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1985 Metabolic Responses of the Estuarine Gastropod Thais Haemastoma to Hypoxia (Energy Charge, Opine Dehydrogenase,survival, Adaptation, Respiration). Martin A. Kapper Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Kapper, Martin A., "Metabolic Responses of the Estuarine Gastropod Thais Haemastoma to Hypoxia (Energy Charge, Opine Dehydrogenase,survival, Adaptation, Respiration)." (1985). LSU Historical Dissertations and Theses. 4095. https://digitalcommons.lsu.edu/gradschool_disstheses/4095

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Kapper, Martin A.

METABOLIC RESPONSES OF THE ESTUARINE GASTROPOD THAIS HAEMASTOMA TO HYPOXIA

The Louisiana State University and Agricultural and Mechanical Ph.D. Col. 1985

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University Microfilms International METABOLIC RESPONSES OF THE ESTHARINE GASTROPOD THAIS HAEMASTOMA TO HYPOXIA

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

Physiology

by Martin A. Kapper B.S., St. Lawrence U niversity, 1976 M.A., State University of New York at Buffalo, 1980 August, 1985 ACKNOWLEDGEMENTS

The process of designing, performing, and writing up a dissertation

project would be impossible without the active help and cooperation of

others. It is therefore my duty and privilege to gratefully acknowledge

the contributions of the following individuals: Richard Roller, who came

through in the clutch with technical help, and was always ready to help

collect snails; to Reed Payne, for his invaluable help with the bioassay

experiments; to Penny Culley and Mark Carls, for help with statistical

analyses and general computer operation; to Shiao Wang and Dr. David

Garton, for help In collecting snails; to Drs. David Livingstone and

Ross Ellington, for introducing me to the biochemical techniques

employed here; to Wilton DeLaunne of the LUMCON Port Fourchon lab, who

helped me above and beyond the call of duty; to Tina Roller, for her

excellent graphics work; and to the past and present members of ray

graduate committee, Drs. William Stickle, Thomas Dietz, Earl Weidner,

John Fleeger, Ernst Blakeney, Robert Allen, and John Lynn, for their

continued support over the years, and for their constructive criticsms

and comments that helped to vastly improve the quality of the final work.

A special debt is owed to Dr. William B. Stickle, Jr, who served as my major professor and directed this project. B ill has been much more

than an academic mentor and friend. Without his faith, encouragement and above a ll patience, I never would have finished. Thanks a lot, Bill.

Parts of the work reported on here were supported by National

Science Foundation Grant #DEB-7921825 and grants from the Louisiana

State University Fisheries Initiative Program and the Petroleum Refiners

Environmental Council of Louisiana (PRECOL) to W.B. Stickle. TABLE OF CONTENTS

Title Page...... i Acknowledgements ...... 11 Table of Contents ...... I l l List of Tables ...... iv List of Figures...... v Abstract...... vl Introduction...... 1 M aterials and Methods...... 4 R esu lts...... 11 Discussion...... 16 Literature Cited...... 28 Tables ...... 37 Figure Legends...... 43 Figures ...... 45 V ita ...... 56 Approval Sheets...... 59 LIST OF TABLES

TABLE PAGE

I. Water quality variables for 30°C-30o/00® hypoxia bioassay...... 37

II. Oxyregulation indices for Thais haemastoma...... 3 8

III. A c tiv itie s of pyruvate oxidoreductase enzymes in foot tissu e of T. haemastoma a fte r 28 days exposure to hypoxia...... 39

IV. Anoxia LT-50's of marine molluscs ...... 40

V. Oxyregulation indices, Kj /Rj and B2 of marine m olluscs ...41

VI. Adenylate energy charge in marine Invertebrates...... 42

iv LIST OF FIGURES

FIGURE PAGE

1. Diagram of hypoxia Bloassay system...... 45

2. 95% confidence bands fo r LC-90, LC-50, and LC-10 over 28 days hypoxia...... 46

3. Oxygen consumption ra te of T. haemastoma at f u ll oxygen saturation and 1 0 , 20 and 30°c...... 47

4. Oxygen consumption rate of T. hemastoma a fte r 28 days exposure to hypoxia...... 48

5. Oxygen consumption rate of T. haemastoma with declining P0 2 ’ .49

6. Oxygen consumption rate of T. haemastoma with declining PO2 before and a fte r 28 days a t 53 mm O2 ...... 50

7. Concentrations of adenylates in foot tissu e of haemastoma exposed to hypoxia for 28 days...... 51

8. Adenylate energy charge and arginine phosphate concentration in foot tissu e of T^. haemastoma exposed to hypoxia for 2 days...... 52

9. Time course of changes in adenylate energy charge and arginine phosphate concentration in foot tissu e of T. haemastoma exposed to anoxia ...... 53

10. Time course of changes in adenylate concentrations in foot tissue of T. haemastoma exposed to anoxia ...... 54

11. Time course of changes in pyruvate oxidoreductase enzyme activity in foot tissu e of T. haemastoma exposed to anoxia...... 55

v ABSTRACT

The hypoxia tolerance (LC-50) of Thais haemastoma was Inversely

related to acclimation temperature but was not related to acclimation salinity between 10 and 30°C and 10 and 3 0 °/ooS. T. haemastoma is moderately tolerant to anoxia, the LT-50 (time to 50% mortality) being

12-22 days. Oxyregulatory ability varies from poor to good, and varies inversely with temperature. The oxygen consumption rate was lower after

28 days exposure to hypoxic conditions than at full oxygen saturation at all salinities at 20 and 30°c. Oxygen consumption was low at 10°C regardless of ambient PO 2* Oxyregulatory ab ility in T. haemastoma did not show acclimation after 28 days exposure to 53 mm 0£ at 30° and

30°/ooS. Likewise, no Induction of the activities of the pyruvate oxidoreductase enzymes in foot tissue occurred after 28 days exposure to

10, 33, and 67% of oxygen saturation. The ratio of mean activities of alanopine dehydrogenase:lactate dehydrogenase:strombine dehydrogenase:octopine dehydrogenase was 3.8:1.2:1.0:0. Alanopine dehydrogenase activity nearly doubled on the first day but had returned to the control level by day 2 , and no change occurred in stromblne dehydrogenase and lactate dehydrogenase activity over 6 days exposure to anoxia. The total concentration of cellular adenylates remained constant, but the adenylate energy charge,

([ATPl+.5[ADP]/([ATP]+[ADP] + [AMP]), was reduced a t low P02~s * A rginine phosphate concentrations were lower in snails exposed to 10 and 33% oxygen saturation than in those at 67 and 100% of oxygen saturation.

Energy charge declined from 0.79 to 0.58 a fte r one day of anoxia at 30°C and 30°/ooS while the arginine phosphate concentration declined by 95%.

There was no further decrease in either variable with longer exposure to anoxia. Hypoxia tolerance of T. haemastoma is due to a reduction in

vi metabolic demand, especially at low temperature, with ATP production maintained at the expense of arginine phosphate while metabolism switches to anaerobic pathways. The opine pathway is probably only used as a transitional mode before use of long-term anaerobic pathways is in itia te d .

vii INTRODUCTION

The primary mode of metabolism of most metazoans is aerobic, yet many marine invertebrates have been recognized as having a substantial capacity for anaerobic metabolism during exposure to hypoxia (de Zwaan and Wijsman, 1976; Livingstone, 1982). There have been se v e ra l recorded instances of hypoxic or anoxic conditions developing In the Atlantic

Ocean off New Jersey (Garlo, et_ a l., 1979), in the Chesapeake Bay

(Officer, et a l., 1984; S eliger, et a l . , 1984) and in the Gulf of Mexico

(Harper et a l., 1981; Turner and Allen, 1982). Although most of these recorded hypoxic events have been offshore, recurrent hypoxic episodes have been documented in the inshore bays and estuaries of the northern

Gulf of Mexico between Texas and Alabama (Rabalais, et_ a l . , 1985).

Although many species of benthic marine molluscs are extremely resistant to anoxia, with LT-50's (time to 50% mortality) of up to 35 days (Theede, et_ a l., 1969), few species can survive indefinitely without oxygen. During a three month hypoxic episode (<2 ppm, or 23 mm of oxygen) off the Texas coast, macrobenthic species diversity and population density declined precipitously, although populations of the bivalve Abra aequalis appeared to be little effected (Harper, e£ al.,

1981).

The respiratory response of marine molluscs to declining oxygen tension and hypoxia has received considerable attention over the past 15 years (Bayne, 1971, 1973; Bayne and Livingstone, 1976; Mangum and van

Winkle, 1973;). Most Investigators have concentrated on either the changes in the rate of oxygen consumption with acutely declining PO2 or on the anaerobic biochemical pathways used during anoxia exposure.

Animals have traditionally been classified as either oxyregulators if their oxygen consumption rate remains constant with declining PO2 or 2 oxyconformers, if the oxygen consumption rate is proportional to ambient

(Mangum and van Winkle, 1973). R ealistically, oxyregulation and oxyconformity (or oxygen independence and oxygen dependence) describe opposite ends of a spectrum.

