Respiratory changes in the euryhaline clam, Mulinialateralis (Say), over a range of temperature and salinity combinations

J. Exp. Mar. fliol. Ecol., 1984, Vol. 81,

pp. 269-280

269

Elsevier

JEM 334

RESPIRATORY LATERALZS

CHANGES IN THE EURYHALINE

CLAM, MULZNZA

(Say), OVER A RANGE OF TEMPERATURE

AND SALINITY

COMBINATIONS

JOHN B. WILLIAMS’ Department

of Zoology. North Carolina State University, Raleigh. NC 27650, U.S.A.

Abstract: The combined effects of five temperatures (15, 20, 25, 30, and 32 “C) and five salinities (10, 15, 20,25, and 35x,) on respiratory metabolism in the euryhaline clam, Mulinia lateralis (Say), were determined from polarographic measurements of oxygen consumption. Although both the effects of temperature and salinity were statistically significant as well as their interaction effects, the effect oftemperature was greater. Euryhalinity (previously ascribed to M. laterulb based only on its geographical distributions) was reflected physiologically by the few significant differences between respiration rates in various salinities at a given temperature. Significant differences were most often between rates in 20s0 and the lo%, or 35%, extremes. Consistently highest rates in 20x,, may reflect a salinity preferendum since this salinity was similar to long-term mean habitat salinity. Key words: euryhaline; salinity; temperature; clam; respiration; polarographic

INTRODUCTION

Physiological mechanisms can exhibit wide variations in rate within a range of tolerance for different physical factors. For example, the effects of temperature on metabolic activity in poikilotherms have been extensively investigated (Prosser & Brown, 1962) as well as the modifying actions of capacity adaptations (Vernberg & Vemberg, 1972). However, fewer studies have addressed the synergistic effects of additional factors, such as salinity, on metabolism in estuarine invertebrates (Dehnel, 1960; Vemberg etal., 1973, 1974; Dimrock & Groves, 1975; Bishop etal., 1980; Shumway & Koehn, 1982). Designation of some estuarine organisms as euryhaline has been based upon the observed distribution of adults in relation to salinity or laboratory tolerance experiments. Does metabolism vary greatly over a similar range of conditions and at what rate does it change as tolerance limits are approached? Investigating this question is important to determine to what extent organisms may be restricted metabolically even though they can tolerate a broad salinity range. Florkin & Schoffeniels (1969) stated the need to differentiate between this “ecological” euryhalinity and “physiological” euryhalinity in their discussion of intracellular volume regulation. Since temperature and salinity are two primary factors controlling species distribution ’ Present address: Department of Natural Sciences, P.O. Box 2061, South Carolina State College, Orangeburg, SC 29117, U.S.A. 0022-0981/84/$03.00

0

1984 Elsevier Science Publishers B.V.

210

JOHN B.WILLlAMS

and physiology in most temperate estuaries, investigators have recognized the need to

