Pallial Fluid Research Articles

The development of multiplex PCR assays for the rapid identification of multiple Saccostrea species, and their practical applications in restoration and aquaculture

BackgroundThe ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted in the misidentification of species, creating challenges for understanding basic species-specific biological information that is required for restoration and aquaculture. Genetic barcoding has proven essential for accurate species identification and understanding species geographic ranges. To reduce the costs of molecular species identification we developed multiplex assays using the cytochrome c oxidase subunit I (COI or cox1) barcoding gene for the rapid identification of five species of oysters within the genus Saccostrea that are commonly found in Queensland, Australia: Saccostrea glomerata, Saccostrea lineage B, Saccostrea lineage F, Saccostrea lineage G, and Saccostrea spathulata (lineage J).ResultsMultiplex assays were successful in species-specific amplification of targeted species. The practical application of these primers was tested on wild spat collected from a pilot restoration project in Moreton Bay, Queensland, with identified species (S. glomerata, lineage B and lineage G) validated by Sanger sequencing. DNA sampling by extraction of oyster pallial fluid was also tested on adult oysters collected from the Noosa estuary in Queensland to assess whether oysters were able to be identified non-destructively. DNA concentrations as low as 1 ng/ μL still amplified in most cases, allowing for identification, and mortality at 6 weeks post pallial fluid collection was low (3 out of 104 sampled oysters).ConclusionThese multiplex assays will be essential tools for species identification in future studies, and we successfully demonstrate their practical application in both restoration and aquaculture contexts in Queensland. The multiplex assays developed in this study outline easily replicable methods for the development of additional species-specific primer sets for the rapid identification of other species of Saccostrea found across the Indo-Pacific, which will be instrumental in unravelling the taxonomic ambiguities within this genus in tropical regions.

A Microcosm Experiment Reveals the Temperature-Sensitive Release of Mucochytrium quahogii (=QPX) from Hard Clams and Pallial Fluid as a Stable QPX Reservoir.

Mucochytrium quahogii, also known as QPX or Quahog Parasite Unknown, is the causative agent of QPX disease in the hard clam (Mercenaria mercenaria). Host-pathogen-environment interactions between M. quahogii, the hard clam, and temperature were explored in a microcosm experiment. Hard clams were housed in individual tanks with sterile seawater under two temperature regimes: low (13 °C) temperature, which is thought to be optimal for QPX disease development, and high (20 °C) temperature, which has been shown to promote "healing" of QPX-infected clams. Hard clam tissue, pallial fluid, seawater, and shell biofilms were collected and assayed for M. quahogii. The release of M. quahogii from naturally infected live hard clams into seawater was detected only in the low temperature treatment, suggesting that temperature influences the release of potentially infectious cells. M. quahogii was commonly found in hard clam pallial fluid, even after 9 weeks in the lab, suggesting pallial fluid is a stable reservoir of M. quahogii within its primary host and that M. quahogii is not a transient component of the hard clam microbiota. Overall, results support a host-specific relationship and that M. quahogii is a commensal member of the hard clam microbiota, supporting its classification as an opportunistic pathogen.

Mucochytrium quahogii (=QPX) Is a Commensal, Opportunistic Pathogen of the Hard Clam (Mercenaria mercenaria): Evidence and Implications for QPX Disease Management.

Mucochytrium quahogii, commonly known as QPX (Quahog Parasite Unknown), is the causative agent of QPX disease in hard clams (Mercenaria mercenaria), but poor understanding of the relationship between host and pathogen has hindered effective management. To address this gap in knowledge, we conducted a two-year study quantifying the distribution and abundance of M. quahogii in hard clam tissue, pallial fluid, and the environment. M. quahogii was broadly distributed in clams and the environment, in areas with and without a known history of QPX disease. M. quahogii in clams was not strongly related to M. quahogii in the environment. M. quahogii was always present in either the tissue or pallial fluid of each clam, with an inverse relationship between the abundance in the two anatomical locations. This study suggests that the sediment-water interface and clam pallial fluid are environmental reservoirs of M. quahogii and that there is a host-specific relationship between M. quahogii and the hard clam, supporting its classification as a commensal, opportunistic pathogen. There appears to be minimal risk of spreading QPX disease to naïve clam populations because M. quahogii is already present and does not appear to be causing disease in hard clam populations in locations unfavorable for pathogenesis.

