CONTRACT REPORT H-73-1 THE BRACKISH WATER CLAM RANGIA CUNEATA AS INDICATOR OF ECOLOGICAL EFFECTS OF SALINITY CHANGES IN COASTAL WATERS by S. H. Hopkins, J. W. Anderson, K. Horvath

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June 1973

Sponsored by Office, Chief of Engineers, U. S. Army

Conducted for U. S. Army Engineer Waterways Experiment Station Vicksburg, Mississippi

Under Contract No. DACW39-7I-C-0007 By Department of Biology, Research Foundation, Texas A

, APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED B è ï ï a r y

Au g 3 1973

Bureau of Reclamation Denver, Colorado

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Destroy this report when no longer needed. Do not return it to the originator.

The findings in this report are not to be construed as an officia Department of the Army position unless so designated by other authorized documents. 92041911

THE ^BRACKISH WATER CLAM RANGIA CUNEATA AS INDICATOR OF ECOLOGICAL EFFECTS OF SALINITY CHANGES IN COASTAL WATERS3

by £ S. H. Hopkins, J. W. Anderson, K. Horvath —

SCI J 101 I0I[ DÌ00101

June 1973

Sponsored by Office, Chief of Engineers, U. S. Army

Conducted for U. S. Army Engineer Waterways Experiment Station Vicksburg, Mississippi

Under Contract No. DACW39-7I-C-0007

By Department of Biology, Research Foundation, Texas A<5kM University, College Station, Texas

ARMY-MRC VICKSBURG. MISS.

APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED L FOREWORD

This report was prepared under Contract No. DACW39-71-C-0007 with the Department of Biology, Texas A&M University Research Foundation, College Station, Texas, for the U. S. Army Engineer Waterways Experiment Station (WES). This report was written by Dr. S. H. Hopkins, assisted by the following people, who participated in the investigation. Dr. Jack W. Anderson directed the work on the physiology of Rangia cuneata and some of the work on reproduction and development; Brian Bedford did the major part of the physiological work for a Ph. D. dissertation; and students Susan Baldwin, Thomas M. Dillon, and Glen Michael Hightower aided in the work on physiology and on reproduction, development, and growth. Dr. Kalman Horvath directed the work on the biochemistry of Rangia, also on the ciliary activity and on the measurements from which the condition index was calculated; he was assisted by students Michael P. Klett and Lynn W. Jagers in the physical measurements and the biochemical work and by Kathleen Hooper Julicher in measurements of ciliary activity. C. A. Bedinger collected many of the clams, made a number of ecological observations, and got well along in a study of some biochemical parameters in relation to season and salinity, but did not have his biochemical results complete enough to include in this re- port; he also participated in the study of reproduction and larval de­ velopment. Melvin R. Frei, working in Dr. Horvath's laboratory, com­ pleted a study of crystalline style enzymes in three different popula­ tions of Rangia cuneata, which he used for his Ph. D. dissertation. Jack M. Neagle studied the parasites of these three populations. The introduction, the review of the natural history, and the overall dis­ cussion and conclusions were written by Dr. Hopkins. The typist was Ruth Peschke. The contract was monitored by Mr. F. A. Herrmann, Jr., Chief of the Estuaries Division, under the general supervision of Mr. H. B. Simmons, Chief of the Hydraulics Laboratory.

iii Director of WES during the conduct of this study and the publica­ tion of this report was COL Ernest D. Peixotto, CE. Technical Director was Mr. F. R. Brown.

iv TABLE OF CONTENTS

Page FOREWORD ...... i ü

SUMMARY ...... ix

INTRODUCTION ...... 1 NATURAL HISTORY ...... 6 Zoology of Rangia ...... 6 Population Density ...... H Biomass ...... 13 Commercial Value ...... 15 Reproduction, Development and Setting ...... 17 Growth ...... 21 Food and Feeding ...... 25 Fauna of the Rangia Zone ...... 27 Predation ...... 34 Parasites ...... 36 Bottom Type Inhabited ...... 37 Temperature Relations ...... 38 Salinity Relations ...... 42 DESCRIPTION OF THE STUDY AREAS ...... 43 Trinity River Delta-Trinity Bay ...... 43 Trinity Bay ...... 44 Trinity River Delta ...... 47 Lake Anahuac ...... 49 The Neches River ...... 50 PHYSIOLOGICAL STUDIES ON EFFECTS OF SALINITY ...... 57 General Introduction...... 57 General Methods and Materials ...... 58 Effect of Salinity on Blood Osmotic Concentration, Per Cent Water, and Per Cent Ash ...... 59 1. Introduction ...... 59 2. Materials and Methods ...... 60

v a. Osmoregulation ...... 60 b. Body water percentage and ash percentage... 61 3. Results 61 a. Osmoregulation ...... 61 b. Per cent body water and per cent ash ...... 66 4. Discussion ...... 70 Effects of Salinity on the Uptake and Accumulation of Glycine ...... 75 1. Introduction ...... 75 2. Materials and Methods ...... 75 a. Uptake of glycine ...... 75 3. Results ...... 79 a. Uptake of glycine by whole animals ...... 79 b. Uptake of glycine by isolated gill tissue.. 84 c. Velocity of glycine uptake ...... 84 d. Fate of accumulated glycine ...... 89 4. Discussion ...... 97 Effects of Salinity and Temperature on Respiration... 100 1. Introduction ...... 100 2. Materials and Methods ...... 100 a. Whole animal respiration ...... 100 b. Gill respiration ...... 102 3. Results ...... 103 a. Whole animal respiration ...... 103 b. Gill respiration...... 112 4. Discussion ...... 117 Effect of Salinity on Glycogen Utilization by Rangia. 120 1. Introduction ...... 120 2. Materials and Methods ...... • 120 3. Results ...... 121 4. Discussion...... 121

vi Page

Effect of Salinity on Feeding ...... 125 1. Introduction ...... 125 2. Materials and Methods ...... 126 3. Results ...... 128 4. Discussion ...... 135 Effects of Salinity on Reproduction, Larval Development and Growth ...... 136 1. Introduction ...... 136 2. Materials and Methods ...... 137 3. Results ...••...... 138 4. Discussion ...... 147 Phytoplankton Studies ...... 149 1. Introduction ...... 149 2. Materials and Methods ...... 149 3. Results ...... 150 4 • Discussion...... 150 SOME INDICES OF SEASONAL AND ENVIRONMENTAL EFFECTS .... 153 Effects of Salinity and Temperature on Ciliary Activity ...... 153 Introduction ...... 153 Methods ...... 153 Results and Discussion ...... 154 Dissolved Carbohydrate Uptake by Rangia ...... 158 Introduction ...... 158 Methods ...... 158 Results ...... 159 Seasonal Physical-Chemical Parameters of the Com­ position of Rangia ...... 163 Introduction ...... 163 Methods ...... 163 Results ...... 164

vii Page COMPARATIVE STUDIES OF CRYSTALLINE STYLE ENZYME ACTIVITY IN POPULATIONS FROM WATERS OF DIF­ FERENT SALINITY ...... 172 Introduction and Literature Review ...... 172 Materials and Methods ...... 174 Results and Discussion ...... 176 Chlorinity ...... 178 Incubation Time ...... 187 Ionic Effects ...... 200 Conclusions ...... 205 OVERALL SUMMARY AND DISCUSSION ...... 212 CONCLUSIONS ...... 220 REFERENCES CITED ..... 224 OTHER RANGIA REFERENCES ...... 245 ADDENDA ...... 250

viii SUMMARY

It was apparent to Dr. S. H. Hopkins, a consultant for the Corps of Engineers on the effects of engineering works on biota of coastal waters, that there was seldom as much information on the ecology of estuarine organisms as was needed to predict the effects of environ­ mental changes. The ecology of oysters comes close to being an excep­ tion. Oysters have been the subject of literally thousands of scien­ tific publications. Enough information has been accumulated on their biology and that of their competitors, pests, predators, and parasites to permit fairly confident predictions as to how and how much they will be affected by a given change in the environment, e.g., a rise or drop of 10 ppt (parts per thousand) in the salinity of the bay they live in. Unfortunately, there is not this much basic knowledge on most estuarine animals or plants. Of all estuarine environments, the least understood is the low- salinity zone inland from the oyster communities where the salinity averages below 10 ppt and may be zero at times. Much of this zone is characterized not only by low but by extremely variable salinity; the water may be fresh on the ebb and definitely salty on the flood tide, or the salinity may be below 1 ppt during half the year and 15 ppt or higher during the other half. This variability excludes most animals, excepting fishes that move back and forth with their preferred salini­ ties. Such an area, where competitors and predators are mostly ex­ cluded, is an ideal habitat for an organism that can live in it and monopolize the abundant food resulting from nutrients brought in by runoff from the surrounding land. There are a few such species, and these few become very abundant in the low-salinity zone. In Gulf coast estuaries the clam Rangia cuneata has long been recognized as the dominant benthic animal in the 0-15 ppt salinity zone. Since about 1955 it has become the dominant species of this zone in Atlantic coast estuaries also, from Georgia to Maryland; so dominant, for instance, that in a long stretch of James River it makes up 99 percent of the benthic biomass. Obviously this is a key species in the 0-15 ppt zone as the oyster is in the 10-30 ppt zone of estu­ aries; if the same sort of information were available for Rangia as for the oyster, we would be well on our way to understanding the ecology of the low-salinity waters and could make predictions on the effects of changes, but it is not available. The biology of Rangia has been studied by only a few dozen investigators, compared with the many hun­ dreds who have studied oysters. The fact that Rangia thrives best in a zone that is unfavorable for most animals would lead one to suspect that its biology includes some unusual adaptations, but what these special features are, and why Rangia is not only adapted but restricted to the apparently hostile environment in which it lives, have remained mysteries. Dr. Hopkins has had a personal interest in Rangia cuneata since 1951 and has studied it as much as time and money would permit. Recently

ix several students and colleagues became interested in using Rangia as an experimental animal. After this group had started working together, a contract with the Army Corps of Engineers enabled them to improve, ex­ pand, and integrate their investigations into an effort to answer the questions: How will a change in salinity of an estuary (which might be caused by engineering projects such as channel dredging or dam building) affect the important biota of the low-salinity brackish waters, and how can Rangia cuneata be used as an indicator of the effects of salinity changes on this biota? In the search for a laboratory test that could be used to deter­ mine when salinity changes were favorable or unfavorable, salinities in the range from 0 to 38 were tested on adult Rangia clams for effects on survival; regulation of internal salinity; intake, use and release of amino acids; respiration; glycogen use under aerobic and anaerobic con­ ditions; feeding rate; ciliary activity; uptake of glucose; glycogen storage and "index of condition11 in natural environments through a sea­ sonal cycle; carbohydrate-digesting enzymes; and reproduction. It was determined that Rangia cuneata has a system of compensating reactions that allows it to adjust to changes in salinity over the range from 0 to 38 ppt and over the temperature range from 10 to 35° C without harm. It was concluded from these and further studies that the key to the welfare of a Rangia population is not the physiology of the adult individuals, but reproduction and recruitment. The keys to the use of Rangia cuneata as an indicator were found to be two facts: (1) a change in salinity, either up from near 0 or down from 15 ppt and above, is necessary to induce spawning; (2) the embryos and early larvae can survive only in salinities between 2 and 10 (or 15) ppt. On the basis of laboratory and field studies, the model proposed for Rangia in estuaries has the population consisting of: (1) a central subpopulation in the most favorable breeding zone where the salinities between 2 and 10 ppt and the changes in salinity necessary for reproduc­ tion occur in most years; (2) a low-salinity population upstream that is made up of one, two, or three year-classes resulting from larvae that were carried up the estuary, set, and survived in infrequent favorable (high-salinity) years; and (3) a similar subpopulation of one or a few year-classes downbay that set in years of freshets. Since a change in salinity, not just a favorable level, is required for reproduction, per­ fect stabilization of salinity at any level will result in dying out of the population in 15-20 years when old clams reach the limits of their life span. To use Rangia as an indicator of salinity climate, a large number of clams of all sizes are collected at random and measured for construc­ tion of a histogram that will reveal the number of modal peaks, which represent year-classes. If there are several such peaks, especially if there is a peak in the lengths below 30 mm, the population is in good shape and can feed many fishes, crustaceans, wild ducks, and other desirable clam-eaters. If there is only one size class, the population

x is in danger unless there is a nearby breeding population to replenish it whenever conditions permit. When Rangia dies out, there will in­ evitably be a decrease in the number of fish, crustaceans (crabs and shrimp), and birds that the brackish-water area can support.

xi THE BRACKISH WATER CLAM RANGIA CUNEATA AS INDICATOR OF ECOLOGICAL EFFECTS OF SALINITY CHANGES IN COASTAL WATERS

Sewell H. Hopkins Jack W. Anderson Kalman Horvath

INTRODUCTION

Many engineering projects have the potentiality of changing the salinity climate in estuaries. Most obviously, diversion of water from one river system to another will cause some increase in salinity of the donor river’s estuary, and some decrease in the estuary which receives the diverted fresh water, even if this water passes through the water supply and sewage systems of a city on the way. Dams on rivers running toward the coast hold the water back only temporarily- (unless there is also a diversion or a great loss by evaporation), but even this may change the climate and hence the ecology of an estuary if it stabilizes the salinity instead of permitting the natural alternation of low flow and flooding. Dredging or enlarging navigation channels may allow saltwater intrusion far inland into the part of an estuary or tidal river where salinity was formerly low. Such changes are not necessarily bad or necessarily good. Salinity is a natural environmental factor, like temperature. Changes in such factors are more adverse for some organisms and more favorable for others; as a general rule, within limits, the biomass or mass of living organisms remains fairly constant while the diversity or number of species changes. The elimination of some species, or the addition of a species new to the area, may be considered beneficial by some people and harmful by others, depending on their interests and points of view. It is not necessary for the biologist to render a judgment on whether the changes in salinity that will result from an engineering project are good or bad, but it is necessary for him to try to predict what the ecological effects of the changed salinity will be. It is easy to predict that most freshwater species will be excluded from waters

1 whose salinity is changed from 0 to 15°/oo (ppt or parts per thousand) and equally easy to predict that most marine species will be eliminated from estuaries or coastal waters whose salinity is lowered from 35 to 15 ppt. With estuarine species, adapted to spend all or part of their lives in waters where salinity is always lower than the oceanic 35 ppt and may change 10 ppt with each tide cycle, the situation is more com­ plex and prediction is more difficult. The !,harsh salinity climate'* of estuaries is not necessarily preferred by the organisms that live in it; some fishes, for instance, will choose a higher or a lower salinity when given a choice. Sedentary animals such as oysters may grow faster, reproduce better, and (if protected from enemies) survive longer in oceanic than in estuarine salinity. Many estuarine plants actually grow better in fresh water if relieved of competition from other plants. The estuarine fauna and flora are made up of some species that physiologically require the intermediate salinities of estuaries, and other species that could live better elsewhere if it were not for the many more competitors, predators, and parasites in the more stable fresh waters and oceanic waters. Also, some species spend their youth in low salinities for food and protection from predators but must go to sea to reproduce. The best-known or at least most-studied American estuarine organism is the oyster (Crassostrea virginica), to which thousands of publications have been devoted. It thrives in the higher salinities near the mouth of the estuary, but reaches maximum numbers and biomass in the zone where frequent lowering of salinity prevents most oyster predators from building up large populations; in recent years it has been dis­ covered that frequent flooding also protects oysters from parasites such as the fungus Labyrinthomyxa marina and the protozoan Minchinia nelsoni. Still farther up the estuary, where the salinity is usually below 10 ppt and none of the oyster's parasites and shell pests can survive, the oyster itself is frequently wiped out by freshets and must be replenished by larvae coming from the downbay population. Predictions of the effects of changes in salinity are relatively easy for the oyster: permanent lowering of salinity will wipe out the