address both variables in combination. Most of these previous studies investigating the combined effects of temperat~e and salinity at diierent levels primarily dealt with survival in embryo and larval stages (Costlow et al., 1962, 1966; Davis & Calabrese, 1964; Zein-Eldin & Aldrich, 1965; Cain, 1973; Lough & Gonor, 1973; Sandifer, 1973; Young & Hazlett, 1978). Additional studies (e.g., Dehnel, 1960; Vemberg et al., 1974; Dimrock & Groves, 1975; Nelson et al., 1977; Shumway & Koehn, 1982) recognized the impo~~ce of understand~g combined temperature and salinity effects on adults since a functional response of an organism exposed to a wide range of values for a given factor (salinity) could vary differently at one level of a second factor (temperature) than at another level. Without an understanding of different physiological changes with temperature and salinity throughout an organism’s tolerance range, incorrect assumptions might be made concerning its energy req~rements and behaviors activity levels. For example, Vemberg & Vemberg (1972) in their review of temperature effects reported that standard metabolism for some species may be more independent of temperature changes than active metabolism. Intuitively one would assume that metabolism would increase directly with temperature, but does this occur at the same rate at all salinities? Bayne et al. (1976) reviewed previous research on temperature and salinity effects on resp~ation in euchre mussels, however, these effects are less well known for infaun~ bivalves. By studying euryhaline species, we can address the important questions of how metabolism changes with temperature at different salinities and the relationship between survival (tolerance) and metabolism in bivalves over a wide salinity range. This investigation examined the combined effects of temperature and salinity on the respiratory metabolism of the euryh~ine clam, ~~Zi~i~l~t~ruli~ (Say). iw: I~ter~Ii~ was selected because: (1) it occurs over a wide salinity range (1.4-75. lx, Breuer, 1957) and populations experience different thermal regimes throughout its latitudinal distribution from Canada to Mexico; (2) it is highly important in estuarine food webs (Morris, 1973); and (3) it is of conveniently small size for laboratory respirometry (< 2.0 cm length). In addition to discussing the physiolo~~al ~ons~uenees of eu~h~~ty, this report evaluates relative effects of temperature and salinity on respiration statistically and presents the need for additional research to relate these results to potential osmoregulation or osmoconformity. METHODS

AND MATERIALS

M. lateralis were obtained from the lower Cape Fear River estuary, North Carolina (33 “58/N: 77”58’W), at a water temperature of 28.0 “C and salinity of 16.0x,. In order to avoid differences in respiration due to extremes in clam size or age, only individuals approximately the same length, 6.8-9.4 mm, were used. Final respiration rates were adjusted for clam dry tissue mass (wei~t-speci~c rates) to produce comparable values which were expressed as ~1 0, - mg - ’ * h - ‘. The experimental design followed in this study was a randomized complete block

RESPIRATORY

CHANGES

IN THE EURYHALINE

CLAM, MChXW4 LATERALIS

271

design using a 5 x 5 factorial analysis with five levels of temperature (l&20,25,30, and 32 ‘C) and five levels of salinity (10, 15,20,25, and 35x,). The highest test temperature (32 “C) approached the upper thermal tolerance limit reported for M. lateralis in Chesapeake Bay (Kennedy & Mihursky, 1971), a thermal regime similar to the Cape Fear River estuary. Initially six salinity levels were included, however, a 5%, solution was discontinued because M. lateralis did not survive > 24h at this salinity. Temperature levels were maintained in five separate controlled temperature water baths A 0.1 ‘C. Each test salinity was mixed from “Instant Ocean” (Aquarium Systems, Inc.) with well water and placed into five 45-l fiberglass tanks in each temperature bath. Air was bubbled continuously into each tank and 3-4 cm of sediment were spread over the tank bottom to allow clam burrowing. No food was provided for the clams throughout the experiment except that present in the initial water column and sediments. For this reason, experimental respiration rates reflected standard metabolism (Vernberg & Vernberg, 1972). Four to five clams were randomly assigned to each test salinity in each temperature bath and separated for identification by fiberglass screens. Water temperatures were lowered or raised to test levels at a rate of 1.O’ C * day - ’ from the initial temperature of 28.0 ‘C. Clams were acclimated to each experimental temperature for at least 2 wk prior to measuring respiration. Three replicate measures of respiration were made for each clam in each treatment combination (except when clams died and were replaced). Oxygen consumption was detected in a Gilson water-jacketed micro-reaction chamber by a Clark-type oxygen electrode with teflon membrane. Changes in dissolved 0, percent saturation were measured on an oxygen meter (Y.S.I. Model 53) calibrated to 100% using a modified Winkler technique (Carpenter, 1965) and were plotted by a recorder (Sargent SR-5). To avoid complications due to potential oxygen-conforming metabolism, respiration was usually recorded until the clam had reduced 0, levels by only 5-10% and no data were used for analyses after 0, levels had been lowered to 70% of calibration levels. In measuring respiration, each clam was positioned on a fiberglass screen inside the 1.5-ml chamber tilled with a test salinity nearly saturated with air. The chamber solution was circulated by a small magnetic stirrer and its temperature was held stable by the constant-temperature water surrounding the chamber. In most cases each replicate for all clams at the same temperature was completed on the same day between 0900 and 2100 with the third replication being made within 2 wk of the first. To minimize the effects of possible die1 metabolic rhythms, the order in which each salinity group was tested was varied between replications. Corrections for non-clam 0, uptake (e.g. bacteria) were determined by mechanically disturbing each clam and measuring 0, change while the clam ceased pumping with its shell tightly closed. M. lateralis whole weights (g) were measured after each replication and converted to dry tissue weights (mg) for final respiration rates. Dry weights were predicted from regressions of dry weight on whole weight for 125 clams measured to the nearest 0.0 1 mg with a Mettler balance (Mettler H5 1). A regression of clam volume on whole weight was

