp. In addition, Pandalopsis glabra Kobjakova, 1936, a species endemic to the Sea of Okhotsk, is supplementary reported. Most specimens were collected from steep slopes or hard bottoms at depths greater than 400 m by commercial shrimp trap or gill nets operated by local fishermen. Differentiation of the new species is primarily based on a morphological comparison, supplemented by a preliminary genetic analysis of partial segments of the mitochondrial 16S rRNA gene. An identification key to the species of Pandalopsis is presented.

Early life stages of Northern shrimp (Pandalus borealis) are sensitive to fish feed containing the anti-parasitic drug diflubenzuron.

Increasing use of fish feed containing the chitin synthesis inhibiting anti-parasitic drug diflubenzuron (DFB) in salmon aquaculture has raised concerns over its impact on coastal ecosystems. Larvae of Northern shrimp (Pandalus borealis) were exposed to DFB medicated feed under Control conditions (7.0 °C, pH 8.0) and under Ocean Acidification and Warming conditions (OAW, 9.5 °C and pH 7.6). Two weeks' exposure to DFB medicated feed caused significantly increased mortality. The effect of OAW and DFB on mortality of shrimp larvae was additive; 10% mortality in Control, 35% in OAW, 66% in DFB and 92% in OAW + DFB. In OAW + DFB feeding and swimming activity were reduced for stage II larvae and none of the surviving larvae developed to stage IV. Two genes involved in feeding (GAPDH and PRLP) and one gene involved in moulting (DD9B) were significantly downregulated in larvae exposed to OAW + DFB relative to the Control. Due to a shorter intermoult period under OAW conditions, the OAW + DFB larvae were exposed throughout two instead of one critical pre-moult period. This may explain the more serious sub-lethal effects for OAW + DFB than DFB larvae. A single day exposure at 4 days after hatching did not affect DFB larvae, but high mortality was observed for OAW + DFB larvae, possibly because they were exposed closer to moulting. High mortality of shrimp larvae exposed to DFB medicated feed, indicates that the use of DFB in salmon aquaculture is a threat to crustacean zooplankton.

Comparative transcriptome analysis of the Pacific White Shrimp (Litopenaeus vannamei) muscle reveals the molecular basis of residual feed intake.

Feed efficiency is an economically important trait in genetic improvement programs of L. vannamei. Residual feed intake (RFI), an ideal measure of feed efficiency, is the difference between observed feed intake and expected feed requirement predicted from maintenance and production. Exploring the molecular basis of RFI is essential to facilitate the genetic breeding of feed efficiency in L. vannamei. However, few studies have been reported in this aspect. In this study, we sequenced muscle transcriptomes of a high-efficiency group, a low-efficiency group and a control group originating from two families, and compared the gene expression patterns between each extreme group and the control group. A total of 383 differentially expressed genes were identified, most of which were involved in cell proliferation, growth and signaling, glucose homeostasis, energy and nutrients metabolism. Functional enrichment analysis of these genes revealed 13 significantly enriched biological pathways, including signaling pathways such as PI3K-Akt signaling pathway, AMPK signaling pathway and mTOR signaling pathway, as well as some important pathways such as ubiquitin mediated proteolysis, cell cycle, pentose phosphate pathway and glycolysis/gluconeogenesis. These genes and pathways provide initial insight into the molecular mechanisms driving the feed efficiency in L. vannamei.

Convergent Evolution of the Osmoregulation System in Decapod Shrimps.

In adaptating to different aquatic environments, seawater (SW) and freshwater (FW) shrimps have exploited different adaptation strategies, which should generate clusters of genes with different adaptive features. However, little is known about the genetic basis of these physiological adaptations. Thus, in this study, we performed comparative transcriptomics and adaptive evolution analyses on SW and FW shrimps and found that convergent evolution may have happened on osmoregulation system of shrimps. We identified 275 and 234 positively selected genes in SW and FW shrimps, respectively, which enriched in the functions of ion-binding and membrane-bounded organelles. Among them, five (CaCC, BEST2, GPDH, NKA, and Integrin) and four (RasGAP, RhoGDI, CNK3, and ODC) osmoregulation-related genes were detected in SW and FW shrimps, respectively. All five genes in SW shrimps have been reported to have positive effects on ion transportation, whereas RasGAP and RhoGDI in FW shrimps are associated with negative control of ion transportation, and CNK3 and ODC play central roles in cation homeostasis. Besides, the phylogenetic tree reconstructed from the positively selected sites separated the SW and FW shrimps into two groups. Distinct subsets of parallel substitutions also have been found in these osmoregulation-related genes in SW and FW shrimps. Therefore, our results suggest that distinct convergent evolution may have occurred in the osmoregulation systems of SW and FW shrimps. Furthermore, positive selection of osmoregulation-related genes may be beneficial for the regulation of water and salt balance in decapod shrimps.