2 beds farthest upbay, but improve conditions for the oysters farther downbay; permanent raising of salinity, or the elimination of frequent flooding by dams upstream, will allow predators and parasites to move farther up the estuary and cause higher mortality of midbay oysters, but improve conditions for upbay oyster populations. The oyster is the dominant organism in the middle region of many estuaries, but like many other estuarine animals it is really a marine species for which estuarine conditions are less than optimal. Farther up the estuary, in the "bay heads" and lower reaches of tidal rivers, the dominant animals are more likely to be "true estuarine species" for which the low salinities are physiologically as well as ecologically optimal. In most estuaries along the coast of the Gulf of Mexico the dominant benthic (bottom dwelling) animal of this zone, where salinity is too low for most animals of marine origin and too high for most freshwater species, is the brackish water clam Rangia cuneata. During the last twenty years this clam has reappeared, after thousands of years of apparent absence, in the lower salinities of estuaries along the Atlantic coast from Florida to Maryland and finding this "ecological niche" unoccupied, has had such a "population explosion" that it may now make up 99 per cent of the benthic biomass in the low-salinity zone of estuaries (Cain, 1972). Rangia cuneata is not only a species for which low salinity, in the range 1-15 ppt, is optimal, it is also a species which evidently cannot maintain a population outside of this range (Hopkins, 1970). Only a few scattered individuals are found in the parts of estuaries where salinities are consistently above 15 ppt, and the Rangia that live in the fresh portions of tidal rivers can be shown to be survivors of larvae that settled there during a year of low river flow when salinity extended that far upstream (Cain, 1972). So this is a species whose local populations can be destroyed in a few years by any change that reduces salinity permanently below 1 ppt or raises it permanently above 15 ppt. (For "permanently" one could substitute "15 years," apparently the maximum lifetime of this species.) Salinity would be expected to fall below 1 ppt above the dam if the lower part of a 3 tidal river is dammed to make a reservoir. Large areas that normally have salinity below 15 ppt might have an increase above this critical limit if there is a diversion of the river at the head of the estuary (to furnish water to a different watershed), or if a deepwater channel dredged through the area allows intrusion of sea water. The low-salinity area being usually the narrowest part of the estuary, as well as the farthest inland, it is likely to be the part first and most affected by an engineering project that causes a change in salinity. Perhaps the ideal way to evaluate the ecological effect of a permanent salinity change would be to study the changes in the entire community before, during, and after construction of an en­ gineering project. However, this would require many man-hours of work, many professional experts, and a study of several years duration. The next best thing would be to study an "indicator species, if there is one, which would represent the community and whose health, vigor, or growth in given environmental conditions would indicate the health of the species associated with it. The most promising candidate for such an "indicator" is Rangia cuneata. It is found in most estuaries of the Gulf coast and of the South Atlantic states from Florida to Maryland, it is a species whose distribution is known to be strictly limited by salinity (1 to 15 ppt), it becomes very abundant where it occurs (sometimes reaching several hundred individuals per square meter), it is easily obtainable the year around, it is a key form in estuarine communities because it converts detritus and phytoplankton into meat that feeds fish and crustaceans, it actually has a high commercial value for its shells, and it potentially has a higher future value for its meat. Before a species can be reliably used as an indicator, many aspects of its biology must be known which have not yet been fully revealed by the few studies published on Rangia cuneata. It is not enough to know that 99 per cent of the population lives in the zone where salinity averages between 1 and 15 ppt. We need to know what salinity conditions result in populations made up entirely of adults several years old, and what salinities are associated with populations where clams less than one year old outnumber all older individuals• If growth is stimulated by one salinity and stunted by another, we need to be able to interpret this in a quantitative way. We need to know for Rangia, as we do for the oyster, what conditions stimulate and what conditions inhibit reproduction; what is used for food at different stages of development of the young; and how salinity differences affect foods and feeding. We need to know whether this species passively conforms to the salinity of the surrounding medium, as most molluscs do, or whether it actively regulates its internal salinity. If it does regulate internal salinity (osmoregulate), some of the energy derived from its food must be used this way; and we need to know how much, at each salinity level. In many animals the rate of respiration or oxygen consumption changes whenever salinity changes; and if salinity is then kept constant, the respira­ tory rate may either continue at the changed rate or gradually return to its former rate. Different respiratory rates imply that food is being used (or fuel is being burned) at different rates, so if changes in these rates are caused by salinity we need to know just how, in a quantitative way. Activity of the cilia is used to draw currents of food-bearing and oxygen-bearing water into the clam’s mantle cavity and onto the gills where food is captured and oxygen absorbed. It is known that ciliary activity of molluscs is affected by salinity (and by other factors); but in the case of Rangia no study of changes in the action of cilia had been made, in relation to salinity or to anything else, prior to this investigation. Changes in chemical content of Rangia, associated with changes in salinity, had been reported by previous investigators but on the basis of such scanty data that their studies could be considered only preliminary. A sound knowledge of the chemical changes associated with salinity differences, based on adequate data, is still lacking. It was the purpose of the present investigation to close the gaps in existing knowledge, to confirm or refute what had previously been reported on the effects of salinity, and to support those reports found correct with more and better data in the hope of making Rangia cuneata more useful as an indicator of the ecological changes 5 resulting from changes in salinities and salinity distribution patterns, whether caused by engineering works or by natural phenomena.

NATURAL HISTORY Zoology of Rangia

The complete classification is phylum Mollusca, class Pelecypoda (bivalves), order Eulamellibranchia, suborder Adapedonta (Veneroida in some classifications), superfamily Mactracea, family Mactridae, subfamily Mactrinae, genus Rangia, subgenus Rangia, species Rangia cuneata Gray in Sowerby, 1831. So far as known the superfamily and family began in Cretaceous times (Dali, 1898; Moore, 1969), later than most other bivalve groups (Shrock and Twenhofel, 1953); the genus Rangia first appeared in the Miocene (Dali, 1898) and II. cuneata in the Pliocene (or Miocene, according to Maury, 1920) division of the Tertiary period. Possibly Praerangia minúscula Cossmann (1908) from the Paleocene of Belgium is an early Rangia, but it differs considerably from American species (Moore, 1969). Most Mactracea are true marine clams. Even in the subfamily Mactrinae, the genera Mactra and Spisula are oceanic clams burrowing in sandy (Mactra) or muddy (Spisula) sea bottoms. (Spisula solidissima, the so-called surf clam, is a valuable commercial marine species.) On the other hand Mulinia and Rangia in the same subfamily are estuarine clams; the typical American species Mulinia lateralis is one of the more abundant molluscs in the low-salinity "bay heads" of the Gulf coast, with the scarcer Rangia (Rangianella) flexuosa Conrad. Rangia (Rangia) cuneata goes beyond being a typical estuarine species, for it is almost entirely restricted to areas where salinity is below 15 ppt most of the time and is most abundant far up the tidal rivers where salinity may stay below 1 ppt continuously for months or even years. Whereas Mactra, Spisula and Mulinia, each with many species, are widely distributed over much of the world, Rangia is strictly a New World genus, according to Dali (1894 a,b, 1898). There is one Pacific coast species, Rangia (Rangianella) mendica Gould, in the

6 brackish water of mangrove swamps in the Gulf of California (Keen, 1958) . Raiigia (Rarigianella) flexuosa lives in estuaries of the Gulf of Mexico from Northern Florida to Vera Cruz, Mexico (Dali, 1894 a). Rangia (Rangia) cuneata in Pleistocene time was found from New Jersey to northern South America (Richards, 1938, 1939, 1962; Moore, 1969) but in Recent time has been restricted to the Gulf of Mexico coast of the United States and Mexico. During the last 20 years, R. cuneata has apparently extended its range again to the estuaries of the Atlantic coast from Florida to Maryland. We say apparently because there is a chance that there was a resurgence from some small pockets of survivors of the Pleistocene Atlantic coast population, e.g., in the low-salinity waters on the border between eastern North Carolina and Virginia (Hopkins and Andrews, 1970). The maps, Figures 1, 2 and 3, show the known distribution of Rangia cuneata as of this date. Table 1 lists localities reported by authors who give some information on the natural history of the species. This information may relate to past or present distribution, type of substrate in which clams are found, food, growth rate, repro­ duction, temperature relations, or other phases of natural history; but the factor most often mentioned is salinity, which is our subject, so this is the phase of ecology mentioned in Table 1.

Table 1. Local studies giving information on natural history of Rangia cuneata, especially information on salinity relations in nature.

Salinity of Rangia Habitat Author ______Locality______(ppfc) ______Date______Upper Chesapeake Bay 1-8 Pfitzenmeyer, 1970 Elk River, Md. 0.3 - ? Gallagher& Wells, 1969 Potomac River, Md. 5.7 - 11.8 Pfitzenmeyer & Drobeck, 1964 James River 0-15 Cain, 1972 0.2 - 13.8 Peddicord, in progress Virginia (distribution) Wass, 1963 7 Table 1 (cont.) Salinity of Rangia Habitat Author ______Locality______(ppt)____ Date

Pamlico River estuary, 0-18 Tenore, Horton N. C. & Duke, 1968 Tenore, 1970

Newport River estuary, 0.6 - 35.4 Wells, 1961 N. C. (mean 19)

Trent River, N. C. 0.01 - 6 Wolfe & Petteway 1968

Neuse River estuary, 1-12 Porter, 1969 N. C. Wolfe, Cobum, Brooks & Lewis, 1969; Wolfe, 1967

Altamaha River estuary, 2 - 11.5 Godwin, 1968 Ga. Godwin, 1967 St. Lucie Estuary, Fla. 0.15 - 26.3 Gunter & Hall, (east coast) (average 9.7) 1963 (shells only recorded) Atlantic coast, Pleistocene Richards (ranged from New Jersey 1936, 1938, 1939, south) 1962 Atlantic coast, Recent 0-15 Hopkins & (since 1955 reappeared, Andrews, 1970 Ga. to Md.)

Caloosahatchie River 0.1 - 33.3 Gunter & Hall, estuary, Fla. (west (average 6.5) 1965 coast) Myakka R. & Peace R., <1 - 23 Woodburn, Charlotte Co., Fla. 1962 Apalachee Bay - St. 5-25 Menzel, 1971 George’s Sound area Fla. (fauna of) Ochlockonee, Carabelle & 0-22 Olsen, in progress Apalachicola Rivers, Fla. Escambia River, Fla. 0.04 - 9.0 Wurtz & Roback, 1955

Mississippi (molluscs f) <15 Moore, 1961

8 Table 1 (cont.) Salinity of Rangia Habitat Author Locality (ppt) Date

Lake Pontchartrain, 1.2 - 18.6 Darnell, 1958, La. (av. <6) 1961, 1962, * 1964 0.3 - 5.8 Fairbanks, 1963 1.2 - 12.2 Suttkus, Darnell Darnell, 1954 (shell dredging) Tarver, 1970 Marshes in Barataria 0.5 - 18 Jaworski, 1970 drainage basin

Grand Lake & White Lake, 0.08 - 2.7 Gunter & La. Shell, 1958 Vermilion Bay, La. 2.5 - 5.0 Gooch, 1971 Southwestern Louisiana 0.5 - 9.0 Hoese, 1972 Sabine Lake, La. - Tex. 0-16 Stevens, 1960, 1962 Kane, 1961 Sabine River, La. - Tex. 0.1 - 11.8 Wurtz & Roback, 1955 Neches River, Tex. 0.1 - 0.4 Wurtz & Roback, 1955 0.05 - 12.9 Baldauf, 1952, 1953 <0.3, 65% of yr. Hopkins, 1970 Trinity River delta, Tex. 0-16 Baldauf, 1970 van Connor, 196 (personal com­ munications) Trinity Bay 1 - 10(1960) Parker, 1966 av. 11 Shidler, 1961 4 - 16(1964) Upper Galveston Bay 10 - 26(1964) Parker, 1966 av. 18 East Bay 7 - 25(1959) Hoese, 1959 1 - 23(1964) Parker, 1966 Clear Lake av. 4.7 (1961) Mock, 1965 17.8 (1963) Mock, 1965 Table 1 (cont.) Salinity of Rangia Habitat Author Locality ____ (ppt)_____ Date (low salinity parts of Galveston Bay system, Tex.) Carancahua Bay 14 - 19(1930) Strecker, 1935 (Karankawa Bay) 11.7 - 13.3(1935) Ladd, 1951 6 - 23(1965) Mitchell, 1894 Martinez, 1966

Lavaca Bay 0 - 20(1930) Strecker, 1935 0 - 10(1935) Mitchell, 1894 14 - 27(1965) Martinez, 1966

Redfish Lake in delta 0-19 Mackin, 1971 of Lavaca River (low salinity parts of Matagorda Bay system, Tex.) Green Lake in delta of 0 - ? Hedgpeth, 1950 Guadalupe River Mission Lake & Guadalupe 0-10 Ladd, Hedgpeth & Bay when Rangia Post, 1957; common; Ladd, 1951

Hynes Bay & Upper 11 - 20 Parker, 1955, 1959 San Antonio Bay plus when Warwick, 1963; scarce Beyers & Warwick, 1968 (low salinity parts of San Antonio Bay system, Tex.) St. Charles Bay, Tex. (0 - 40) Ladd, Hedgpeth & (extremely vari- Post, 1957 ble) Martinez, 1966 (Aransas Bay system)

10 Table 1 (cont.)

Salinity of Rangia Habitat Author ______Locality______(ppt)_____ Date

Neuces Bay at mouth of 0-20 Singley, 1893 Nueces River, Tex. (Corpus Christi Bay system)

Florida & Gulf states Hedgpeth, 1953 Pleistocene distribution (Fig. 8, p. 129, 130)

Population Density

Rangia cuneata is the most widely distributed and by far the most abundant species of brackish water clam. The following reports give recent figures on density of some populations (Table 2):

Table 2. Population densities: numbers of Rangia cuneata per square meter recorded by various authors in different areas, 1963-1972.

Locality Author Clams/M^ Max Mean Maryland Upper Chesapeake Bay Pfitzenmeyer, 10,000* 1,200* 1970 Potomac River Pfitzenmeyer and Drobeck, 1964 Upper Cedar Point 226 Lower Cedar Point - 11 Virginia James River, Hog Cain, 1972 600 — Island James River, mile Peddicord, 1971** - 130 25 James River, mile Peddicord, 1971** - 100 31 James River, mile Peddicord, 1971** - 20 39 North Carolina Neuse River Porter, 1969 300

11 Table 2 (cont.) ______Locality Author Clams/M^ Max Mean Georgia Altamaha River, inter- Godwin, 1968 14 tidal Florida Ochlockonee River Olsen, 1972** 52

Louisiana L. Pontchartrain, Fairbanks, 1963 North

"Juveniles" (< 23 mm) 1,806 "Adults" ( >23 mm) 31 L. Pontchartrain, Fairbanks, 1963 South "Juveniles" (< 23 mm) 1,881 "Adults" (> 23 mm) 2.6 Vermilion Bay Gooch, 1971 Fearman Lake 3.6 North Mud Point 7.4 South Mud Point 756 180 Southwestern La. Hoese, 1972 Atchafalaya Bay 69 6.1 East Cote Blanche Bay 34 7.0 West Cote Blanche Bay 130 8.5 Vermilion Bay 238 26.6 Grand Lake 116 16.9 "Miscellaneous" 97 11.8 Entire area, west of 11.1 Atchafalaya River Texas Neches River Hopkins, 1970 496 250 Trinity River delta Van Conner, 1968** 9.7 Trinity Bay Bedford, 1972 47 Hynes Bay Beyers and Warwick 130

12 Table 2 (cont.) *This population, first to appear in upper end of Chesapeake Bay, was one year class; all died before 4 years old, presumably from cold (see also Gallagher & Wells, 1969; Cain, 1972).. **Personal communication.

Biomass Hopkins (1970), on the basis of the estimated average of 250 4-year-old clams per square meter in the Neches River above the port of Beaumont, Texas, calculated that this population would produce annually 12,400 pounds of shell and 2,560 pounds of wet meat per acre (13,900 kilograms of shell and 2,900 kilograms of meat per hectare). Biomass of Rangia cuneata here (based on standing crop) was 227.5 grams (dry weight of meat) per square meter. However, it is evident from Table 2 that the Neches River popula­ tion had more than average density. Hoese (1972) estimated that the brackish waters of southwestern Louisiana (Atchafalaya River to Calcasieu Lake) averaged 11.1 Rangia clams (approximately 11 g dry meat) per square meter; this included two water bodies, White Lake and Calcasieu Lake, where no live clams were found although many shells indicated that there had been populations in earlier years. On the basis of these figures, and populations indicated by dredging and tube samples, Hoese estimated the number of living Rangia cuneata in the study area by three different methods, getting totals of 32.3 billion to 48.5 billion clams. On the basis of the intermediate figure of 38.5 billion, the living clams would have a shell weight of 1.86 billion pounds and a wet meat weight of 390 million pounds. Hoesefs study area of course did not include the active Mississippi River delta, Lake Pontchartrain, Lake Maurepas, and many other low- salinity brackish water bodies east of Atchafalaya River; that eastern area is an important source of commercial clam shell production and has large populations of live Rangia as well as great accumulations of shells. In the densely populated area from Vermilion Bay to 2 western Atchafalaya Bay, the population of 17.2 clams/m would 2 yield a biomass of 17g/m .

