Lewis E. Deaton

Osmoregulation and Excretion

The article discusses advances in osmoregulation and excretion with emphasis on how multicellular animals in different osmotic environments regulate their milieu intérieur. Mechanisms of energy transformations in animal osmoregulation are dealt with in biophysical terms with respect to water and ion exchange across biological membranes and coupling of ion and water fluxes across epithelia. The discussion of functions is based on a comparative approach analyzing mechanisms that have evolved in different taxonomic groups at biochemical, cellular and tissue levels and their integration in maintaining whole body water and ion homeostasis. The focus is on recent studies of adaptations and newly discovered mechanisms of acclimatization during transitions of animals between different osmotic environments. Special attention is paid to hypotheses about the diversity of cellular organization of osmoregulatory and excretory organs such as glomerular kidneys, antennal glands, Malpighian tubules and insect gut, gills, integument and intestine, with accounts on experimental approaches and methods applied in the studies. It is demonstrated how knowledge in these areas of comparative physiology has expanded considerably during the last two decades, bridging seminal classical works with studies based on new approaches at all levels of anatomical and functional organization. A number of as yet partially unanswered questions are emphasized, some of which are about how water and solute exchange mechanisms at lower levels are integrated for regulating whole body extracellular water volume and ion homeostasis of animals in their natural habitats.

Hyperosmotic volume regulation in the gills of the ribbed mussel, Geukensia demissa: rapid accumulation of betaine and alanine.

The content of betaine and alanine in gills of the ribbed mussel Geukensia demissa increases rapidly following transfer of the tissues from 250 to 1000 mOsm seawater (SW). Increases in alanine, proline and glycine account for most of the increase in the amino acid pool. The betaine content increases from 45 to 150 &mgr;mol/g dry weight within 12 h. Transfer of isolated gills from 250 to 1000 mOsm SW results in a temporary cessation of all ciliary activity. Within 20-40 min following transfer, ciliary activity has recovered. Recovery of ciliary activity precedes recovery of tissue hydration. The uric acid content of gills is unchanged by exposure to hyperosmotic media, suggesting that uric acid is not a store of nitrogen for alanine synthesis from pyruvate. In other organisms, the accumulation of betaine in response to hyperosmotic stress is a slow (days to weeks) process that probably involves changes in gene expression. The rapid, large increases in betaine reported here suggest that gene expression is not a factor in volume recovery by euryhaline bivalve tissues exposed to acute hyperosmotic stress.

CHARACTERIZATION OF PHENOLOXIDASE FROM CRASSOSTREA VIRGINICA HEMOCYTES AND THE EFFECT OF PERKINSUS MARINUS ON PHENOLOXIDASE ACTIVITY IN THE HEMOLYMPH OF CRASSOSTREA VIRGINICA AND GEUKENSIA DEMISSA

Abstract The enzyme phenoloxidase is believed to be a component of internal defense in invertebrates. We detected phenoloxidase activity in the membrane fraction of hemocytes from the eastern oyster, Crassostrea virginica. The activity is associated with a protein with a molecular weight of 133 kDa. That the enzyme is a phenoloxidase and not a peroxidase was shown by inhibition of the activity by diethyldithiocarbamic acid. The phenoloxidase displayed a preference for diphenol substrates and was inactivated by incubation at temperatures above 70°C. The activity was highest in a pH range of 6.0–7.5. The oyster pathogen P. marinus causes serious disease in the commercial oyster, Crassostrea virginica, but not the mussel Geukensia demissa. We measured the effect of P. marinus cells on the phenoloxidase activity in hemolymph from C. virginica and G. demissa over a 6-h time course. The presence of P. marinus significantly suppressed the phenoloxidase activity of both species at the 2-h time interval. The phenoloxidase activity seemed to increase at the 4-hr and 6-h time intervals. These data suggest that a transient inhibition of host phenoloxidase may play a role in the study of P. marinus infection.

Osmotic regulation and salinity tolerance in the freshwater snail Pomacea bridgesi and the freshwater clam Lampsilis teres

