VOLUME REGULATION AND NITROGEN ...

Reference: Biol. Bull. 169: 458—475.(October, 1985)

VOLUME REGULATION AND NITROGEN METABOLISM IN THE MURICID GASTROPOD THAIS HAEMASTOMA M. A. KAPPER'@, W. B. STICKLE', AND E. BLAKENEYZ* ‘¿Department ofZoology and Physiology,2Departmentof Biochemistry, Louisiana State University. Baton Rouge,Louisiana 70803 ABSTRACT

Ammonia and primary amine excretion and concentrations of intracellular nm hydrin-positive substance (NPS) and free amino acids (FAA) were measured in Thais haemastoma acclimated to salinities between 5 and 35%oand over 14 days following direct transfer from 10 to 3O%oor from 30 to lO%o. There was no trend in excretion

rates with acclimation salinity. Intracellular NPS and FAA levels were directly related to acclimation salinity, with amino acids constituting over 90% ofthe NPS at salinities greater than lO%o.The intracellular free amino acid pool of T. haemastoma was not dominated by any single amino acid but glycine, alanine, aspartate, taurine, proline, and glutamate (in decreasing order) each contributed more than 5% of the FAA. Alanine and glycine were the major intracellular osmotic effectors during both the high and low salinity transfers. Taurine levels did not change in the hyperosmotic transfer, but taurine was lost from the foot over the course ofthe hyposmotic transfer, suggesting that it behaves as a passive osmolyte. Snails are capable of taking up ex ogenous ammonia from seawater during a 10 to 3O%otransfer, suggesting that ammonia is being used as an aminating

source. INTRODUCTION

The southern oyster drill Thais haemastoma (Gray, 1839) is exposed in the field to both diurnal salinity fluctuations between 15 and 30%oand extended periods of relatively constant salinity (Hewatt, 1951; Barrett, 197 1). Even though its low salinity distributional limit in nature is lS%o, it will survive for over four weeks at salinities as low as 5—7.5%o (Garton and Stickle, 1980; Hildreth and Stickle, 1980) and maintains a positive energy budget throughout the salinity range at temperatures greater than 15°C(Stickle, 1985a). As is true of other marine molluscs, the hemolymph of T. haemastoma remains isosmotic to ambient seawater (Hildreth and Stickle, 1980; Stickle and Howey, 1975). The predominant labile intracellular osmolytes in marine molluscs are organic com pounds. In many species studied to date, these are free amino acids (Burton, 1983), but changes in the intracellular free amino acid pool of several gastropods appear to be insufficient to account for the changes in intracellular osmolality following a change in ambient salinity (Schoffeniels and Gilles, 1972; Polites and Mangum, 1980). In organic ions and quaternary ammonium compounds such as glycine betaine and

proline betaine have recently been identified as the primary labile intracellular osmolyte in some species (Pierce et a!., 1983). The objectives of the present study were to (1) determine the degree of volume regulation and the changes in the patterns of nitrogen excretion in T. haemastoma Received 25 February 1985; accepted 2 June1985. S Current *5 Current

address: address:

Department Zoology

ofChemistry, Department,

Centenary Iowa

State

458

College, University,

Shreveport,

Louisiana

Ames,

50011.

Iowa

71104.

VOLUME REGULATION IN THAIS

459

during high and low salinity adaptation, (2) determine the changes in the free amino acid pool during adaptation to altered saliities, and (3) determine the extent to which changes in the free amino acid pool are responsible for salinity adaptation. MATERIALS AND METHODS

Collection and acc!imation of anima!s Snails were collected from pilings and bulkheads in the vicinity ofCaminada Pass near Grand Isle, Louisiana, transferred to Baton Rouge, and placed into 38-liter aquaria containing artificial seawater (ASW; Instant Ocean, Mentor, Ohio) of the same tem perature and salinity (30°C,27%o)as in the field. Salinity adaptation was accomplished by adding either deionized water or concentrated ASW to change salinity by 2%oper day. Animals were held at the final salinities for two weeks before being used. Small oysters (Crassostrea virginica) were provided as prey. NPS andfree amino acids Ninhydrin-positive substances (NPS) and free amino acid levels were measured in the foot tissue ofsnails acclimated to 5, 7.5, 10, 15, 20, 25, 30, and 35%oat 30°C, and directly transferred from 10 to 30%o or from 30 to lO%o.Measurements were taken on transferred snails (n = 10) on days 0, 1, 2, 3, 7, 10, and 14 after transfer. NPS and free amino acids were also measured in the foot tissue of snails used in the ammonia-loading experiments described later. Foot tissue was excised, then frozen in liquid nitrogen and lyophilized. After grind ing in a Wiley mill, 10 mg of tissue were leached in 5 ml of 5-sulfosalicylic acid for 48 h. Samples were centrifuged at 20,000 X g for 15 mm and the supernatant was assayed for NPS according to Rosen (1957). Concentrations ofindividual amino acids were determined on a Beckman Model 119 amino acid analyzer. Ammonia and primary amine excretion and activity The rate of ammonia and primary amine exchange was measured by incubating snails in 150 ml of ASW for 60 mm and analyzing the incubation medium by the Solorzano