13 Cain (1972) stated that Rang id cuneata accounted for 99% of the total benthic biomass in the oligohaline (low salinity) part of James River, Virginia, He did not give figures for this biomass, but reported 0 to 600 live clams per square meter. Peddicord (personal communication) counted averages of 20, 100 and 120 clams per square meter in James River, which at 1 gram dry meat per clam would give biomasses of 20, 100 and 120 2 grams/m ; these are extraordinarily high figures even for estuarine benthic biomass. James River heretofore has not been noted for high productivity. Probably the best data ever obtained on the contribution of Rangia cuneata to the total benthic biomass are those reported by Pfitzenmeyer (1970). During an intensive field study of the effects of channel dredging in upper Chesapeake Bay off the mouth of Sassafras River, Maryland, in salinity of 0-8 ppt, R. cuneata suddenly appeared for the first time in the autumn of 1966 and continued to increase in numbers and biomass until the end of December 1968 when the entire population began to die off, apparently killed by cold. At the initiation of the study in September 1966 the average dry weight of the benthic biomass was 0.90 grams per square meter. Then channel dredging started 2 and another series of samples showed 0.67 g/m in December 1966. 2 In April 1967 biomass averaged 1.91 g/m , in August 1967 the 2 2 mean was 2.34 g/m , and in January 1968 biomass averaged 2.33 g/m . The major contributor to this larger biomass was Rangia which was increasing in numbers and size. In December 1968, just before the die-off, the benthic biomass had an average dry weight per square meter of 6.42 grams, 2.76 times the January 1968 biomass, 9.6 times the biomass of December 1966, and 7.1 times the figure for September 1966 before channel dredging began. Even though two other molluscs, Macoma phenax and M. balthica, decreased as Rangia increased, there could be no doubt that R. cuneata greatly increased the benthic biomass in the study area while it was present, and thereby added greatly to the food available for fishes, crustaceans, and wild ducks. In a recent letter (8/29/72) Pfitzenmeyer tells

Ik us that this population still exists, in spite of repeated winter kills, with a current density of 1 clam per square meter.

Commercial Value At present the principal commercial value of Rangia cuneata is for the shells. Fresh shells and the much larger accumulations of old shells, some thousands of years old, are "mined" with bull­ dozers and draglines from deposits on land and with shell dredges from deposits in water bottoms, and used in place of gravel for paving roads, driveways, parking lots, etc.; also as a source of calcium carbonate in various manufacturing and water purification processes. Among the industries using clam shell are steel and aluminum producers, paper mills, glass factories, cement plants, and poultry feed manufacturers, according to Gooch (1971). Data obtained from various sources in 1970 indicated that common retail prices were $3.50 per cubic yard at Louisiana shell piles and $4.25 delivered in Texas. In recent years the official figures for Louisiana, based on severance taxes collected from producers, have ranged around 10 million cubic yards of shell produced and sold per year, of which 5 million is oyster shell and 5 million is clam (Rangia) shell. According to the Twelfth Biennial Report, Louisiana Wildlife and Fisheries Commission, the total production of clam shell for the years 1966 and 1967 was nearly 9.5 million cubic yards. At $3.50 per cubic yard this was worth $33.25 million, or $16.6 million per year. Tarver (1970) claimed that Lakes Maurepas and Pontchartrain annually produce 5 million cubic yards of clam shell worth over $12 million. It should be emphasized that it is the accumulation of centuries that is being exploited. As valuable as Rangia cuneata is for its shell, it has a much greater potential value for its meat. In the years 1966 and 1967 (Lyles, 1969) a North Carolina company paid fishermen 30 cents a pound and marketed 138,000 pounds of Rangia meat canned with ocean clam juice. We had heard that this business was discontinued because of legal difficulties involving labeling but Chestnut (letter, 8/29/72) tells us that it has continued; in a 6-year

15 period, 1966-1971, 325 thousand pounds have been produced, valued at 34 cents a pound when landed. The report of Lyles (1969) was the basis for the figure of 30 cents per pound used by Hopkins (1970) in calculating the value of thé Rangià cünéàta population in Neches River above Beaumont. This population consisted mainly of 4-year-old clams, estimated to average 250 per square meter of bottom. It was calculated that an acre of such a bed would produce $25 worth of shell and $750 worth of meat per year, or a total value of $775 per acre per year, but the total producing area in the Neches was small, possibly less than 100 acres. In southwestern Louisiana Hoese (1972), on the basis of an average of 11.1 live clams per square meter over an area of over 7.5 million acres, including two large water bodies which contained no living Rangia, estimated a total shell weight (for living clams) of 1,864 million pounds, which we calculate to be worth $3.7 million at $3.50 per cubic yard. The wet meat weight of these clams was estimated by Hoese to be 390 million pounds, which we calculate to be worth $117 million at 30 cents per pound; thus the standing crop of Rangia cuneata in this area had a total value of over $120 million. The time required to produce this crop is unknown, but was almost certainly less than 10 years, as this is believed to be close to the usual duration of life of Rangia cuneata. Even if production required 10 years, the annual crop would be worth $12 million, and this is for only one part of the Rangia producing area of Louisiana. Hoese (1972) recommended harvesting only 5 per cent of the standing crop annually until more is known about replacement rates, but even this would yield an annual harvest worth over $6 million. The calculations on value of Rangia meats are worthless if the meat cannot be sold, so the desirability of this clam as food must be considered. There can be no doubt that Rangia flesh is edible, for this clam was for thousands of years a main­ stay in the diet of many tribes of Indians along the Gulf coast. Over the centuries huge mounds or middens of shells, among which Rangia often dominated, were built up at favorite camping grounds

16 and village sites, especially in Louisiana and Texas (Mclntire, 1958; Ambler, 1967; Story, 1968). The Houma Indians in the swamps and marshes east of Houma, Louisiana, were eating quantities of Rangia in 1941 (Speck and Dexter, 1946) and probably still are. In collecting Rangia in Trinity Bay, Texas, we had to compete with several families of Orientals who were taking clams, bushels at a time, for home use; they considered them excellent food. Many individuals of all races eat Rangia frequently, some by choice and some by necessity. Harold Harry (personal communication) reports seeing these clams sold for food on the streets of New Orleans in the 1930?s. Besides the North Carolina episode, there have been other cases of commercial marketing of Rangia cuneata in Texas as "hard clams" or "littleneck clams" beginning in the nineteenth century (Singley, 1893) and continuing sporadically to at least within the past ten years (O’Heeron, personal com­ munication) . We personally know many people who eat or have eaten Rangia; all consider it at least edible, but opinions of taste and texture run from "tough and strong" to "delicious." No doubt, as in most foods, preparation and seasoning as well as individual taste preferences determine how it is rated. However, it must be admitted that at present there is not a large established market demand for this species. On the other hand, the same has been true in the past for many other seafoods that are now in great favor. The demand does not exist in advance of supply; it must be developed by the right kind of promotion, like the inland demand for blue crabs and shrimps. So the present commercial value of Rangia as food is slight, but its potential value may be quite high, possibly much higher than the current North Carolina price of 34 cents a pound would indicate.

Reproduction, Development and Setting

Fairbanks (1963) examined gonads of Rangia cuneata in Lake Pontchartrain throughout the year. He found gonads developing

17 prespawning condition and "imminent ripeness" in February and March, production of ripe gametes in April and May, a short period of recovery from this spawning in early June, and then a longer period of gamete production from late June to October or early November. Natural spawning was not observed, but eggs taken from females in March, June and October were fertilized when placed with ripe sperm (also removed from gonads) and develop­ ment to the veliger stage was observed at temperatures of 22.8° to 26.65° C. Salinity was not mentioned but apparently lake water was used, in which case salinity was 5.5 - 6.0 ppt in October; in March and June 1957 salinities were 5.5 - 6.0 ppt and 4.5 - 5.5 ppt, respectively, but in March and June 1958 they were only 1.4 - 1.8 ppt and 1.0 - 1.2 ppt; Fairbanks does not say in which year he did these March-June experiments, but it seems from the context to have been 1958. Lake temperatures (monthly means) in 1958 were: February 10° C, March 15 C, and 21 - 29.5° C, April through July (end of study period). In December 1947 and January 1948 Fair­ banks recorded minima of approximately 8° C. Water temperatures in 1957 were somewhat higher than in 1948, 17.5° C in February, 16° C in March, 20-30° C April through October, and 17° C in November from our reading of the graph, Fairbanks’s Fig. 3. Fairbanks paid little attention to salinity but considered tem­ perature to be the factor controlling the annual reproductive cycle. The eggs fertilized by Fairbanks were 69 microns in diameter. In 34.25 hours they developed into veligers 93 microns long, which swam rapidly. Later larvae were not obtained in experiments or seen in plankton catches. The smallest juvenile clams found in bottom sediments were 0.375 mm so Fairbanks guessed that this was the size at which setting (the final settling of swimming larvae to the bottom) occurred. Chanley (1965) transferred Raiigia curie at a from James River, where they grew in salinities of about 5 ppt, to the laboratory at Wachapreague on the Virginia Seaside, where he kept them in a salinity of 15 ppt (prepared by mixing sea water and pond water), 18 and at a temperature of 23° C. The clams were taken on April 22, when gonads were ripe. Spawning was induced by rapidly raising water temperature to 30° C and adding sperm stripped from a male. Larvae were fed a mixture of unicellular algae. At 22 - 24.4° C they developed to the setting stage (176 x 160 microns) in 7 days. Chanley was interested only in describing and picturing the swimming larvae so they could be identified in plankton; this he did well. Gooch (1971) and Hoese (1972), working in southwestern Louisiana, were impressed by the fact that in many places only one size class, evidently representing a single year class, is present. Hopkins (1970) had suggested that successful reproduction might occur only at intervals of several years. Gooch (1971) pointed out that R. cuneata, on the Gulf coast at least, is ready to spawn (contains mature gametes) most of the year; he also found that young Rangia, less than 8 mm long, were present in all populations in his Vermilion Bay study area, and yet no recruitment was observed (i.e., these very young clams usually did not grow up). Gooch suggested that some successful spawning might occur annually, but successful recruitment might have no specific pattern and occur only at intervals of several years, because in most years predators and/or parasites wipe out small annual crops; true recruitment occurs only when there is phenomenally prolific spawning involving the vast majority of adults, over­ whelming the destructive capacity of the predators, and thus giving rise to one of the dominant year classes so characteristic of the species. Cain (1972) found that in James River, Virginia, during summer months 50 per cent of the Rangia cuneata in the 14-20 mm length class and 70 per cent of those in the 21-30 mm length group had gonads containing recognizable sex products, as did all clams longer than 30 mm. He estimated that in James River sexual maturity was usually attained at the age of one year. Gametogenesis began in early April and continued through the summer. Ripe gametes were observed from May to late November. Slight spawning peaks were noted during the summer, but the major peak occurred in autumn. The gametogenic cycle was basically the same in high 19 and low salinities. Temperature was more important; gametogenesis occurred at all stations when water temperature rose in spring to 15° C. Gametogenesis occurred at a slower rate in clams in fresh water, and more clams were found in the "spent" phase than in the higher salinities. Cain (1972) found that ability to spawn as shown by possession of abundant ripe eggs or sperm cells did not mean that a Rangia would spawn. He was unsuccessful in attempts to stimulate spawn­ ing in the laboratory by a variety of methods that had been found to work for other bivalve molluscs. His first real success was with clams from salinities lower than 1 ppt that were brought to the laboratory and placed in water of 5 ppt salinity, then fed algae, and finally stimulated by adding ripe sperm to the water near the siphons; this usually caused spawning in 1-2 hours. Other changes were later found to stimulate spawning. Cain concluded (p. 94) that: "Evidently a change in salinity either up from 0°/oo (= 0 ppt) or down from 10°/oo or 15°/oo is necessary for spawning. The laboratory study indicated that a rise from near 0°/oo to 5°/oo was the best stimulus for spawning, especially after the clams had remained overnight in 5°/oo. This is also shown in the field where clams at station C spawned in fall 1970 with a 5°/oo rise in salinity and failed to spawn when the salinity remained low during 1971." He also cited a tremendous spawning and recruitment of R. cuneata that occurred in Back Bay and Currituck Sound when a storm raised the salinity from the normal less than 1 ppt to about 4.5 ppt. Cain (1972) fertilized many eggs and reared larvae through to the setting stage under a variety of temperature and salinity conditions. He found 32° C to be optimum for growth in a salinity of 14 ppt, but 32° C was not good for survival of embryos and early larvae, which lived best in 8 ppt salinity and a temperature of 24° C. Larvae were reared to setting size, 0.150 — 0.180 mm, in 7 days. There was some survival at all of the 16 temperature-salinity combinations tested (from 8 to 32 C and 2 to 20 ppt); at 8 and 16° C and 2, 8 and 14 ppt some lots had

20 90 to 100 per cent survival for 7 days. Effects of temperature and salinity shocks on growth and on survival were also tested. Cain’s experiments were too extensive and too complex to summarize, but we can say that this was the best study yet made of the repro­ ductive cycle and larval tolerances of Raiigia. Cain also partially solved the puzzle of populations made up of one year—class by finding that such a population in the upper James River estuary resulted from an intrusion of salt water into this area in 1965, which allowed larvae to be carried up river and set nearly 60 miles from the mouth, in an area where the salinity had not since been high enough for reproduction, so that the 1970-72 population consisted entirely of clams 5-7 years old and 53-63 mm long.

Growth

Fairbanks (1963), on the basis of the distribution of length modes in clams of two Lake Pontchartrain populations, estimated that North Shore clams attained an average length of 15 mm by the end of the first year of growth, 20 mm by the end of the second year, and 24 mm by the end of the third year, while South Shore clams grew to 20 mm in the first, to 29 mm in the second, and to 34 mm in the third year of growth. He did not try to determine growth rates beyond the third year. Differences in growth of the two populations were attributed to a combination of several differences in local conditions. Salinity was somewhat lower and more variable at the North Shore station (1.1 - 5.9 compared with 1.5 - 5.8 ppt) but Fairbanks tended to put more weight on the slightly higher organic content of the brownish North Shore water. Williams (1972) used the same method to estimate the growth rate of Rangia cuneata on the west side of Trinity Bay near our McCollum Park collecting area. By his estimates, Trinity Bay clams are 19.5 mm long when 1 year old, 31 mm at the end of their second year, 41 mm at the end of the third year, 48.5 mm when 4 years old, and 51.5 mm at 5 years of age. These estimates were based on rather small numbers of individuals. Salinity here is 4 - 16 ppt in most years.