Unlike most vertebrates, lactate is not the sole end product of anaerobiosis in marine molluscs. Depending on the species and the duration of anoxia, such varied molecules as alanine, succinate, and proprionate w ill accumulate (Fields, 1983; Gade, 1983; Livingstone,

1982). Recently, another class of molecules, the opines, have begun to attract attention. These molecules are formed by the condensation of an amino acid with pyruvate, oxidizing NADH. The opine dehydrogenase enzymes along with lactate dehydrogenase are classified as pyruvate oxidoreductases. Octopine dehydrogenase, which uses arginine as the amino acid substrate, alanopine dehydrogenase which uses alanine, and stromblne dehydrogenase, which uses glycine, are functionally analogous to lactate dehydrogenase, and result in the same total ATP formation

(Livingstone 1983). High a c tiv itie s of these enzymes are have been found in many species (Livingstone eit al., 1983).

The adenylate energy charge represents the state of energetic balance in the cell. It's value can be thought of as being related to the balance between energy-yielding and energy-requiring metabolic processes (Atkinson, 1968). When the concentration of ATP is high relative to the concentrations of ADP and AMP, key enzymes in glycolysis and the Krebs cycle are allosterically inhibited. Conversely, when ATP concentrations are low with the resultant increase in ADP and AMP concentrations, these same enzymes are allosterically activated

(Atkinson, 1968). In normally metabolizing cells, adenylate energy charge is regulated to an average value of 0.85 (Ivanovici, 1980). 3

The southern oyster d rill, Thais haemastoma is the dominant subtidal gastropod on oyster reefs and other hard substrates in the northern Gulf of Mexico. It is responsible for losses of up to half the yearly oyster crop in Louisiana (Cake, 1983). T. haemastoma is a remarkably euryhaline species, the adults surviving for at least four weeks in the lab at 5°/0oS (Garton and Stickle, 1980; Hildreth and

Stickle, 1980), and maintains a positive energy budget from 15 to 30°C and 7.5 to 35°/ooS (Stickle, 1985) although its low-salinity distributional limit in the field is 15°/ooS (St. Amant, 1938).

JT. haemastoma is exposed to diurnal fluxes of dissolved oxygen; for example, a the daily range from 25 to 120% of saturation was observed over a 24-hour period (Stickle, unpublished data). The snail w ill burrow into the azoic zone of sediments in the winter, and intermittently in the summer. The only previous reference regarding the ability of T. haemastoma to survive hypoxia is from a fisheries report that states that the snail w ill "survive, experimentally at least, oxygen levels too low to support most other marine life" (Butler, 1954).

The specific objectives of this study are 1) to determine the hypoxia tolerance of the southern oyster drill, Thais haemastoma over its normal temperature and salinity range, 2) to determine the degree of regulation of the aerobic respiratory rate of T. haemastoma subjected to acutely declining oxygen tension, and to determine if this response will acclimate to prolonged hypoxia, 3) to determine the activities of the pyruvate oxidoreductase enzymes, and determine if these enzymes are induced over the course of chronic hypoxia, and 4) to determine the changes in the phosphoadenylate and arginine phosphate pool and adenylate energy charge as an indicator of physiological condition in T. haemastoma exposed to chronic hypoxia. These data w ill serve as a 4

s ta rtin g point for understanding the mechanisms by which T. haemastoma

is able to withstand variation in one of the most important abiotic

facto rs in i t s environment, oxygen.

MATERIALS AND METHODS

Oyster D r ill C o llectio n and Maintenance

Thais haemastoma were c o lle cte d throughout the year from bulkheads

and j e t t i e s in the v ic in ity of Caminada Pass, Grand I s le , La. The sn a ils

were brought back to the lab and placed in 38-liter glass aquaria using

Instant Ocean^^ artificial seawater at the same temperature and

salinity as occurred at the collection site. Aquaria were housed in a

temperature-controlled environmental room under constant Illumination.

Live oysters (Crassostrea vlrginica) obtained from local seafood

suppliers were continually available to the snails as prey items. When necessary, acclimation temperatures of 1 0 , 20 and 30°c were reached by

changing the temperature of the environmental room by 1°C per day. Final acclimation salinities of 10, 20, and 30°/ooS were reached by adding either 40°/ooS artificial seawater or deionized water to the aquaria to change salinity by 2°/oo per day. Snails were held for two weeks at the final temperature-salinity combination before being used in experiments.

Hypoxia Exposure System

The hypoxia exposure system (Fig. 1) consisted of three sets of five flow-through aquaria, one set each at 10, 20 and 30°/ooS. Water was pumped from a large filtration unit into each of five 38-liter glass tan k s a t each s a l i n i t y by p e r i s t a l t i c pumps a t2 0 -5 5 ml*min“ ^, a r a te sufficient to ensure one to two complete turnovers of the water in each tank In a day. The water level was maintained in each tank by a constant-level siphon, which drained back into the filtration unit. The filter units were large (55-gallon) partitioned plywood boxes lined with 5 fiberglass resin. The boxes contained two beds of crushed oyster shell as the filtration material. Effluent water from each tank passed over both beds in succession before being returned to the tanks.

Target oxygen tensions of 107, 53, 10, and 0 mm 0 2 corresponding to

67, 33, 10 and 0% of oxygen saturation were created and maintained by mixing oxygen and nitrogen in Matheson model 7402T gas mixers along with

0.03% carbon dioxide to maintain pH near 7.8. Each gas mixer was connected to an outlet manifold and fed three tanks, one at each salinity. The outlet manifolds were connected to the standpipes of the standard under-gravel filters of each tank. Aeration of control tanks

( a i r —sa tu r a tio n « 156—157 mm 0 2 ^ was by diaphragm aquarium air pumps.

Each tank was covered by a form fitting plexiglass lid with holes d r il le d in i t to accommodate gas lin e s, water lin e s , and the siphon. A ll tanks were located in a temperature-controlled water bath. P02, pH, and ammonia concentration were monitored daily as indices of water quality.

P02 was always within 10-15% of the target value at the three higher levels, while the 10% oxygen saturation tank was within + 5 mm 02 targ e t, and the P02 of the 0% oxygen saturation tankB was usually in the range of 3-8 ram 0£* The pH was in the range 7.6-8.1, and ammonia lev els were consistently below 25 pmoles*l”l (Table I).

Hypoxia Bioassays

Survival at each P0 2 was determined daily for 28 days at each temperature-salinity combination. A snail was considered dead if both the siphon and the foot failed to respond to tactile stimulation. LC-10,

LC-50, and LC-90 valu es, the PO2 a t which 10, 50, and 90% of the s n a ils had died after a defined period of time, were calculated dally by the

SAS Probit procedure (SAS Institute, 1982), or by the Spearman-Karber technique (Hamilton et al., 1977) if mortality in at least two of the 6

fiv e P02 s was not between 0 an^ 100%.

Oxygen Consumption and Indices of Oxyregulation

Oxygen consumption rates at constant P02""s wsr® measured In a flow­

through system as described by Stickle et^ al. (1985), with the following

modifications: incurrent water was bubbled with a gas mixture of the

appropriate PO2 frdm a gas mixer identical to those used in the hypoxia

exposure system. Water was pumped directly from the reservoir Into the

distribution manifold at a low flow rate. Flow through the individual

glass animal chambers was regulated to 5-1 0 ml * min~* by regulating the

speed of the pump and by using different size hypodermic needles on the outlet ends of the animal chambers. The entire apparatus was placed

inside a temperature controlled environmental room. After the snails were placed in the chambers and the flow rates adjusted, one hour was

allowed for the animals to acclimate to the apparatus. Water samples were taken anaerobically with a 5-ml glass syringe, and the PO2 was measured using the Radiometer thermostatted cell housing a Radiometer model E-5046 oxygen electrode connected to a Radiometer PHM71 Mk II acid-base analyzer. Oxygen consumption rates were calculated by subtracting the PO 2 from the outlet of an animal chamber from the average of the P02's of the two blank chambers in the system divided by the air-saturated PO2 at that temperature and salinity. That value was m u ltip lied by the oxygen content of a ir saturated water as determined from the nomograph of Strickland and Parsons (1968) and the individual chamber's flow rate in liters • hr~^ to yield microliters of oxygen consumed per hour.

Dry snail weight was determined by cracking the snail's shell with a hammer and removing the soft tissues which were then frozen in liquid nitrogen, lyophilyzed, and weighed. The relationship between oxygen 7

consumption rates and dry weight at constant PO2 was described by the

allo m etric form:

jil O2 • h r - ' « a * (dry weight)'*

where a and b are the slope and intercept of the regression of logjp

oxygen consumption on logjQ dry weight. For Qjq determinations the

individual regression lines at 100% oxygen saturation were compared by

analysis of covariance with temperature and salinity as main effects and

weight as the covariate. The salinity term was non-significant, and the

weight effect was Identical for all three temperatures, therefore, the

data were pooled and a common slope was calculated. Oxygen consumption

rates at 100% saturation are expressed as jil 0 £ • gram dry tissu e - ®*^-*^.

To compare rates of oxygen consumption at different PO2"6 w ith in

temperatures, a second set of logjg oxygen consumption to logjg dry

weight regressions was calculated and the lines subjected to analysis of

covariance with temperature, salinity, and PO2 as main effects and dry weight as the covariate.