(1969) phenol-hypochlorite

method

(ammonia)

and North's

(1975) flu

orescamine technique (amines). Ammonia exchange is defined as the sum of NH3 and NH4@exchange. Since urea makes up an appreciable fraction of the excreta in some carnivorous marine invertebrates (Stickle, l985b), further samples ofthe incu bation medium were analyzed for urea by the Sigma urea assay (Sigma technical bulletin #640) at 10 and 30%o.All glassware used in excretion measurements had been baked at 450°Cin a muffle furnace to eliminate exogenous amines. Excretion was measured

for snails acclimated

to each steady state salinity and at hours 3, 6, 9, and

12, and days 1, 2, 3, 4, 5, 6, 7, 10, and 14 after transfer from 10 to 30%oor from 30 to 10%o. Further experiments were designed to test the ability of T. haemastoma to take up exogenous ammonia from the medium for use as a possible aminating source during high salinity acclimation. The ammonia excretion rate of snails acclimated to 10 and 3O%owas measured using incubation water containing various concentrations of NH4C1 up to 350 @sM. These served as a control to see if snails would normally take up ammonia from ambient seawater. Then snails acclimated to lO%owere placed in a chamber through which 30%owater was pumped. The high salinity water flowing through the cell contained 0, 35, 45, 100, 175, or 350 @LMNH@IC1. Ammonia

excretion

460

M. A. KAPPER ET AL.

or uptake was measured after 24 h for the 35, 45, and 100 zM spiked animals, and in the time intervals of 8—12h and 20—24h after the transfer for animals subjected to either 0, 175, or 350 @[email protected] tissue was sampled for NPS and FAA de termination from lO%oacclimated snails (controls) and at h 12 and 24 after transfer from snails in the 0, 175, and 350 @MNH@C1spiked transfers. In both sets of salinity transfer experiments, a snail's activity was assigned a value of 1.0 ifits foot was extended and attached to the substrate, 0.5 ifthe foot was extended but not attached, and 0 if the foot remained withdrawn. Body water determination The amount of water in the soft tissues at each salinity was determined as the difference in the weight of the soft parts before and after lyophilyzation. Oglesby's (1975) beta value was calculated at each steady state salinity as an indicator of the degree of regulation

of body water content.

Statistical analyses The General Linear Model procedure and Duncan's Multiple Range option of the Statistical Analysis System (SAS Institute, 1982) were used in data analysis. A prob ability level of 0.05 was significant. RESULTS

Steady state experiments Levels of ninhydrin-positive

substances

in foot tissue of Thais haemastoma

were

directly related to the acclimation salinity over the range of 5—35%o when expressed in terms of @moles . g dry tissue weighr' or @tmoles . g tissue water@ (Fig. 1).

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VOLUME REGULATION IN THAIS

461

The free amino acid composition ofthe foot tissue of T. haemastoma at different acclimation salinities is given in Table I. Free amino acids comprise over 91% of the NPS pool at acclimation

salinities of 10%oand above. At 5 and 7.5%o free amino acids

account for 72 and 61% of the NPS, respectively. Other non-amino acid nitrogenous substances make up a significant portion of the NPS pool at very low salinities. Excretion rates of snails acclimated to constant salinities varied among salinities (Fig. 2). Ammonia excretion rates were significantly greater than zero, but did not show a linear trend across salinities. The rate ofammoma excretion was higher at 15 and 2O%othan at any of the other acclimation salinities. The rate of primary amine exchange in snails acclimated to 5, 7.5, and 30%owas in the uptake direction from the incubating medium. There was no significant exchange of amines between the animals and the medium at either 10 or 3S%o,and the snails excreted amines at a constant rate ofO. 17 @smole •¿ g ‘¿ . h' between 15 and 25%o.The rates ofurea excretion at 10 and 30%owere 0. 17 ±0.07 and 0.20 ±0.06 @moles •¿ g dry weight' ‘¿ h', re spectively, representing a minimal contribution to total nitrogen excretion. Thais haemastoma is an excellent regulator of tissue water content. When acci mated

to constant

salinity

between

5 and 30% the percentage

of the fresh weight

of

the soft tissues consisting of water is not significantly different, and is only slightly lower at 35%o(Table II). Direct transfer experiments Changes in percent body water over the 14 days ofa 10 to 30%oand a 30 to lO%o transfer are shown in Figure 3. During the 10 to 3O%otransfer, body water declined from 79.3 ±0.5% on day 0 to 72.7 ±0.9% on day 2. By day 3 the percent body water had stabilized, and was not significantly different from the 3O%ocontrol. For the 30 to 10%otransfer, percent body water increased from 71. 1 ±38% on day 0 to 80.2 ± 1.3% on day 2, was not significantly different from the lO%ocontrol by day 3. The beta value was 0.13 two weeks following transfer to either high or low salinity. Unlike the animals used for steady-state determinations of body water content, the snails used in these experiments

