During 2006 and 2007, we documented the re-emergence of severe episodes of vibriosis caused by Vibrio tubiashii in shellfish hatcheries on the west coast of North America. Lost larval and juvenile production included 3 previously undescribed hosts, Pacific (Crassostrea gigas) and Kumamoto (C. sikamea) oysters and geoduck clams Panope abrupta, with a 2007 decline in larval oyster production of ~59% in one hatchery. Losses of larval and juvenile bivalves were linked to V. tubiashii blooms in the coastal environment, which were associated with the apparent mixing of unusually warm surface seawater and intermittently upwelled cooler, nutrient-and Vibrio spp.-enriched seawater. The ocean temperature elevation anomaly in 2007 was not clearly linked to an El Niño event, as was a similar episode in 1998. Concentrations of the dominant shellfish-pathogenic vibrios were as high as 1.6 × 10 5 cfu ml -1 in the cold, upwelled water. The bacteria possessed the genes coding for a protease and hemolysin described for V. tubiashii, and pathogenic isolates secreted these peptides. Lesions resulting from a classic invasive disease and a toxigenic noninvasive disease occurred in oyster and geoduck clam larvae. Management and prevention require reduction of incoming concentrations of the bacteria, reduction of contamination in water and air supplies and in stock chemical solutions, removal of bacterial toxins, and interruption of the cycle of bacterial amplification in the hatchery and in microalgal food supplies.

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Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD 50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD 50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 × 10 4 colony forming units (CFU) ml -1 after 24 h and between 3.2 × 10 2 and 1.9 × 10 3 CFU ml -1 after 48 h.KEY WORDS: Pacific oyster larvae · Vibrio · Bacteria · Pathogenicity testing · Shellfish hatchery Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: [223][224][225][226][227][228][229][230] 2004 genicity of bacteria isolated from bivalve hatcheries during routine monitoring and disease outbreaks. MATERIALS AND METHODSIsolates and reference strains. Bacterial isolates collected from Pacific oyster Crassostrea gigas larvae (n = 16) and juveniles (n = 12), European flat oyster Ostrea edulis juveniles (n = 1), and hatchery environments (n = 4) from the Pacific Northwest of the United States (Table 1) were stored at -80°C in Marine 2216 broth (Difco Laboratories) with 10% (v/v) glycerol. Subsequently, they were shipped on dry ice from AquaTechnics (Sequim) to the University of Washington (Seattle, Washington). The following control strains were included in the pathogenicity assays: RE 14 (nonpathogenic or negative control), RE 82 (pathogenic or positive control), and previously described larval bivalve pathogens Vibrio alginolyticus American Type Culture Collection (ATCC) 19108 and V. tubiashii ATCC 19106 (Tubiash et al. 1965, 1970). All cultures were maintained at -80°C for long-term storage.Isolates were grown for 24 h on Marine 2216 agar and incubated at 20 and 26°C in the first and second set of experiments, respectively. Experiments were conducted using sand-filtered Puget Sound seawater collected at the Seattle Aquarium (Seattle, Washington). Suspensions of the bacterial strains in filter-sterilized (0.22 µm) seawater were adjusted to an optical density (600 nm) of 0.2 to attain a uniform concentration of bacteria for the experiments. Bacterial density was confirmed by standard plate-count assays using Marine 2216 agar.Pathogenicity testing. Experi...

Larval oysters were experimentally infected with isolates of pure cultures of marine Vibrio sp. These animals were studied live, histologically, with the immunofluorescent test and with electron microscopy. All inoculated groups, but no control groups, demonstrated decreased growth and/or high mortality.One bacterial isolate attached preferentially to larval shell and destroyed mantle tissue as it grew along the internal surface of the shell. Phagocytes consumed invading bacterial cells but were ultimately overwhelmed by the infection.The second bacterial isolate caused velar damage in young larvae that remained active. In these larvae, the velar cells became detached or internally disorganized and lost their rectractor muscle insertions. In more advanced larvae, detachment of absorptive cells of the digestive gland was the earliest change observed and seemed to result from attachment of bacterial antigens to the cell surface. In both younger and more advanced larvae, food cycling and nutrient utilization were disrupted early in the disease process. The older larvae showed a general atrophy.Clinical signs such as mantle cell detachment could be detected early in the disease. Other clinical observations and the immunofluorescent test were also useful in the early diagnosis of vibriosis in larval oysters.Key words: vibriosis, larval oysters, Crassostrea virginica, histology, electron microscopy, ultrastructure, immunofluorescence, oyster hatcheryDes larves d'huîtres ont été infectées expérimentalement par des isolats de cultures pures de Vibrio sp. marins. Ces animaux furent étudiés en vie, histologiquement, à l'aide du test d'immunofluorescence et enfin au microscope électronique. Tous les groupes inoculés, mais non les groupes témoins, accusent un déclin de croissance et/ou une mortalité élevée.

Microblade technology was important in hunter‐gatherer adaptations throughout northern Asia from the late Pleistocene through the Pleistocene/Holocene transition and beyond. To date, most studies from the region are concerned with origins, technological lineages, and culture history. In contrast, we direct attention to issues involving the role of microlithic technology in adaptive strategies and problem solution among north Asian hunter‐gatherers by looking at artifact design and risk analysis. First we discuss the function of Asian microblades and outline the general costs and benefits of organic points with microblade insets over simple organic points and flaked stone points, as well as the relative advantages of wedge‐shaped and split‐pebble microcores in terms of the Z‐score model. We conclude with a review of the role of microlithic technology as a risk‐minimizing strategy of Arctic and sub‐Arctic large‐game hunters in northern Asia and suggest further lines of inquiry.

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