21 Gooch (1971) planted Vermilion Bay clams (R. cuneata) of local populations in marked one-square-meter plots on natural bottoms and measured the growth of clams of various sizes in a period of one year. At South Mud Point he got virtually no growth, at North Mud Point an increase in length of 3.0 to 6.1 mm, and in Fearman Lake increases of from 1.5 (for very large clams) to 9.7 mm (for 34.5 mm clams). Study of size distribution in large samples obtained by dredging indicated that the North and South Mud Point populations had only one modal peak and probably con­ sisted of a single year class. At South Mud Point the modal class was 31 - 32 mm in January and remained at nearly the same point during the next 12 months; by December the mode had moved only to 33 - 34 mm. Each monthly sample contained 485 to 1105 clams, so the data should be reliable. The poor growth at this station was blamed by Gooch on the substrate of 4—6 inches of soft mud over hard clay and the exposure to much wind and wave action. The large samples taken by dredge at North Mud Point had a single mode at 44—45 mm in 1968 and this had increased only to 44 - 47 mm by 1969. The bottom at North Mud Point consisted of hard clay with numerous large and small depressions filled with an almost liquid sediment of high organic content. Gooch considered this type of bottom, and the exposure of the location to wind and wave action, to be adverse to rapid growth. Dredged samples taken in Fearman Lake, on the other hand, had several modal peaks, presumably representing several year classes: 40 - 41 mm, 46 — 47mm, a very large peak at 60 — 61 mm which might have masked smaller peaks at 52 - 53 and 56-57, high peaks at 64 - 65 and 70 - 71, and smaller peaks at 74 - 75 and 78 - 79 mm. A population from Delta Bend in the Mississippi River delta also showed peaks at 70 - 71, 74 - 75, and 78 — 79 mm. Gooch con­ sidered Fearman Lake to be an excellent growth area because it was protected from wind and wave action and had a firm mud bottom with a relatively high organic content. Putting together all samples in his study area (Vermilion Bay, Louisiana, plus

22 Delta Bend on the Mississippi River) Gooch found modal peaks which might represent year classes at 34 - 35, 44 47, 60 - 61, 70 - 71, 74 75, and 78 - 79 mm, or 6 in all. Of course this did not include clams of the first year class, and perhaps not the second, as Gooch found few clams smaller than 30 mm. Gooch considered his year class measurements to agree well with Wolfe and Pettewayfs growth curve. Salinity in western Vermilion Bay usually ranged around 2.5 - 5 ppt. Wolfe and Petteway (1968) studied growth in one population of Rangia cuneata in Trent River, a branch of Neuse River, North Carolina. This population was sampled at the same station 12 times between November 9, 1965 and July 6, 1967. Length fre­ quency analysis of 6,287 clams revealed 5 distinct size groups that were recognizable in 3 or more consecutive samples. From the progression of modes, von Bertalanffy curves were constructed and growth equations were derived. This is the most elaborate and mathematically sophisticated of the growth studies on Rangia cuneata and has received good acceptance. According to the composite growth curve, R. cuneata reaches a maximum length (for Trent River) of 70 - 75 mm in approximately 10 - 11 years. Trent River has quite low salinity, 0.01 - 6.0 ppt during the study period, and Rangia reached the end of its range 2 miles farther upstream. Even at the sampling station a few fresh­ water clams, Anodonta imbecilis, were found living among the Rangia. Cain (1972) accepted the growth data of Fairbanks (1963) and Wolfe and Petteway (1968) and obtained no data of his own on growth of adult Rangia cuneata in James River, Virginia. However, he found that 50 per cent of the clams in the 14 - 20 mm length group had gonads containing "recognizable sex products," i.e., egg or sperm cells, and pointed out that according to Fairbanks and to Wolfe and Petteway, these sexually mature individuals would be only one year old. Cain (1972) did extensive experimentation on survival and growth of embryos and larvae of James River Rangia cuneata, finding that the best growth occurred at 32° C and 14 ppt and the best survival at 24° C and 8 ppt; the

23 best combination for growth and survival was 24° C and 14 ppt salinity. At lower temperatures (16° C) and salinity of 8 ppt there was good survival but little growth. The best setting (settling of larvae to bottom to become young clams 0.2 mm long) occurred in fall and winter. Tenore, Horton and Duke (1968) used shell growth (increase in length) as one criterion of substrate effects on Ráiig i a cuné at a. Clams gathered locally were measured, placed in boxes bottomed with different types of sediment, allowed to grow in Pamlico River estuary from June to November, and then measured again. Although they found statistically different growth rates showing that sand was a more favorable substrate than clay- sand and that organic matter or phosphate increased growth in sand but hindered growth in clay-sand, the increases in shell length were quite small; 0.33 mm in clay-silt compared with 0.53 mm in sand; 0.37 in clay-sand with 0.1% organic matter compared with 0.28 in clay-sand with 1.0% organic, and 0.44 in sand with 0.1% organic compared with 0.62 in sand with 1.0% organic matter. The sizes of clams were not given. Pfitzenmeyer (1970) observed the first known appearance of Rangia cuneata in upper Chesapeake Bay, off the mouths of Sassafras River and Elk River, Maryland, during a study of effects of channel dredging in which many bottom samples were routinely examined each month. Salinity in this area ranged from 0 to 8 ppt during the study period. Rangia was first seen in autumn 1966 and continued to increase in numbers and biomass until late December 1968 or January 1969, when nearly all died, apparently killed by cold. This kill was also observed in Elk River nearby, and reported by Gallagher and Wells (1969). Rangia reached its maximum biomasss in December 1968, just before the die-off. No data on measurements were reported by Pfitzenmeyer, but he stated that three distinct year-classes could be distinguished and that the largest individual was 40 mm long, which would be 3 to 4 years old by Wolfe and PettewayTs (1968) curve. However, Gallagher and Wells (1969) measured 244 Elk River shells and

2 k found a high peak at 25 mm and a lower one at 28 mm; the existence of a 19-26 mm size class was indicated. Other possible peaks were shown, but the sample was too small to permit any certainty in identifying size classes. The largest individual was 48 mm long and the smallest 11 mm. From the data it hardly seems possible that there were less than 4 size classes (year- classes) in this population. Elk River winter and spring salinities are commonly below 1.0 ppt (0.3 ppt in January 1969).

Food and Feeding One of the greatest gaps in our knowledge of Rangia curieata is the lack of any quantitative data, and any but the most meager qualitative data, on the food it ingests, digests, and utilizes. Lacking even this basic information, of course there is no knowledge of how nutrition is affected by changes in the environment. Shellfish biologists remember, though, that a similar gap existed in knowledge of the oyster, often touted as our most studied marine animal, until quite recently; even now, our knowledge of oyster nutrition is far from satisfactory. Detritus, particulate matter resulting from breakdown of dead plants, has been often mentioned as a major source of nutrition for Rangia. The earlier references to detritus were based on Darnell’s papers (1958, 1961, 1964) on the food of animals in Lake Pontchartrain. Darnell (1958) examined the stomach contents of 3 specimens of R. cuneata and found them to consist of 70 per cent unidentifiable detritus, 10 per cent sand, 17 per cent "small round bodies," and traces of eggs, bottom diatoms, foraminif- erans, and vascular plant debris. On the basis of these scanty observations he classified Rangia as a typical filter feeder which fed mainly on detritus, pointing out that these clams are most abundant on the muddiest bottoms and in waters of maximum turbidity. Darnell (1961) said the "small round bodies," prob­ ably derived from disintegrated colonies of the blue-green algae Anabaena or Microcystis, made up a significant though minor part (one-sixth) of the food ingested. The typical detritus feeders among bivalve molluscs, such

25 as Yoldia, feel around on the surface of the bottom with palp probosces, collecting light particles which are carried by cilia in a groove on the proboscis to a sorting apparatus; there large particles are rejected and small ones directed into the mouth. The Mactracea to which Rángia belongs have no such structures, but instead have the gills adapted for capturing food, so they are filter feeders like most bivalves. In such a mollusc, currents created by the many large cilia on mantle and gill surfaces draw suspended particles, including small planktonic organisms, into the mantle cavity; those particles that touch the gills are trapped in mucus and carried by ciliary action toward the mouth. Along the way they come in contact with palps which may divert them away from or direct them toward the mouth. Oysters of the genus Crássostrea, which includes our Gulf and Atlantic coast commercial oyster, are adapted to live in estuaries, in turbid water and often on muddy bottoms; they have an especially efficient system for rejecting not only large particles but small mud particles that have no food value, and directing nutri­ tive particles to the mouth, so they do not starve with stomachs full of mud as do the oysters of the genus Ostrea. Biologists examining stomach contents of oysters found diatoms to be most prominent among the identifiable contents, and for many years diatoms were considered to be the oyster’s most important food. Diatoms have an indigestible covering and remain identifiable all the way through the digestive tract. Many of them also remain alive all the way through, and they have been cultured from fresh oyster feces. Other food particles and organisms such as naked flagellates may be digested so quickly that they never make up a large part of the identifiable material in the stomach. So better methods than microscopic examination of stomach contents must be employed to find out what a filter-feeding mollusc actually digests and assimilates. One of the best methods is to tag test materials with radioactive isotopes and then test the mollusc’s tissues for radioactivity.

26 Tenore, Horton and Duke (1968) used radioactive tags to show that Rangia cuneata actually assimilates food from detritus. However, their fldetritusn was a heat-killed culture of Chlamydomonas, a flagellated unicellular alga, which had been grown in water containing zinc-65, so their findings do not necessarily apply to detritus of non-algal origin. They also showed that Rangia assimilates phosphates containing phosphorus-32 from "sediment,11 nature not specified, when the tagged sediment particles were mixed with sand in the bottoms of experimental trays. Clams burrowing in bottom sediments accumulated radioactivity in adductor muscle tissue and clams suspended above the bottom did not, in both the zinc-65 and the phosphorus— 32 experiments. In contrast to the doubtful situation with adult clams, there is no doubt that the swimming larvae of Rangia live and grow well when fed only flagellated unicellular algae. Chanley (1965) reared R.. cuneata larvae to setting stage in 7 days by feeding them "a mixture of unicellular algae," which by reference to earlier papers we can guess to be the naked chrysomonads Isochrysis and Monochrysis. Cain (1972) added no food during the 24-hour period during which Rangia larvae developed from the fertilized egg to the straight-hinged veliger stage, but fed them a mixture of Isochrysis galbani and Monochrysis lutheri during the remainder of the 7-day period required to rear the larvae to the setting stage. Cain terminated his experiments when the larvae set (settled to the bottom as young clams), leaving us without information on food requirements of the minute juveniles.

Fauna of the Rangia Zone

The low-salinity brackish water zone occupied by Raiigia cuneata slightly overlaps the range of the typical freshwater clams, the Unionidae, on its inland border, and slightly over­ laps the inland edge of the oyster zone on its seaward border. Therefore it is associated with freshwater and highly euryhaline estuarine animals on one side, and on the other with the most euryhaline members of the oyster community and with estuarine fishes and crustaceans.

27 In the farthest inland, fresh (< 0.5 ppt) to brackish (= 5 ppt) part of the Rangia zone, R. cuneata is usually the most abundant species or makes up the largest part of the biomass in an assemblage that includes the following list (those marked with asterisk are most important, A means "in Atlantic coast estuaries" and G means "in Gulf coast estuaries"): Porifera (sponges): Trochospongilla horrida (G). Coelenterata (jelly animals): Cordylophora lacustris (A,G). Platyhelminthes (flatworms): unidentified Rhabdocoelida and larval trematodes. Rotifera (wheelworms): Brachionus bidentata (G), JB. calyciflorus (G), Rotaria neptunia (G), Anuraeopsis fissa (G). Bryozoa, Entoprocta: Urnatella gracilis (G). Bryozoa, Ectoprocta (moss animals): Plumatella repens (G), Paludicella sp. (G), Fredericella sultana (G). Annelida, Oligochaeta: Pristina longiseta (G), Stylaria sp. (G), Enchytraeidae (A, G), Tubificidae (A, G), Lumbriculidae (G). Annelida, Polychaeta: Laeonereis culveri (A, G), Polydora sp. (A, G) . Annelida, Hirudinea (leeches): Helobdella sp. (G), Piscicola sp. (G). Mollusca, Pelecypoda (bivalves): Congeria leucophaeta (A, G), *Rangia cuneata (A, G) ,*Polymesoda caroliniensis (A, G), Unionidae, e.g. Anodonta (A, G). Arthropoda, Crustacea: *Crayfishes Procambarus clarkii (G) and Procambarus blandingii acutus (G) (also unidentified species, (A, G). Shrimps *Palaemonetes kadiakensis (G), Palaemonetes paludosus (A, G), Palaemonetes pugio (A, G), *Macrobrachium ohione (G). Crabs *Callinectes sapidus (A, G), *Rhithropanopeus harrisii (A, G). Barnacles Balanus improvisus, B . amphitrite. Amphipods Corophium lacustre (A, G), Gammarus spp. (A, G). Arthropoda, Insecta: Ephemerida (may fly nymphs), several genera especially Hexagenia; Odonata (dragonfly and damsel fly nymphs); Diptera, Culidae (mosquitoes), Chironomidae (midges;

28 aquatic larvae called blood worms). Coleoptera, Gyrinidae (whirligig beetles) and aquatic larvae. Chordata, Vertebrata, Osteichthyes (bony fishes): *Menidia beryllina (A, G), *Dorosoma petenense (G), *Cyprinodon variegatus (A, G), *Gambusia affinis (A, G); *Notropis, several species, e.g., N. venustus (G) , N. lutrensis (G), N. maculatus (G), N. chalybaeus (A); *Hybognathus nuchalis (A, G), *Notemigonus crysoleucus (A), *Poecilia latipinna (G), *Dorosoma cepedianum (G), *Centrarchus macropterus (A, G), *Pomoxis nigromaculatus (A, G), *Pomoxis annularis (G), *Micropterus salmoides (A, G), *Lepomis macrochirus (A, G), Lepomis gibbosus (A), Enneacanthus gloriosus (A), Enneacanthus obesus (A), *Aphredoderus sayanus (A, G), Etheostoma barratti (A), Lucania parva (A, G), Perea flavescens (A), Esox americanus (A), Morone americana (A), Ictalurus furcatus (G), Ictalurus punctatus (A, G). The above lists were compiled from the following sources: Baldauf (1952, 1953) on Neches River, Texas; Wurtz and Roback (1955), Neches and Sabine River, Texas, and Escambia River, Florida (invertebrates only); Gunter and Shell (1958), Grand Lake and White Lake, Louisiana; Gunter and Hall (1965), Caloosahatchie Estuary, Florida; Keup and Bayless (1964), Neuse River, North Carolina. They are not intended to be complete, but to show that this region has a fauna that is predominantly fresh water in origin and affinities, but is invaded by a few species of marine origin, some of which (the bivalve molluscs Rangia cuneata and Congeria leucophaeta and the crab Rhithropanopeus harrisii) may reach their maximum abundance here in the tidal rivers and brackish lakes. Fishes that are voracious predators of molluscs and crustaceans, such as the freshwater catfishes, Ictalurus spp., the freshwater drum, Aplodinotus grunniens, the saltwater drums Sciaenops ocellata, Pogonias cromis, Micropogon undulatus.and Leiostomus xanthurus, and the gars, Lepisosteus spp., do enter this zone but do not become as abundant here as in other zones of more suitable salinity, allowing the prey species that can live here to build up dense populations.

29 The lower part of the Rangla zone, farther seaward, is the zone designated by various authors as "bay heads" or "river- influenced estuaries". The salinity usually averages between 5 and 10 ppt but may become fresh (<0.5 ppt) during periods of high river flow and rise above 15 ppt in periods of low freshwater inflow. The benthos, predominantly invertebrate animals living on the bottom, is most characteristic of this zone. The list of fishes is more confusing than enlightening unless one knows that species of freshwater origin are found here during periods of high river flow and low salinity, while marine species predominate during periods when salinity is near or above the 5-10 ppt "normal" range. A list of characteristic animals of this zone follows: Porifera: Cliona truitti, boring sponge, found only on seaward edge of zone (A, G). Coelenterata: *Hydroids Bimeria franciscana (G), Obelia spp. (G), Cordylophora lacuStris (A, G); anemones Fagesia lineata (A), Diadumene leucolena (A) or Aiptasia pallida (G). Ctenophora: *Mnemiopsis leidyi (A), *M. mccradyi (G) . Platyhelminthes: Turbellaria Hydrolimax grisea (A) and Stylochus elllpticus (A, G); many larval and adult trematodes in invertebrate and vertebrate hosts; some cestodes ditto. Nemertea: Micrura leidyi (A), Tubulanus pellucidus (A). Rotifera: Brachionus plicatilis (G), Filinia longiseta (G), Synchaeta bicornis (G), Synchaeta littoralis (A, G), Keratella cochlearis (G). Nematoda: Many, for lists see Chitwood (1951) and Timm (1952). Bryozoa, Ectoprocta: *Acanthodesia tenuis (A) and *Membranipora crustulenta (A, G) especially on oysters; Membranipora membranacea (A) on submerged seed plants; Electra crustulenta (A), Nolella sp. (G), Victorella pavida (A, G), Bowerbankia gracilis (A, G). Annelida, polychaeta: Polydora websteri (A, G), Polydora ligni (A, G), Loandalia fauveli (G), Loandalia americana (G), *Laeonereis culveri (A, G), *Streblospio benedicti (A, G), *Paraprlonospio pinnata (A, G), Capitella capitata (A, G),

30 Heteromastus filiformis (A, G), Scolecolepides viridis (A, G), Pectinaria gouldi (A, G), Glycinde solitaria (A, G), *Neanthes succinea (A, G) , Hypaniola grayi (A), Mediomastus californiensis (G), Sigambra spp. (G), Autolytus sp• (G), Eteone heteropoda (G), Glyptis vittata (G), Piopatra cuprea (G), Amphicteis gunner! floridus

(6). Annelida, Oligochaeta: Tubifex tubifex (A), Peloscolex gabriellae (G) , Enchytraeus albidus (A, G). Annelida, Hirudinea: Myzobdella lugubris (A, G) on blue crabs, oysters, etc. Mollusca, Pelecypoda: *Rangia cuneata (A, G), Rangia (Rangianella) flexuosa (G), *Macoma mitchelli (A, G), *Macoma balthica (A), ^Macoma phenax (A), *Mulinia lateralis (G), Mulinia pontchartrainensis (G), ^Congeria leucophaeta (A, G), *Mya arenaria (A), Gemma gemma (seaward edge of zone on sand, A), Tagelus plebeius (seaward edge of zone only, G), *Brachidontes recurvus (usually attached to oysters, A, G), *Polymesoda carolinensis (mostly in edge of marsh, intertidal, A, G), Modiolus demissus (seaward edge of zone, in marsh, intertidal), *Crassostrea virginica (seaward edge of zone), Amygdalum aborescens (G). Mollusca, Gastropoda: *Littoridina sphinctostoma (very abundant, thousands per square meter in places, G), *Vioscalba louisianae (thousands per square meter in some Louisiana waters, G), *Neritina reclivata (common on low-salinity oysters, feeding on algae, G), Doridella obscura (-Corambella baratariae, nudibranch on low-salinity oysters, G), Odostomia barretti (G), ^Retusa canaliculata (only snail in Rangia zone, Neuse R., A), Nassarius acutus (seaward edge of zone, G), *Littorina irrorata (on Spartina alterniflora in marsh, lower edge of zone), Epitonium rupicolum (seaward edge of zone, A), Hydrobia sp. (A), Sayella chesapeakeia (A), Acteon punctostriatus (A), Odostomia impressa (A). In addition, in Louisiana the two mollusc-drilling snails Thais haemastoma and Polinices duplicata occasionally invade the seaward edge of the Rangia zone during high-salinity periods.

