Microbicidal phagocytic function and oxidative metabolism of the hemocytes of 2 oyster species, Ostrea edulis and Crassostrea gigas, were investigated using a luminol-enhanced chemiluminescence (CL) technique. First, experimental parameters adapted to marine bivalve hemocytes were established on hemolymph pools of C. gigas in order to obtain qualitatively and quantitatively homogeneous samples and so to perform statistically comparable assays. The use of Modified Alsever Solution (MAS) allowed hemocytes to b e kept non-aggregated and in a non-stimulated state until starting the assays. A number of aliquots of 2 X lo5 hemocytes with a n MAS final concentration of 3.5 % showed high chemiluminescent response after stimulation by zymosan particles A partic1e:hemocyte ratio of 80:1 gave optimal CL activity. Activity was inhibited by cytochalasin B, a phagocyt~c inhibitor, and by sodlum azide, the latter indicating the involvement of oxygen products. Having thus defined suitable parameter values, the CL protocol was then applled to 0. edulis for qualitative a n d quantitative analyses of respiratory burst capacity at the species and individual levels. Results suggested inter-and intra-specific variability of CL responses. Under the same experimental conditions, 0. eduhs hemocytes generally displayed higher CL activities than C. gigas. Moreover, clear individual vanabllity was demonstrated. The same experimental number of hemocytes showed great differences in CL responses between individuals suggesting a posslble correlation with hemogram characteristics such a s cell type percentages.
Myxosporeans in the genus Kudoa infect the flesh of many marine fishes and often cause unsightly lesions and softening of the flesh texture. We are particularly interested in K. thyrsites because it is associated with soft flesh in the Atlantic salmon (Salmo solar), an important commercial species in Canada. Sequences of the small-subunit (SSU) rDNA (about 1600 base pairs) were obtained from K. miniauriculata, K. amanuensis, and K. poniformis. We aligned these sequences with one obtained from coho salmon (Oncorhynchus kisutch) and designed "Kudoa general" primers (KUD1f and KUD2r). These primers, in combination with other general primers, were then used to obtain the SSU rDNA sequence of K. thyrsites from two host species, Atlantic salmon and tubesnout (Aulorhynchus flavidus), from British Columbia, Canada. Sequence comparisons of these isolates indicated that Kudoa species cluster by geographic location rather than by morphology of spores. The three species from the eastern Pacific were approximately 97% identical, whereas K. amamiensis (from Japan) was about 91% identical with these species. Sequence comparisons of K. thyrsites from Atlantic salmon and tubesnout revealed a difference of only 0.07% between these isolates. Comparison of SSU rDNA sequences from the four Kudoa species and Henneguya salminicolo analyzed in this study with those from other available myxosporean genera (Myxidium and Myxoholus) showed that taxonomic divisions at the order and suborder levels were consistent with classical views of the taxonomy of the Myxosporea. Using specific regions of the SSU rDNA, we also developed a sensitive and specific polymerase chain reaction test for detection of K. thyrsites.
In addition to Crassostrea gigas, Mikrocytos mackini Farley 1988, a pathogenic intracellular protistan of unknown taxonomic affiliations, produces disease and mortalities in other species of economically important oysters (Crassostrea virginica, Ostrea edulis and Ostrea conchaphila). Preliminary evidence suggests that these alternate species may be more susceptible to infection and the resulting disease than the usual host C. gigas. M. mackin1 ~solated from C. vlrginica and 0. edulis were infective for oysters. Warm temperatures (above 15°C) prevented the development of M. mackini in C. gigas, C. virginica and 0. edulis.
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