Respiration rates under conditions of acutely declining oxygen

tension were measured in a closed system respirometer. The respirometer

vessel used at 20 and 30og consisted of a 1000 ml Kontes reaction flask which had a plexiglas platform covering a magnetic stirring bar on the

bottom. The vessel was completed by attaching a four-holed flask top to

the bottom. The ground glass joint between top and bottom was sealed with a small amount of silic o n vacuum grease and secured by a clamp. The

center hole of the respirometer top was occupied by a Radiometer model

E-5046 macro oxygen electrode mounted in a rubber stopper. The other

three holes contained an inlet port that delivered water to the bottom of the chamber and two outlet ports, providing exhaust from the top. The

in le ts and o u tle ts were equipped with stopcocks. The oxygen electrode 8 was attached to a Radiometer PHM71 Mk II acid-base analyzer. Because the respiratory rate of T . haemastoma Is extremely low at 10<>c, it was necessary to construct a 500 ml respirometer.

A declining oxygen tension experiment consisted of placing a sn ail in the bottom of the vessel, sealing the vessel, and placing it into a temperature controlled water bath over a magnetic stirrer. Air-saturated water was pumped into the vessel from a continually aerated reservoir.

Once the respirometer vessel was filled with water and all air bubbles removed, the magnetic stirrer was turned on and water was allowed to flow through the system for an hour. After this period of acclimation to the apparatus, the pump was turned off, a ll stopcocks were closed, and the decline in PO2 in the chamber was followed on an Omniscribe Model

5212-15 strip chart recorder connected to the Radiometer PHM71 Mk II

Acid-Base analyzer. Oxygen consumption rates were calculated as follows:

The total amount of oxygen (ml) In the respirometer vessel at full oxygen saturation was calculated by taking the volume of the chamber minus the volume of the sn a il and m ultiplying by the oxygen s o lu b ility in u l 0 2 *ml water- * (Strickland and Parsons, 1968). By dividing this value by the PO2 at full saturation, )il O2 * mm °^ t a *nec** T^e decline in PO2 for a 30-minute interval was converted to oxygen consumption in fi 1 O2 * hr-1. The respiration rate thus calculated is considered to be the oxygen consumption rate at the midpoint PO2 of the in te rv a l. The response to acutely declining oxygen tension was measured in sn a ils acclimated to 10, 20 and 30°c and 10, 20 and 30°/ooS (n=10).

A second set of experiments was conducted to determine if the response to hypoxia is altered after chronic exposure. The response to acutely declining PO2 was f*rst measured on ten snails acclimated to

30°C and 30°/ooS. These animals were then marked with a non-toxic 9

indelible marker and placed into an aquarium being maintained at 51.6 +

3.3 (x 9 5 % C.I., n*=28) mm O 2 . The response to d e c lin in g PO2 of each

snail was measured again after exposure to chronic hypoxia for 28 days.

Three indices of respiratory response to declining PO2 were

calculated for each temperature-salinity combination run. The critical oxygen tension, or Pc, is the PO2 where the respiratory rate changes from being relatively oxygen-independent to being relatively oxygen- dependent. The Pc was calculated for each experiment by running an iterative partial linear regression program on the P0 2 “ oxySen consumption rate data. Briefly, the program assumes that the P02-oxygen consumption r a te curve can be approxim ated by two s tr a ig h t lin e s , representing the P0 2's where the individual reacts more as an oxyconformer (below the Pc) and more as an oxyregulator (above the Pc).

Suggested values for the Pc were supplied, and linear regressions calculated based on the data points lying between the origin and the putative Pc. This procedure was iterated until a maximum coefficient of determination (R2) was obtained.

The second calculated index of respiratory oxygen dependence is

Kj/K2 (Bayne, 1971; Tang, 1933). Ki and K2 are respectively the Y- intercept and slope of a linear-transforraed hyperbola fit of weight- sp ecific oxygen consumption to PC>2 :

P02 /V02 = Kj + (K2 x P02).

When Kj is large compared to K2 > the oxygen consumption rate is proportional to PO2 , oxygen dependence. When Kj is small compared to K-2 > the oxygen consumption rate approaches a constant, oxygen independence.

The magnitude of the index Kj/k 2 is inversely proportional to the degree of oxygen independence of the aerobic respiration rate.

When the oxygen consumption rate at different oxygen tensions is 10 expressed as a percentage of the respiratory rate at full saturation,

the data often fit the quadratic curve defined by: 2 S tandardized V02 = BO + (B1 x P02) + (B2 x PO 2 )■

When plotted in this manner, B2, the coefficient of the P0 2^ terra describes the departure from linearity. The stronger the degree of oxygen independence, the more negative B2 w ill he (Mangum and van

Winkle, 1973).

Each of these indices was calculated for each declining oxygen tension experiment. Two way analyses of variance for temperature and salinity for each index were performed using the.General Linear Models procedure of SAS (SAS Institute, 1982), with individual differences detected using the Duncan's Multiple Range Test and least squares means options. Correlation and regression analyses were performed to compare the three indices of respiratory response to declining 0 P2; pc, K|/K2, and B2. Paired-sample t-tests on the differences between the values of each index before and after chronic exposure to hypoxia were made by the

SAS procedure Means (SAS In stitu te , 1982).

Biochemical Variables

A ctivities of the pyruvate oxidoreductase enzymes lactate dehydrogenase (LDH-E.C. 1.1.1.27), octoplne dehydrogenase (ODH-E.C.

1.5.1.11), alanopine dehydrogenase (ADH-E.C. 1.5.1.x), and strombine dehydrogenase (SDH-E.C. 1*5.1.x) were determ ined in fo o t tis s u e of snails exposed for 28 days at 156-157, 107, 53, and 16 mm 0 2 a t 30° and

3 0 °/ooS using an NADH-linked spectrophotometric assay technique

(Livingstone et al., 1983). Enzyme activities were also measured in foot tissu e of sn a ils on days 0 , 2 , 4, and 6 of exposure to anoxia.

Concentrations of ATP, ADP, AMP, and arginine phosphate were measured in foot tissue according to the methods in Lowry and Passonneau 11

(1972), substituting lobster arginine phosphokinase for creatine phosphokinase (Nicchitta and Ellington, 1983). Arginine phosphate and adenylates were measured after the same experimental conditions as used fo r the opine dehydrogenase enzymes. The adenylate energy charge (AEC) was ca lc u la te d as ([ATP]+.5 [ADP]) / ( [ATP] + [ADP] + [AMP]). H exokinase was purchased from Boehringer, and a ll other biochemicals were purchased from Sigma. Foot tissue was used for the biochemical analyses because muscle tissues have the highest activities of opine dehydrogenase enzymes (Eberlee, et a l., 1983; Plaxton and Storey, 1982) and because the foot of _T. haemastoma is the easiest tissue to dissect rapidly before proteolysis and ATP degradation set in.

RESULTS

Tolerance

Hypoxia tolerance Increased with decreasing temperature, but there was no clear trend across salinities (Fig. 2). As shown by an increase in the LC values over time, hypoxia tolerance decreased with increasing length of exposure to low oxygen tension.

Thais haemastoma is very to le ra n t of moderate hypoxia fo r more than

28 days within the seasonal temperature and salinity range to which it is is normally exposed. There were only six deaths recorded at 67% oxygen saturation over the entire course of the study, and those were a ll at 30°. At 10°, the snails tended to be moribund regardless of the

P0 2 > and it was difficult to determine when an individual had died. Only the snails at 67 and 100% oxygen saturation had their foot extended at

10°. No feeding was observed at 10°t although fresh oysters were continually made available. At 20 and 30°C snails had their foot extended, were attached to the substrate and were actively crawling about the tanks at PO 2 exposures down to 33% oxygen saturation for a ll 12

28 days of the hypoxia exposure. The snails exposed to 0 and 10% of

oxygen saturation became inactive after two to three days of exposure

and did not feed. These snails either had the foot extended, but limp

and unattached to the substrate, or had the opercula tightly closed with

the siphon showing in the siphonal canal of the shell. Occasionally the

foot of a snail in a 0 or 10% oxygen satu ratio n tank would appear to be

swollen for several days before the snail died. A snail was considered

to be alive if the foot or siphon would retract after tactile

stimulation.

Oxygen Consumption and Indices of Oxyregulation

The rate of oxygen uptake at fu ll oxygen saturation was directly

related to the acclimation temperature (Fig. 3). At 10°c, the oxygen consumption rate was very low, indicating cold torpor, and the animals

tended to be moribund. Q10's were 3 .8 8 between 10 and 2 0 °, and 1.95 between 20 and 30°. The o v e ra ll Qjq between 10 and 30° was 2.75.

After 28 days exposure to low PO2 , the relationship between log^Q oxygen uptake and logjg ^ry weight was different at each temperature, but was consistent across salinities and P 0 2 's within temperatures.

Oxygen consumption was directly related to acclimation temperature, and except for the group at 30° and 10°/oo, the tendency was either for oxygen consumption to be d ire c tly rela te d to PO2 or ^or there to be no statistically significant difference in oxygen consumption rates across

P02^s (FlS* 4).