31 Arthropoda, Crustacea: Copepods *Acartia tonsa, *Eurytemora affinis, Oithona brevicornis and Paracalanus crassirostris were found to be most abundant in Lake Pontchartrain zooplankton by Suttkus, Darnell and Darnell (1954), and many harpacticoids occur in bottom sediments and among oysters. Barnacles *Balaiius improvisus, *B. amphitrite and *B. ebumeus are abundant in this zone, both A and G. Amphipods include *Corophium lacustre (A, G), *Corophium louisiananum (G), Ampelisca abdita (G), *Gammarus spp. (A, G), *Carinogammarus mucronatus (A, G), *Leptocheirus plumulosus (A), Erichthonius brasiliensis (G), Melita nitida (A, G), Haustorius arenarius (A), Cyamadusa compta (A), Caprella acutifrons (A), Grubia compta (A), and on shores Orchestia agilis. Isopods include Edotea triloba (A, G), Aegathoa oculata (G), Nerocila munda (G), Cassidisca lunifrons (A, G), *Cyathura polita (A), Chiridotea almyra (G), and Erichsonella attenuata. The Mysid shrimps Mysis stenolepis (G), *Neomysis americana (A), Mysidopsis almyra (G), and Mysidopsis bahia have been reported from at least the margins of this zone. Decapods include the shrimps *Penaeus setiferus, *Penaeus duorarum and *Penaeus aztecus in Gulf coast and southern Atlantic estuaries, *Palaemonetes pugio (A, G) and Crangon septemspinosus (A), the crabs *Callinectes sapidus (A, G), *Rhithropanopeus harrisii, (A, G), *Eurypanopeus depressus (A, G), *Neopanope texana texana (G), Ogyrides limicola (G), Pinnotheres ostreum (in oysters, A), and on shore several fiddler crabs, Uca spp. (A, G) . Chordata, Urochordata: The estuarine sea squirt Molgula manhattensis may appear in the seaward edge of the Rangia zone in periods of raised salinity, A and G. Chordata Vertebrata, Chondrichthes: The stingray Dasyatis sabina (not abundant). Chordata, Vertebrata, Osteichthes: *Menidia beryllina (A, G), *Mugil cephalus (A, G), *Anchoa mitchilli (A, G), *Leiostomus xanthurus (A, G), *Micropogon undulatus (A, G), *Brevoortia patronus and *B^. gunteri (G), *Brevoortia tyrannus (A),

32 Alosa pseudoharengus (A), *Galeichthys fells (A, G), *Lagodon rhomboides (A, G), *Bairdiella chrysura (A, G), *Cynoscion arenarius (G), *iC. nebulosus (A, G), *Cynosclon regalis (A), *Paralichthys lethostigma (A, G), *Gambusia affinis (A, G), *Cyprinodon variegatus (A, G), Lucania parva (A, G), Eucinostomus gula (G), Eucinostomus argenteus (G), *Sciaenops ocellata (G), *Fundulus grandis (G), *Fundulus heteroclitus (A), Fundulus majalis (A), Morone americana (A), Morone saxatilis (A), Perea flavescens (A), Chasmodes bosquianus (A, G), Gobiesox strumosus (G), Gobiosoma bosci (A, G), Opsanus beta (G), Opsanus tau (A), *Pogonias cromis (A, G), *Anguilla rostrata (A), Gobionellus hastatus (G), Hypsoblennius hentzi (A, G), Lepisosteus osseus (A, G), Lepisosteus spatula (G), Lepisosteus productus (G), Membras martinica (G). Chordata, Vertebrata, Reptilia: Malaclemys terrapin, diamond-back terrapin, (A, G). Chordata, Vertebrata, Mammalia: Tursiops truncatus, bottlenose dolphin (A, G). The above list, incomplete but including most of the common and most characteristic species, was compiled from Suttkus, Darnell and Darnell (1954) for Lake Pontchartrain; Gunter and Shell (1958), Gooch (1971) and Hoese (1972) for southwestern Louisiana; Gunter and Hall (1965) for Caloosahatchie Estuary, Florida; Williams (1972 unpublished thesis), Mackin (1971 unpublished report), Reid (1955, 1956, 1957), and various un­ published reports of Texas Parks and Wildlife Department biolo­ gists of which Shidler's (1960) are especially noteworthy, on the low-salinity parts of the Galveston Bay system; unpublished reports of Baldauf (1952, 1953) and Stevens (1960, 1962, 1963) on lower Neches River and Sabine Lake, Texas; Frey (1946) and Pfitzenmeyer and Drobeck (1963, 1964) on Potomac River; Pfitzenmeyer (1970) and Ritchie (1970) on upper Chesapeake Bay; Cain (1972 unpublished dissertation) on James River, Virginia; Keup and Bayless (1964), Porter (1969), and Wolfe and Petteway (1968) on the Trent- Neuse River system of North Carolina; a number of unpublished 33 Project 9 reports (1947 - 1952) of Texas A&M Research Foundation on the low-salinity oyster bed fauna of Louisiana; and Hopkins's firsthand knowledge.

Predation

Predation must be considered from two points of view. People interested in Rangia cuneata as a harvestable crop, either wild or cultivated, look on predators as enemies and on predation as one of the hazards to production. Other people are interested in the benthos as a source of feed for game and food fishes, crustaceans (crabs, shrimps), or wildfowl such as ducks, geese, or even whooping cranes; the predators are the species they want to increase, So the more predation the better, as long as the prey can maintain enough population to continue feeding the desired predator. Most of the information relative to the first point of view comes from the Lake Pontchartrain studies of Fairbanks (1963), Suttkus, Darnell and Darnell (1954), and especially the reports of Darnell (1958, 1961). Fairbanks (1963) showed that there was a rapid decrease in numbers of Rangia as they grew older: the number per square meter decreased from 1806 "juveniles" (below 23 mm long) to 31 "adults" (above 23 mm) at North shore, while at the South Shore station the decrease was from 1881 juveniles to 2.6 adults. Fairbanks suspected that at least part of this depletion in the Rangia population was caused by predation, but he did not study any predators or conduct any experiments on predation. Darnell (1958) reported finding the shells of young Rangia in the stomachs or intestines of stingrays (Dasyatis sabina), three species of gars (Lepisosteus), blue catfish (Ictalurus furcatus), freshwater drum (Aplodinotus grunniens), spot (Leiostomus xanthurus), croaker (Micropogon undulatus), black drum (Pogonias cromis), and less frequently in pinfish (Lagodon rhomboides), flounder (Parallchthys lethostigma), gizzard shad (Dorosoma cepedianum), and even in anchovies (Anchoa mitchilli). Shells of small clams were also found in the guts of river shrimp

3 ^ (Macrobrachium ohione) , white shrimp (Peiiaeus setiferus) and blue crab (Callinectes sapidus). Darnell did not record the freshwater mud crab, Eh ithrop anopeus harrisii , as a predator of Rangia but it probably is as it is known to eat other small clams. Only two species were found to ingest large "Rangia clams," Aplodiiiotus grunriiens and Pogonias cromis, both of which are noted as voracious eaters of molluscs of many kinds. The blue crab, Callinectes sapidus, is a notorious enemy of quahog clams, Mercenaria merceriaria: it can clean out a planted bed of small clams in a few days, and even opens and devours half-grown quahogs larger than the average adult Rangia, so it is probably a more important predator of R. cuneata than has yet been realized. Gunter and Shell (1958) reported that during periods of low salinity (high freshwater runoff) freshwater catfish, Ictalurus furcatus, entered brackish lakes of southwestern Louisiana and lived on Rangia cuneata and the mussels, Congeria leucophaeta, attached to the clams. 0*Heeron (1966) planted R. cuneata in lower Galveston Bay and showed that in high-salinity waters they were attacked and killed by the drilling snail or oyster conch Theis haemastdma. His theory was that R. cuneata was limited to the lower salinities because it was only there that the clams could live to maturity without being eaten by Thais and other high-salinity predators. It is true that an occasional pre­ dacious snail, Thais haemastoma or Polinices duplicata, does get into the thinly populated seaward edge of the Rangia zone; but in most places, most of the time, the ranges of Rangia and of these snails are separated by several miles. Furthermore, many other molluscs including dense populations of oysters and such clams as Macoma, Mulinia, Gemma, Mercenaria, and in Chesapeake Bay My a, live within the normal salinity range of Thais, Polinices and Urosalpinx, so it seems that some factor in addition to predation must be limiting Rangia populations. From the other point of view, the usefulness of Rangia as food for desired species of predators, Cain (1972) quotes a 1965 report of the U. S. Bureau of Sport Fisheries and Wildlife on food consumed by wild ducks in Back Bay and Currituck Sound

35 (Virginia-North Carolina border) in 1962. In various species of ducks, Rangia ciiiieata shells made up 8.2 to 24.4 per cent of the volume of the gizzard contents. The wildlife biologists estimated that 83,000 pounds (dry weight) of Rangia meat was consumed by the ducks of this area during the one-year period. According to Cain, Back Bay and Currituck Sound normally have salinity of less than 1 ppt, but a storm had put ocean water into the bay and raised the salinity to about 4.5 ppt which caused successful spawning and setting of R. cuneata, hence the large quantities of small Rangia available to the duck population. The estimated 83,000 pounds dry weight would be equivalent to 415,000 pounds wet weight, which is more than the total commercial production (325,000 pounds) of North Carolina during the 6-year period 1966-1971. Evidently, as of now, Rangia cuneata as a natural resource is more important as food for many game fishes, food fishes, shrimps, crabs, and water fowl than it is as food for man. However, potentially man may be the greatest predator of all. Even now, many thousands of living Rangia are being taken and killed unintentionally or incidentally by the shell dredges in Lake Pontchartrain.

Parasites

Fairbanks (1963) found trematode sporocysts and cercariae parasitizing the gonads of 4.45 per cent of the adult Rangia at his North Shore station and 1.91 per cent of the South Shore adults in Lake Pontchartrain; the larger size classes of clams had the highest incidences of infection. The parasitologist Sogendares thought these trematode larvae belonged to the family Fellodistomatidae but could not identify them further. Many of our R. cuneata were examined for parasites. Texas A&M student Jack M. Neagle in a student project report for a course in helminthology, submitted May 1972, described two kinds of larval trematodes found in these clams; one has unbranched sporocysts which give birth to nonoculate trichocercous

36 cercariae with V-shaped excretory bladder and excretory duct opening on end of tail; the other has branched sporocysts from which cercariae of the bucephaloid type emerge. The first type of larva would be expected by a parasitologist (Hopkins) to encyst in an invertebrate host such as a mollusc, and when this second intermediate host is eaten by a fish the larva should develop into an adult in the intestine. This may be the same as Fairbanks’s larvae which Sogendares tentatively assigned to Fellodistomatidae. The second type of larva must belong to the family Bucephalidae, all of which penetrate and develop further in small fishes; when these second intermediate hosts are eaten by predacious fishes the infective larvae develop into adults in the intestine. Different incidences of infection were found in the three collecting sites: 0 of 125 Rangia from the Neches River site 5 miles upriver from the Port of Beaumont, 4 of 125 from McCollum Park, west shore of Trinity Bay, and 70 of 125 from Lake Anahuac on the east side of the Trinity River delta. All of the McCollum Park infections were found in one lot of 25 which led Neagle to suspect that this collection had been mixed with some clams from the lake. All 4 infected clams from McCollum Park and 51 of the 70 infected clams from Lake Anahuac contained trichocercous cercariae; 19 clams from Lake Anahuac produced bucephaloid cercariae. For purposes of this investigation, the parasites found are of interest for 3 aspects: (1) the high incidence of parasitism in the isolated Lake Anahuac population, (2) the fact that parasitized clams produced few or no gametes because of "parasitic castration," and (3) the warning that the possibility of parasitism must be considered in physiological and biochemical studies, as parasitized clams might well react differently from nonparasitized ones.

Bottom Type Inhabited

Rangia, being a burrowing clam but not a very active burrower, is never found on hard-packed sand, rock or hard clay bottoms, though it is occasionally found in soft pockets or silt-filled

37 depressions in hard bottoms. Scanning 25 reports in which the type of sediment inhabited by Rangia cuneata is mentioned, we find "mud" listed 12 times, "sand" 3 times, "sandy mud" 4 times, "clay-silt" 5 times, and "silt, clay, fine sand" twice. Substrates mentioned once include "clay with some sand" and "black, soft clay." Parker (1966) is the only author who made any attempt to analyze quantitatively the substrates occupied by Rangia cuneata. He concluded that wherever this clam was abundant in the Galveston Bay system, the percentage of sand was between 12 and 85, clay was between 14 and 65 per cent, and silt was less than 30 per cent. There is much disagreement about the effects of high organic content in Rangia-bearing substrates. Working in the same general area, southwestern Louisiana, Gooch (1971) concluded that high concentrations of coarse organic matter produced the most success­ ful populations, while Hoese (1972) stated that Rangia was scarce in very highly organic sediments (over 10 per cent carbon). However, Gooch was judging success not by number of clams per square meter, but by number of modal peaks which indicate successful sets of young clams, and by growth rate. Tenore, Horton and Duke (1968) found in field experiments that organic matter was beneficial to Rangia cuneata in sand substrates, but harmful to clams in clay-silt substrates; they also found that clay-silt was less favorable than sand to survival and growth of adult clams. They did not test sediments for suitability as a substrate for setting. Several authors have suggested that sediments best for the setting of young clams are not the same as those most favorable for adults. Tenore (1970) found adult Rangia in Pamlico Sound in sand substrates only, and stated that young clams set on mud substrates but usually did not survive because of the high organic content, which he considered adverse.