Abstract Unionid clams ( Lampsilis teres) and apple snails ( Pomacea bridgesi ) were gradually acclimated to media ranging in osmotic concentration from fresh water to 400 mOsm. There was no mortality among L. teres in all media from fresh water to up to dilute sea water with an osmolality of 200 mOsm; mortality was 20% in dilute sea water with an osmotic concentration of 400 mOsm. The survival of P. bridgesi was 80–100% in media ranging from fresh water to 200 mOsm. All snails exposed to media of 400 mOsm died within a few days. L. teres is a hyperosmotic and hyperionic regulator when the ambient osmolality is less than 50 mOsm and an osmotic and ionic conformer when the environmental osmolality is above 50 mOsm. The hemolymph of P. bridgesi is hyperosmotic and hyperionic in animals acclimated to osmolalities below 100 mOsm; the animal is an osmoconformer in higher ambient osmolality. The concentration of Ca 2+ is higher in hemolymph from P. bridgesi than in blood from L. teres. Tissue hydration in L. teres does not change in animals acclimated to higher salinities, suggesting that the animals are good volume regulators. Tissue hydration is unchanged in P. bridgesi gills in animals acclimated to osmolalities from fresh water to 150 mOsm, but falls in animals acclimated to 200 mOsm. The amino acid content of the gills of both species increases with acclimation to high salinity, but in media of 200 mOsm, the increase in L. teres is larger than that in P. bridgesi . In L. teres , alanine, glycine and β-alanine are responsible for most of the increase; glutamic acid, alanine and glycine show the largest increases in gills from P. bridgesi . Our analysis of these data is consistent with the hypothesis that freshwater gastropods have a lower capacity for increasing the amino acid pool in response to increases in the ambient osmolality than do freshwater bivalves.

Osmoregulation and epithelial permeability in two euryhaline bivalve molluscs : Mya arenaria and Geukensia demissa

Abstract Mya arenaria (subclass Heterodonta) survives for at least 5 wk in media of 100–1000 mOsm; Geukensia demissa (subclass Pteriomorphia) survives in media of 50–1000 mOsm. Tissue hydration does not increase either in G. demissa or in M. arenaria acclimated to diluted seawater (SW). The concentrations of Na + , K + , Mg 2+ , and Cl − in the blood of G. demissa conform to ambient over the entire salinity range. The concentration of Ca 2+ in the blood of animals acclimated to media of ≤200 mOsm is ≈2.5 mM. Blood concentrations of Na + , Mg 2+ , and Cl − largely conform to ambient in M. arenaria . In M. arenaria acclimated to SW of >200 mOsm, blood Ca 2+ conforms to ambient, but is significantly hyperionic in animals acclimated to 50 mOsmSW. The diffusional water, Na + , and Cl − permeabilities of isolated mantle tissue from Mya acclimated to 1000 mOsm SW are, respectively, 3.8 × 10 −4 , 5.7 × 10 −5 , and 2.7 × 10 −5 cm·s −1 . The permeabilities of the mantle of G. demissa acclimated to 1000 mOsm SW are: H 2 O, 1.3 × 10 −4 , Na + , 4.0 × 10 −5 , Cl − , 2.9 × 10 −5 cm·s −1 . There is no consistent trend of a decrease in epithelial permeability in animals acclimated to dilute SW. The hypercalcemia that occurs in Mya acclimated to dilute SW also occures in other brackish water heterodontid clams. This response does not occur in brackish water pteriomorph bivalves. The relatively low water permeability of Geukensia is an adaptation to the high intertidal habitats preferred by this species. Mya , which has a higher permeability to water, is protected from severe desiccation stress by its deep burrowing habit.

Oxygen uptake and heart rate of the clam Polymesoda caroliniana Bosc in air and in seawater

Abstract The rates of oxygen uptake and heart beat, and tissue levels of alanine and succinate of the marsh clam Polymesoda caroliniana Bosc were measured in animals exposed either to air and or isosmotic, aerated seawater (SW). The oxygen consumption rate was variable, the mean rate was approx. 380 μl/h/g dry weight in SW and in air. Animals exposed to air for 24 h did not accumulate an oxygen debt. The heart rate of animals with the valves ajar was 13 in SW and 11 in air. In clams exposed to air for 24 h, the levels of alanine and succinate in the gill were, respectively, 6.5 and 3.9 μmol/g wet weight. The alanine content of gills from animals maintained in SW was 5.6; succinate was 4.4 μmol/g wet weight. The adductor muscles of clams in SW contained 8.9 μmol/g wet weight alanine and 2.8 μmol/g succinate. The adductor muscles of animals exposed to air for 24 h contained 9.7 μmol/g alanine and 2.4 μmol/g succinate. Polymesoda caroliniana is capable of the same rate of O 2 uptake wether in air or submerged in SW. There is no marked bradycardia during emersion, and no evidence of the accumulation of metabolites indicative of anaerobic metabolism. While these response are typical of those found in other bivalve species that occur in high intertidal habitats, the air-breathing capacity of P. caroliniana is exceptional.

