Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Light intensity determines temporal niche switching of behavioral activity in deep-water Nephrops norvegicus (Crustacea: Decapoda).

The temporal distribution of behavioral programs throughout the 24-h day, known as temporal niche of a species, is determined by ecological factors that directly affect the adaptive value of the timing of specific behaviors. Temporal niche switching has been described in several species and is likely adaptive in habitats where the daily timing of those factors changes. Benthic species whose habitats span a wide range of water depths are exposed to considerable depth-dependent environmental changes. Temporally scheduled trawl surveys of the Norway lobster, Nephrops norvegicus, reveal that animals emerge from burrows at night on the shallow shelf (10-50 m deep), at crepuscular hours on the lower shelf (50-200 m), and at daytime on the slope (200-400 m). The mechanisms underlying nocturnality/diurnality switches are chiefly unknown, and Nephrops offers a unique model for their study. The depth-dependent decrease in luminance is a likely candidate determining the temporal distribution of behavior. The authors explored this possibility in the laboratory by exposing Nephrops to light:dark (LD) cycles of 470-nm monochromatic lighting that mimic conditions at the 100-m-deep shelf (10 lux) or the 300-m slope (0.1 lux). Two groups of animals were respectively exposed to each light intensity according to the following protocol: an initial 12:12 LD stage followed by constant darkness (DD), followed in turn by a second 12:12 LD stage. Activity at the burrow opening (door-keeping = DK), as well as full emergence (E), was continuously monitored. Under 10-lux LD cycles, most animals showed nocturnal DK activity-with some being crepuscular or diurnal-and all animals showed nocturnal E activity. In contrast, both behaviors were clearly diurnal in animals under 0.1-lux LD cycles. The phase of the nocturnal and diurnal DK rhythms detected respectively at 10 and 0.1 lux upon release into DD revealed that these rhythms are entrained circadian rhythms. The present data indicate that nocturnality/diurnality switches in Nephrops in its natural habitat, evidenced by captures at different depths, are likely determined by light intensity. This temporal niche switching involves different patterns of photic entrainment, leading to bona fide circadian diurnal or nocturnal phenotypes, as well as exogenous masking of behavioral outputs.

65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus.

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 muM verapamil, nifedipine, and the calcium ionophore A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1,000 microM) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas.

This investigation combines confocal microscopy with the cation-specific fluorescent dyes Fluo-3 and BTC-5N to localize calcium and heavy metals along the length of intact lobster (Homarus americanus) hepatopancreatic tubules and isolated cells. A metallothionein-specific antibody, developed in mollusks with cross-reactivity in crustaceans, showed the tissue-specific occurrence of this metal-binding protein in several organ systems in lobster and in single cell types isolated from lobster hepatopancreas. Individual lobster hepatopancreatic epithelial cell types were separated into pure single cell type suspensions for confocal and antibody experiments. Intact hepatopancreatic tubules showed high concentrations of both calcium and heavy metals at the distal tips of tubules where mitotic stem cells (E-cells) are localized. In addition, a concentrated distribution of calcium signal within isolated single premolt E-cells in solution was disclosed that might suggest an endoplasmic reticulum compartmentation of this cation within these stem cells. Both E- and R-cells showed significantly (P < 0.05) greater intracellular calcium concentrations in premolt than intermolt, suggesting the accumulation of this cation in these cells prior to the molt. Antibody studies with lobster tissues indicated that the hepatopancreas possessed 5-10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells. These results suggest that crustacean hepatopancreatic stem cells (E-cells) and R-cells play significant roles in calcium and heavy metal homeostasis in this tissue. Interactions between the four hepatopancreatic cell types in this regulatory activity remain to be elucidated.