Temperature Relations

The range now occupied by Rangia cuneata extends from 39° 30TN (Northeast River, Elk River, and upper end of Chesapeake Bay, Maryland) to somewhere in the vicinity of 20° N (Vera Cruz or Campeche, Mexico, according to "personal communications" cited 38 by various authors). In this latitudinal range of approximately 20 degrees, or even in the 12 degrees between northern Maryland and southern Texas, one would probably expect great differences in water temperatures. The differences may be less than expected, as the following data show. Biggs (1970) reported 1966 water températures in the vicinity of the most northern Rangia beds in the upper Chesapeake ranging from 1 to 28.3° C. Rangia evidently survived well in 1966, but there was a winter kill in 1968-69 and later winters when surface water temperatures were 0° C or below (Gallagher and Wells, 1969; Pfitzenmeyer, 1970). Frey (1946) assembled records on Potomac River for several past years. The lowest temperature recorded anywhere in the estuary during the years of records was 0.05° C in the mouth of the river at a depth of 37 feet. In the part of the estuary where Rangia now lives the lowest record was 4° C. The highest water temperatures ever recorded in the Potomac, according to Frey, were 31.3° C on surface and 28.9° C on bottom, July 21, 1943. Cain (1972) presented a graph of bottom temperatures near Hog Island in James River, Virginia, indicating a 1970-1971 range from about 0.05° C in February 1971 to approximately 29° C in August 1970 and 1971. He did not mention any mortality of adult clams occurring during the periods of extreme temperatures. Porter (1969) working in Neuse River and Wolfe and Petteway (1968) in Trent River recorded water temperature ranges of 6 - 29° C and 4.8 - 34.7° C, respectively, on North Carolina Rangia beds. Godwin (1967) while collecting clams along 50 miles of Georgia coastline from August 1966 to June 1967 recorded bottom temperatures from 11.5 to 30° C. Fairbanks (1963) and Suttkus, Darnell and Darnell (1954) found surface temperatures ranging from 8.5 to 33° C (February 1957 - July 1958) and from 9 to 34° C (1953 - 1954) in Lake Pontchartrain, Louisiana. Gooch (1971) cited a 1970 study by Dugas as showing "mean water surface temperatures" of 10.5 to 31.1° C in Vermilion Bay, Louisiana. Menzel and Hopkins (1952) recorded bottom temperatures 39 of 3.4 - 36° C in Bayou BasBleu, Terrebonne Parish, La., during the period September 1947 - February 1949. Mackin and Hopkins (1962) reported bottom temperatures of 7.7 - 35° C in Barataria Bay, July 1948 - August 1949, mentioned a February 1951 record of 2° C in Bayou Rigaud (Grand Isle harbor) when many spotted trout (Cynoscion nebulosus) and other fish died, and reported occurrence of 40° C temperatures in water ebbing off marshes and flats into small bayous. In Texas, Stevens (1960) recorded "average monthly water temperatures" of 9 to 29° C in Sabine Lake, 1958 - 1959. In the Galveston Bay system, water temperatures ranging from 2.9° C to 34° C were found by Mock (1965) in shallow waters of Clear Lake, and in open waters of the bay 6.2 to 35° C temperatures were reported by Pullen (1961), Shidler (1961), Stevens (1963), Hofstetter (1959) and Johnson (1966). In Neches River, 1951 - 1953, Baldauf (1952, 1953) recorded surface temperatures of 10.6 - 39.2° C and bottom temperatures of 11.9 - 32.5° C. Collier and Hedgpeth (1950) assembled several "runs" of records for various past periods in the Aransas Bay region, including ranges of 10.5 - 30.5° C (surface) and 10.1 - 27.3° C (bottom) in Copano Bay, 7.5 - 31.4° C in Aransas Bay, 4.4 - 30.5° C at Harbor Island near Port Aransas, 5.0 - 29.4° C at Corpus Christi, and 4 - 35° C in Upper Laguna Madre; except for the Copano records, these were all surface temperatures, and all covered at least a year and included all seasons. Childress (1966) assembled San Antonio Bay system records for all months in the period 1959 - 1964, during which water temperatures ranged from 6.5 to 32.2° C. Simmons (1957), working in Upper Laguna Madre during the period October 1951 - September 1955, found bottom temperatures ranging from 4 to 35° C. Summarizing, the ranges of water temperatures recorded in these reports run from 0.05 - 31.3° C in Maryland to 2 - 40° C in Louisiana and 4 - 35° C in southern Texas. Gunter (1945), Collier and Hedgpeth (1950) and Simmons (1957) have pointed out that on the Gulf coast a water temperature of 4° C, if continued through low tide periods, will cause massive kills of fishes in the shallow bays. However, there seem to be no reports of winter kills of Raiigia except in northern Maryland, and no reports from anywhere of mass mortality during high temperature periods. Incidentally, R. cuneata collected from Trinity Bay (over 30° C) in summer have been put on ice and kept at 0.2° C for over 24 hours without apparent harm, and afterward kept in laboratory aquaria for weeks at 22.5° C. Such temperature shocks apparently do not kill adult clams. Cain (1972) in preliminary tests found that "survival of embryos and larvae was low (less than 10 per cent) and erratic at 35° C." In his subsequent experiments he tested tolerance of embryos and early larvae only between 8 and 32° C, getting no survival at 8° C in any salinity, but some survival in salinity of 8 ppt at 32° C. Later stages of larvae had some survival to setting in all temperature-salinity combinations tested (8, 16, 24 and 32° C and 2, 8, 14 and 20 ppt). Besides possible lethal effects of extreme highs and lows, temperature is also important in controlling reproduction, as first reported (for R. cuneata) by Fairbanks (1963). Cain (1972) found gametogenesis beginning in spring when water temperatures in James River reached 10° C, and reaching completion (i.e., gametes being produced) when water temperature was 16° C. Spawning, or release of gametes, required a change in salinity and could not be caused by temperature alone, but it occurred when temperatures were between 12 and 22° C. Fall spawning began when water temperature had fallen to 22° C and continued until the water had cooled to 17° C. Larvae grew fastest at the highest temperature tested, 32° C, but survived best at lower temperatures (24° C). Setting was most prolific in fall and winter, but could occur at any season. The principal observed effect of lower temperatures (8 - 16° C) was to slow or stop growth of larvae, and the principal effect of higher temperatures (above 24° C) was to increase growth rate but decrease percentage survival.

hi Salinity Relations

The role of salinity as a major factor in the ecology has been discussed under several headings and will not be gone over again at this point. However, it may be pertinent to say here that the role of salinity in the natural environment is not a simple one that can be defined by a few laboratory studies, as shown by the fact that the effects of salinity on oysters are still being tested and discussed after many years of studies in both field and laboratory. For instance, Peddicord (personal communications, 1971 - 1972) and Cain (1972) in James River, Virginia, have found that the density of Rangia populations decreases as you go up river, as follows: At mile 25 (25 miles above mouth of river), salinity 4-13 ppt, clams average 130 per square meter. At mile 31, 2 salinity 1.2 - 11.2 ppt, the average density is 100 clams/m . 2 At mile 39, salinity 0.4 - 5.0 ppt, there are 20 clams/m . On the other hand, in Ochlockonee River, western Florida, Olsen (personal communication) has found that population density of R. cuneata increased upriver: at mile 1, salinity up to 22 ppt, 2 less than 1 clam/m . At mile 4, salinity 0-11 ppt, clams average 12 per square meter. At mile 5, salinity 0 - 9 ppt, 2 28 clams/m . At mile 6, salinity 0 - 9 ppt, clams average 52 2 per m . Rangia is present, in undetermined densities, up to mile 12, salinity 0-1.7 ppt. In the present state of our knowledge it does not seem possible to explain the above apparently contradictory data on the basis of any of the laboratory studies so far reported, but if we also knew the seasonal changes in salinity, the changes with tides, the current patterns, etc., along with the distribution of bottom types and a few things we do not yet know about the influence of sediments on setting and survival of juvenile clams, a reasonable explanation might become clear. Even on the basis of the few published and unpublished reports now available, the pieces are beginning to fit together to make some pictures. After adding our own findings, we will attempt to put together as complete an overall picture as possible. Accounts of our own separate studies follow here, and then the overall discussion, summary, and conclusions.

DESCRIPTION OF THE STUDY AREAS

Field studies were made, and Rangia cuneata were collected for laboratory studies, in two main areas, the Trinity River Delta - Trinity Bay area and the Neches River above Beaumont, Texas (Figs. 2 and 3).

Trinity River Delta - Trinity Bay

Trinity River, the main source of fresh water running into the Galveston Bay system, empties at the northeast end of Trinity Bay, in which it has built up an extensive delta with many distributaries (called bayous in this part of the world). Trinity Bay is the northeast branch of Galveston Bay. The Trinity River delta and Trinity Bay are surrounded by Chambers County, a rice farming and cattle ranching Coastal Prairie county that is thinly populated (1970 population 12,187) and has no large towns (Anahuac with 1,881 people is the largest), thus contrasting strongly with the neighboring counties, Jefferson on the east with 0.25 million people and Harris on the west with 1.75 million (Texas Almanac for 1972 - 1973, The Dallas Morning News). Although Chambers County has a large mineral production (oil, natural gas, salt, sulphur) and there are some 300 oil and gas wells in the bay itself, Trinity Bay remains one of the least polluted parts of the Galveston Bay system. Galveston Bay, proper, receives much pollution from the heavily populated and highly industrialized areas on its west side. The northwest arm through which the Houston Ship Channel runs is called Upper Galveston Bay, a name that also applies to all of that part of Galveston Bay north of a line from Smith Point to Eagle Point. Fifty years ago there was an almost continuous

^3 reef of oysters and shells, Redfish Reef, across the bay on this narrow "waistline," a reef so large that it nearly divided Galveston Bay into two bays. Rathbun (1895) described an August 1892 investigation by Battle of conditions for oysters in Galveston Bay; according to this account, at that time all of the bay above Redfish Reef was too fresh for oysters, and the reef itself was composed mainly of empty shells. The famous oyster biologist Galtsoff (1926, 1931) said that Redfish Reef and other such barriers prevented "the mingling of fresh and sea water. In these cases cutting through the reefs and digging passes will materially improve conditions." He suggested that the desired reef cutting "can be accomplished without expense to the States by granting to private concerns the right to dredge on certain dead or unprofitable reefs," among which he evidently intended to include Redfish Reef. During the next 30 years millions of cubic yards of oyster shell was removed from Galveston Bay bottoms by shell—dredging companies• Redfish Reef was lowered in several places and had a large channel cut through it, and evidently the increased "mingling of fresh and sea water" that Galtsoff advocated has had some effect, for Upper Galveston Bay, above Redfish Reef, is now one of the commercial oyster- producing areas.

Trinity Bay

This bay, the northeastern arm of Galveston Bay into which Trinity River flows, has an area of approximately 100 square miles. It is flatbottomed and shallow, only 9 feet deep in the center and usually about 5 feet deep 1000 yards from shore; the upper quarter is even more shallow, 6 feet deep in the center of the bay. Oysters grow in Trinity Bay, but in most years only in the lower end; the upper end is usually too fresh for oysters, so its benthic fauna is dominated by Rangia ciineata. Like other Texas "bayheads," Trinity Bay is characterized by extremely variable and unpredictable salinity - unpredictable a month in advance, that xs; of course when there is a flood on its way down Trinity River one can predict lowering of bay salinity. To pick a few years at random, Hofstetter (1959) reported that only 6 of 33 water samples taken in Trinity Bay from January 1958 to May 1959 had salinity as high as 10 ppt. Shidler (1961) presented a graph showing average salinities at a number of Trinity Bay stations during the period July 1, 1959 to June 30, 1960; all stations averaged 1-10 ppt. Stevens (1963) presented a graph of salinities at the same Trinity Bay stations, based on monthly samples taken, September 1, 1961 to December 3, 1962; all stations, averaged together, ranged from 3 — 4 ppt in September 1961 to approximately 12 ppt in January and November 1962, and salinity averaged below 10 ppt in 9 of the 15 months. O'Heeron (1966) averaged all salinity data at each station sampled by Texas Parks and Wildlife and U. S. Bureau of Commercial Fisheries biologists and located the mean annual 10 ppt and 15 ppt isohalines during each of three years, 1963, 1964, and 1965. In 1963 the 10 ppt isohaline crossed the middle of Trinity Bay and the 15 ppt isohaline was outside the mouth of Trinity Bay. In 1964 the 10 ppt isohaline was in the upper quarter of Trinity Bay and the 15 ppt isohaline crossed its middle. In 1965 the 5 ppt isohaline was in the upper third of Trinity Bay, the 10 ppt at the mouth of Trinity Bay, and the 15 ppt isohaline ran north and south through the center of Galveston Bay. O'Heeron (1966) found living Rangia cuneata only in parts of the Galveston Bay system where salinity was usually less than 15 ppt, mostly within the mean annual isohaline of 15 ppt, and especially in Trinity Bay. This was supported by findings of Texas Parks and Wildlife biologists such as Shidler (1960) and Pullen (1961). Jack C. Parker (1966) of the U. S. Bureau of Commercial Fisheries made a more intensive and detailed study of the distribution of Rangia cuneata in the Galveston Bay system in relation to bottom sediments and salinities. He found these clams to be "many" to "scarce" in Upper East Bay (salinity 11 — 23 ppt, mean 16 ppt), absent in Lower East Bay and Lower

1+5 Galveston Bay, "none" to "scarce" in Upper Galveston Bay (salinity 9-25 ppt, mean 18 ppt), "none" to "scarce" in Lower Trinity Bay (10 - 24 ppt, mean 17 ppt), and "many" in Upper Trinity Bay (4 - 16 ppt, mean 11 ppt). Parker never found living Rangia cuneata at any point where the salinity at the time exceeded 18 ppt. Monthly collections of Rangia Cuneata, to be used in studies conducted by several workers on our project, were made at and near McCollum Park, a quarter mile upbay (northeast) from Point Barrow on the west shore of Trinity Bay. The water here is shallow and clams were collected by hand or with the aid of a garden rake while wading from the shoreline out to about 400 meters from shore. The bottom is black, firm silty mud in a layer about 50 cm thick over gravelly clay; the mud layer is covered by a 2 - 5 cm thick layer of silty sand. The mud contains hydrogen sulfide which blackens shells of the clams, but the black turns to brown after a few hours exposure to air. Old Rangia shells occur in the mud and underlying clay, along with shells of Macoma and Tagelus. Living Rangia are found in the bottom from the surface to a depth of over 15 cm, but the great majority lie just under the surface of the bottom sediments, anterior end down, with the posterior end projecting just enough to allow the siphons to extend above the surface layer of sand, according to C. A. Bedinger. McCollum Park clams seemed to fall into three size classes: 10-20, 30-40, and 50-60 mm. The young (10 - 20 mm) individuals seemed to be most numerous in the deeper part of the collecting area, farthest offshore, and the medium (30 - 40 mm) size were most numerous in Diplanthera beds less than 100 meters from shore, in water about 20 cm deep, while the largest clams were irregularly distributed. Predation by laughing gulls was observed, but far more clams were taken by several families of Orientals. Salinity at McCollum Park has ranged from near 0 to 16 ppt, with lower salinities in winter and spring and higher in summer, increasing toward autumn. Water temperatures during the study period ranged from 7° C to 35° C. When water was 7° C, mud

k6 temperature was 10° C, so clams were not as cold as the overlying water. Tides were determined more by wind than by lunar influence; the highest water level observed was on January 3, 1971, with a strong south wind blowing; then a cold front or "norther1* came in and water level dropped 4 feet, so that the lowest tide was observed on January 4, with most of the collecting area laid bare. Dissolved oxygen content was highest in cold weather, especially on windy days, and lowest on hot, calm days.

Trinity River Delta

The delta of the Trinity River with its distributaries or bayous, and Lake Anahuac, are shown in Fig. 3. This area is described from the biologist’s view point by Baldauf (1970). Much of the field work for this report was done by John Van Connor who has given us some detailed information on the salinity dis­ tribution, the Rangia population, and bottom types in these waterways. A very sparse population of Rangia cuneata is found in the main channel of Trinity River up to a point 3.8 miles from its mouth; at this point salinity is less than 0.5 ppt. The bayous have much larger populations of R. cuneata, extending much farther inland: above Interstate Highway 10 there are Rangia in Old River, Lost River, Lost River Bayou, Lost Lake, Wiggins Bayou, and even Lake Charlotte where salinity was only 0.3 - 0.8 ppt during the study period. The barnacle Balanus improvisus was found on cypress trees in Lake Charlotte; this was the most inland point where it occurred. Other salinities recorded by Van Connor include 0.1 to 6.3 ppt in Lost River Bayou at its junction with Wiggins Bayou. In Old River, R. cuneata were found at a point 1.3 miles above Interstate 10 where the salinity ranged from 0.0 to 7.9 ppt, and 6 or 7 miles upstream from this point living Unionidae (freshwater clams) were collected. There were Indian shell mounds of impressive size at the lower end of Lost Lake and at the junction of Wiggins and Lost River Bayous. Although large quantities of shell had been taken from these mounds for commercial uses, there was enough left to provide interesting archaeological studies (Shafer, 1966). Most of the shells were from Rangia cuneata, showing that this clam was a mainstay in Indian diets. Dense populations of R. cuneata were found in Old and Lost River Lake, below Interstate 10; also in Round Lake and Mud Bayou. In Cotton Lake and in Cross Bayou, R. cuneata was mixed with R. flexudsa. The densest Rangia populations were found in sandy mud or muddy sand bottoms in the lower end of Cross Bayou, where salinity ranged from 0.1 to 15.7 ppt. Cross Bayou runs directly into Trinity Bay and is the main outlet for the bayou-and-lake system west of the main channel of Trinity River. The most inland point where living Rangia were found was 10.65 miles, by water, from the mouth of Cross Bayou. Except in Cross Bayou, the Rangia in these delta waterways are living in mud bottoms. For our laboratory studies, Trinity River delta clams were collected under the bridge on Interstate Highway 10, on the east side of the waterway marked on the highway signs as "Old and Lost River" on the west side and as "Lost and Old River" on the east side. This collecting site could be designated more exactly as "east side of Lost River at its junction with Old and Lost River Lake." Salinity here was measured at 1.5 ppt. The clams are found in a muddy bottom and are collected by hand while wading. These were the largest Rangia cuneata available to us; the average volume was twice that of McCollum Park clams. The Trinity, the main source of fresh water for the Galveston Bay system, is the third largest river flowing to the Gulf in Texas; with a length of 550 miles and a drainage basin of 17,969 square miles, much of it in high-rainfall areas, it has an annual runoff of 5,800,000 acre-feet (Texas Almanac for 1972-73, The Dallas Morning News). Baldauf (1970, p. 132) presents a bar graph showing average flow each month during the period March 1966-May 1968. The highest flow rate (16 million cubic feet per second) was in May 1966 and the lowest (25,000 cf/s) was in August 1967. The Trinity River delta is a center of controversy over

1+8 the Wallisville Dam project and its possible ecological effects, Baldauf (1970) was not concerned with Rarigia, but centered his attention on the importance of the Trinity delta system of water­ ways as a nursery area for white and brown shrimp, blue crabs, and Gulf menhaden. He estimated that the dam (actually a permanent saltwater barrier more than a dam, most of it an earth levee only about 4 feet high) and the reservoir it would back up behind it would "destroy11 approximately 12,500 acres of nursery ground, probably cause some change in the nursery areas below the damsite (which would include Cotton Lake, Red Bayou, Cross Bayou, and the last 3.8 miles of Trinity River) and would consequently "cause permanent declines in the numbers of these species" (i.e., shrimp, crabs, and menhaden). At this writing construction of Wallisville Dam is being held up awaiting final court decisions.