In the declining oxygen tension experiments, snails extracted all detectable oxygen from the respirometer vessel in all nine temperature- s a U n ity conditions used. Aerobic shutdown, as described by Mangum and van Winkle (1973) never occurred. At 20 and 30°C» the responses of individual snails to declining PO2 ranged from excellent oxyregulation, 13

with P c ' s as low as 5 mm 02 to Poor oxyregulators with P c ' s as high as

97 mm 02. At 20 and 30° s n a ils were able to extract a l l oxygen from the

re sp iro m e te r in 8-12 hr. The 2-way a n a ly s is of v a ria n ce of Pc on

temperature and salinity indicated no significant difference in Pc with

salinity. Pc was significantly higher at 10° than at either 20 or 30°C» where Duncan's Multiple Range Test Indicated no significant difference

(Table II). Qualitatively, the overall response patterns of the oxygen

consumption rate to acutely declining P02 were id en tic a l at 20 and 30°

(Table II). For both the relatively good regulator (Fig. 5a) and the relatively poor regulator (Fig. 5b) as P02 decreases, the oxygen consumption rate remains relatively constant until the Pc is approached, and as P02 continues to decrease, oxygen consumption becomes proportional to the oxygen partial pressure.

When the pump was turned off after the acclimation period in the declining PO2 experiments at 10°, the indicated PO2 dropped by 10-20 mm

°2 within the first 10-15 minutes. This decline in P02 translated to an oxygen consumption rate in excess of 1000 fil'g dry weight“^,hr, nearly ten times the average oxygen consumption rate at 10° determined in the flow through system at f u l l oxygen saturation. The rapid decline in PO2 is very likely an artifact due to the change in hydrostatic pressure on the oxygen electrode membrane. For the calculation of the oxyregulation indices Pc, B2, and K j/r2 at 10°C, full oxygen saturation was considered to be in the range of 130-140 mm 0 2*

At 10° oxygen consumption remained f a ir ly constant between 50 and

150 pl"g dry weight~^*hr“^ over most of the PO2 range (Fig. 5C), Pc's varied widely among different individuals, some as low as 10 mm 0 2 and some as high as 130 mm 02 (Table II). The K^/K 2 values were uniformly low at 10° (Table II), indicating good oxyregulation, as could also be 14

inferred from the consistency of oxygen consumption rates over the large

range of PC>2. This consistency, however, made the relationship between

PO2 and standardized oxygen consumption linear, the average Blope being

0.12, with the result that the quadratic term, or B2 value at 10° was not significantly different from 0 (Table II), and would indicate that

the snails are not regulating at this temperature. There was not a significant difference in any of the calculated oxyregulation indices with s a lin ity at 10°.

According to a two-way an aly sis of variance, oxyregulatory a b ility as measured by the quadratic coefficient of the second degree polynomial, B2, relating standardized oxygen consumption to PO2 was highly significant for temperature (P».0001), but not significant for salinity (P=.1402) when considered across a ll temperature-salinlty combinations. Duncan's M ultiple Range test for temperature indicated that B2 was not different between 20 and 30°, but that a significant difference existed between the two higher temperatures and 10°*

The magnitude of Kj/i^ is inversely related to oxyregulatory ability. There was a highly significant (P<0.0001) difference in the mean value of K j/^ with temperature, the best regulation occurring at

10°, the poorest at 30°. There was also a salinity factor, regulation at

20 and 30°/oo being poorer (P=0.0061) than a t 10°/ooS.

The only correlation between oxyregulation indices was between Pc and B2, (P=0.0002) with a correlation coefficient (r) of 0.39 (n=90).

There was not a significant correlation between Pc and Kj /K2 or between

B2 and K1/ k2.

Eight out of ten snails showed no evidence of acclimation to hypoxic conditions at 30° and 30°/oo as a shift of the P02“oxyfien consumption curve to the left. The oxygen tension-oxygen consumption 15

curves generated before and after 28 day's exposure to 53 mm 02 were either superimposed or slightly shifted to the right (Fig. 6). Paired- sample t-tests indicate no significant differences in either the B2 or

K1/K2 index before and after chronic hypoxia regardless of whether the index for the pre-exposure run had been calculated over the PO2 ran6e 0-

156 mm O2 or over the range of 0-53 mm O2 .

Biochemical Variables

The total adenylate pool ([ATP]+[ADP]+[AMP]) remained constant in foot tissue of Thais haemastoma at 30°C and 3 0 °/ooS a f t e r 28 days exposure to hypoxia between 10 and 100% of oxygen saturation (Fig. 7).

ATP concentrations were significantly lower, and ADP and AMP concentrations were significantly higher in the hypoxia-exposed animals than in the normoxic controls (Fig. 7). There was a slight, but statistically significant decrease in the AEC in the hypoxia-exposed snails relative to the controls (Fig 8). There was not, however, a significant difference in AEG among the three groups exposed to 10, 33, and 67% of oxygen saturation (Fig. 8). The concentration of arginine phosphate was the same in animals exposed to either 100 or 67% oxygen saturation, but had declined by half at 53 and 10% of oxygen saturation

(Fig. 8).

In snails exposed to anoxia (<5 mm 0£) both the AEC and arginine phosphate concentration showed a significant decline within one day

(Fig. 9). After the first day of anoxia, concentrations of arginine phosphate were not significantly different from zero. The AEC also did not show significant decreases after day one. The total adenylate pool remained constant over the six days of anoxic exposure (Fig. 10). The concentration of ATP declined over the first two days of exposure, while

ADP and AMP concentrations continued to rise, with the greatest increase 16

occurring over the first two days of exposure (Fig. 10).

Alanopine dehydrogenase had the highest activity among the opine

dehydrogenase class of pyruvate oxidoreductases regardless of either the

duration or degree of hypoxia. Octopine dehydrogenase activity was not detected in any treatment. After 28 days exposure to 33, 67, or 100% of oxygen saturation, a n aly sis of variance showed no sig n ifican t difference in any of the pyruvate oxidoreductase activities with P02 (Table III).

The analysis of variance for alanopine dehydrogenase activity was not significant, P=0.0547. None of the snails exposed to 0 or 10% oxygen saturation survived for the 28 day exposure period.

During six days exposure to anoxia, the lactate dehydrogenase activity showed a slight, but significant increase in activity on the fourth and sixth day. There was no change in stromblne dehydrogenase activity, and alanopine dehydrogenase activity nearly doubled on day one, but had returned to the control activity by day two and did not change over the next four days (Fig. 11).

DISCUSSION

Thais haemastoma su rv iv es for more than 28 days at oxygen tensions approaching zero, placing it in about the mid range of molluscan anoxia tolerance (Table IV). It is extremely tolerant of mild hypoxia, surviving Indefinitely at P0 2 's as low as one-third of oxygen saturation. There is no apparent difference in hypoxia tolerance with salinity, and tolerance is inversely related to temperature (Fig. 4).

The Camlnada Pass-Barataria Bay estuary, where this population of T, haemastoma is found, is characterized by seasonal and diurnal fluctuations of temperature, salinity, and dissolved oxygen (Barrett,

1971). Over a 24-hour period, the dissolved oxygen levels at one of the collection sites varied from a low of 25% saturation at 6:30 A.M. to 17

nearly 120% saturation in the early afternoon of September 27, 1980

(Stickle, unpublished data). Seasonally, the snails are subject to long­

term hypoxia, burrowing into the sediment in the winter, when the

ambient temperature falls below 20c>c. Snails emerge from the sediment

during the winter during intermittent warm spells which last long enough

to increase the temperature in this shallow estuarine system. They will

a ls o o c c a s io n a lly burrow in to the sedim ents in th e summer when the

temperature exceeds 30°C (personal observations). Snails have been

collected in mid-August covered with black, hydrogen sulfide-laden mud

from around the bases of pilings.

The hypoxia tolerance of T. haemastoma at 10° does not vary much

with duration of exposure (Fig. 2), the 28-day LC-50 is 10-16 mm 02» aiu*

the anoxia LT-50 (time to 50% m ortality) is 12-22 days. These data are

indicative of the high degree to which T. haemastoma is adapted to its

environment. The ability of the snail to overwinter in anoxic sediments

is determined by its high hypoxia tolerance and low metabolic demand

(100 p i O2 • g dry weight- * * hr- !) at ^ow temperature. It is during the winter months that T. haemastoma is the most likely to encounter chronic

hypoxia. The response of T. haemastoma to anoxia while buried in

hydrogen sulfide-laden sediments may be different than the response to

anoxia in the water column due to a possible negatively synergistic

interaction of anoxia and hydrogen sulfide on survival. Theede et a l.

(1969) found that the anoxia tolerance of several benthic species declined when hydrogen sulfide was added to the system.

In an encyclopedic study, Theede et a l. (1969) determined the anoxia LT-50 in several species of benthic invertebrates from the Baltic and North Seas under anoxic conditions. They found that species from soft muddy substrates such as the bivalves Cyprina is landica, 18

Scroblcularia plana, and Mya arenarla, or those intertidal species that were regularly exposed to air, such as Mytllus edulis were able to

survive longer in the absence of oxygen than were species inhabiting hard, submerged substrates such as the asteroid, Asterlas rubens, or the shrimp, Crangon crangon. In general, the hypoxia tolerance of species from microhabitats where the dissolved oxygen concentration varies Is greater than the hypoxia tolerance of species living in environments where dissolved oxygen is stable and at high concentrations over the long term (Vernberg, 1972). Habitat differences appear to be more closely related to differences in hypoxia tolerance than are phylogenetic relationships (Theede, et^ a l., 1969; Vernberg, 1972).