JOHN B. WILLIAMS

272 also

developed to correct reaction chamber volume for clam displacement volume. Factorial analyses and regressions were conducted on an IBM-370 computer using the Statistical Analysis System (SAS) (SAS Institute Inc., 1979). RESULTS

During respiration meas~ements, M. IateraI~ often exhibited ~scont~uous pumping activity of irregular duration. Clams inverted the extreme distal portion of the excurrent siphon and ceased pumping even while the main siphon still remained extended. Pumping cessation resulted in an immediate slowing of 0, uptake and interruptions usually lasted a few minutes or less followed by a forceful exhalation through the incurrent siphon. In&ally chamber 0, concentration declined sharply due to the reduced oxygen tension of this exhaled water, but soon 0, uptake assumed a more steady rate (Fig. 1). Brand & Taylor (1974) observed similar behavior in the clam, Arctica islandica, and found that 0, consumption returned to fairly constant levels following the initial exhalation. The number of stops per minute by M. lateralis was irregular and did not differ greatly over the temperature range of 15-32 “C. In order to describe M. later& resp~ation for the total measurement interval (including stopped intervals as well as during periods of steady pumping), two respiration values were determined. Total clam 0, uptake for the measurement time period, including the non-pumping intervals as well as the initial exhalation and constant pumping phases, was called TRESP (Fig. 1). Respiration during the constant pumping phase-only was designated PRESP (Fig. 1). -

PUMPING BEGINS

\ PUMPING STOPS

CONSTANT 02 UPTAKE

-i3 P

PUMPING STOPS

05

1.0 TIME

1.5

2.0

(minutes)

Fig. 1. Exampie recording plot of 0, uptake and clam pumping behavior in the respiration chamber indicating PRESP and TRESP: PRESP is respiration during active pumping only; TRESP is respiration for entire interval including pumping and nonpumping periods.

RESPIRATORY CHANGES IN THE EURYHALINE CLAM, MULINIA LATERALIS

213

Respiration rate means at all temperature and salinity combinations were slightly higher for PRESP than for TRESP. Mean PRESP ranged from 1.68 to 5.64 ~1 0, f mg- ’ . h - ‘, while mean TRESP ranged from 0.89 to 4.77 ~1 0,. mg - ’ . h - ‘. The regression used in converting clam whole weight to dry tissue weight for these rates was significant at the P = 0.0001 level and had an r2 of 0.97. The resulting equation expressing dry tissue weight in grams (DRY) on whole wet weight (WHOLE) in grams was: DRY = 0.03 x WHOLE. Claxn displacement volume (VOL) in ml was determined from the regression equation: VOL = 0.05 + 0.522 x WHOLE. This regression was significant at the P = 0.0001 level and had an 12 of 0.73. In all test salinities except 20x,, both TRESP and PRESP increased with temperature up to 30 “C, but were depressed at 32 “C (Fig. 2a,b). As described in Steel & Torrie

Fig. 2. Response surface for ~uZj~ja luieralix mean PRESP and TRESP &IO,. mg - ’ . h - ’ ) at 25 combinations of temperature and salinity: PRESP is respiration during active pumping only; TRESP is respiration for entire interval incIuding pumping and nonpumping periods.

