Molecular characterization of two ferritins of the scallop Argopecten purpuratus and gene expressions in association with early development, immune response and growth rate.

Ferritin is involved in several iron homoeostasis processes in molluscs. We characterized two ferritin homologues and their expression patterns in association with early development, growth rate and immune response in the scallop Argopecten purpuratus, a species of economic importance for Chile and Peru. Two ferritin subunits (Apfer1 and Apfer2) were cloned. Apfer1 cDNA is a 792bp clone containing a 516bp open reading frame (ORF) that corresponds to a novel ferritin subunit in A. purpuratus. Apfer2 cDNA is a 681bp clone containing a 522bp ORF that corresponds to a previously sequenced EST. A putative iron responsive element (IRE) was identified in the 5'-untranslated region of both genes. The deduced protein sequences of both cDNAs possessed the motifs and domains characteristic of functional ferritin subunits. Both genes showed differential expression patterns at tissue-specific and early development stage levels. Apfer1 expression level increased 40-fold along larval developmental stages, decreasing markedly after larval settlement. Apfer1 expression in mantle tissue was 2.8-fold higher in fast-growing than in slow-growing scallops. Apfer1 increased 8-fold in haemocytes 24h post-challenge with the bacterium Vibrio splendidus. Apfer2 expression did not differ between fast- and slow-growing scallops or in response to bacterial challenge. These results suggest that Apfer1 and Apfer2 may be involved in iron storage, larval development and shell formation. Apfer1 expression may additionally be involved in immune response against bacterial infections and also in growth; and thus would be a potential marker for immune capacity and for fast growth in A. purpuratus.

Biological effects of mechanically and chemically dispersed oil on the Icelandic scallop (Chlamys islandica).

This study aimed to simulate conditions in which dispersant (Dasic NS) might be used to combat an oil spill in coastal sub-Arctic water of limited depth and water exchange in order to produce input data for Net Environmental Benefit Analysis (NEBA) of Arctic and sub-Arctic coastal areas. Concentration dependent differences in acute responses and long-term effects of a 48h acute exposure to dispersed oil, with and without the application of a chemical dispersant, were assessed on the Arctic filter feeding bivalve Chlamys islandica. Icelandic scallops were exposed for 48h to a range of spiked concentrations of mechanically and chemically dispersed oil. Short-term effects were assessed in terms of lysosomal membrane stability, superoxide dismutase, catalase, gluthatione S-transferases, glutathione peroxidases, glutathione reductase, glutathione, total oxyradical scavenging capacity, lipid peroxidation and peroxisomal proliferation. Post-exposure survival, growth and reproductive investment were followed for 2 months to evaluate any long-term consequence. Generally, similar effects were observed in scallops exposed to mechanically and chemically dispersed oil. Limited short-term effects were observed after 48h, suggesting that a different timing would be required for measuring the possible onset of such effects. There was a concentration dependent increase in cumulative post-exposure mortality, but long-term effects on gonadosomatic index, somatic growth/condition factor did not differ among treatments.

Molecular cloning and transcriptional regulation of an allograft inflammatory factor-1 (AIF-1) in Zhikong scallop Chlamys farreri.

The allograft inflammatory factor-1 (AIF-1) is one of the key factors associated with inflammatory response. In the present study, the full-length cDNA of AIF-1 was identified from Zhikong scallop Chlamys farreri (named as CfAIF-1) by EST (expressed sequence tag) analysis and RACE (rapid-amplification of cDNA ends) approaches. The cDNA of CfAIF-1 consisted of a 5-terminal untranslated region (UTR) of 58 bp, a 3-UTR of 607 bp with a poly (A) tail, and an open reading frame (ORF) of 468 bp encoding a polypeptide of 155 amino acids with the putative molecular mass of 17.8 kDa. There was an EF hand Ca(2+)-binding motif in the deduced amino acid sequence of CfAIF-1 which was conserved in other AIF-1s. CfAIF-1 shared closer phylogenetic relationship with invertebrate counterparts than vertebrate. The mRNA transcripts of CfAIF-1 were dominantly expressed in hepatopancreas, hemocytes and adductor. During scallop ontogenesis, the CfAIF-1 mRNA was expressed at a low level at early developmental stages from eggs to blastula, and then increased significantly from gastruta to late veliger larvae (P<0.05). Moreover, the mRNA expression levels of CfAIF-1 in the hemocytes of adult scallop were significantly up-regulated during 12-48 h after LPS, PGN and poly I:C stimulation (P<0.01), but there was no significant fluctuation detected after glucan stimulation. Furthermore, the challenge of bacteria Vibrio anguillarum remarkably induced the mRNA expression of CfAIF-1 in hemocytes at 6h (P<0.05) and 12h (P<0.01). All these results collectively indicated that CfAIF-1 might be involved in the immune response during the ontogenesis and contribute to the defense against microbe infection in scallops.

A selenium-dependent glutathione peroxidase in the Japanese scallop, Mizuhopecten yessoensis: cDNA cloning, promoter sequence analysis and mRNA expression.

Glutathione peroxidase (GPx) is an antioxidant enzyme that protects cells from oxidative damage in the innate immune responses against bacterial infections. GPx is also involved in immune defenses. In this study, we report cloning and characterization of a GPx (designated as MyGPx) coding sequences and promoter from Japanese scallop, Mizuhopecten yessoensis. The full-length 1081 nt MyGPx mRNA contained a 28 nt 5' untranslated region (UTR), a 603 nt open reading frame and a 450 nt 3' UTR containing a polyadenylation signal (AATAAA). Multiple sequence alignment revealed that amino acids essential to enzymatic function of MyGPx proteins were highly conserved. A 1628 nt 5'-flanking sequence of MyGPx was identified by genome walking. Here, several potential transcription factor binding sites were detected in the putative promoter region, and nine single nucleotide polymorphisms (SNPs) were found in the 5' sequence flanking the promoter region. Quantitative Real time PCR (qRT-PCR) was employed to measure GPx mRNA expression in adult tissues and monitor mRNA expression patterns during embryonic development and following stimulation by the bacteria Vibrillo anguillarum. Collectively, the results suggest that MyGPx fulfills an important function during M. yessoensis development and may be an important immune effector in adult molluscs.

Immunotoxicity and oxidative stress in the Arctic scallop Chlamys islandica: effects of acute oil exposure.

With increasing oil exploration in Arctic regions, the risk of an accidental oil spill into the environment is inevitably elevated. As a result, concerns have been raised over the potential impact of oil exposure on Arctic organisms. This study assessed the effects of an acute oil exposure (mimicking an accidental spill) on the immune function and oxidative stress status of the Arctic scallop Chlamys islandica. Scallops were exposed to the water accommodated fraction of crude oil over 21 d (maximum SigmaPAH 163 microg l(-1)) and immune endpoints and oxidative stress parameters were measured. Mortalities were recorded during the exposure and reductions in immunocompetence were observed, with significant impairment of phagocytosis and cell membrane stability. Scallops were also subjected to oxidative stress, with a significant reduction in glutathione levels and induction of lipid peroxidation. After the acute oil exposure had subsided, no recovery of immune function was observed indicating potential for prolonged sublethal effects.