Gary H. Wikfors

Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst

Abstract The capability of an oyster to respond to environmental stresses, such as periodically high summer temperatures, as well as disease or parasite infections, depends, in large measure, upon the viability and functional capability of haemocytes. Eastern oysters ( Crassostrea virginica ) were subjected to a sudden increase in temperature from 20 to 28 °C for 1 week, and several haemocyte functions were determined before and after the temperature elevation using the flow cytometer. Previously, we described the characterization of different haemocyte types using new and modified flow cytometric methods. In this report, we provide detailed protocols for flow cytometric methods to: (1) determine haemocyte aggregation using paired samples with or without an antiaggregant solution; (2) assess haemocyte viability using propidium iodide (PI); (3) quantify haemocyte phagocytosis with fluorescent microbeads; and (4) measure the respiratory burst response of individual haemocytes using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and zymosan to activate the release of reactive oxygen species (ROS). The temperature increase caused no significant change in haemocyte aggregation, although there was a trend of increasing aggregation in granulocytes and small granulocytes, but a slight decrease in hyalinocyte aggregation. Phagocytosis of all haemocyte types decreased after the temperature increase. Significantly higher percentages of dead haemocytes in all haemocyte types (attributable to a large increase in mortality of hyalinocytes, the most numerous cells) were found after the temperature increase, suggesting generally less capable immune function. Numbers of dead small granulocytes and granulocytes tended to decrease, but this was not statistically significant. Effects of temperature elevation upon respiratory burst were not statistically significant; however, a trend of increased ROS production after temperature elevation was consistent for all haemocyte types. Granulocytes, hyalinocytes, and small granulocytes showed increased production of ROS in the presence of zymosan; granulocytes showed the highest induced fluorescence.

Flow-cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: I. Haemocyte types and morphology

In this report, we provide detailed protocols for flow-cytometric characterization of haemocytes from the eastern oyster, Crassostrea virginica, and report effects of sudden temperature elevation upon haemocyte characteristics. Haemocytes were differentiated from other particles in the haemolymph using a combination of the DNA-binding fluorochrome SYBR Green and internal cell complexity. In formalin-fixed haemolymph, four distinct subpopulations of haemocytes were found: small hyalinocytes, large hyalinocytes, small granulocytes, and granulocytes. Hyalinocytes were numerically dominant, small and large together accounting for 60% of the haemocytes; granulocytes accounted for 30–35%, and small granulocytes were least numerous at <5%. These percentages were the same for haemolymph analysed immediately after it was withdrawn from living oysters (without fixation) as for haemolymph fixed with formalin, although small and large hyalinocytes were not always clearly differentiated into two distinct populations. There was a general trend for the fixed cells to appear in the flow cytometer to be larger and more complex than the fresh ones (except for granulocyte complexity). Oysters subjected to a sudden increase in temperature from 20 to 28 °C for 1 week showed significant changes in haemocyte morphology. Sudden temperature elevation was associated with a general decrease in size of all haemocyte types. The ability to detect changes in individual haemocyte types for many oysters by these flow-cytometric methods should improve progress in understanding oyster mortality associated with combined effects of environmental stresses, disease, and parasites.

Impact of algal research in aquaculture

Algal aquaculture worldwide is estimated to be a

INFLUENCE OF CHEMICAL COMPOSITION OF ALGAL FOOD SOURCES ON GROWTH OF JUVENILE OYSTERS, CRASSOSTREA VIRGINICA

5–6 billion U.S. per year industry. The largest portion of this industry is represented by macroalgal production for human food in Asia, with increasing activity in South America and Africa. The technical foundation for a shift in the last half century from wild harvest to farming of seaweeds lies in scientific research elucidating life histories and growth characteristics of seaweeds with economic interest. In several notable cases, scientific breakthroughs enabling seaweed‐aquaculture advances were not motivated by aquaculture needs but rather by fundamental biological or ecological questions. After scientific breakthroughs, development of practical cultivation methods has been accomplished by both scientific and commercial‐cultivation interests. Microalgal aquaculture is much smaller in economic impact than seaweed cultivation but is the subject of much research. Microalgae are cultured for direct human consumption and for extractable chemicals, but current use and development of cultured microalgae is increasingly related to their use as feeds in marine animal aquaculture. The history of microalgal culture has followed two main paths, one focused on engineering of culture systems to respond to physical and physiological needs for growing microalgae and the other directed toward understanding the nutritional needs of animals—chiefly invertebrates such as mollusks and crustaceans—that feed upon microalgae. The challenge being addressed in current research on microalgae in aquaculture food chains is to combine engineering and nutritional principles so that effective and economical production of microalgal feed cultures can be accomplished to support an expanding marine animal aquaculture industry.

