Terry L. Cucci

Particle selection, ingestion, and absorption in filter-feeding bivalves

Abstract Measurements were made of the clearance rate of six bivalve species each in the presence of mixed cell suspensions of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller (clone Exuv), the diatom Phaeodactylum tricornutum Bohlin (clone Phaeo), and the cryptomonad flagellate Chroomonas salina (Wislouch) Butcher (clone 3C). Use of flow cytometry allowed estimation not only of the clearance rate of individual cell types, but also of their proportional occurrence in the pseudofaeces and faeces. It has been recognized that at least three mechanisms of selecting suspended particles may be present in isolation or in combination. These are: (a) preferential clearance on the ctenidia: Ostrea edulis L., for example, preferentially clears the dinoflagellate Exuv compared with similar sized cells of the diatom Phaeo and the cryptomonad flagellate 3C; (b) preingestive selection on the labial palps: the diatom Phaeo was consistently and preferentially rejected in the pseudofaeces of Ensis directus Conrad, Placopecten magellanicus (Gmelin) and Arctica islandica (L.), (c) differential absorption in the gut i.e., post-ingestive selection: of the mixed diet which was ingested, there is clear evidence of a preferential absorption of the cryptomonad flagellate 3C in the majority of the bivalves from which we obtained faecal material. The possibility of selective removal of particular components of the available food resource, especially in the case of our experiments with the cryptomonad flagellate 3C, suggests that such organisms may be quantitatively more important in the diet of bivalves than their relative abundance under natural conditions might lead us to suppose. The ability of the oyster Ostrea edulis to selectively clear the dinoflagellate Exuv from mixed cell suspensions also may have important implications in our understanding of how toxic dinoflagellates may be concentrated on the ctenidia of commercially significant bivalves.

CELLULAR DNA CONTENT OF MARINE PHYTOPLANKTON USING TWO NEW FLUOROCHROMES: TAXONOMIC AND ECOLOGICAL IMPLICATIONS1

Two new fluorochromes, PicoGreen® and SYTOX Green™ stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument configuration of modern low‐power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes)examined. The major advantages of these dyes over others are 1)suitability for direct use in seawater, 2)green fluorescence emission of the DNA‐dye complex (wavelength 525 ± 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA‐dye complex with an increase in fluorescence > 100‐fold compared to the unstained cell, and 4)dyes can be used to quantify double‐stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phyto‐plankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustagmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, preservation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence.

The effects of the toxic dinoflagellate Protogonyaulax tamarensis on the feeding and behaviour of bivalve molluscs

A series of experiments was carried out to assess the effects of the toxic dinoflagellate Protogonyaulax tamarensis on shell-valve activity, rates of particle clearance, preingestive, and postingestive particle selection in seven species of bivalve molluscs from three geographic localities: Maine, Rhode Island, and Spain. The responses observed were species-specific and varied with collection locality. Responses included shell-valve closure and/or siphon retraction (Mya arenaria, Mytilus edulis, Geukensia demissa), reduced rates of particle clearance (M. arenaria, G. demissa), increased rates of particle clearance (Ostrea edulis), production of mucus (M. edulis from Spain and Rhode Island, Placopecten magellanicus, G. dernissa). Mortalities were noted in M. edulis from both Spain and Rhode Island. Two species (Modiolus modiolus and Spisula solidissma) exhibited no effects of the toxic dinoflagellates. Data are also presented for particle selection, clearance and filtration rates for the seven species. It is suggested that species which are periodically exposed to dinoflagellate blooms may have evolved mechanisms permitting them to exploit the toxic organisms as food with no ill effects.

Impact of fouling organisms on mussel rope culture: interspecific competition for food among suspension-feeding invertebrates☆

The clearance rate of natural planktonic assemblages was measured for the blue mussel Mytilus edulis (L.) and a co-occurring fouling community from mussel rope cultures using flow cytometry. Blue mussels had significantly higher clearance rates for all particle types and size classes. In addition, blue mussels showed selective feeding in favor of small phytoplankton (3–5 μm), whereas the solitary ascidian Ciona intestinalis (L.) and the suspension-feeding gastropod Crepidula fornicata (L.) showed preferential selection for large phytoplankton (> 16 μm). Clearance rates for large phytoplankton by these members of the fouling community were, however, always lower than blue mussels. Under conditions where food is not a limiting factor, interspecific competition for food by the associated fouling community should not significantly limit the yield of mussels.

Site of particle selection in a bivalve mollusc

Bivalve molluscs form dense populations that exert profound effects on the particle loads and phytoplankton composition of coastal waters. It has long been known that bivalves can select among different particle types, including selecting against those of poor nutritional value, but because of difficulties in observing particle transport processes in the pallial cavity in vivo, the mechanism of selection was not known. We now use a combination of video endoscopy and flow cytometry to show that oysters can select living particles from non-living detritus on the gills. Our methods could aid the study of suspension feeding in many animal groups.