showed a significant (a = 0.05) difference in percent body

water on day 0 (before being transferred) between 10 and 30%oyielding a beta value of 0. 18. This is still a very low value for beta and is indicative of excellent regulation of body water content. Fourteen days after being directly transferred from 30 to 10%o,the concentration offoot NPS was not significantly different from the lO%osteady state value of 161 ±3 @tmoles . g dry weight@ (Fig. 1). Two weeks after transfer from 10 to 3O%othe con centration of NPS in foot tissue was significantly higher than the steady state value at 30%o, but not from the steady

state value at 35%o (Fig. 1).

With the exception of glutamate and taurine, the concentrations of each amino acid in foot tissue of snails directly transferred from 10 to 30%owas higher 14 d after the transfer than in foot tissue ofsnails acclimated to the 3O%osteady state (Table III). The largest discrepancy between the concentrations of any amino acid between the steady state and post-transfer

conditions

occurred with alanine, whose concentration

reached 237 smoles •¿ g dry weighr' on day 3 after the transfer, as compared to only 55 @tmoles . g dry weighr' in animals acclimated to 30%o. Arginine

and aspartate

were the only two amino

acids whose

concentration

did

not decline over the course ofthe 30 to lO%otransfer (Table IV). For each ofthe other amino acids, the concentration two weeks after the hyposmotic transfer was similar to the concentration in animals maintained at 10%o,except for serine and threonine, which were not detected in the lO%oacclimated animals, but were present in small amounts in the 30 to lO%otransfers. Alanine, glycine, and glutamate all showed tran

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(@±S.E.,n = 12)of Thaishaemastomaacclimatedto steadystatesalinities. sient increases in concentration (@imoles. g dry weighr1) over the first one to two days of the 30 to lO%otransfer. This time corresponds to the time when the snails were withdrawn into their shells with the opercula closed (Fig. 4A). Motor activity, and primary amine and ammonia excretion all dropped to nearly zero during the 12 hours immediately following a salinity transfer from 10 to 30%o

[email protected] II Percenttissuewaterin (n)]Sal%Thais haemastomaas afunction ofacdimation sailnity. [1±S.E. waterDMR572.9 (12)A7.574.2 ±0.74(12)A1070.98 (12)A1567.43±0.57(12)A2072.34

Body ±1.88 ±0.47

(30)A2569.77±0.85(12)A3072.01±0.51(24)A3563.51±0.84(11)B ±0.50

Percent tissue water is not significantly different for those salinities sharing a common letter according to Duncan's Multiple Range Test (DMR).

464

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DAYS POST-TRANSFER FIGURE3. Changes in the percent body water (@±S.E., n = 6) of Thais haemastoma over time after direct transfer from 30 to lO%o(0), and from 10 to 30%o(•).

(Fig. 4) or from 30 to 10%o(Fig. 5). After transfer from 10 to 30%o, activity of the snails remained low over the first 24 h. By day 3 all of the snails had reattached to the substrate indicating a normal activity pattern (Fig. 4A). Primary amine excretion fell during the 12 hours after the 10 to 3O%otransfer and slowly rose over the next 6 days. Amine loss fell to zero on day 10, and was not significantly different from control on day 14 (Fig. 4B). Ammonia excretion dropped precipitously immediately following the transfer and remained low for three days. Ammonia loss increased on days 4 and 5, and had returned

to the control

level by day 6 (Fig. 4C).

Snails remained unattached and withdrawn for the first 24 h after the 30—10%o transfer (Fig. 5A). All had reattached by day 3 indicating a normal activity pattern (Fig. 5A). Amine excretion peaked on day 2 after transfer and returned to the control level by day 3 where it remained for the rest of the experiment (Fig. 5B). Ammonia excretion peaked on days 2—3after transfer and remained fairly constant over days 4—7 before

rising

again

by day

14 (Fig.

5C).

Ammonia loading In snails acclimated to either 10 or 30%o,ammonia exchange with the medium was always in the direction ofammonia release, regardless ofthe amount of exogenous ammonia present. Twenty-four hours after snails were transferred from 10 to 3O%o there was a linear dose-related uptake of ammonia at exogenous ammonia concen trations up to 100 @M.At concentrations greater than 100 @iMammonia there was no change in the uptake rate (Fig. 6). Over the first 24 hours of direct transfer from 10 to 3O%othe only amino acid to show any change in concentration was alanine (Table V), which, without exogenous ammonia in the high-salinity water, rose from 10 to 87 smoles . g dry weight@. When either 175 or 350 sM ammonia was added to the high-salinity water, alanine levels rose at the same rate as in the control transfers for the first 12 h, but had leveled off

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