Experimental and Histological Studies of Four Life- History Stages of the Eastern Oyster, Crassostrea virginica, Exposed to a Cultured Strain of the Dinoflagellate Prorocentrum minimum

Two algal flagellates, Dunaliella tertiolecta Butcher and Tetraselmis maculata Butcher, harvested in the stationary phase from a semi-continuous carboy culture apparatus, were analyzed for dry weight, total carbohydrate, total protein, and total lipid. Each species was cultured in three different growth media. The growth response of D. tertiolecta was similar in all three formulations but populations of T. maculata were considerably limited in the reduced-nutrient medium, X1. Both algal species cultured in the X1 medium had significantly greater dry weights and contained more carbohydrate and less protein than cells cultured in the standard formulation (E). A third formulation (N/P), in which all medium components were reduced except nitrate and phosphate, produced algae with reduced carbohydrate and increased protein as compared with E medium. The total lipid content of D. tertiolecta was significantly less than that of T. maculata regardless of the culture medium.Algae cultured in the three formulations...

DIVERSE FEEDING RESPONSES OF FIVE SPECIES OF BIVALVE MOLLUSC WHEN EXPOSED TO THREE SPECIES OF HARMFUL ALGAE

Effects of the dinoflagellate Prorocentrum minimum (strain EXUV) upon four life-history stages of the eastern oyster--embryos, feeding larvae, newly set spat, and juveniles--were investigated in laboratory exposure studies. Embryonic development was not affected significantly by living, heat-killed, or sonicated cells, or by growth-medium extracts from P. minimum cultures. Feeding larvae, however, showed poor growth and poor development of the digestive system when fed P. minimum, as compared with larvae fed Isochrysis sp. (strain T-ISO). Growth of larvae fed mixed P. minimum + Isochrysis diets was intermediate. Larvae and newly set spat that had been fed a diet of 1/3 P. minimum + 2/3 Isochrysis exhibited distinctive changes in digestive-system anatomy. Spat showed an abnormal accumulation of lipid in the stomach epithelium. Absorptive cells in the digestive glands of both larvae and spat contained accumulation bodies, often with a laminated, fibrous appearance in preparations for transmission electron microscopy. These accumulation bodies were PAS (periodic acid-Schiff) positive and may correspond to autolysosomal bodies within P. minimum cells. Juvenile oysters developed the ability to digest P. minimum, but only after a refractory period of about 2 weeks, during which most P. minimum was filtered but rejected as pseudofeces. The linking of accumulation bodies within absorptive cells of oyster digestive diverticula with dinoflagellate autolysosomal bodies suggests a mechanism by which some dinoflagellates interfere with feeding in phytoplankton grazers.

The relationship between gross biochemical composition of cultured algal foods and growth of the hard clam, Mercenaria mercenaria (L.)

Abstract Shell closure and restriction of filtration are behavioral responses by which bivalve molluscs can limit exposure of soft tissues to noxious or toxic agents, including harmful microalgae. In this study, we assessed the clearance rates of five species of bivalve mollusc—the northern bay scallop Argopecten irradians irradians, the eastern oyster Crassostrea virginica, the northern quahog Mercenaria mercenaria, the softshell clam Mya arenaria, and the blue mussel Mytilus edulis—exposed for one hour to each of three harmful-algal strains: Prorocentrum minimum, Alexandrium fundyense, and Heterosigma akashiwo. Clearance rates of harmful-algal cells were compared with clearance rates of a benign microalga, Rhodomonas sp., and to a Mix of each harmful alga with Rhodomonas sp. Qualitative observations of valve closure and production of biodeposits were also assessed during the exposure experiments. Feces and pseudofeces were collected and observed with light and fluorescence microscopy for the presence or absence of intact, potentially-viable algal cells or temporary cysts. Results increase our understanding of the high variation between the different bivalve/harmful alga pairs. Responses of bivalve species to the different harmful algae were species-specific, but in most cases indicated a preferential retention of harmful algal cells, probably based upon different characteristics of the algae. Each shellfish species also reacted differently to the harmful-algal exposures; several remained open; whereas, others, such as oysters exposed to the toxic raphidophyte Heterosigma akashiwo, closed shells partially or totally. Similarly, production of feces and pseudofeces varied appreciably between the different bivalve/alga pairs; with the exception of softshell clams Mya arenaria, intact cells of most harmful-algal species tested were seen in biodeposits of the other four bivalve species. These results extend our understanding of the high species specificity in the interactions between harmful algae and bivalve molluscs and confirm that generalizations about feeding responses of bivalves to harmful algae cannot easily be made. In most cases, however, there was at least some ingestion of the harmful algae leading to exposure of soft tissues to the algal cells.