Microbial Inhibition by Bacteria Isolated from Pallial Cavity Fluids and Associated Mucus of the Eastern Oyster Crassostrea virginica (Gmelin)

Thirteen aerobic, mesophilic marine bacterial strains were isolated from the pallial (mantle) cavity fluids and associated mucus of the eastern oyster Crassostrea virginica Gmelin. All strains were Gram-negative and identified to genus using 16S RNA gene sequence analysis. Each isolated strain was tested for its ability to inhibit growth of six Gram-negative and five Gram-positive bacterial tester strains, as well as the yeast Candida albicans, using an in vitro agar diffusion screening method to detect antimicrobial activity (Romenenko et al. 2008). All of the marine bacteria isolated from the oyster pallial cavity fluids showed some ability to inhibit tester strains. Two isolates, F (red) and M, inhibited the greatest variety of indicator strains and produced the largest zones of inhibition. Gram-negative indicator strains were more susceptible to antimicrobial activity of pallial fluid isolates than Gram-positive strains. None of the isolated bacteria was shown to inhibit C. albicans. In addition, targeted 16S metagenome libraries from the Chesapeake Bay were screened for the presence of bacteria isolated from this study. Results indicate that there are commonalities between bacteria associated with oysters from Long Island Sound and the Chesapeake Bay systems.

Antimicrobial Activity in the Pallial Cavity Fluids of the Ribbed Mussel Geukensia Demissa from a Highly Impacted Harbor in Western Long Island Sound

Fluid and its associated mucus from the pallial (mantle) cavity of the ribbed mussel Geukensia demissa (Dwillyn) from Black Rock Harbor, Bridgeport, CT, inhibited the growth of both Gram-positive (Staphylococcus aureus) and Gramnegative (Escherichia coli) bacteria in antimicrobial assays. A significant reduction in size after 24 h was noted in E. coli grown in the presence of ribbed mussel fluid. Mussel-soluble lysozyme levels in the pallial cavity fluid averaged 0.019 + 0.018 relative fluorescence units/mg protein, and no seasonal pattern of lysozyme activity was found (P = 0.522). During the course of the study, copper concentrations ranged from 0.09 to 0.37 ppm and zinc concentrations from 0.17 to 0.66 ppm in the pallial cavity fluid. These values were only slightly higher than the concentrations of these metals found in seawater samples taken at the site, indicating only very low levels of sequestration of heavy metals by G. demissa in the pallial fluid cavity. A comparable study of oysters (Crassostrea virginica) from the same site found lysozyme levels 10 times higher and zinc concentrations two orders of magnitude greater than in that in mussels reported here (Brousseau et al. 2014). These results suggest substantial interspecies variation in profiles of defensive agents involved in antimicrobial activities of marine bivalves and highlight the need for additional studies to characterize these differences.

EFFECTS OF CHROMIUM ON PROTEIN AND CARBOHYDRATE LEVELS IN THE MANTLE OF FRESHWATER MUSSEL Anodonta woodiana (Lea, 1834)