Sessile or slow-moving species show a greater tolerance to hypoxia than do highly active pelagic species (Stickle and Kapper, unpublished data;

Vernberg, 1972).

One response of mobile animals to declining oxygen tension is to migrate from the area. A common behavior of T. haemastoma during the declining oxygen tension experiments was to crawl to the top of the respiroraeter and remain there for the duration of the experiment.

Southern oyster d rills are often found in the field close to the air- water interface, where dissolved oxygen levels are at a maximum. T. haemastoma is only rare ly found out of the water.

Acclimation can be defined as the return of a rate function towards its original value after an alteration in some environmental parameter

(Prosser, 1973). For oxygen consumption under chronic hypoxia, acclim ation occurs i f the oxygen consumption rate at low P(>2 is higher after the acclimation period than before. If the oxygen consumption rates before and after chronic exposure to hypoxia are the same, then complete (100%) acclimation has occurred. 19

The oxygen consumption rates of T. haemastoma exposed to long-term hypoxia stress exhibit the same general pattern as the oxygen consumption rates with acutely declining oxygen tension (Figs. 4, 5); as the PO2 declines, so does the rate of oxygen consumption. This pattern i s most pronounced a t 30°C, and i s suggested a t 20°C. At 10°C, the snails are in a state of cold torpor and do not respond to most stimuli.

The fact that the oxygen consumption rate of oyster d rills exposed to low PC>2 for 28 days is lower than that at full oxygen saturation and the fact the oxygen consumption-P02 curve does not shift to the left after

28 days exposure to 53 mm 02 at 30°C and 30°/ooS indicates that T. haemastoma does not exhibit acclimation to moderate hypoxia. _T. haemastoma is slightly heat-stressed at 30°c, the scope for growth

(excess of energy absorbed over energy expended through metabolic processes) under normoxic conditions at 30° and 30°/oo is approximately

350 joules*day“ l for a 1 g dry weight sn a il compared to 1500 joules*day“

* at 20o an(j 30°/oo (stickle, 1985). Even though the oxygen consumption rate of T. haemastoma did not acclim ate to moderate hypoxia, the sn ails did not show any behavioral evidence of physiological stress at 53 mm

02* They continued to move about the aquaria and feed throughout the four week experiment, even with the reduction in the adenylate energy charge from 0.86 in the controls to 0.68 after 28 days exposure. Total metabolic demand of the snail at 33 and 10% oxygen saturation is likely decreased, as reflected by a reduced rate of oxygen consumption (Gade,

1983). Reduction of the total metabolic output of marine molluscs during anoxia can be considerable, anaerobic heat production of Littorina lrro ra ta (Pamatmat, 1978) and L itto rin a lltto r e a (Hammen, 1908) is only

5% of aerobic heat production, Modiolus demissus produces 7.5% of its aerobic heat output while under anoxia (Pamatmat, 1979), and Mytilus 20

edulis produces only 7-10Z of its aerobic heat production when kept

under a nitrogen atmosphere (Shick et al., 1983).

In contrast to the lack of acclimation of oxyregulation by T.

haemastoma, Bayne and Livingstone (1977) noted a d istin c t sh ift of the

oxygen consumption-P02 curve of Mytilus edulis to the left after five

days at 53 mm O2 . The s h i f t was accompanied by a decrease in the oxyregulation index B2 from -0.049 x 10-^ to -0.345 x 10“^ over the

course of 17 days. Acclimation of the rate of oxygen consumption in M. edulis was evident at both 10 and 17°C. In contrast, Bayne et^ al.,

(1976) found no evidence of acclim ation of oxygen consumption in Mytilus californianus maintained at 58 ram O2 ^or UP to 13 days, a response sim ila r to th at of T. haemastoma.

One strategy used to maintain an adequate oxygen supply to the g ills during hypoxia is increased ventilation (Herreid, 1980). Increased energy use for ventilation results In an increased rate of oxygen consumption, which, as PO2 continues to decline, leads to further increases in ventilation. Eventually (at the Pc) the benefit of the greater effort put into ventilation is exceeded by its the energetic cost and both v e n tila tio n and oxygen consumption decline. An increase in ventilatory effort might explain the transient increase in the oxygen consumption rate at the Pc of the snail in Fig. 5A.

The measurement of v e n tila tio n and excurrent PO2 ga s tr opods is difficult because of the continual movement of the siphon and the diffuse nature of the exhalant stream. One cannot put a respiratory mask on a snail as is commonly done with crabs (Sabourin, 1984) or place flow sensors and oxygen electrodes by a w ell-defined exhalant siphon, as has been done with bivalves (Taylor and Brand, 1975a). In larger snails, these technical problems are surmountable, and Mangum and Polites (1980) 21

were able to measure ventilation and the P02 exhalant current in

the whelk Busycon cana lieu latum under declining oxygen tension. II. canal leu latum is a very large snail, the dry soft tissue weight of a d u lts reaching 20 grams or more as compared to Thais haemastoma, where the dry weight of the soft tissues of a large Individual may be 3-4 g.

Oxygen consumption in B. canalleulatum is regulated between 40 and 120 mm O2 and is proportional to the PO2 on e^-bher side of that range. The changes in oxygen consumption are reflected in the ventilation rate and percent oxygen extraction. As the oxygen consumption rate declines with declining P02 from 150 to 120 ram 02 ventilation and percent extraction both decline. As the PO2 continues to decline, the percentage of oxygen extracted from the ventilatory current rises concurrent with an increase in the ventilation rate, having the effect of maintaining the overall rate of oxygen consumption constant (Mangum and Polites, 1980).

Among the factors that have been shown to affect oxyregulatory ability in molluscs are temperature, salinity, and size (Herreid, 1980).

Many authors have found that within a species large individuals are b e tte r regulators than are sm all in d iv id u als (Bayne, 1971; Famme, 1980;

Murdoch and Shumway, 1980; Taylor and Brand, 1975a,b). This relatio n sh ip cannot be made for th is set of experiments with T. haemastoma since the snails used for the declining oxygen tension experiments were selected for a uniform, large size.

The lower mean value for at 20° than at 30° (Table II) indicates that T. haemastoma is better able to regulate its oxygen consumption at 20°C than at 30°C, where it is under a slight degree of heat stre ss (S tick le, 1985). The average B2 values of T. haemastoma at

10°C are not significantly different from zero, indicating minimal oxyregulation. The oxygen tension-oxygen consumption curve at 10° has a 22

slope of 0.12 pi * g dry weight-1 * hr-1 * mm 02_1 for nearly the entire

range of P02 (Fig. 6C), which, along with the low value of (Table

II) indicates excellent regulation.

At 20°C, both Kj/K 2 and B2 indicate better oxyregulation in _T.

haemastoma at 10 and 30°/ooS than at 20°/ooS (Table II). In the marine

pulmonate Amphibola crenata, which occurs in salinities between fresh

water and 40°/ooS, K|/K2 1s lowest and B2 is the most negative between 0

and 25°/ooS Indicating the best oxyregulation at the lowest salinities

(Shumway, 1981). In contrast, the oxyregulatory ability of the bivalves

Anadra granosa and Gelonia ceylonica were reduced at the lower end of

th e ir s a lin ity ranges (Bayne, 1973).

It should be kept in mind that the terms "oxyregulation" and

"oxyconformity" do not describe a dichotomy, but rather the two extremes

of a continuum (Mangum and van W inkle, 1975), and th a t th e two oxyregulation indices, Kj /K2 and B2 are only mathematical constructs

that indicate approximately where an animal's response pattern lies on

that continuum. Each index appears to have validity, and has been calculated for several molluscan species (Table V). Only rarely, however, have both indices been calculated from the same declining oxygen tension experiments as in these experiments. The failure of these oxyregulation indices to show strong correlations in this study dictates that care must be used in their interpretation. In some species, the oxygen consumption rate under declining P02

Sometimes, as with Busycon canaliculatum , a region of strong oxyregulation w ill be "sandwiched" in between two regions of oxyconformity (Mangum and P o lite s, 1980). Whenever the actu al response of the respiration rate to declining oxygen tension departs sufficiently 23

from the in itial assumptions of the model that the index is based on,

the calculated result w ill be meaningless (van Winkle and Mangum, 1975).

The opine dehydrogenase enzymes are functionally equivalent to

lactate dehydrogenase, anaerobically condensing an amino acid with

pyruvate to produce an opine compound while oxidizing NADH (Livingstone,

1983). Their occurrence is extremely widespread throughout the animal

kingdom (Livingstone, et_ al., 1983). Strombine, the condensation product

of glycine and pyruvate, has been demonstrated to accumulate in the

posterior adductor muscle of Mytilus edulis during air exposure (de

Zwaan and Zurburg, 1981), and in the adductor muscle of Crassostrea

virginica exposed to anoxia (Eberlee, et a l., 1983), although in both

cases the increase in the concentration of strombine was less than that

of alanine, indicating that the opine pathway is quantitatively less

important during anaerobiosis than the alanine pathway. The

concentration of strombine continued to increase in M. edulis (de Zwaan and Zurburg, 1981) and C. virginica (Eberlee et a l., 1983) adductor muscle during recovery before returning to control levels. Similarly, alanopine accumulated in the adductor muscle of Modiolus squamosus during air exposure, and after a brief drop at the onset of recovery, continued to accumulate for six hours of a twelve hour recovery period

(Nicchitta and Ellington, 1983). Continued use of anaerobic pathways during aerobic recovery has been hypothesized by de Zwaan et_ al. (1983) to supplement aerobic pathways during the replenishment of the arginine phosphate pool when ATP requirements are high.

