Phylogenetic and morphological characterisation of the green algae infesting blue mussel Mytilus edulis in the North and South Atlantic oceans.

Blue mussels Mytilus edulis with shell deformations and green pustules containing parasitic algae were collected at 3 coastal sites (Burøy, Norway; Bockholm, Denmark; Goose Green, Falkland Islands). A comparative study, including mussel histopathology, algal morphology, ultrastructure and phylogenetic position was performed. Green pustules were mainly located in the posterior portion of the mantle and gonad tissues and the posterior adductor muscle. Electron microscopy confirmed the presence of algal cells with similar morphology to Coccomyxa parasitica. Algae were oval shaped with a single nucleus and chloroplast, 1 or 2 mitochondria and a dense granular cytoplasm with a lipid inclusion body, Golgi apparatus and small vesicles. Partial small subunit (SSU) rRNA phylogeny confirmed the inclusion of parasitic algae into the Coccomyxa clade. However, the sequence identity between almost full SSU rRNA sequences of parasitic algae and others in this clade yielded an unexpected result. Green algae from mussels were distant from C. parasitica Culture Collection of Algae and Protozoa (CCAP) strain 216/18 (94% identity), but very similar (99% identity) to C. glaronensis (a lichen endosymbiont) and green endophytes from the tree Ginkgo biloba. The CCAP strain 216/18 was a sister sequence to Nannochloris algae, far from the Coccomyxa clade. These results suggest a misidentification or outgrowth of the original CCAP strain 216/18 by a different 'Nannochloris-like' trebouxiophycean organism. In contrast, our sequences directly obtained from infested mussels could represent the true C. parasitica responsible for the green pustules in blue mussels.

Vibrio tapetis-like strain isolated from introduced Manila clams Ruditapes philippinarum showing symptoms of brown ring disease in Norway.

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.

Picoeucaryot alga infecting blue mussel Mytilus edulis in southern Norway.

During summer 2001, blue mussels Mytilus edulis with abnormal shell growth were collected near Kragerø, southern Norway. The mussels had green spots in their mantle tissues, mainly posteriorly and ventrally, and in the adductor muscle. Mussels from 4 sites had a prevalence of green spots varying from 2 to 71% that correlated well with shell deformities. Histological examination revealed the presence of round or ovoid algae, 0.9 to 1.5 x 1.2 to 2.4 microm, free within haemocytes and in the lesions, characterised by an inflammatory response and the presence of cellular debris. The alga contain a relatively large nucleus, 1 chloroplast and 1 mitochondrion. Size and morphology suggest that the alga might be a picoeucaryot green alga. Infection of mussel tissues appears to start in the posterior mantle edge, near the siphons, and spread anterior-ventrally in the mantle connective and storage tissues-occasionally spots were also found in the gonad follicles. Large infected areas were also observed in sinuses within the adductor muscle. Only mussels that were 3 yr old or more were infected. Deformations apparently resulted from years of continuous shell formation by a contracted, partly deformed mantle. Most deformed mussels had eroded shells, allowing some light penetration through the exposed, thin nacre. Young, thin-shelled mussels were not infected. The present work suggests that the alga has, at least partially, a parasitic relationship with the mussels, and is associated with pathological alterations in mussel tissues.