<p>Chromium is one of the technical importance metals found in the aquatic environment at increasing concentrations, resulting from metallurgic, refractory, chemical, and tannery in­dustries (Satyaparameshwar et al., 2006). The metal is known to be essential for all animals such as maintenance of normal glucose tolerance (Chassard-Bouchaud et al., 1989). It also involves in metabolisms of lipids, proteins, nucleic acids, and mineral substances (Pechova & Pavlata 2007). At elevated concentrations, chromium is toxic to mussels, resulting in changes of mRNA expression, immunomodulation, DNA stability, and stress response path­ways (Franzellitti et al., 2012). Swan mussels (Anodonta woodiana) living at the interface of the free-flowing water and the sediment phase of many lotic freshwater ecosystems are known to accumulate metals in their body. Mussels require carbohydrates as main energy source for their metabolic processes (Honkoop et al., 1999). Proteins are also required by mussels for catalyzing biochemical reactions, transport and storage of molecules in and out, or within cells, and have structural and mechanical functions (Albert et al., 1994). Proteins and carbohydrates are also important components in the shell formation (Marie et al., 2007; Marin & Luquet 2004). Mantle secretes proteins and carbohydrates into extra pallial fluid, which is directly adjacent to the shell. The objective of this research was to study the effects of Cr on the protein and carbohydrate levels in the mantle of A. woodiana.</p>

The influence of site and season on the gut and pallial fluid microbial communities of the eastern oyster, Crassostrea virginica (Bivalvia, Ostreidae): community-level physiological profiling and genetic structure

The microbial communities and overall health of the eastern oyster, Crassostrea virginica, have long been topics of interest due to the fundamental economic and ecological roles this species maintains. A broad scale characterization of the oyster microbiome over spatial and seasonal scales, however, has never been carried out. The primary goal of this study was to examine the factors mediating microbial communities of the gut and pallial fluid of C. virginica at three sites within the Long Island Sound estuary, with a focus on both genetic structure (T-RFLP) and physiological profiling (EcoPlates) of the microbiome. Results indicated that the genetic structure of microbial communities of oysters was minimally separated across sites, but was influenced by season. Although the microbial community structure was similar, the number of carbon sources utilized by these communities (richness) varied across site, season, and anatomical location within the host. Parameters including oyster condition index, Dermo disease, and ambient water temperature were measured to assess their influence on the oyster microbiome. Only water temperature was found to have a significant relationship with microbial community structure and richness. Results suggest that a core microbiome may exist within the eastern oyster, specifically for those populations that are not genetically distinct.

Brooding in the Chilean oyster Ostrea chilensis: unexpected complexity in the movements of brooded offspring within the mantle cavity.

Brooding in invertebrates serves to protect embryos from stressful external conditions by retaining progeny inside the female body, effectively reducing the risk of pelagic stages being exposed to predation or other environmental stressors, but with accompanying changes in pallial fluid characteristics, including reduced oxygen availability. Brooded embryos are usually immobile and often encapsulated, but in some Ostrea species the embryos move freely inside the female pallial cavity in close association with the mother’s gills for as long as eight weeks. We used endoscopic techniques to characterize the circulation pattern of embryos brooded by females of the oyster, Ostrea chilensis. Progeny at embryonic and veliger stages typically circulated in established patterns that included the use of dorsal and ventral food grooves (DFG, VFG) to move anteriorly on the gills. Both embryos and veligers accumulated around the mother’s palps, and remained there until an active maternal countercurrent moved them to the gill inhalant area. Both food grooves were able to move embryos, veligers, and food-particle aggregates anteriorly, but the DFG was more important in progeny transport; early embryos were moved more rapidly than veligers in the DFG. A microcirculation pattern of embryos was apparent when they were moved by gill lamellae: when they were close to the VFG, most embryos lost gill contact and ´´fell´´ down to the DFG. Those that actually reached the DFG moved anteriorly, but others came into contact with the base of the lamellae and again moved towards the VFG. The circulation pattern of the progeny appears well-suited for both cleaning them and directing them posteriorly to an area where there is more oxygen and food than in the palp region. This process for actively circulating progeny involves the feeding structures (gill and palps) and appears to be energetically costly for the female. It also interferes with feeding, which could explain the poor energy balance previously documented for brooding females of this species.

Ammonia accumulation in the brood chamber of the estuarine gastropod Crepipatella dilatata: How big a problem for mothers and brooded embryos?