Selective feeding by Balanion sp. (Ciliata: Balanionidae) on phytoplankton that best support its growth

Abstract The planktonic ciliate, Balanion sp., feeds preferentially on certain dinoflagellates when confronted with mixtures of microalgae. The preferred prey are species which support its fastest growth (≈ 3 div./day at 15°C). At high food densities, Balanion can grow as rapidly in algal mixtures which contain its preferred prey as its corresponding monocultures. Balanion are found year-round in estuaries in which dinoflagellates are common but are only occasionally the dominant phytoplankters. Preferential ingestion of suitable dinoflagellate prey may thus increase the growth rate of this ciliate in situ.

Overestimation of heterotrophic bacteria in the Sargasso Sea: direct evidence by flow and imaging cytometry

Abstract Accurate measurements of bacterial biomass in the ocean are needed for modeling marine microbial food webs and global biogeochemical cycling. We present direct evidence that previous estimates of heterotrophic bacteria biomass in the oligotrophic ocean are confounded by the presence of the abundant photosynthetic procaryote, Prochlorococcus . The chlorophyll autofluorescence of these photosynthetic bacterial cells is very faint and fades rapidly under epifluorescence microscopy. Detection and enumeration of these cells thus far has almost exclusively been by flow cytometry. Using a cooled, charge-coupled device (CCD) camera we were able to image these cells for direct biovolume measurements. A double-exposed image of DAPI-stained Prochlorococcus cells shows that they are indistinguishable from heterotrophic bacteria in standard slide preparations. At two Sargasso Sea stations Prochlorococcus could cause an overestimation of surface (top 150 m) integrated heterotrophic bacterial biovolume (biomass) of 18 and 22% determined by standard microscope methods. At the subsurface chlorophyll maximum Prochlorococcus was 33 and 43% of the heterotrophic bacterial biovolume (biomass) at these stations. Prochlorococcus cell size increased from 0.05 μm 3 in the surface mixed layer to about 0.2 μm 3 below 100 m, confirming previous interpretations of flow cytometric light scatter measurements. Shifting biomass from the heterotrophic bacteria pool to the primary producer compartment has significant implications for ecosystem structure and trophic transfer in marine food webs.

Optical backscattering by calcifying algae : Separating the contribution of particulate inorganic and organic carbon fractions

Light scattering properties of biogenic CaCO3 particles [particulate inorganic carbon (PIC)] were determined on cultured calcifying algae and field-derived CaCO3 particles. The particles were separated from particulate organic carbon (POC) with a flow cytometer, volume-scattering functions were measured with a laser light-scattering photometer, and particle composition was measured using atomic absorption spectrometry. Small calcite coccoliths were best sorted by gating on the ratio of horizontally polarized forward light scattering and vertically polarized forward light scattering; plated coccolithophores could be sorted by gating on side scattering and forward angle light scattering. Normalized volume-scattering functions for the culture-derived calcite particles varied by a factor of 2 for the different species. Backscattering cross sections (m2 particle−1) for calcite particles varied by ∼35 times and were generally a function of size. Backscattering efficiencies were ∼2–4 times higher for cells with CaCO3 than without it. CaCO3-specific backscattering showed much less variability across various species; the calcite-specific backscattering coefficient varied by only ∼38% for both cultured coccolithophores and field-derived CaCO3 particles. Organic carbon-specific backscattering of “naked” coccolithophores was highly consistent within all coccolithophores used in our experiments, as well as with values in the literature. Our results suggest that both POC and PIC can be optically estimated, the former by measuring backscattering of decalcified phytoplankton as well as their size distribution, and the latter is proportional to acid-labile backscattering. These results show the feasibility of a rapid optical technique for measuring two biogeochemically important carbon fractions in the sea.

COCCOLITH PRODUCTION AND DETACHMENT BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)

Laboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. 14C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. 14C‐labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a 14C‐based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell−1·d−1 while net detachment rates were as high as 15 coccoliths· cell−1·d−1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.

Particle clearance and selection in three species of juvenile scallops

Juvenile scallops (<2 mm shell height) of three species (Placopecten magellanicus, Patinopecten yessoensis, Argopecten irradians) were fed mixed, unialgal cultures. Scallops were fed a total of six algal clones simultaneously and clearance rates were monitored using flow cytometric techniques. In another experiment, scallops were presented with natural assemblages of particulate matter as a food source. Data are presented on differences in clearance rates for the individual algal species as well as size-related differences of algal clones, and uptake of chlorophyll vs. non-chlorophyll cells, both within and between scallop species. Significant differences in clearance rates of individual algal species have been found within and between scallop species. Particle selection does not appear to be based upon size alone and is apparently based on other characteristics of the algae as well. The results demonstrate pre-ingestive sorting.