Mapping of the immunodominant regions of shrimp tropomyosin Pan b 1 by human IgE-binding and IgE receptor crosslinking studies.

BACKGROUND: Epitope mapping of an allergen is generally done by IgE-binding assays with short synthetic peptides, but this provides little information about which domains are responsible for IgE receptor crosslinking on effector cells. Our aim was to map the immunodominant regions of shrimp tropomyosin by both IgE-binding and IgE-receptor crosslinking studies. METHODS: Five overlapping fragments covering Pandalus borealis tropomyosin were cloned, expressed in Escherichia coli and characterized by circular dichroism spectroscopy, native PAGE and bis(sulfosuccinimidyl) suberate-crosslinking. IgE binding was detected by Western blot, indirect ELISA and inhibition ELISA, and IgE receptor crosslinking was investigated by basophil activation test and skin prick test with Norwegian shrimp allergic adults. RESULTS: The N- and C-terminal fragments of tropomyosin showed the highest amount of secondary structure. Western blot studies showed preferential binding to the terminal fragments, while indirect and inhibition ELISA studies showed binding to all fragments, but with individual variations. Basophil CD63 expression was upregulated by all fragments at high concentrations (1 µg/ml) and showed individual variations comparable to ELISA results. A mixture of the fragments with equal molar ratios induced comparably strong CD63 activation as for tropomyosin. Skin prick test studies showed positive responses to the terminal and middle fragments and increased responses to the fragment mixture compared to whole tropomyosin. CONCLUSIONS: The terminal and middle fragments of tropomyosin had the highest IgE reactivity, but overall no clear immunodominant region was observed in this study. These results correlated well with previous studies with short peptides. Dividing shrimp tropomyosin into five fragments did not reduce the allergenicity of the protein.

Differences in gene organization between type I and type II crustins in the morotoge shrimp, Pandalopsis japonica.

Crustins are cysteine-rich cationic antimicrobial peptides (AMPs) found in decapod crustaceans. Six novel crustin genes (Paj-CrusIc, Id, Ie, If, IIb and IIc) were identified in the morotoge shrimp, Pandalopsis japonica. Deduced amino acid sequences of isolated Paj-Crus genes ranged from 99 to 178 amino acid residues (10.6-17.8 kDa). Sequence analysis of nine isolated Paj-Crus genes and 100 different crustins from various decapod crustaceans revealed that a splice site and KXXXCP motif within the WAP domain may be the main criteria for classifying type I and II crustins, suggesting that the two types of crustin genes may have been generated by different processes. We also identified three intron-less crustin I genes (Paj-Crus Id, Ie and If) for the first time, which may have been generated by gene duplication. The tissue distribution profiles showed that Paj-CrusI genes were expressed predominantly in the gill and epidermis, whereas Paj-CrusII genes were expressed ubiquitously, suggesting that the two types of crustins may play different roles in various tissues or under different physiological conditions. Differing from previous results, hemocyte-specific crustin was not isolated from Pandalopsis japonica. This study showed that both types of crustin genes (types I and II) exist in decapod crustaceans and their primary structure and expression profiles differ from each other, suggesting that they may play different biological roles. This will help to extend our knowledge of the crustacean innate immune response, which will provide important basic information of shrimp immunity against various pathogens.

Maladaptive sex ratio adjustment by a sex-changing shrimp in selective-fishing environments.