Lake Anahuac Possibly more could have been learned about the future effects to be expected in the area behind Wallisville Dam by a study of the history and present status of Lake Anahuac. This 5,300 acre water body was formerly an embayment at the upper end of Trinity Bay and was known as Turtle Bay. Continued extension of the Trinity River delta, a natural process, eventually blocked it off from the bay and made it a delta flank lake, similar to Lake Pontchartrain, Lake Maurepas, Lake des Allemands and Lake Salvador flanking the lower Mississippi River. Some salt water still entered Turtle Bay through a narrow pass. This was blocked by a saltwater barrier or dam in 1951, making Turtle Bay a reservoir, Lake Anahuac, storing 35,300 acre-feet of water which is still brackish but fresh enough for use in irrigating rice. Trinity River water is pumped into the northern end of the reservoir, keeping the salinity below 1 ppt, except that Hurricane Carla in 1961 put saltier water (4.7 ppt) into the lake before "all" water drained out through breaks in the levee, according to Mr. LaFlore at the pump station. In spite of its low salinity, Lake Anahuac contains a large population of Rangia ciineata, reported by C. A. Bedinger to be as dense as anywhere in Trinity Bay or in the Trinity delta distributaries, and several size classes are present indicating different age groups. The lake also contains blue crabs, Callinectes sapidus, and Atlantic croakers, Micropogort undulatus, both of which reproduce only in salty water (in or near the Gulf). Baby crabs and croakers, and swimming larvae of Rangia, could have been pumped into the lake from Trinity River. An investigation might reveal a number of other "marine" species living in this reservoir, which is perhaps as fresh as the Wallisville Reservoir will be, along with many freshwater species. Several lots of Rangia cuneata were collected from Lake Anahuac and used in physiological, biochemical and parasitological studies.

The Neches River

The Neches has the second largest flow among Texas rivers, 6 million acre-feet annually (Texas Almanac 1972-1973). Only its nearby twin, the Sabine, has a larger flow (6.8 million acre- feet). Each river is approximately 400 miles long and has a drainage area of about 10,000 square miles, nearly all in the 45-60 inch rainfall zone. These two rivers run into Sabine Lake, a brackish "lake estuary" which opens by a narrow pass into the Gulf. The bottom fauna of Sabine Lake is dominated by living Rangia cuneata and the bottom sediments contain dense concentrations of

Rangia shells, the accumulated product of centuries, which are being dredged by shell companies. The principal towns are Orange (population 24,000) on the Sabine River, Beaumont (116,000) on the Neches, and Port Arthur (57,000) on Sabine Lake. There is much tidal marshland, populated by muskrats, in the surrounding area, and much prairie farmland used mostly for rice and cattle. The lower 20 miles of the Neches River has on its west bank one industrial establishment after another and receives from them industrial effluents, including wastewater drainage from the old Spindletop oilfield and from petroleum refineries, as well as domestic sewage from Beaumont and several smaller towns below it. The east bank, on the other hand, has almost 50 no human population and consists mostly of swamp and marsh, with some grazing land. This polluted 20-inile stretch of river has an impoverished aquatic fauna and flora and frequent small fish kills, although conditions are nearly normal in the last mile or two above Sabine Lake. At the mouth of the Neches River, the ship channel and intracoastal waterway intersect. During a 1951-1953 study conducted by the Texas A&M Research Foundation (Project 32) conditions of low oxygen and no oxygen were frequently found in the Neches below Beaumont, and no living molluscs of any kind were in this 20-mile stretch, but in the 5-mile stretch from the Port of Beaumont to the northern city limits dense populations of Rangia cuneata were found, along with the attached mussel Coitgeria leucophaeta clustering on shells, cypress trees, logs and boat bottoms; and abundant crustaceans of a few species, the barnacle Balanus improvisus, the shrimps Macrobrachium ohione and Palaemonetes kadiakensis, and the crabs Rhithropanopeus harrisii and Callinectes sapidus; along with the species of marine origin were the freshwater crayfishes Procambarus blandingii acutus and Procambarus clarkii, and many freshwater fishes. Particularly dense populations of Rangia were found 0.5 miles below the boat-landing at the end of the road between Beaumont Country Club and Forest Lawn Memorial Park (cemetery) where Collier’s Ferry used to cross the river. This station is marked by a large sign on each bank warning that the Fresh Water Canal crosses under the river as a pipeline. In 1951-52 and 1952-53 a number of 1-square-foot sample areas were marked out on the Rangia beds here during "northers" when river level was lowered enough to expose the clam flats, and all clams within each sample area were removed, counted, and measured. For comparison, similar measured areas of this and nearby clam beds were sampled by Richard Harrel and Gilbert Gatlin of Lamar State College in 1969. Later a group from Texas A&M, including Hopkins, visited this site to collect clams. Environmental conditions were amazingly little changed after the lapse of 18 years, and the

51 clam samples were nearly identical in both size (length measure­ ments) and density (number per square foot or per square meter). The length range was 37-56 mm in 1951-52, and 35-55 mm in 1969, which according to the Wolfe-Petteway growth curve would indicate an average age of approximately 4 years on both occasions. The sample areas indicated an average of about 250 clams per square meter in both years. On the basis of these figures and weights of shells, wet meats and dry meats, Hopkins (1970) estimated that this area would produce annually 12,400 pounds of shell per acre and 2,560 pounds of wet meat per acre (13,900 kilograms of shell and 2,900 kg of wet meat per hectare), or 500 pounds of dry meat per acre (2,275 kg per hectare); this would be worth $775 per acre ($750 for meat at 30 cents per pound, $25 for shell) or $1914 per hectare, per year. The Neches River Rangia grew in a fairly firm sandy mud bottom sprinkled with shells and were found from just below surface down to 5 inches below the surface of the mud, on the nearly flat shoulders on the east side of the channel. They were exposed to air only during "northers" when water level was lowered a foot or two feet below normal; at usual low tide level the clams would be covered with 0.5 - 1.5 feet of water. On later trips to collect clams for laboratory studies, Rangia cuneata was found to be more widely distributed than had been thought; clams were collected on both sides of the channel in all depths that could be reached by wading or skin-diving. They probably do not live in the center of the channel, which is approximately 18 feet deep here, for during periods of low river flow salt water pushes up past Beaumont to Collier's Ferry and above, bringing with it pollution from the city, and oxygen disappears at the bottom of the channel while this condition persists. At such times of low river flow, salinity reaches 16 ppt in the bottom of the channel and 13 ppt at the surface, but during most of the year the river is fresh (salinity below 0.5 ppt). The conditions of high salinity and pollution, with low oxygen in the deeper water, are intensified by withdrawal of fresh water

52 Explanation of Figs. 1, 2, and 3

Fig . 1. Southeastern United States.

1 . Upper end, Chesapeake Bay, Md. i i . Caloosahatchie R. 2. Potomac River, Md. Estuary, Fla. 3. Rappahannock River, Va. 12. Peace & Myakka R., Fla. 4. James River, Va. 13. Ochlockonee R., Fla. 5. Back Bay, Va. 14. Apalachicola R., Fla. 6. Currituck & Albemarle Sounds, 15. Mobile Bay, Ala. N. C. 16. L. Pontchartrain, La. 7. Pamlico River, N. C. 17. Atchafalaya R. and Bay, La. 8. Newport River, N. C. 18. Vermilion Bay, La. 9. Neuse River, N. C. 19. Grand & White Lakes, La. CM o

10. Altamaha River, Ga. • Sabine Lake, La. - Tex.

Fig . 2. Texas Gulf Coast.

1 . Sabine River 7. Carancahua Bay 2. Neches River 8. Lavaca Bay 3. East Bay 9. San Antonio Bay 4. Lake Anahuac (Hynes Bay & Guadalupe Bay 5. McCollum Park at upper end) on Trinity Bay 10. St. Charles Bay 6. Clear Lake 11. Nueces R. & Bay

Fig.► 3. Galveston Bay and Trinity Bay.

1 . Trinity Bay 4. Lake Anahuac 2. McCollum Park 5. East Bay 3. Lost River 6. Clear Lake

Redfish Reef, across the "waist" between Smith Point on the east and Eagle Point on the west, separates Upper and Lower Galveston Bay.

53 «O oo oo «O ^ 0»tn

FIG. 1. - Southeastern United States.

5 ^ 55 FIG. 3. - Galveston Bay and Trinity Bay.

5 6 upriver by rice farmers, and temporary installation of a salt­ water barrier upstream from Coller’s Ferry. The barrier serves to keep fresh water upriver as well as to prevent salt water from going up as far as the pumping station. The Corps of Engineers has a project to build a permanent saltwater barrier in the Neches, with facilities to permit passage of boats. The present status of this project is not known to us, nor do we know enough about the plans to guess what its effects ori Rangia or its other ecological effects might be.

PHYSIOLOGICAL STUDIES ON EFFECTS OF SALINITY

General Introduction

Most of the published work on Rangia has been concerned with field ecology, taxonomy, and systematics. This literature has been reviewed in an earlier section. The only published physiological studies on Rangia are a group of papers on the amino acid and sugar metabolism of Rangia (Allen, 1961; Allen and Awapara, I960; Chen and Awapara, 1969; Simpson and Awapara, 1966; Stokes and Awapara, 1968) and a paper on the CO^ production of Rangia (Beyers and Warwick, 1968). Apparently no studies have been conducted regarding the effects of salinity on temperature tolerance, osmoregulatory capabilities, amino acid uptake or respiration of adult Rangia. It was believed that an understanding of Rangia* s physiological processes was necessary in order to evaluate the effects of coastal zone modifications. Rangia can survive for over 8 months in salinities ranging from almost 0 (tap water) to 38 ppt (= 38°/oo) in the laboratory (Bedford, preliminary observation), but is not reported in nature from waters that are continually above 15 ppt (Hopkins, 1970; Hopkins and Andrews, 1970; Moore, 1961; and Ladd, 1951) . Parker (1955) has suggested that an increase in the salinity (to 20 ppt) caused the virtual disappearance of Rangia from upper San Antonio Bay. A knowledge of the physiological ecology of Rangia not only will be of value for a more complete understanding

57 of the effects of the environment on estuarine organisms but will have social and economic value as well. As Hopkins (1970) has demonstrated, the annual production of shell and meat by Rangia of a Neches River population is worth $775 per acre. In addition to this direct harvest value to man, Rangia is an ecological asset because it converts detritus and phytoplankton into tissue that feeds many fishes and crustaceans.

General Methods and Materials

The Rangia cuneata used in this study were collected from McCollum Park, Trinity Bay (population A ) , and Old River Lake (population B), both in Chambers County, Texas. Population A was used in all experiments unless stated otherwise. The salinity for the upper Trinity Bay region is quite variable, with the yearly average differing as much as 10 ppt from one year to the next. The usual salinity range is between 5 and 15 ppt. The salinity at the McCollum Park site was taken at monthly intervals for the extent of the present study. Salinity to the nearest 0.5 ppt (o/oo) was measured with an optical refractometer. For February 1970 - February 1971 the range was from 1 to 20 ppt with a mean of 8 ppt. For February 1971 - February 1972 the range was from 5 to 18 ppt with a mean of 14.5 ppt. The salinities for Old River Lake were not taken at regular intervals during this study; however, monthly data by Baldauf (1970) for the period of March 1966-May 1968 give a range of 0-8 ppt and a mean of 1.6 ppt. See Figure 3. The McCollum Park area had a very uniform hard sandy-mud bottom with an average depth of 45-95 cm extending out from shore for 300 meters. The Old River Lake collection area differed in its softer bottom and its lower salinity, usually one-third to one-half that of the McCollum Park region. The drainage from Old River Lake enters Trinity Bay near McCollum Park. Population densities of Rangia at McCollum Park were checked three times during the second year of the study and the density was consistently 45-49 live clams per square meter.

58 After collection the clams were transported to the laboratory and placed in large aerated aquaria containing water of the same salinity as at the collection site. The water used for holding and experimental work was prepared with distilled water and "Instant Ocean" (Aquarium Systems, Inc., East Lake, Ohio). The clams were held in the holding aquaria for two days prior to experimentation. Rangia were acclimated to the experimental salinity in steps of 5 ppt per 2 days. Preliminary experiments showed that if this acclimation procedure was followed, a survival rate of 98 per cent or better was achieved. In most experiments the clams were held at the stated experimental salinity for at least 7 days before use. Rangia were used in experiments after a period of between 10 and 24 days of maintenance in the laboratory. The minimum of time was necessary for acclimation and the maximum was dependent on the time required to conduct the various experiments. In every case, when the response to salinity was tested, all clams utilized in a given experiment were from the same collection. In earlier experiments (Bedford unpublished) Rangia have survived in the laboratory for over 2 months without food. Allen (1959) has also reported that Rangia survive long periods of starvation and that no appreciable changes occur in the amino acid composition after 21 days without food. The clams were held and experiments were conducted in an air-conditioned laboratory at 22+1° C.

Effect of Salinity on Blood Osmotic Concentration, Per Cent Water, and Per Cent Ash

1. Introduction The concept that most marine bivalves are isosmotic with their environment has been well reviewed in a recent printing (1965) of Krogh’s classic work and in later papers by Robertson (1964) and Potts (1968). Recently this concept has been reinvestigated by Pierce (1970) in a study of six bivalves which exhibited tolerances from euryhaline to stenohaline in character. His results showed all six species to be osmoconformers, but they

59 were not isosmotic with the environment. In all cases the osmotic concentration of the internal fluid was hyperosmotic to the environment by a slight but constant amount over the nonlethal salinity range of the species. He concluded that this hyperosmotic condition appeared to be the result of passive equilibrium rather than active regulation and was probably characteristic of all osmoconformers.

2. Materials and Methods a. Osmoregulation Both populations of Rangia were examined for osmoregulatory ability. The salinity at the time of collection was 15 ppt for McCollum Park and 7 ppt for Old River Lake. The two populations were kept separated during the experiments, but otherwise treated in an identical manner. Blood for analysis was removed by directly puncturing the ventricle with a Pasteur pipette, drawn to a fine point, using a slight suction from a rubber bulb. An initial attempt to utilize a hypodermic syringe proved to be unsuccessful, because of the difficulty in applying the slight amount of suction needed while keeping the needle in the ventricle. Removal of the blood was facilitated by puncturing the ventricle just as it expanded to its maximum size. Unless this procedure was followed, the required 0.2 ml of blood could not be obtained. The blood samples and samples of the medium for each group at a specific salinity were measured on a Fiske osmometer which was found to have an accuracy of + 1 milliosmole per liter H^O (mOsm/liter). The blood samples were analyzed immediately after removal, and were not centrifuged prior to analysis, as previous tests showed no change in the osmotic concentration after centrifugation. To test for the effect of the artificial sea water on Rangia, the clams’ osmoregulatory ability was examined in both natural water from the collection site, and in Instant Ocean diluted to the equivalent salinity. The clams were acclimated in each test salinity for 7 days before analysis.