214

JOHN B. WILLIAMS

(1960), the shape of these response surfaces (Fig. 2a,b) implied some form of interaction between the effects of temperature and salinity. These interaction effects were more clearly distinguished by plotting separately the changes in mean TRESP and PRESP with salinity at each temperature (Fig. 3a,b) and changes with temperature at each salinity (Fig. 4a,b). In both instances respiration in each of the five levels of one factor changed at a different rate across the other factor’s range of values. For example, the slope of PRESP in 25 “C rose more steeply from 10 to 207& and declined more steeply from 20 to 35x,, than did PRESP in 15 or 20 “C (Fig. 3a). The main effects of temperature and salinity, as well as their interaction effect, were all determined to be highly significant for both PRESP and TRESP (Table I) using the aA a

x---x=

5

o---o= 20°C +-q--Q= 25°C e---a= 30°C c -. = 32°C -----__

15°C

------_____,

0

-----_____ X

1

,,,,,,.,, 10

_,,,,_,,, 15

,,,,,,,,,

,_,,,,,,,

20 SALINITY

25

__ 30

Fig. 3. PRESP

35

%.

x---x= v-o= *-----*=

SALINITY

““‘,~

15°C 20°C 25°C

0 A..

PRESP and TRESP (@Oz. mg- ’ h _ ‘) for MU&I lateralis at different salinities for five temperatures: is respiration during active pumping only; TRESP is respiration for entire interval including pumping and nonpumping periods.

RESPIRATORY CHANGES IN THE EURYHALINE CLAM, MULINIA

LATERALIS

275

general linear model (GLM) procedure of SAS (SAS Institute Inc., 1979) for analysis of variance, Among these factors, temperature had a greater effect on M. lff~e~~l~s respiration than salinity alone or the temperature x salinity interaction effect (Table I). No significant differences were found between the three replications (Table I). For all temperatures except 15 “C, both PRESP and TRESP were higher in 20%, than the other salinities (Fig. 3a,b). In most comparisons, however, there were few significant differences between salinities, depending upon temperature, using Duncan’s multiple range test (Steel & Torrie, 1960) (Table II). PRESP and TRESP were generally si~~c~tiy higher in 20x,, than in either lo%,, or 35x,, at the upper temperatures.

6

a x---x= -o= *-----it= c--.-e=

5 Y. I:

m---m=35%

,-1

,_,,,~,,~ 15

10

16 15 26 25

c* I’ ,,*’

,% z /g ,g

X

__.-W------_~

.‘,I,,,,,L,,,_,,_._ 20

_.l,,.,,, 25

TEklPERATURE

x---x= W=15%

10

30

,,,,‘,,,,r

35

“C.

,/’ ,/’

%

7%~--- _a= 20 /& t--4=252 m---8’35%

_,_*e$ _,-_rl-_-_

l

>_.--0

04......,.. 10

/--=a’ m

‘.‘b

,,* -

-

..l..,,.,),,,...,,, 15

20 TEMPERATURE

Fig. 4.

PRESP

and

/

25

,,,,.,,,,

,, 30

,,.,.

35

“C

mg - ’ *h - ’ ) for ~~li~~u luteruEs at different temperatures for five salinities: during active pumping only; TRESP isrespiration for entire interval including pumping and nonpumping periods.

TRESP ($0,.

PRESP isrespiration

y

JOHN B. WILLIAMS

276

TABLEI Analysis of variance for Muliniu later& PRESPand TRESPin a randomized complete block design using a 5 x 5 factorial analysis for temperature (T) and salinity (S): PRESPis respiration during active pumping only; TRESP is respiration for entire interval including pumping and nonpumping periods; ** significant at P = 0.001 level; ns, not significant. Source PRESP Treatments T

s TxS

Replications Error Total TRESP Treatments T

s TxS

Replications Error Total

d.f.