Crepipatella dilatata is an estuarine gastropod that broods its encapsulated embryos in the pallial cavity for several weeks before releasing juveniles. When environmental quality declines (e.g., in response to decreased salinity), females seal themselves against the substrate and isolate the pallial cavity from surrounding seawater, altering the chemistry of the pallial fluid surrounding the embryos. We documented rates at which total ammonia concentrations (NH 4 +–N + NH 3) increased in the pallial cavity during these extended periods of isolation, as well as excretion rates for both adult females and developing embryos (veligers). Also, we quantified the effects of elevated ammonia concentrations on both adults (mortality, egg capsule abandonment, and neck-radular activity) and veligers (mortality, and activity of the velum and velar cilia). In the pallial fluid, ammonia concentration increased about 150% during the first 16 h of female isolation from about 7–17 μg ammonia l −1, while excretion rates decreased nearly 85%, from an average of 5.5 to about 0.85 μg ammonia h −1 g −1 after 12 h of isolation. Brooded veligers also decreased their individual excretion rates over time, from 0.0035 to 0.0007 μg ammonia h −1 veliger −1 over 12 h. Ammonia accumulation eventually caused females to abandon their egg capsules and to detach from the substrate; in the field, this would probably lead to the death of both the encapsulated embryos and the detached females, but this happened only at concentrations of ≥ 0.1 mg ammonia l −1. In contrast, veligers withstood concentrations up to 34 mg ammonia l −1 for 72 h without dying, although ciliary activity and velar lobe activity decreased at concentrations of ≥ 0.1 mg ammonia l −1. Ammonia tolerance did not seem to change with continued development. Although previous studies have shown that prolonged isolation causes dangerous declines in oxygen and pH in pallial fluid for this species, the accumulation of ammonia in itself seems too low to cause direct harm to adults or developing embryos.

Effect of reduced pH on shells of brooded veligers in the estuarine bivalve Ostrea chilensis Philippi 1845

The oyster Ostrea chilensis develops in estuaries and incubates its embryos inside the pallial cavity. Reduced external salinity triggers valve closure and isolation of the brood chamber, which in turn alters conditions in the pallial fluid, including pH reductions ranging from 7.65 (SD = 0.05) to 6.97 (SD = 0.02) in non-brooding females and 6.96 (SD = 0.10) in brooding females after 12 h of isolation. Exposing veliger larvae to acidic pH's (5 and 3) decreased the thickness of the embryonic shell valves. Calcium, sodium, chlorine, tin, sulphur, and magnesium were the most important (> 99%) elements constituting the veliger shells. Calcium content in the pallial cavity fluid increased with continued isolation in both brooding and non-brooding females, but calcium remained as the primary component (about 94%) of embryonic shells and maintained its normal proportion with respect to the rest of the elements at all tested pH's. The increased calcium in the pallial fluid was identified mainly as coming from the shells of brooding females. If any neutralizing elements were coming from embryonic shell valves, all elements in those shells must have been solubilising to the same degree. Veligers ceased all further shell growth while isolated within the female's brood chamber. However, veliger shell growth resumed once females were returned to high-salinity seawater. Our study shows that female isolation of the mantle cavity, often considered an adaptive response to the periodically reduced salinities of estuarine waters, can in fact generate adverse effects on brooded embryos.

Brood chamber isolation during salinity stress in two estuarine mollusk species: from a protective nursery to a dangerous prison