Growth of post-set oysters, Crassostrea virginica, on high-lipid strains of algal flagellates Tetraselmis spp.

Under controlled laboratory conditions, young, post-set hard clams, Mercenaria mercenaria (L.), were reared for at least 12 weeks on 19 axenically-cultured unialgal diets; unfed controls were included. Daily algal rations were adjusted to have equivalent packed cell volumes but varied considerably in cell number (inversely related to cell size), dry weight, protein, lipid, and carbohydrate. Growth rates of hard clams fed experimental diets were calculated from weekly live-weight determinations; large differences in clam growth were observed. Of all algal ration characteristics measured, protein and lipid were the only two to show significant correlations (Spearman rank procedure, P < 0.05) with clam growth rate. A multiple linear regression model relating dietary protein, lipid, and carbohydrate with clam growth rate was highly significant (P < 0.0001), and explained 69% of the differences in clam growth on the 19 algal diets. Three algal species were cultured in different media to alter gross biochemical composition; results of feeding these algal strains confirmed that both dietary protein and lipid must be present in sufficient quantities to support rapid growth of hard clams. These findings have direct application to experimental and commercial clam rearing, and also further our understanding of the role of nutrition in population biology of clams in the field.

Immunological responses of the Manila clam (Ruditapes philippinarum) with varying parasite (Perkinsus olseni) burden, during a long-term exposure to the harmful alga, Karenia selliformis, and possible interactions.

Abstract Nine microalgal strains from the prasinophyte genus Tetraselmis that were chosen for high total lipid content, and one marine strain of the chlorophyte Chlamydomonas , were compared with Isochrysis sp., strain T-ISO, as diets for oyster spat in a controlled laboratory feeding experiment. Five of these high-lipid Tetraselmis strains supported significantly faster oyster growth than an equivalent ration of T-ISO. Doubling times for oyster weight, volume, and shell height were in the range of 1.5–3 weeks on single daily feedings of the best diets. Tetraselmis strains yielding the most rapid oyster growth contained higher contents of the essential fatty acid 20:5 n − 3 and of the sterols 24-methylcholesterol and/or 24-methylenecholesterol. These data are consistent with our earlier findings that these compounds appear to be deficient in most phytoplankton for optimal growth of Crassostrea virginica spat.

Sterols ofChaetoceros andSkeletonema

The present study evaluated the possible effects of a toxic dinoflagellate, Karenia selliformis, upon immunological hemocyte functions of the Manila clam Ruditapes philippinarum, and on the progression of infection by Perkinsus olseni. Clams with variable levels of perkinsosis were exposed for 6 weeks to simulated blooms of cultured the K. selliformis (10(2) and 10(3)cell ml(-1)). Samples were collected after 0, 2, 3, and 6 weeks of exposure. The following hemocyte parameters were measured by flow cytometry: percentage of dead cells, cell size and complexity, apoptosis, phagocytosis, and production of reactive oxygen species. Agglutination activities of K. selliformis on horse erythrocytes, serum protein concentration, and condition index of clams were also assessed. The harmful alga K. selliformis caused a significant decrease in hemocyte size and percentage of apoptotic cells. In contrast, P. olseni did not affect clams strongly; the only significant effect was an increase in hemocyte size in heavily infected clams. After 2 and 3 weeks, the prevalence and burden of P. olseni decreased in clams exposed to K. selliformis, but after 6 weeks, and a diminution in K. selliformis cell density in the exposure, this effect disappeared. In vitro tests exposing P. olseni to K. selliformis showed direct algal toxicity to the parasite (increased percentage of dead cells and altered morphology). Initial exposure of P. olseni-infected clams to K. selliformis appeared to modify the host-parasite interaction by causing effects in both organisms.