Although octopine, alanopine, and strombine were not assayed for, some preliminary hypotheses may be drawn regarding the possible use of these m olecules during exposure to hypoxia or anoxia In T. haemastoma.

Firstly, unlike Thais laplllus, which has a very high octopine 24

dehydrogenase activity (Livingstone, 1982), the absence of any

detectable octopine dehydrogenase activity in haemastoma (Table III)

rules out octopine as a metabolite during either anoxia or a subsequent

recovery period. Secondly, since the activities of the opine

dehydrogenases are the same in snails that have been exposed to low

oxygen levels for 28 days as in snails maintained for 28 days at

norraoxia, (Table III) it is unlikely that the synthesis of these enzymes

is induced as a response to chronic hypoxia. Thirdly, the transient

increase in alanopine dehydrogenase activity on the first day of anoxia

(Fig. 11) suggests that alanopine Is being produced during the

transition from the use of aerobic to anaerobic metabolic pathways.

Alanine, the amino acid substrate of alanopine dehydrogenase, is a major end product of anaerobic metabolism in haemastoma (Ellington, personal communication). If, in fact, the alanopine dehydrogenase pathway is activated during the onset of hypoxia, It may be one of the mechanisms by which T. haemastoma is able to successfully cope with the

large diurnal fluxes In PO2 tl*at characterize Its environment during the spring and summer. Since the detection of an enzyme activity in vitro does not verify its role in vivo, these hypotheses remain to be verified experim entally for T.haemastoma.

Although the adenylate energy charge has not fulfilled its early promise as an absolute quantitative Indicator of stress (Ivanovici,

1979), it is still useful quantitatively for Intraspecific and qualitatively for interspecific comparisons when coupled with knowledge of muscle phosphagen concentrations. That T. haemastoma is slightly stressed by mild hypoxia is evident from the decline in energy charge from 0.86 in snails remaining at 100% oxygen saturation to 0.72 in snails exposed to 67% oxygen saturation for 28 days (Fig 8). Use of 25

energy charge alone can be deceiving. When oxygen saturation is less

than 100%, there is not a significant difference in either the

concentrations of adenylate phosphates or in the adenylate energy charge

(Fig. 7) which would suggest that there is no difference in the

magnitude of stress incurred by snails exposed to several partial

pressures of dissolved oxygen. Use of the adenylate energy charge as a

stress index is misleading without concurrent knowledge of the

concentration of the muscle phosphagen. There is a significant 59%

decrease in the concentration of arginine phosphate between 67 and 33%

oxygen saturation which indicates that the snails are at a lower energy

level in the more hypoxic condition.

In snails exposed to anoxia (<5-8 mm O 2 ) the decline in energy

charge and arginine phosphate concentrations occurred within one day, an

identical response as observed in the whelk, Nassa mutabilis (Gade, et

a l., 1984) the mussel Mytilus edulis (Ebberink and de Zwaan, 1980;

Wijsman, 1976), the cockle, Cardium tuberculatum (Gade, 1980), the

anemone Bunodosoma cavernata (Ellington, 1981) and others (Table VI).

During this in itial exposure to anoxia, metabolism is changing from

dependence on high output aerobic pathway to low output anaerobic

pathways (de Zwaan, 1977; Gade, 1983). Evidently th is tra n s itio n takes

some time, because ATP levels are maintained at the expense of arginine

phosphate. Arginine phosphate is the primary source of high-energy

phosphate during muscular activity in molluscs (Gade, 1980; Grieshaber,

1978). The extremely low level of motor activity in T. haemastoma at

very low oxygen tensions may be related to the depletion of the

phosphagen pool.

The energy charge in foot tissu e of T. haemastoma a fte r one day of anoxia was 0.58, a 28% decline from the control while arginine phosphate 26

concentration had fallen by 95%. In contrast, in snails exposed to 10%

oxygen saturation for 28 days, energy charge had declined by 20% and

arginine phosphate concentration by only 54%. The threshold between the

zone of capacity adaptation (the environmental conditions where the

animal can survive indefinitely) and the zone of resistance adaptation

(where, unless environmental conditions change for the b etter, death is

inevitable) (Vernberg and Vernberg, 1972) can be better predicted by a

significant decline in the concentration of the phosphagen, arginine

phosphate, than by a decline in the adenylate energy charge in foot

tissu e of T. haemastoma.

A temporary period of anoxia or hypoxia often results in a

transient Increase in both the aerobic respiration rate and degree of

oxyregulation during the recovery period (Herreid, 1980). Both length

and intensity of hypoxia exposure are correlated with the magnitude of

increase in oxygen consumption and oxyregulation re la tiv e to the pre-

exposure s ta t e (Shumway, 1981; Bayne and L iv in g sto n e, 1977). The

presence of an elevated rate of oxygen consumption after anoxia and

increased evidence of oxyregulation is most pronounced in animals living

in microhabitats regularly exposed to hypoxic conditions (McMahon and

Russel1-Hunter, 1978). Higher than normal oxygen consumption rates after

a hypoxic episode have been compared to the classical oxygen debt

(Herreid, 1980), where the increased oxygen consumption is used to metabolize the accumulated end products of anaerobiosis. If such an oxygen debt is present in T. haemastoma after short term exposure to anoxia it would Indicate that the responses to acute and chronic hypoxia are very different. It would be useful to determine if jC. haemastoma incurs an oxygen debt after acute hypoxia, and if so, if it is associated with an accumulation of alanopine or strombine during the 27

recovery phase. Better understanding of the oyster drills response to

short term anoxia would be gained by a fine tuning of the time course of

change in tissue arginine phosphate concentration and adenylate energy

charge and the recovery of these parameters during re-exposure of oyster drills to normoxic conditions.

I t has been established that Thais haemastoma is very to leran t to

low oxygen conditions within its normal range of temperatures and s a l i n i t i e s . At 20 and 30°c the pattern of oxygen consumption with d e c l i n i n g PO2 *s variable, but most individuals tend to be oxyconformers. At 10°c, the aerobic respiration rate is extremely low

(100 jil O2 * g dry weight- * • hr- *), but is constant over the PO 2 range of 20-140 mm 0£ reflecting the torpid state of the snail at low temperatures.

Although T. haemastoma survives for 28 days at 33% of oxygen saturation, the animals do not show acclimation of the aerobic metabolic rate during hypoxia. Finally, the opine dehydrogenase pathway is probably not being used to maintain ATP production during chronic hypoxia, but may be active during the in itial transition to anaerobic metabolism at the onset of hypoxic stress. 28

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TABLE I

Mean weekly water quality variables for the 30°C-30°/ooS bloassay experiment. Values are running means from day 0 up through the Indicated day. Underlined v a lu es for ammonia and pH are th e extrem es recorded. These are r e p r e se n ta tiv e of the complete set of hypoxia tolerance experiments.

P02 (mm 02 ) Target Day P02 0-7 0 -1 4 0-21 0-28

0 8 9 8 8 16 18 18 21 21 53 56 52 47 46 107 92 93 94 96 156 151 145 141 140

AMMONIA (pM) Target Day po2 0-7 0-14 0-21 0-28

0 7 22 18 18 16 5 9 7 8 53 23 17 13 11 107 TS 13 11 10 <■» • 156 20 16 12 11 pH Target Day po2 0-7 0-14 0-21 0-28

0 7 .9 7 .9 7 .8 7.7 18 7 .8 7 .8 7 .9 7 .8 53 7 .6 7 .5 7.5 7 .5 107 7 .6 7 .6 7.6 7 .5 156 7 .6 7 .5 7 .5 7 .4 3B

TABLE XI

Indices of oxyregulation In Thale haenastoaa exposed to acutely declining oxygen tension. Pc, the critical oxygen tension, was calculated fron the breakpoint of the curve relating PO2 to oxygen consumption as calculated fron the Iterative partial regression technique. B2 x 10"* la the quadratic coefficient relating standardized oxygen consumption to POj (Mangum and van Winkle, 1973). Kj/Kp 18 calculated as the slope divided by the y-lntercept of the llnear-transforned hyperbola relating weight-specific oxygen consumption to PO2 (Bayne, 1971). Data are given as x+S.E., n«10.