Sums of squares

Mean squares

F

(24) 4 4 16 2 245 271

(289.48) 170.24 63.51 55.73 1.87 323.17 614.52

42.56 15.88 3.48 0.93 1.32

32.27** 12.04** 2.64** 0.71””

(24) 4 4 16 2 246 272

(213.43) 131.43 42.91 39.09 0.64 220.36 434.43

32.86 10.73 2.44 0.32

36.68** 11.98** 2.73** 0.36””

TABLEII Comparisons of differences in PRESP and TRESP between salinities (10, 15, 20, 25, and 35%,) for each temperature using Duncan’s multiple range test: any two means not underlined by the same line are significantly different at the P = 0.05 level; PRESP is respiration during active pumping only; TRESP is respiration for entire interval including pumping and nonpumping periods, Ranked respiration means (~10,. mg- ’ h- ‘) for salinities (%,,) Temp. (“C)

PRESP

TRESP

15

20 2.37

15 2.24

10 2.20

25 2.00

35 1.68

15 1.56

10 20 25 1.51 1.49 1.09

35 0.89

20

20 25 3.21 2.42

10 2.38

15 2.31

35 1.94

20 2.17

15 1.61

10 35 25 1.56 1.40 I .20

25

20 4.65

15 25 3.85 3.51

10 3.07

35 1.85

20 3.51

15 2.85

10 2.40

25 2.37

35 1.11

30

20 4.92

15 4.51

25 4.47

35 4.02

10 2.76

20 3.67

25 3.41

15 3.28

35 2.94

10 2.12

32

20 5.64

35 3.59

15 2.70

25 2.26

10 1.83

20 4.77

15 2.29

35 2.22

10 25 1.68 1.62

RESPIRATORYCHANGESIN THE EURYHALINECLAM,MULINIA

LATERALIS

211

DISCUSSION M; ia~raIj~ has been identified as an eurytopic oppo~unist (Boesch et al., 1976) and exhibited broad euryhalmity in Alazan Bay, Texas, where it was observed in salinities ra.q$ng from 1.4 to 75.1x, (Breuer, 1957). Oxygen consumption rates for extremely euryhahne species (reviewed by Kinne, 1971) have previously been described as temporarily remaining unaffected within tolerable ranges of salinity. In my study of A&.~atera~~~, this gen~~~ation could be applicable to clams at lower temperat~es, but, at 25 oC and above, respiration varied more widely between some salinities (Fig. 4a,b). While the overall effect of salinity on clam respiration was statistically smaller than temperature, this variability in respiration with salinity at higher temperatures was reflected in the significant temperature x salinity interaction effect (Table I). Oxygen consumption rates measured in my experiment represented metabolic processes subjected to long-term stable conditions of temperature and salinity. Rates within each temperature and salinity treatment did not change significantly between replications (indicating salinity acclimation had occurred) even though wide differences in respiration were observed between the different treatments. Because respiration measurements were not conducted during the initial acclimation period, it could not be detents whether M. laterals oxygen consumption was temporarily un~ected by salinity changes as in the extremely euryhaline species reviewed by Kinne (1971). These M. lateralis experimental respiration rates would probably also differ from individuals in nature due to a more plentiful food supply in the estuary compared to that in the experimental tanks. One potential difference might be between the temperature-induced rate of change for these standard metabolic rates and the active metabo~sm of clams in nature (Vemberg & Vemberg, 1972). The euryhalme qualities ascribed to M. lateralis by previous researchers (Breuer, 1957; Wass, 1972) based upon its natural distributions were reflected in its physiological responses measured in my experiment. Oxygen consumption rates increased significantly with rising temperature, but displayed fewer significant differences between most salinities within each temperature. Among these significant differences for respiration rates at the same temperature, most were between an intermediate salinity (20%,) and an extreme salinity (35 or lo%,). My findings suggest that euryhalinity in M. lateralis enables these clams not only to survive in a wide range of salinities, but also to maintain statistically similar metabolism. Bayne et al. (1976) described a similar capability for the exhume mussel ~~~~~~ educe which exhibited comparable respiration in “field ambient” salinities from 6 to 30x,. Hypothetically these patterns of respiration for Mulinia lateralis could be linked to its degree of osmoconformity or osmoregulation. Bivalve molluscs in general have been described as osmoconformers (Gardiner, 1972) with respiration declining as they were exposed to salinities higher or lower than that in their normal habitat. However, Bayne et al. (1976) hypothesized that this reported decline in respiration from laboratory studies for at least one species, Mytih edulis, may have been produced by inadequate