The Effect of Cadmium Exposure on Digestive Enzymes in the Eastern Oyster Crassostrea virginica

ABSTRACT The Eastern oyster Crassostrea virginica is a marine bivalve that has been used extensively in metal bioaccumulation studies. We exposed C. virginica to 0 mg/L cadmium, 0.1 mg/L cadmium, or 0.5 mg/L cadmium in seawater for 96 h and then measure the activity of enzymes (amylase, laminarinase, and protease) in the digestive gland. The levels of cadmium in the gills and digestive glands of the animals were also determined. Exposure of the animals to 0.5 mg/L cadmium resulted in a significant decrease in the activities of amylase, laminarinase, and protease enzymes compared with oysters exposed to either 0 mg/L cadmium or 0.1 mg/L cadmium. This decrease corresponds to significantly higher cadmium levels in the gills and digestive glands of oysters exposed to 0.5 mg/L cadmium. The results of this study suggest that exposure to cadmium affects the ability of the animals to process ingested food.

Physiology of the Invasive Apple Snail Pomacea maculata: Tolerance to Low Temperatures

ABSTRACT Apple snails of the genus Pomacea native to South America have invaded and become established in Europe, Asia, and the United States. Both the channeled apple snail Pomacea canaliculata and the island apple snail Pomacea maculata have been reported in theUnited States. The two species are difficult to distinguish usingmorphological characters, leading to uncertainty about the identity of the animals from populations in the United States. Because the snails are subtropical, their tolerance of low temperatures is a critical factor in limiting the spread of the animals from present localities along the coast of the Gulf ofMexico to more northern areas. The tolerance of P. maculata collected in Louisiana to temperatures as low as 0°C was examined. There was no mortality among animals maintained in water at temperatures of 20°C or 15°C for 10 days. Survival of animals during a 10-day exposure towater at temperatures 10°C and 5°C was 50%. The LD50 for a 10-day exposure was 7°C. Snails did not survivemore than 5 days in liquid water at 0°C. Ammonia excretion by animals in temperatures of 20°C and 15°C was comparable to values reported for freshwater gastropods; at very lowtemperatures, excretion of ammoniawas decreased.Therewas no difference in themean values of the osmolality of the hemolymph of animals exposed to 20°C, 15°C and 10°C for 10 days. Sequencing of mitochondrial cytochrome oxidase 1 identified the animals in the Louisiana population used in this study as P. maculata.

Modulation of pumping rate by two species of marine bivalve molluscs in response to neurotransmitters: Comparison of in vitro and in vivo results.

Most studies regarding the neuroanatomy and neurophysiology of molluscan ctenidia have focused on isolated ctenidial tissue preparations. This study investigated how bivalve molluscs modulate their feeding rates by examining the effects of a variety of neurotransmitters, including serotonin, dopamine, and the dopamine agonist apomorphine on both isolated ctenidial tissue and in intact members of two commercially important bivalve species: the blue mussel, Mytilus edulis; and the bay scallop Argopecten irradians. In particular, we examined the effect of changes in: 1) beat of the lateral cilia (in vitro), 2) distance between ctenidial filaments and/or plicae (in vivo), and 3) diameter of the siphonal openings (in vivo) on alteration of bulk water flow through the mantle cavity. Important differences were found between isolated tissue and whole animals, and between species. Drugs that stimulated ciliary beat in vitro did not increase water processing rate in vivo. None of the treatments increased water flow through the mantle cavity of intact animals. Results suggest that A. irradians was primarily modulating lateral ciliary activity, while M. edulis appeared to have a number of ways to control water processing activity, signifying that the two species may have different compensatory and regulatory mechanisms controlling feeding activity.

Physiology of the Apple Snail Pomacea maculata: Aestivation and Overland Dispersal

Apple snails, in the genus Pomacea, have gained considerable notoriety for their impact on invaded habitats. Louisiana is currently under invasion by Pomacea maculata, which represents a potential threat to the state’s valuable plants and cash crops. Insight into the physiology of the invasive snail may assist in developing control measures and enhance our understanding of the processes of adaptation and coevolution that accompany introductions. This paper addresses the capacity, extent, and means by which aquatic apple snails in Louisiana tolerate aerial exposure, as well as the factors that contribute to desiccation tolerance in P. maculata. Invasive P. maculata in Louisiana survived about 10 months of aestivation before 50% mortality was incurred, during which body mass was reduced by only about 30%; mortality and loss of body mass were positively correlated during aestivation. Size affects the loss of body mass in snails under 20 grams. Relative humidity interfered with the induction of aestivation, but it did not significantly affect the loss of body mass. Invasive apple snails in Louisiana also demonstrated the inclination and ability to sustain travel for at least three hours over dry land, at a rate of two meters per hour. The results of this study show that P. maculata is well adapted for survival in the absence of water. The ability to sustain travel over land and to endure long periods of aerial exposure suggests that the dry-down of infested bodies of water would not significantly impact populations of P. maculata in Louisiana.