1. Selective harvesting is acknowledged as a serious concern in efforts to conserve wild animal populations. In fisheries, most studies have focused on gradual and directional changes in the life-history traits of target species. While such changes represent the ultimate response of harvested animals, it is also well known that the life history of target species plastically alters with harvesting. However, research on the adaptive significance of these types of condition-dependent changes has been limited. 2. We explored the adaptive significance of annual changes in the age at sex-change of the protandrous (male-first) hermaphroditic shrimp and examined how selective harvesting affects life-history variation, by conducting field observations across 13 years and a controlled laboratory experiment. In addition, we considered whether plastic responses by the shrimp would be favourable, negligible or negative with respect to the conservation of fishery resources. 3. The age at sex-change and the population structure of the shrimp fluctuated between years during the study period. The results of the field observations and laboratory experiment both indicated that the shrimp could plastically change the timing of sex-change in accordance with the age structure of the population. These findings provide the first concrete evidence of adult sex ratio adjustment by pandalid shrimp, a group that has been treated as a model in the sex allocation theory. 4. The sex ratio adjustment by the shrimp did not always seem to be sufficient, however, as the supplement of females is restricted by their annual somatic growth rate. In addition, adjusted sex ratios are further skewed by the unintentional female-selectivity of fishing activity prior to the breeding season, indicating that the occurrence of males that have postponed sex-change causes sex ratio adjustment to become unfavourable. 5. We conclude that the plastic responses of harvested animals in selective fishing environments must be considered in efforts to conserve wild animal resources, because such responses can become maladaptive.

Molecular cloning and characterization of three cDNAs encoding allatostatin-like neurosecretory peptides from Pandalopsis japonica.

Three cDNAs encoding allatostatin-like peptides (two myoinhibitory peptides; Paj-MIPI and Paj-MIPII, and one C-type AST; Paj-ASTC) were identified from Pandalopsis japonica through a combination of bioinformatic analysis and PCR-based gene cloning strategy. Paj-MIPI and Paj-MIPII encoded proteins with 189 and 117 amino acid residues, respectively, and a total of 10 mature peptides are putatively produced from the two MIP cDNAs (seven from Paj-MIPI and three from Paj-MIPII). Among the MIPs from various arthropods, their size and organization varied and it was unable to establish the monophyletic evolutionary relationship, which is mainly due to difference in the number and location of the mature peptide W(X(6))W motif of each MIP gene. Based on the sequence similarity of six residues flanked by two conserved tryptophan (W) residues, crustacean MIPs could be further classified into at least four groups. Paj-ASTC cDNA (648bp) encoded a protein with 143 amino acid residues. The prepropeptide of Paj-ASTC showed conserved C-type AST characteristics including a signal sequence, two dibasic cleavage sites, and a mature peptide sequence with two cysteine residues at the 7th and 14th positions, creating a disulfide bridge. Based on the sequence similarity in the mature peptides, the ASTCs in arthropods could be further classified into two subgroups, AVSCF-ASTC and PISCF-ASTC. Phylogenetic and sequence similarity analysis showed that Paj-ASTC belonged to the PISCF-ASTC subgroup. Expression studies revealed that AST-like peptides from P. japonica were mainly expressed in neuronal tissues, and the expression of Paj-ASTC was also detected in the intestine. Eyestalk ablation (ESA) altered the mRNA expression levels of both Paj-MIPs and Paj-ASTC, suggesting that factors from the sinus gland/X organ complex had a transient effect on AST-like peptide transcription. Correlation analysis of three allatostatin-like peptides revealed a strong positive correlation in brain tissues, suggesting that transcriptional regulation of three allatostatin-like peptides from P. japonica is influenced by the similar physiological condition.

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides.

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis.

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.

Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression.

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5' end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.

Expression profiling without genome sequence information in a non-model species, Pandalid shrimp (Pandalus latirostris), by next-generation sequencing.

