Cysteine protease activities have been characterized with benzyloxycarbonyl-lysine p-nitrophenyl ester as a synthetic substrate and E64 as a specific inhibitor in the hepatopancreas of the shrimp Penaeus vannamei. An optimum pH of 5.1 has been measured. To characterize these cysteine proteases, a hepatopancreas cDNA library was screened by hybridization to a Norway lobster cysteine protease cDNA fragment. Two cDNAs encoding P. vannamei cysteine protease precursors have been cloned and sequenced. The encoded polypeptides have 326 and 322 amino acid residues, respectively, each consisting of partial signal sequences (15 and 10 residues), a pro-region (93 and 94 residues), and a mature enzyme polypeptide (218 residues). Cys25, His159 and Asn175 form the catalytic triad in the putative active site of the mature enzymes. Compared with invertebrate cysteine proteases (Homarus and Fasciola), each of the two shrimp enzymes shows 70 and 52% amino acid sequence identity, respectively; 63% identity is shown with rat cathepsin L. Northern hybridization analysis showed the same size for the different cysteine protease transcripts in hepatopancreas tissue (approximately 1.1 kb). During intermolt cycles, variations in cysteine protease activity were correlated with the variations in the levels of specific mRNA.

The Manila clam Ruditapes philippinarum was introduced to Norway in 1987 and was produced in 2 hatcheries until 1991. Clam seed was planted at 6 sites. Two sites were on the Island of Tysnes, south of Bergen. Surviving adult Manila clams were recovered in 1995 and 1996. In the present study, Manila clams from the original seeding that displayed morphological signs of brown ring disease (BRD) were recovered in June 2003 (n = 7) and in June 2004 (n = 17). Samples from extrapallial fluid, tissues and haemolymph were inoculated on marine agar. Replicate subcultures on selective media were used to select potential Vibrio tapetis strains, and in total, 190 bacterial strains were isolated. One of these strains clustered within the V. tapetis clade and was named NRP 45. DNA:DNA hybridisation with the type strain CECT4600 showed 52.7 and 57.3% DNA:DNA similarity. Hybridisation of NRP 45 and the V. tapetis LP2 strain, isolated from corkwing wrasse Symphodus melops, produced 46.6 and 44.4% re-association. Partial gene segments encoding 16S rRNA, gyrase B protein (GyrB) and chaperonin 60 protein (Cpn60) were characterised and compared to CECT 4600. NRP 45 showed 5 differences in the 1416 nucleotides (nt) of the 16S rRNA encoding gene (99.6% similarity), while the GyrB encoding gene had 62 substitutions of 1181 nt compared (94.8% similarity) and the Cpn60 encoding gene had 22 substitutions out of 548 nt compared (96% similarity). This is the first finding of BRD and the first isolation of a V. tapetis-like bacterial strain from a bivalve in Norway. KEY WORDS: Brown ring disease · Vibrio tapetis · Bivalve · Manila clams · Bacteria · NorwayResale or republication not permitted without written consent of the publisher Dis Aquat Org 81: [153][154][155][156][157][158][159][160][161] 2008 disease has subsequently spread along the East Atlantic coast from England to North Africa (Paillard 2004). BRD was first observed in Spain in 1994, in England in 1997 and in Ireland in 1998 (Paillard & Maes 1994, Castro et al. 1996, Novoa et al. 1998. The highest prevalence is encountered in the northernmost countries, and BRD is generally classified as a coldwater disease (Paillard et al. 1994). In Norway, the Manila clam was introduced in 1987 (Mortensen & Strand 2000). Approximately 500 broodstock specimens were imported from Scottish Seafarms Ltd to 2 Norwegian hatcheries, located in Espevik on the Island of Tysnes, south of Bergen, and in Vallersund in South Trøndelag County (Fig. 1A). From 1987 to 1991, more than 200 million seed were produced, with ongrowth done in nurseries at Espevik, Rong, Vallersund and Vågstanda. Most of the Manila clam seed were exported to Spain and Ireland. In Espevik, the native clam R. decussatus was also produced by the same methods and during the same period as the Manila clams. Clam production was terminated in 1991. Between 1988 and 1991, Manila clam seed were released on beaches at 6 sites. At Seløy (Fig.1B), approximately 2000 Manila clam spat were released in 1988, and a total of 28 speci...

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