Faced with environmental stress, the females of many marine invertebrate species can isolate their brood chambers from the external environment for prolonged periods. Although brood chambers have therefore typically been assumed to protect the brooded embryos, the effects of continued isolation on conditions within brood chambers have not been examined. In the present research, we investigated the effects of external stress on conditions in the brood chambers for 2 sympatric species, the gastropod Crepipatella dilatata and the bivalve Ostrea chilensis. Both species incubate their embryos in the pallial cavity for at least 4 wk before the young emerge as larvae (oyster) or juveniles (gastropod). Brooding and non-brooding females were stimulated to isolate the pallial cavity from the external environment by exposing them to water of reduced salinity (<22 to 24 psu). We then recorded subsequent changes in salinity, dissolved oxygen, pH, and ammonium ion concentration within the pallial fluid. Although salinity within the pallial cavity remained high for both species, oxygen availability for brooding females of C. dilatata and O. chilensis dropped to hypoxic levels (<1.5 mg O2 l -1 ) within 12 h and 20 min, respectively. In addition, pH of the intrapal- lial fluid dropped substantially with prolonged maternal isolation, suggesting a possible impact on shell formation for brooded veligers. Indeed, in the case of C. dilatata, the pH of pallial fluid initially fell to 6.4, but then tended to become more basic after 6 h of maternal isolation, a possible conse- quence of shell dissolution. On the other hand, ammonium ion concentration increased progressively with prolonged maternal isolation. Our data show that, whereas initially the isolation mechanism protected incubated embryos from exposure to low salinity stress, continued isolation from the exterior transformed the brood cavity from a protective environment to an oppressive one, i.e. from a protective nursery to more of a prison.

Isolation of incubation chambers during brooding: effect of reduced pH on protoconch development in the estuarine gastropod Crepipatella dilatata (Calyptraeidae)

The sedentary gastropod Crepipatella dilatata encapsulates its embryos and incubates them in the pallial cavity. Females can deliberately isolate their embryos from the external environ- ment for many hours at a time by pressing their shells tightly against the substrate. We documented the effects of such isolation on pH in the pallial cavity and the effects of reduced pH on changes in shell thickness and the proportion of calcium in embryonic shells. We also quantified the concen- tration of calcium in the water retained in the pallial cavity during isolation, as well as the growth of encapsulated veligers. Average protoconch thickness decreased by as much as 50% at reduced pH (pH = 6 to 3). During 24 h of exposure to water at the different pHs, calcium was lost from shells, but in same proportion as the loss of other components. Calcium in the pallial fluid increased during the first 5 h of isolation for both brooding and non-brooding females, suggesting shell dissolution. The calcium content of pallial cavity fluid for brooding females differed from that of non-brooding females, but did not differ from that in the intracapsular fluid, suggesting that calcium diffuses freely through the egg capsule wall. Isolation of the incubatory chamber impeded embryonic shell growth, possibly because the calcium was acting as a 'buffer' to regulate pH changes in the pallial fluid. The isolation of embryos by incubating females, usually viewed as an adaptive benefit of brood- ing, can cause pronounced negative effects on embryonic development during the period of shell formation.

Haemocyte response associated with induction of shell regeneration in the deep-sea vent mussel Bathymodiolus azoricus (Bivalvia: Mytilidae)

This study reports on the haemocyte responses after induction of shell regeneration in the hydrothermal mussel Bathymodiolus azoricus. Haemolymph was drawn from live mussels collected at Menez Gwen hydrothermal vent site (850 m depth) at the Mid Atlantic Ridge (MAR) and was compared with those collected following laboratory acclimatisation (1 atm and Ca-rich algal diet) and also with induced specimen for up to 30 days. Simultaneously, histological changes in mantle micro-morphology with the histochemical detection of Ca mobilisation in tissues were conducted. On the basis of light- and transmission electron microscopy, it is concluded that the physiological equipment involved in shell regeneration in the deep sea bivalve closely resembles that in littoral mytilids, a group that B. azoricus is closely related. This in spite of previously alleged molecular and cellular adaptations to extreme conditions typical at deep sea hydrothermal vents. Three types of blood cells were identified sharing various morphological similarities with those in many non-vent bivalves. Significant increase in the number of circulating haemocytes was detected from day 5 after induction shell regeneration. It is suggested that the increase may be a result of migration of haemocytes from the connective tissue, probably to the shell growth frontline. It is alleged that a first peak in haemocyte number is a non-specific immune response related wound healing, which renders changes in the pallial fluid that are favourable for CaCO 3 deposition. The conspicuous presence of an unidentified, acid soluble, highly refractive structure in the haemolymph of induced mussels was detected, which may play a role in Ca nucleation. This study has set the stage for investigations underway on the influence of hydrostatic pressure on shell biomineralisation in B. azoricus subjected to post-capture hyperbaric simulations.