278

JOHNB.WILLIAMS

acclimation. Gsmoregulation has not been investigated for Mulinia lateralis, however, osmoregulatory ability has been observed in a closely related bivalve of the same Mactridae molluscan family, Rangia cuneata (Bedford & Anderson, 1972; Fyhn, 1976; &&sing & Towle, 1978; Mangum et al., 1979). Further research should investigate internal osmotic changes in Mulinia lateralis in combination with respiration measurements to determine to what degree osmoregulation or osmoconformity is responsible for its euryhalinity. No phytoplankton food sources were provided to these filter-feeding clams during my experiment leaving only sediment organic matter, sediment microflora or bacteria as food sources. Hence, no significant clam growth was observed during the experiment. By contrast, A4. lateralis held in natural estuarine waters were capable of nearly doubling their length in 2 wk depending upon food abundance and temperature (Williams, 1978). Measurements of food availability and clam growth in my experiment indicated that little food was available for any osmoregulatory energy demands. It is difficult to determine whether the observed patterns of A4. lateralis respiration might be linked to either osmoregulation or osmoconformity. If osmoregulation were hypothesized, then exposure to salinities increasingly higher or lower than some preferred range would require additional energy. As available energy reserves were depleted, osmoregulation and general metabolism would be reduced; resulting in lower oxygen uptake. Respiration was consistently highest in 20x,, with PRESP and TRESP being significantly lower than these 20x,, rates most often in either the 35 or lo%,, solution (Table II). The higher rates in 20& might reflect an inherent salinity preferendum. Birkhead et al. (1979) reported that salinity in the Cape Fear River estuary in the vicinity from which these M. lateralis were collected varied widely (from 4-30x,), but had a mean between 15 and 19z0 for a 5-yr period. Alternatively, if M. lateralis were strictly an osmoconformer, then its similar respiration over a range of salinities might reflect an ability to tolerate wider osmotic changes in its tissues and still actively metabolize as described by Gardiner (1972) for euryhaline species in general. Bayne et al. (1976) reported that Mytilus edulis, an osmoconformer (Gardiner, 1972), also exhibited similar respiration for a wide salinity range. For species exposed to periodic broad salinity fluctuations this adaptation would be energetically efficient. This study has established that the consequences of euryhalinity in AL lateralis are not only reflected in its broad salinity tolerance, but also by its similar metabolic rates over a range of salinities. Additionally, the finding that significant differences in respiration between salinities changed with temperature emphasized the importance of studying combined factor effects on adult organism physiology. Most previous multifactor experiments with estuarine species primarily dealt only with tolerance in embryo and larval stages. As estuaries become increasingly exposed to man-induced stresses, knowledge of how organisms function under naturally interacting conditions will be essential for evaluating potential impacts superimposed by man. Thermal loadings, river flow alteration, or other perturbations could affect metabolic activity differently at one level

RESPIRATORY CHANGES IN THE EURYHALINE CLAM, MULINIA LATERALIS

279

of a second factor than at another, as observed for clam respiration in these difI’erent combinations of temperature and salinity. ACKNOWLEDGEMENT

This work was partially supported by a research grant from the Carolina Power and Light Company. REFERENCES B. L., R.J. THOMPSON& J. WIDDOWS, 1976. Physiology I. in, Marine mussels: their ecology and physloZogy, edited by B.L. Bayne, Cambridge University Press, Cambridge, pp. 121-206. BEDFORD, W. B. & J. W. ANDERSON,1972. The physiologic~ response of the estuarine clam ;pangia ~uneatff (Grey) to salinity. I. Osmoregulation. Physiol. ZooI., Vol. 45, pp. 255-260. BIRKHEAD, W. A., B. J. COPELAND& R. G. HODSON, 1979.Ecological monitoring in the lower Cape Fear River estuary 19714976. Carolina Power 8c Light Co. Rept. 79-1, Raleigh, North Carolina, 292 pp~