While the study of phenotypic variation is a central theme in evolutionary biology, the genetic approaches available to understanding this variation are usually limited because of a lack of genomic information in non-model organisms. This study explored the utility of next-generation sequencing (NGS) technologies for studying phenotypic variations between 2 populations of a non-model species, the Hokkai shrimp (Pandalus latirostris; Decapoda, Pandalidae). Before we performed transcriptome analyses using NGS, we examined the genetic and phenotypic differentiation between the populations. Analyses using microsatellite DNA markers suggested that these populations genetically differed from one another and that gene flow is restricted between them. Moreover, the results of our 4-year field observations indicated that the egg traits varied genetically between the populations. Using mRNA extracted from the ovaries of 5 females in each population of Hokkai shrimp, we then performed a transcriptome analysis of the 2 populations. A total of 13.66 gigabases (Gb) of 75-bp reads was obtained. Further, 58,804 and 33,548 contigs for the first and second population, respectively, and 47,467 contigs for both populations were produced by de novo assembly. We detected 552 sequences with the former approach and 702 sequences with the later one; both sets of sequences showed greater than twofold differences in the expression levels between the 2 populations. Twenty-nine sequences were found in both approaches and were considered to be differentially expressed genes. Among them, 9 sequences showed significant similarity to functional genes. The present study showed a de novo assembly approach for the transcriptome of a non-model species using only short-read sequence data, and provides a strategy for identifying sequences showing significantly different expression levels between populations.

Two juvenile hormone esterase-like carboxylesterase cDNAs from a Pandalus shrimp (Pandalopsis japonica): cloning, tissue expression, and effects of eyestalk ablation.

Methyl farnesoate (MF), a crustacean juvenile hormone (JH) analog, plays important roles in the regulation of a number of physiological processes such as molting, metamorphosis, and reproduction. Understanding its metabolic pathway is a key for various potential applications in crustacean aquaculture, including artificial seed production and enhancement of growth. Although the synthetic pathway of MF is well established, little is known about its degradation and recycling in crustaceans. In insects, juvenile hormone esterase (JHE), a carboxylesterase, is responsible for JH inactivation. Two cDNAs, encoding JHE-like carboxylesterases (CXEs) from the hepatopancreas and ovary of Pandalopsis japonica, were isolated by using a combination of in-silico data mining from an expressed sequence tag (EST) database and traditional PCR-based cloning. The full length Pj-CXE1 (2084bp) and Pj-CXE2 (1985bp) cDNAs encoded proteins composed of 584 and 581 amino acids, respectively. The active site sequence and domain organization of the Pj-CXEs were highly conserved, including the catalytic triad and other motifs, which suggested that both Pj-CXEs are biologically active carboxylesterases. Phylogenetic analysis of the deduced sequences of Pj-CXEs showed that both were most closely related to the JHEs from non-lepidopteran insects. End-point RT-PCR showed that Pj-CXE1 was expressed primarily in the gonad, whereas Pj-CXE2 was expressed in both the hepatopancreas and hindgut. Quantitative PCR showed that Pj-CXE1 was upregulated in the gonads by eyestalk ablation (ESA). In contrast, ESA had no significant effect on Pj-CXE2 expression in hepatopancreas or gonad. This is the first report of the cloning of two JHE-like CXE cDNAs in decapods and the upregulation of Pj-CXE1 by acute withdrawal of eyestalk neuropeptides. Further study is needed to understand the function of CXEs in MF metabolism and its regulation by eyestalk neuropeptides.

Characterization of two vitellogenin cDNAs from a Pandalus shrimp (Pandalopsis japonica): expression in hepatopancreas is down-regulated by endosulfan exposure.

Endosulfan is a neurotoxic organochlorine insecticide of the cyclodiene family of pesticides that inhibits molting and reproduction in aquatic crustaceans. In order to determine the molecular mechanism of endosulfan as an endocrine disrupting chemical (EDC), differential display RT-PCR (DDRT-PCR) was used to isolate genes in the shrimp, Pandalopsis japonica, affected by endosulfan exposure. PCR screening of cDNA from the hepatopancreas from control and endosulfan-exposed animals, using 120 sets of random primers, yielded partial cDNAs encoding two vitellogenin-like proteins (Pj-Vg1 and -Vg2). Complete sequences were obtained using a combination of RT-PCR and RACE-PCR. Pj-Vg1 (7883bp) encoded a protein composed of 2533 amino acid residues (283.27 kDa estimated mass), whereas Pj-Vg2 (7792 bp) encoded a protein composed of 2537 amino acids residues (284.87 kDa estimated mass). Alignment of the Pj-Vgs with those of other vitellogenins identified a conserved subtilisin cleavage site (RQKR) and the lipoprotein N-terminal (vitellin), DUF1081, and von Willebrand factor type D domains, indicating both genes encoded functional proteins. Phylogenetic analysis showed that Pj-Vg1 and -Vg2 were most similar to Pandalus hypsinotus Vg. Both Pj-Vg1 and -Vg2 were expressed primarily in the hepatopancreas, although the Pj-Vg2 transcript was also detected in the ovary. The effects of the 3-day endosulfan exposure (2.5 microg/L and 25 microg/L) on Vg expression in the hepatopancreas were determined by quantitative RT-PCR. Expression of both transcripts was significantly inhibited at 25 microg/L suggesting that Pj-Vgs can be used as indicator for endosulfan exposure.