Fluid and osmolyte recovery in the common pond snailLymnaea stagnalisfollowing full-body withdrawal

The common pond snail Lymnaea stagnalis sacrifices 40-60% of its extracellular fluid (ECF) including solutes to the surrounding environment to fully retract into its shell. Our objectives were to determine recovery time following such ECF loss and characterize mechanisms involved in recovering Na(+), the primary cationic osmolyte in this snail. Pallial fluid was initially collected post-stimulation and again after a second stimulation on sub-groups of those snails 2 h to 10 weeks after initial sampling. Samples were analyzed for pH, osmotic pressure, and individual solute concentrations. Lost volume was recovered within 8 h with no significant changes in pH or total CO(2). Significant decreases in osmotic pressure, [Na(+)] (43% loss), and [Cl(-)] recovered in 48 h as a result of enhanced uptake from the water. Copper and total extracellular proteins took 5 weeks to recover. Measurements of Na(+) transport kinetics completed before and immediately after fluid loss revealed a near threefold increase in both affinity and capacity of the Na(+) uptake system. Sodium uptake was independent of ambient Cl(-) and HCO(3)(-) in both control and fluid-depleted snails. Amiloride significantly reduced recovery-phase Na(+) uptake rates but did not influence baseline Na(+) flux. Recovery uptake was significantly reduced by amiloride, ethylisopropylamiloride, bafilomycin and ethoxzolamide indicating dependency upon Na(+)/H(+) exchange, H(+) pump activity and H(+) from carbonic anhydrase-catalyzed CO(2) hydration. Thus enhanced uptake during recovery is likely via electrogenic Na(+)/H(+) exchange and/or possibly a cation channel.

Localization of the bacterial agent of juvenile oyster disease ( Roseovarius crassostreae) within affected eastern oysters ( Crassostrea virginica)

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters ( Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.

Effect of polycyclic aromatic hydrocarbons on the pallial fluid buffering capacity of the marine mussel, Mytilus galloprovincialis

Pallial fluid buffering capacity of the sea mussel Mytilus galloprovincialis was investigated to establish the potential of this biological parameter to serve as a biomarker. Four groups of 15 animals were used in a 72-h toxicity test. Group 1, the aerobic control group, was placed in a filtered aerated natural seawater aquarium. Groups 2–4 were subjected to hypoxic conditions by removal from water after animals were injected with a single dose of the following: group 2 (anaerobic control) was administered 10 μl of UV-treated filtered natural seawater; group 3 (anaerobic solvent control) was injected with 10 μl of acetonitrile and group 4 (PAH exposed group) with 10 μl of 2 mM anthracene. Pallial fluid was taken from all the animals following seawater immersion or air exposure. Pallial fluid from each individual was extracted, adjusted to pH 5.0 and titrated with NaOH until reaching pH 6.0. The buffering capacity index (β), defined as the amount of μequivalents of NaOH needed to change in one unit the pH of a 5-ml sample of pallial fluid, was calculated for each group. Values were: for group 1, β=3.17 (±0.782); for group 2, β=15.713 (±2.992); group 3 was β=18.124 (±2.288); and group 4 was β=28.109 (±11.398). The statistically significant increase ( P<0.05) in group 4 compared with the other groups indicates that the buffering capacity index (β) is a worthy biological parameter to be further explored as a biomarker for ecotoxicological monitoring programs. The increase in buffering capacity is discussed and a biochemical link between anaerobic metabolism and the exposure to PAH is suggested to explain changes of this biological parameter.

Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri- n-butyltin chloride

Induction of apoptosis by tri- n-butyltin (TBT) in gill tissue of the mussel Mytilus galloprovincialis was investigated. The terminal dUTP nick-end labeling technique (TUNEL) was used to detect cells displaying DNA fragmentation within gill structures. Genomic DNA fragmentation was detected as characteristically ladder-like pattern of DNA fragments induced by single injection of different doses of TBT (1–5 μg/g) below the mantle, directly into the pallial fluid, after 24 h of incubation. DNA degradation of higher order DNA structure, as well as reduced G 0/G 1 cell cycle region (the sub-G 1 region) was detectable after 1.5 h of TBT incubation. Presence of apoptotic cells in mussels’ gills was indicated by the selective loss of G 2/M cells concomitant with the appearance of cells with decreased DNA content in S and G 0/G 1 cell cycle regions. The effect of the TBT on cell cycle in a mussel gill was a dose related and exposure time depending. The possible mechanism of induction of apoptosis in vivo in gill tissue of mussel treated with TBT is discussed.

Quantified Aspects of Pallial Fluid and Its Affect on the Duration of Locomotor Activity in the Terrestrial Gastropod Triodopsis albolabris

Triodopsis albolabris accumulated large quantities of fluid within the mantle cavity, i.e., pallial fluid, as a result of hydration. During periods of dehydration, pallial fluid quantities decreased significantly and the rate of pallial fluid weight loss reflected the pattern of body water (weight) loss. The contribution of pallial fluid weight loss to total animal weight loss during dehydration was relatively constant. Pallial fluid osmolality remained between 38 and 58 mOsm during dehydration of the animals, far below that of the hemolymph of snails both with and without pallial fluid. Hemolymph osmolality of snails retaining pallial fluid during dehydration was significantly lower than that of snails forced to expel pallial fluid prior to exposure to dehydrating conditions. Direct measurements of locomotor activity of individual snails using a treadmill apparatus showed that snails with pallial fluid were active significantly longer than snails without pallial fluid. The results show a direct relationship between the possession of pallial fluid and prolonged locomotor activity of T. albolabris. The present evidence strengthens the hypothesis that pallial fluid acts as a water reserve to replenish water lost to evaporation and mucus production as a result of exposure and locomotor activity.

Ion relations of haemolymph, palliai fluid, and mucus of Lymnaea stagnalis

The concentrations, and where possible, activities of the main inorganic ions were measured in haemolymph, palliai fluid, and mucus of Lymnaea stagnalis appressa Say (Gastropoda: Pulmonata), and in the artificial pond water (APW) in which the snails were stored. Activity coefficients of Na, K, Ca, and Cl in pallial fluid and in APW were compared with values calculated from Debye–Hückel theory. Some Na and Ca in pallial fluid, and Ca in APW are probably bound in organic or inorganic complexes. Comparison of the Nernst potentials with the transepithelial potential difference supports the idea that Na, K, Ca, and Cl must be actively taken up from APW by the snail. Pallial fluid is not identical to haemolymph and is probably a mixture of urine and haemolymph. Pallial fluid contaminates mucus during mucus collection. A method of correcting for this contamination is given. After correction, mucus had higher Ca and K, and possibly higher Mg concentrations than did haemolymph. Mucus had a high concentration of NH4+. Possible sources of ions in mucus are discussed. Mucus secretion rates are low enough that this is not an important cause of ion loss to the snail. Using ether to stimulate mucus secretion alters the water and K content of the mucus.

Anaerobic uptake and utilization of glycine-2- 14C by the estuarine bivalve Rangia cuneata at three salinities

1. 1. During facultative anaerobiosis Rangia cuneata acclimated at 5, 10 and 15‰ were injected with 0.1 μCi glycine-2- 14C into the pallial fluid and incubated for 3, 12, 24 and 72 h. 2. 2. Rangia rapidly accumulated glycine from the pallial fluid. 3. 3. Accumulation was not significantly different at 5, 10 and 15‰. 4. 4. Glycine was only metabolized into proteins, and at a slow rate which increased as salinity decreased. 5. 5. Glycine was not involved in production of ATP during facultative anaerobiosis. 6. 6. Results suggest both a general decrease in ATP utilization and selective inhibition of certain pathways in order to conserve ATP for the more essential pathways.