TEMP. INDEX SALINITY (^ooS) ° 0 ___ ~ 10 20 30 "1 0 PC 99 + 13 86 + 16 95 + 15 B2 -0.022 + 0.022 -0 .0 3 2 + 0 .022 0.010 + 0.016 Kt /K2 17 + 5 25 + 10 34 + 6

20 Pc 57 + 5 50 + 5 60 + 9 B2 -0 .1 0 0 + 0 .0 1 6 -0.047 + 0.007 -0.073 + 0.015 Kj/Kz 43 + 6 B2 + 13 41 + 8

30 Pc 50 + 7 55 + 5 59 + 7 B2 -0.069 + 0.010 -0.043 + 0.007 -0.073 + 0.010 ki / k 2 62 + 10 95 + 12 99 + 15 39

TABLE II I

A ctivities (pmolen'g wet tlsaue"l*nin_l) of the pyruvate oxldoreductase enzymes In foot tissue of Thais haemastoma exposed to hypoxia at 30° and 30°/°° for 28 days (x +_ S.E. , n-5)

Enzyme Z Oxygen S atu ration 100 67 33 Lactate Dehydrogenase 2.08 + 0.13 1.95 + 0.23 1.95 + 0.16 Octopine Dehydrogenase 0.02 + 0.02 0 0 Alanopine Dehydrogenase 6.73 + 1.34 7.59 + 0.86 4.26 + 0.73 Strombine Dehydrogenase 2.00 + 0.24 1.57 + 0.32 1.31 + 0.16 TABLE IV

Time Co SOX mortality (LT-50) in marine molluscs subjected to anoxia.

Temperature LT-50 Species (°C) (Days) Reference

Gastropods Littorina littorea 10 15 Theede et al., 1969 Littorina saxatllls 10 6 H 11 II H Nasaarlus obsoletus 22 9 Kashins 6 Hangua, 1971 HassariuB trivittatus 22 9 a M ii Thais haemastoma 10 22 T his study ■ ii" —i f 20 18 TT II 1 1 it 30 12 II t|

B ivalves Cardlum edule 10 4 Theede et at., 1969 Crasaostrea virginica 5 >40 Dunnlngton, 1968 10-15 20-25 ii ti rt n 15-20 10-15 ir If rt ii 25 5 ii tr Craaaostrea vlrRlatca 10 35 Stickle 6 Kapper, unpublished data II ir 20 20 t« n ir n II II 30 7 H H ti ii Cyprina ls la n d lc a 10 55 Theede et a l., 1969 Mulinia lateralis 10 11 Shuavay e t a l . , 1983 ii -----n ■ 20 4-8 II if "TT it ii ii 30 2 II I! M ti Mya arenarla 10 21 Theede et a l., 1969 M ytilu6 e d u lis 10 35 ti tt m n Scroblcularla plana 10 21-25 ti tr tt H 41

TABLE V

Values of the uxyregulatlon Indices B2 and Kj/ k 2 In tarlne toUuscs.

S p ecies B2 Ki ^2 R eference Gastropoda Acta teatudlnalls -0 .8 1 8 0 HcKahon 6 Rusaell-Hunter, 1978 Aaphlbola crenata -0 .0 4 9 7 96 Shuaway, 1981 Crepldula fornicate 9-26 Newell e£ a l. , 1978 Lacuna vlncta -0 .0 8 6 8 HcHahon 6 R u saell-H unter, 1978 Llttorlna llttorea -0 .0 6 0 3 Llttorlna obtusata -0 .0 4 0 3 Llttorlna aaxatllls -0 .0 5 1 3 W ltrella lunata -0 .0 5 0 3 Thais haeaastota (10°) -0.0320 to +0.0100 17-34 This study (20°) -0.0466 to -0.1001 41-82 (3 0 °) -0.0428 to -0.0734 62-99 Uroaalplnx clnerea -0 .1 1 8 0 Hangu* & van W inkle, 1973

B iv a lv es Anadra granges <33°/oo) 30 Bagne, 1973 " " (20°/oo) 150 Chlatys dellcatula 116 Hackay 4 Shuaway, 1980 Corblcu’la flualnea -0.0097 to +0.0575 HcHahon, 1979 Ccrastoderta edule -0.0400 to -0.0756 HcHahon 6 W ilson, 1981 Crassostrea vlrglnlca -0.1260 to -0.2520 Shutway 6 Koehn, 1982 Gelonla ceylonlca (33°/oo) 15 Bayne, 1973 " " (20°/«O 44 Hacota balthlca -0 .0 1 0 6 to + 0.0489 HcHahon 6 W ilson, 1981 Hod1o lu s d eaiasu s -0 .2 2 8 0 Mangua 6 van W inkle, 1973 Hullnla lateralis (10°) -0 .1 8 3 4 to -0 .0 7 3 5 Shutway, 1983 h (20o } -0 .1 2 0 0 to -0 .0 4 3 5 *9 n » « f30oj -0 .0 0 1 7 to -0 .0 0 2 6 m n Hytllua callfornlanua 63 Bayne et a l-, 1976 Hytllus edulls (30°/oo) 65 Bayne* 1973 M " (20%>°) 160 Hytllua edulls (152 -0 .0 4 2 3 Bayne & Livingstone* 1977 °2> H *1 II <120 -0 .0 6 5 O2) n t» tt ( 83 02) -0 .1 1 9 ii li tt < 52 02) -0 .2 7 9 5 Kucula turglda -0 .0 2 3 4 to +0.0754 Wilson & Davis* 1984 Kangla cuneaca -0 .0 6 9 5 Hangun 6 van Winkle* 1973 Telllna tenuis -0 .0 4 1 2 to +0.0062 HcHahon t W ilson, 1981 42 TABUS IX

Adenylate energy charge (EC) In various narlne tnvertebratee under control and •treeeed condition*

*• Control Stressed Specie* Stressor T issue EC EC Kefarenc* Gastropods Haaaa a u ta b lll* anoxia fo o t 0.88 0 .7 4 Cade e t a l . , 1984 Pyrazua ebenlnus low salinity entire 0.88 0.61 Rainer e t a l ., 1979 Thai* haenastoaa anoxia fo o t 0 .80 0 .5 0 This study

B ivalves Anadra trap ezia low salinity P.A.H. 0.85 0.69 Rainer et al., 1979 Cardlua tuberculatun anoxia foot 0 .9 3 0.91 Gade, 1980 Chlanys opercularls ■wining adductor 0.93 0.42 Grleshaber,11978 Ceukensla dealssa air exposure P.A.H. 0 .7 5 0.75 Hlcchltta 6 Ellington, 1983 Hytllus edulls air exposure e n tir e 0 .8 5 0.76 Wljsaan, 1976 ii ii“ a n o x ia P.A.H 0 .9 2 0.56 Ebberlnk 6 de Zwaan, 1980 Hodlolu* aquaaosua air exposure P.A.H. 0.80 0.50 Hlcchltta 6 Ellington, 1983 Sacco*tree low salinity P.A.H. 0 .7 6 0 .5 3 Rainer e t al., 1979 ■ ercla ll*

Coelenterata Bunodoioaa anoxia e n tir e E llin g to n , 1981 cave m a te

P.A.H.>posterlor adductor a iiclt

i 43

FIGURE LEGENDS

Diagram of the hypoxia bloassay system showing path of water through

filte r unit, pump, exposure tank, and aquarium; and gas mixer

connected to aquarium under-gravel filter.

95% confidence bands around LC-90 (E3), LC-50 (£53), and LC-10 (TTTTTft

of Thais haemastoma over 28 days exposure to hypoxia at 10, 20 and

30°C and 10, 20, and 3 0 ° /° ° S .

Rates of oxygen consumption in Thais haemastoma acclimated to

different temperatures, (pi O2 ’ 8 dry weight- ^*954 • hr-1. x+^S.E.

n=30).

Rates of oxygen consumption (pi 02 • g dry weight-b* hr-1, x+S.E.,

n-10) at 10, 20, and 30°C and 10, 20, and 30°/°oS of Thais

haemastoma after 28 days exposure to hypoxia. Points within

tem perature-salinity groups with the same letters from Duncan's

M ultiple Range test are not significantly different from each

other. For 10°, b=1.0024, for 20°, b=0.640Q, for 30°» b=»0.4800.

Oxygen consumption rate (pi 02 • g dry weight-1* hr-1) of Thais

haemastoma as a function of ambient P02 (mm 02) for (A) a

relatively good oxyregulator; K j/^-lS, B2xl03—0.121, Pc-31; and

(B) and a relatively poor oxyregulator, K^/K2=140, B2x103=-0.026,

Pc=55. These are typical of snails run at 20 and 30°c. (C) Typical

curve relating oxygen consumption rate (oxygen consumption in pl*8

dry weight-1*hr) as a function of ambient P02 (ram 02) at I0°C;

K1/K2“ b, B2xl03=0.005, Pc=*134. 44

6. Representative response of oxygen consumption to declining oxygen

tension before .(# ) and after (■) 28 days exposure to 53 mm 0£ at

30°C and 30°/°°S. The points are superimposed on each other,

Indicating no acclimation of the respiratory rate chronic hypoxia.

This pattern was seen In 8 out of 10 individuals.

7. Concentrations of total adenylates, ATP, ADP, and AMP in foot tissue

(praoles * g wet weight-1) of Thais haemastoma exposed to 10, 33,

67, and 100% oxygen saturation at 30°C and 30°/ooS for 28 days, (x

+S.E., n=5).