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BISHOP,J. M., J. G. GOSSELINK& J. H. STONE, 1980. Oxygen consumption and hemolymph osmolality of brown shrimp, Penueus aztecus. Fish. Bull. NOAA, Vol. 78, pp. 741-758. BOESCH,D. F., M. L. WASS & R. W. VIRNSTEIN,1976. The dynamics of estuarine benthic communities. In, Esfuuri~e processes, Vol. 1, edited by M. Wiley, Academic Press, New York, pp. 177-196. BRAND, A.R. & A. C. TAYLOR,1974. Pumping activity of Arctica islandicu (L.) and some other common bivalves. Mar. Behav. Physiol., Vol. 3, pp. l-15. BREUER,J.P., 1957. An ecological survey of Baffin and Alazan Bays, Texas. Publ. Inst. Mar. Sci. Univ. Texas., Vol. 4, pp. 134-155. CAIN, T. D., 1973. The combined effects of temperature and salinity on embryos and larvae of the clam Rangia cuneata. Mar. BioZ., Vol. 21, pp. l-6. CARPENTER,J. H., 1965. The Chesapeake Bay Institute technique for the Winkier dissolved oxygen method. Limnol. Uceunogr., Vol. 10, pp. 141-143. COSTLOW,J.D., C. G. BOOKHOUT& R. .I. MONROE, 1962. Salinity-temperature effects on the larval development of the crab, Panopeus herbstii Mime-Edwards, reared in the laboratory. Physiol. Zoo/., Vol. 35, pp, 79-93. COSTLOW,J. D., C. G. BOOKHOUT& R. J. MONROE, 1966. Studies on the larval development of the crab Rhithropanopeus harrisii (Gould). 1. The effect of salinity and temperature on larval development. Physioi. Zool., Vol. 39, pp. 81-100. DAVIS, H. C. & A. CALABRESE,1964. Combined effects of temperature and salinity on development of eggs and growth of larvae of M. mercenaria and C. virgin&z. Fish. Bull., Vol. 63, pp. 643-655. DEHNEL,P. A., 1960. Effect of temperature and salinity on the oxygen consumption of two intertidal crabs. Biol. Bull. (Woods Hole, Mass.), Vol. 118, pp. 215-249. DIMROCK,R. V. & K. H. GROVES, 1975. Interaction of temperature and salinity on oxygen consumption of the estuarine crab Panopeus herb&i. Mar. Bioi., Vol. 33, pp. 301-308. FLORKIN, M. & E. SCHOFFENIELS,1969. Mo~e~~~urapproaches to ecology. Academic Press, New York, 203 pp. FYHN, H. J., 1976. A note on the hyperosmotic regulation in the brackish-water clam Rurtgib curzeata. J. Comp. Physiol., Vol. 107, pp. 159-167. GARDINER,M. S., 1972. The biology of invertebrates. McGraw-Hill, New York, 954 pp. KENNEDY, V.S. & J.A. MIHURSKY, 1971. Upper temperature tolerances of some estuarine bivalves. Chesapeake Sci., Vol. 12, pp. 193-204. KINNE, O., 1971. Salinity-invertebrates. In, Marine ecology, Vol. I, purt2, edited by 0. Kinne, WileyInterscience, New York, pp. 821-995. LOUGH, R. G. & J. J. GONOR, 1973. A response-surface approach to the combined effects of temperature and salinity on the larval development of Ad&u cal&miensis (Pelecypoda : Mytilidae). I. Survival and growth of three and tifteen-day-old larvae. Mar. BioL, Vol. 22, pp. 241-250,

280 MANGUM, C.P.,

JOHN B. WILLIAMS

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