The enzyme and the cDNA sequence of a thermolabile and double-strand specific DNase from Northern shrimps (Pandalus borealis).

BACKGROUND: We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies. METHODOLOGY/PRINCIPAL FINDINGS: A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65 degrees C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases. CONCLUSIONS/SIGNIFICANCE: The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.

Molecular characterization of a cDNA encoding vitellogenin in the coonstriped shrimp, Pandalus hypsinotus and site of vitellogenin mRNA expression.

In order to determine the primary structure of vitellogenin in a protandric species, the coonstriped shrimp Pandalus hypsinotus, we previously purified four vitellin components (designated as VnA, VnB, VnC, and VnD, respectively), and chemically analyzed their partial amino acid sequences. In this study, we subsequently cloned a cDNA encoding vitellogenin in this species based on the N-terminal and internal amino acid sequences of VnA, as well as the N-terminal sequence of VnC. The open reading frame of this cDNA encoded a pro-vitellogenin in which vitellins were arranged as follows: NH2-VnA-VnB-VnC/D-COOH. The deduced amino acid sequence possessed a single consensus cleavage sequence, R-X-K/R-R, along the lines of vitellogenins reported in other crustaceans and insects, and the N-terminal sequence of VnB was immediately preceded by this sequence. The comparison of primary structures revealed the existence of a basic and characteristic structure for the vitellogenin molecule in decapod crustacean species, and phylogenetic analysis reflected the current taxonomic classifications of Crustacea. An approximately 8 kb-long transcript of the vitellogenin gene was detected in the hepatopancreas of female shrimps having a gonadosomatic index higher than 1.0 by Northern blot analysis, but was not observed in the hepatopancreas and gonads of male shrimps and the hepatopancreas of female shrimps having a gonadosomatic index lower than 1.0. These results indicate that the hepatopancreas is responsible for vitellogenin synthesis.

Molecular and enzymatic properties of a cathepsin L-like proteinase with distinct substrate specificity from northern shrimp (Pandalus borealis).

We purified a cathepsin L-like proteinase to homogeneity from the hepatopancreas of northern shrimp Pandalus borealis by several chromatographic procedures. The purified proteinase showed the highest specificity for leucine residue at P2, a specificity pattern similar to cathepsins S and K whereas proline and arginine residues were not suitable as P2 substrates. However, unlike these proteinases, it accepted valine almost equally to the phenylalanine residue at P2. The shrimp cathepsin was strongly inhibited by E-64, leupeptin and antipain, while benzyloxycarbonyl-Phe-Tyr(t-Bu)-CHN2, a specific inhibitor of cathepsin L, remained largely ineffective. Next, we determined the primary structure of the shrimp enzyme by molecular cloning and investigated the residues constituting the S2 subsite, which is possibly involved in its unusual substrate specificity. The deduced amino acid sequence of the shrimp proteinase shared the highest identity of 65% with a cathepsin L-like proteinase from lobster, but its identity to the well-characterized mammalian cathepsins S, L, and K fell within narrower ranges of 52-55%. However, the shrimp proteinase differed from these cathepsins in some key residues including, for example, the unique occurrence of cysteine and glutamine residues at the structurally important S2 subsite. Interestingly, transcripts of this proteinase were exclusively detected in the shrimp gut coinciding with its broad pH activity and stability profiles, which is also unusual as a cysteine proteinase. These results suggest that the shrimp enzyme is homologous to mammalian cathepsins S, L, and K, but is distinct from each of these proteinases in both enzymatic and structural properties.