8. Adenylate energy charge (AEC) and arginine phosphate concentration in

foot tissue (pmoles * g wet weight-1) Thais haemastoma exposed

to 10, 33, 67, and 100% oxygen saturation at 30°c and 30°/ooS for

28 days. (x^S.E., n=5).

9. Adenylate energy charge (AEC) and arginine phosphate concentration

in foot tissue (pmoles * g wet weight-1) of Thais haemastoma

exposed to anoxia for 0, 1, 2, 4, and 6 days at 30°C and 30°/ooS

for 28 days. (x^S.E., n=5).

10. Concentrations of total adenylates, ATP, ADP, and AMP concentration

(pmoles * g wet weight-1) In foot tissue of Thais haemastoma

exposed to anoxia for 0, 1, 2, 4, and 6 days at 30°c and 30°/ooS

for 28 days. (xi^.E., n=5).

11. A ctivities of pyruvate oxidoreductase enzymes (pmoles • g wet

weight-1 • min-1) In foot tissue of Thais haemastoma at 30° and

30°/oo after 0, 1,2, 4, and 6 days exposure to anoxia (<5 mm O2 ).

(x ^ S .E ., n = 5). ADH=a l a n o p i n e d e h y d r o g e n a s e ; L D H = * lactate

dehydrogenase; SDH-stromblne dehydrogenase. F ig . 1.

POg CONTROL

WATER TABLE

„ —* — ^ II OYSTER OYSTER CHIPS CHIPS

BIOFILTRATION UNIT 46 Fig. 2

107co o 2 0 7co o 307cOO

100-

\* 80 -

30° 6 0 -

4 0 -

CM o 2 0 -

CP - X 0 - E E 8 0 -

CM O 0 6 0 -

4 0 -

2 0 -

0 - 2 0 - 10°

10 20 10 20 10 20 OXYGEN CONSUMPTION (yu.1*g dry weight"954-hr-1) ig. F ro 4* o> oo o o o o o o o o o o o

H _ m o

XI m x j > ro —I o c XIm

o 0 4 O o

•c* 48 Fig. 4 % OXYGEN SATURATION

i o 7 o o s 50 100 60

1000

3 0 °C 500 AB

CO . £ z & o © o ?

000

20°C 400

200

200

1 0 “ C too AB

0 30 00 00 120 150 0 00 00 00 120 150 0 30 00 00 120150 P0 2 (mm Qj) Fig. 5

1000 800 600

400 200

50 100 150 1000 800

600

400 200

50 100 150 200

150

100

50 1 5 0 OXYGEN CONSUMPTION (ul*g dry weighH.hr I)

*n era ON

- o o ro 00 o 3 3 o ro ro o

o

Cn O CONCENTRATION (qmoles-g wet weight 0.5 ~ 0.5 2.0 2.5 1.0 i. 7 Fig. 0 OYE SATURATION OXYGEN % 20 50 O (m Hg) (mm POg 40 080 60 100 TOTAL ATP AMP ADP 150 100 51 52

Fig. 8

% OXYGEN SATURATION 20 40 60 80 100 DNLT EEG CHARGE ENERGYADENYLATE

3.0 AEC

Ui I 0.75 i 2 a. 4) CO £ 2.0 ARG-P 0.50

D> 1.0 ID 0.25 4> o I-

0 50 100 150 POg (mm Hg) T 01 T O CONCENTRATION (qmoles (qmoles g wet weight )

TIME (DAYS) ARGININE PHOSPHATE (qmoles*g wet weight-1)

3 0 UVH0 ADH3 N3 3 1 V1 AN3 QV Fig. 11 CD o ENZYME ACTIVITY (//.moles*g in.-1) wet weight_1-m c o o o ro o DAYS OF ANOXIA 56

VITA MARTIN A. KAPPER

P erso n al:

Date of Birth: August 10, 1954 Birthplace: Bay Shore, N.Y. Not Married

Home Address: Business Address: 824 W. Chimes S t. #3 Department of Zoology and Physiology Baton Rouge, La. 70802 Louisiana State University Baton Rouge, La. 70803 (504) 343-1258

(504) 388-1132

Education:

B.S. Biology, 1976. St. Lawrence University, Canton, N.Y. 13617

M.A. Biological Sciences, 1980. State University of New York at Buffalo, Buffalo, N.Y. 14260. Major Professor, C.A. Privitera. Project title: "The ability of the chiton Mopalla muscosa to extract oxygen from water and from air"

Ph.D. Physiology, August, 1985 (anticipated). Louisiana State •University, Baton Rouge, La. 70803. Major Professor, W.B. Stickle, Jr. Dissertation title: "Metabolic responses of the estuarine gastropod Thais haemastoma to hypoxia"

Professional Experience:

1976-78: Teaching Assistant SUNY/Buffalo: Animal Physiology, Comparative Anatomy, Embryology, General Biology

1978-79: Technical Specialist, SUNY/Buffalo: Neurophysiology

1979-Present: Teaching Assistant, LSU: Comparative Physiology, Environmental Physiology of Estuarine Organisms, Elementary Physiology (lab), Human Physiology (lecture), General Zoology (majors), General Biology (non-majors)

May-August 1982: GS-7 Physiologist, National Marine Fisheries Service Auke Bay L ab o rato ry , Auke Bay, A laska. Research in to e f f e c ts of oiled sediment and the water soluble fraction of crude oil on the energy budgets of pink shrimp and juvenile king crabs

Awards & Honors:

Robert A. LeFleur/Petroleum Refiners Environmental Council of Louisiana Graduate Fellow, 1980-84 57

Professional Societies:

American Society of Zoologists American Society for the Advancement of Science

Papers Presented:

1. Kapper, M.A. & C.A. Privitera. 1978. Ability of the chiton Mopalia muscosa to extract oxygen from water and from air. Am. Zool. 18: 605.

2. Stickle, W.B. & M. Kapper. 1980. Effects of salinity on nitrogen metabolism in the Southern Oyster D rill Thais haemastoma. Am. Zool. 20: 852.

3. Kapper, M.A. & W.B. Stickle. 1981. Respiratory responses of an estuarine gastropod to declining oxygen tension. Am. Zool. 21: 983.

4. Kapper, M.A., R.C. Chauvin and W.B. Stickle. 1983. Water and ion balance in the six-armed starfish Leptasterias hexactis as a function of salinity. Am. Zool. 23: 995.

5. Kapper, M.A. & W.B. Stickle. 1984. Survival and respiratory rate of Thais haemastoma exposed to chronic hypoxia. Am. Zool. 24: 137A.

Publications:

Stickle, W.B., M.A. Kapper, E. Blakeney and B.L. Bayne. 1985. Effects of salinity on the nitrogen metabolism of the muricid gastropod Thais (Nucella) lapillus. Journal of Experimental Marine Biology and Ecology (in press).

Kapper, M.A., W.B. Stickle and E. Blakeney. 1985. Volume regulation and nitrogen metabolism in the muricid gastropod Thais haemastoma. Biological Bulletin (in press).

L iv in g sto n e , D .R ., W.B, S tic k le , M.A. Kapper and S.Y. Wang. 1985 Microsomal detoxification enzyme responses of the marine snail Thais haemastoma to laboratory oil exposure. Bulletin of Environmental Contamination and Toxicology (in press).

Publications in Preparation:

S h irle y , T .C ., M.A. Kapper, W.B. S tic k le , C. B rodersen, and S.D. R ice. Tolerance and Bloenergetics of Alaskan king crab, Paralithodes camtschatica, exposed to the WSF and oil contaminated sediment of Cook Inlet crude oil.

Stickle, W.B., M.A. Kapper, T.C. Shirley, M.G. Carls, and S.D. Rice. Tolerance and Bioenergetics of the pink shrimp (Pandalus borealis) during long terra exposure to the water soluble fraction and oil contam inated sedim ents o f Cook I n le t crude o i l .

Kapper, M.A. and W.B. Stickle. Osmoregulation in the six-armed starfish, Leptasterias hexactis. 58

Stickle, W.B. and M.A. Kapper. Tolerance limits of several species of Northern Gulf of Mexico invertebrates to chronic hypoxia.

Kapper, M.A. and W.B. Stickle. Metabolic responses of the muricid gastropod Thais haemastoma to hypoxia.

L iv in g sto n e, D .R ., W.B. S tic k le , M.A. Kapper, and S.Y. Wang. Ind u ctio n and activity of the mixed-function oxygenase system in Thais haemastoma after chronic exposure to the water soluble fraction of South Louisiana crude oil.

L iv in g sto n e, D .R ., W.B. S tic k le , M.A. Kapper, and S.Y. Wang. Bioaccumulation of petroleum hydrocarbons In Thais haemastoma after chronic exposure to the water soluble fraction of South Louisiana crude o i l .

L iv in g sto n e, D .R ., A. de Zwaan, W.B. S tic k le , and M.A. Kapper. Phylogenetic distribution and activity of the opine dehydrogenase enzyme system.

I DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Martin A. Kapper

Major Field: Physiology

Title of Dissertation: Metabolic Responses of the Estuarine Gastropod Thais haemastoma to Hypoxia

Approved:

Major Professor and Chairman

Dean of the Graduate School

EXAMINING COMMITTEE:

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t/Q ■ ------

Date of Examination:

July 22, 1985