6o To determine the time necessary for the blood to reach a new steady state of osmotic concentration after transfer to a new environment, a specific experiment was conducted. Rangia were acclimated at 11.3 ppt for one week, after which the water and the blood of 3 animals were analyzed for osmotic concentration. Of the remaining animals, 15 were placed in each of two aerated aquaria, containing dechlorinated tap water or water of 19.4 ppt. At intervals of 2,5,10,18, and 29 hours the media and the blood osmotic concentration of 3 clams in each of the two aquaria were measured as described above.

b. Body water percentage and ash percentage To determine the per cent water and the per cent ash in Rangia tissue at different salinities the clams were acclimated for 10 days at salinities ranging from 1 to 32 ppt. After acclimation the clams were shucked, rinsed with tap water for a few seconds, blotted on "Kimwipes," weighed, and "wet weight" recorded. The tissue was dried to a constant weight (approximately 72 hours at 95° C) and then combusted at 55° C for 24 hours. From these data it was possible to compute the per cent water and the per cent ash in the tissue at different salinities, and then correct wet weight or dry weight to ash-free dry weight in those experiments when direct determinations were not possible. In conjunction with the above experiment the time necessary for water regulation after a salinity shock was examined. After 90 Rangia were maintained at 15 ppt for two weeks, 40 clams were moved directly to a salinity of 5 ppt and 40 others to a salinity of 25 ppt. Ten control clams were left at 15 ppt and analyzed for per cent E^O. At 5,15,30, and 45-hour intervals, 10 clams each from the 5 and 25 ppt groups were analyzed for percent H^O.

3. Results a. Osmoregulation Table 3 lists the salinities over which the osmoregulatory abilities of Rangia were tested. The minimum value of 16 mOsm/liter

6i Table 3. Relationship between media osmolarity and the osmolarity of Rangia blood.

Blood Average Media Standard Number of Population mOsmoles/ mOsmoles/ Deviation Liter T Clams Tested Liter

16 82.0 1.6 3 B 20 91.3 0.9 3 B 32 95.0 5.0 2 A 33 81.0 1.4 3 B 47 100.3 4.1 3 A 79 121.0 1.0 2 B 85 152.0 4.3 3 B 118 154.0 2.8 3 A 118 149.2 4.5 3 B 143 157.3 0.9 3 B 148 163.5 3.5 2 A 170 189.5 1.5 2 B 210 220.0 2.2 3 A 215 229.5 0.5 3 B 265 272.3 2.9 3 A 303 309.3 0.9 3 B 315 319.3 4.2 3 A 465 466.5 0.5 2 B 477 481.7 2.9 3 A 635 631.3 1.3 3 B 640 643.7 3.3 3 A 972 970.0 1.5 2 B

62 is the osmotic concentration of tap water and the maximum value of 972 mOsm/liter is equal to 33.2 ppt, or approximately full strength sea water. It should be noted that no appreciable differences were found in the osmoregulatory abilities of populations A and B. In addition, there were no statistically significant differences between the osmoregulatory capacities of Rangia held in dilutions of natural estuarine water and in the various concentrations of Instant Ocean (Table 4).

Table 4. A comparison between the osmoregulatory abilities of Rangia held in natural estuarine water and in Instant Ocean.

The values listed are in mOsm/Liter.

Natural Media 141______231 Instant Ocean Media 135______238 Avg. Clam Blood 161.3 236.3 157.3 244.7

SD ± 1.25 2.80 2.05 1.26 Blood Media 20.3 5.3 22.6 6.7

In Figure 4, the osmotic concentration of the blood has been plotted against that of the external medium and it can be seen from this figure and Table 3 that the internal concentrations approximate those of the environment at levels greater than 10 ppt. However, at environmental concentrations below this point the blood was found to be hyperosmotic. The hyperosmocity of the blood began to increase rather sharply at an external concen­ tration of approximately 5 ppt (145 mOsm/liters), reaching a maximum differential at a medium concentration of approximately 85 mOsm/liter. A blood-medium differential of 55 to 65 mOsm/liter was maintained by clams in media ranging between 16 and 100 mOsm/liter. When Rangia were taken from 11.3 ppt salinity (331 mOsm/liter) and placed in either a salinity of 19.4 ppt (568 mOsm/liter) or in tap water, it was found that they reach a new steady state of blood osmotic concentration in approximately 18 hours (Figure 5). Those organisms placed in the higher salinity water did not attain osmotic equilibrium until after nearly 29 hours. From the slope

63 I. 4.FIG. -therelationshipofPlotthe osmolarity and media between blood Blood Osmotic Cone. (mOsm.) osmolarityofRangia. M da soi Cone. Osmotic (mOsm.)edia 6U

%0Salinity

FIG. 5. - Media and Blood Osmotic Cone. (mOsm.) hr =. S.D.for18-29-hourintervalsand <2.5 mOsm/L. n=3. where and the medium as a function of time. Vertical bars = S.D., Vertical = bars theand afunctiontime.asof medium thePlotrelationshipofosmolarity of between Rangia blood T i me (hours) 65 >10 ■15 ■

20

inity %oSal of the lines in Figure 5, it is apparent that the osmotic concentration of Rangia placed in fresh water changed faster over the first ten hours than those in 19.4 ppt salinity. In addition, the rate of change is irregular when salinity is increased, but linear during dilution. These findings correlate nicely with the laboratory observation that Bangia placed in a more dilute environment began siphoning vigorously and steadily within approximately 15 minutes. This reaction occurred even when the animals were taken from 32 ppt (°/oo) salinity and placed in fresh water. However, when changed to more concentrated media, Rangia siphoned only intermittently for the first several hours.

b. Per cent body water and per cent ash The regression line for the per cent body water versus salinity is plotted in Figure 6. The body water (volume of water in the tissue) is expressed as a percentage of the wet weight. The body water increased by approximately 7 per cent as the acclimation salinity was decreased from 32 to 1 ppt ( /oo) representing a salinity dilution of 97 per cent. In Figure 7 the percentage of dry weight that is ash is plotted against salinity. The linear fit of the data was very good in both cases. The correlation coefficients were 0.964 for per cent body water and 0.995 for per cent ash. In those few experiments where it was not practical to take the ash-free dry weight directly the above figures were used for conversion. When Rangia acclimated to 15 ppt were salinity-shocked by moving them to 5 ppt and 25 ppt the clams transferred to the lower salinity again demonstrated a quicker response. The clams transferred into 5 ppt (°/oo) had made their major change in per cent body water by the 5-hour interval, while those transferred into 25 ppt (°/oo) did not exhibit any appreciable change until after the 5 hour interval (Figure 8). By the 15-hour interval both sets of clams had reached a level approximating their final steady state. However in both instances there was a slight overshoot in the adjustment of per cent body water, with the

66 FIG. 6. - Percentage body water as a function of salinity. Solid line: straight line of regression. Dotted line: standard error of estimate (n=5 for each point).

67 I. 7. FIG. % OF DRY WEIGHT THAT IS ASH 1 . 8 4 3 9 5 2 0 1 /_ _ - - - . _ . 0

I T 1

2 Per cent of dry weight that is ash as a function of salinity. of function a as ash is that weight dry of cent Per dard error of estimate (n=5 for each point). each for (n=5 estimate of error dard oi ie srih ln o ersin Dte ie stan­ line: Dotted regression. of line straight line: Solid ------

1 5 ------

1 1 ------0

%oSALINITY ,68 1 15 ------

1 20 ------

1 25 ------

32 1

TIME (hours)

FIG. 8. - Volume regulation as a function of time. Vertical bars indi­ cate standard deviations (S.D.), where n=10 (except where noted).

69 oscillations becoming smaller with time. This overshoot- undershoot type of response is very typical of physiological responses to a new environmental condition.

4. Discussion The results of this portion of the study indicate that Rangia are osmoregulators. They are apparently unique among the marine and estuarine bivalves in their osmoregulatory ability, since Robertson (1964) stated: "Unequivocal evidence of osmotic control in brackish-water bivalves is absent." Although the freshwater bivalves have been found to osmoregulate, maintaining their blood concentration slightly above that of the environment (Picken, 1937; Hiscock, 1953a and 1953b; and Potts, 1954a), only two of the euryhaline bivalves investigated have been reported to have osmotic control. These are Scrobicularia plana (Freeman and Rigler, 1957) and Crassostrea virginica (Fingerman and Fairbanks, 1956) . In Scrobicularia, the blood remained in osmotic equilibrium at dilutions of sea water down to 19.2 °/oo (ppt) salinity (A° C - 1.05), but in sea water of 10.8 °/oo ppt salinity (A° C - 0.59) the blood had a mean salinity of 13.5 °/oo(pptJ(A° C - 0.74). However, it was not clear in this case whether the animals were showing active control or had resisted final dilution to the outside level by keeping the valves closed (Robertson, 1964). Gilles (1972) reported a transitory (96 hours) hyperosomotic state in both the blood and perivisceral fluid of Glycymeris glycymeris and Mytilus edulis when the two species were rapidly acclimatized to a dilute medium. However, this was not due to physiological osmoregulation but to the ability of these bivalves to isolate themselves from the external environment by closing their valves tightly. Fingerman and Fairbanks (1956) stated that the oyster Crassostrea virginica has a limited ability to osmoregulate. This evidence was based on blood analysis only 4 to 8 hours after transferring oysters adapted to 17 ppt salinity into salinities ranging from 10 ppt to 36 ppt. Galtsoff (1964) reported that a salinity change of about 10 ppt, continued for several hours,

70 reduced both the rate of water transport and the amount of time oysters remained open. When oysters were placed in a low-salinity environment (13 ppt), very little water was transported even after several days of adaptation. Anderson and Prosser (1953) found that the blue crab Callinectes sapidus showed considerable variation in its os­ moregulatory ability according to where it lived in the estuary. The crabs inhabiting higher salinity environments could not regulate in as low a salinity as those crabs from regions of lower salinity. They also showed that the blood of the quahog (clam) Venus mercenaria conformed to the external environment while the clam pumped water, but Venus from less saline regions tended to pump at lower concentrations than those adapted to more saline regions. It appears that these populations differ in their state of osmotic adaptation. Rangia were not found to exhibit this variation in osmo­ regulatory ability according to the environmental conditions of their populations. Although there were wide fluctuations in salinity in both environments, the salinity that population A was subjected to was always two to three times higher than that of population B (Baldauf, 1970; C. A. Bedinger, personal communication). From the results of this study it appears that Rangia were osmoconformers at salinities above 10 (293 mOsm/ liter) and they began to osmoregulate in more dilute environments, reaching a significant degree of hyperosmocity at approximately 5 ppt (145 mOsm/liter) . As noted in the results, Rangia transferred to a lower salinity adjust their internal osmotic concentrations faster than those subjected to an increased salinity. The clams also demonstrated a more rapid change in per cent body water when moved to a lower salinity than when transfered to higher salinities. This behavior may be a functional adaptation to natural environmental fluctuations. In the upper estuary, rapid dilutions as a result of rain and runoff are common, but concentration is generally a relatively slow process related to evaporation. An exception might be a rapid salinity

71 increase due to storm tides. It is likely that it is this rather unique capacity of os­ moregulation that allows Rangia to exist as a dominant organism in areas of extreme (0 to 25 ppt) salinity fluctuation. A Rangia-dominated estuarine ecosystem has been discussed by Odum (1967) as an area of very low diversity of biological components. It is possible that the dominant organisms in the estuarine ecosystem are those that have been successful in using a portion of their energy budget in salinity adaptation. A recalculation of the data presented by Picken (1937) and Potts (1954b) for the concentration of the blood obtained from the fresh­ water bivalve Anodonta shows that it maintains its blood at a concentration 36.4 mOsm/liter above that of the ambient medium. Potts (1954b) reported that the energy required to maintain this differential represented 1.2 per cent of the total metabolic energy. If we assume this relationship to be true for Rangia, then 2.4 per cent of the metabolic energy would be used in os­ moregulation at salinities of 3 ppt or less. These figures are minimum values, as Potts (1954b) assumed the energy expended to equal the thermodynamic minimum. By conforming to the ambient medium at higher salinities energy is conserved. It is interesting to note that clams from population B (0-8 ppt, mean 3 1.6 ppt) had a mean total volume of 102.0 cm while those of population A (salinity 1-20 ppt, mean 8 ppt) exhibited a mean 3 total volume of 47.5 cm . It does not appear that this use of energy in osmoregulation is limiting to growth of the population. Other workers have also reported finding larger Rangia in the areas of very low salinity (Gunter, 1961). In addition to this osmotic control Rangia demonstrated considerable capacity for volume control. As mentioned in the results, the volume of the tissue water changed only 7 per cent despite a 97 per cent salinity dilution. This change is much smaller than would be expected if the organisms were behaving as simple osmometers (Pierce, 1971). Therefore, volume control

72 must be occurring throughout the salinity range even though Rangia only osmoregulated at the lower salinities. The concept of euryhalinity has been defined by Florkin and Schoffeniels (1969) as "the extent to which an animal is able to maintain constant its cell volume when placed in media of various salinities." If cell swelling occurs to any extent the organism must suffer some loss of its mechanical functions (Lange, 1964). Thus one would expect to find a mechanism for volume control in those organisms faced with fluctuating salinities even though they may not exhibit complete osmoregulation or indeed may not osmoregulate at all. Examples of the last-mentioned situation are mussels of the genus Modiolus. As Pierce (1970) has shown the osmotic concentrations of the body fluids of Modiolus vary linearly with those of the environment (osmotic conformity). Yet Modiolus is also a volume regulator in dilute salinities (Pierce, 1971). The volume response of Modiolus proved to be unidirectional. After a group of Modiolus demissus granosissimus were acclimated to 3°/ooS for 3 weeks they were returned to 36°/ooS and within 12 days all of the mussels had died (Pierce, 1971) . The explanation for this was a loss of cellular solute by the mussels while acclimating to a low salinity, resulting in a compounded water loss when returned to the normal high salinity. It appears that acclimation to very low salinities is a physiological commitment by M. cl. granosissimus, the unidirectional volume response making it irrevocable (Pierce, 1971). Rangia demonstrates no such limitations on its acclimation ability, but it does take longer to acclimate to higher salinities than to lower salinities. It may retain this flexibility in acclimation by virtue of its unique ability to osmoregulate at the low salinities, thus reducing a significant portion of the water loss when exposed to higher salinities. In addition Rangia1s habit of closing its valves and only siphoning for short intervals for the first 2 or 3 hours after exposure to more saline media may allow time for an increase in the intracellular solute concentration.

73 Allen’s (1961) study suggests that the loss of tissue water and gain of inorganic ions by Raiigia reaches a maximum in an environment of 20°/ooS, however 25°/oo was the highest salinity used in this earlier work and the subject was not pursued in detail. The present study demonstrates that this is not the case, as the loss of tissue water and the gain of inorganic constituents continued up to the highest experimental salinity, 32°/oo (32 ppt) . This increase in the percentage of ash in the tissue agrees with a series of investigations by Fredericq (1904), Krogh (1965), and Fox (1941). These workers demonstrated that euryhaline molluscs establish an equilibrium with their environment with respect to inorganic ions. Such an equilibrium suggests that if intracellular osmoregulation is to occur, the solute being used must be organic in composition. Recent data from several studies on euryhaline bivalves have indicated that the source of this organic solute is the intracellular free amino acid pool (Awapara, 1962; Florkin and Schoffeniels, 1969). Allen (1961) has further supported this concept with the evidence that as the salinity of the medium is increased, the concentration of the amino acids present in Rangia also increases. He also found a definite pattern in the relative abundance of the individual amino acids. The major amino acids in order of decreasing con­ centration were always: alanine, glycine, glutamic acid, and aspartic acid. Effects of Salinity on the Uptake and Accumulation of Glycine

1. Introduction The concepts and literature concerning uptake and utilization of dissolved organic materials by estuarine and marine inverte­ brates have been reviewed by Stephens (1968). Stephens (1964) found that the capacity of two polychaetous annelids, Nereis 14 limnicola and N_. succinea, to take up C -labeled glycine was related to thevchlorosity of the exposure media. The uptake of glycine by N. limnicola was significantly increased at salinities above the range at which ion regulation begins (Smith, 1959; Stephens, 1964). The literature regarding the utilization of free amino acids in the osmoregulation of lamellibranch molluscs has been reviewed by Virkar and Webb (1970). Gilles (1972) showed that amino acids played a part in the cellular osmoregulation process in the intertidal bivalves studied. The concentration of free amino acids increased with increasing salinities. Allen (1961) demonstrated that Rangia cuneata taken from different salinities contained quantities of alanine, aspartic acid, glutamic acid, and glycine which increased as the salinity increased from 3 to 17°/oo (ppt). There was a decrease in the concentration of these amino acids as the environmental salinity increased from 17 to 25°/oo S.

2. Materials and Methods a. Uptake of glycine In all of the following experiments the clams were selected for uniform size (55 + 3 mm) to reduce variation due to weight. A random selection of 44 clams within these size limits gave an ash-free dry weight of 1.31 + 0.27 grams. 14 Whole animals were tested for their ability to remove C labeled glycine at various salinities by exposing them for 5 hours in separate 250 ml beakers with 200 ml of water of each 5 12114 salinity, containing 1 x 10 molar C -glycine (10 yc

75 of C /liter). After exposure they were shucked and the meat rinsed for approximately 5 seconds in tap water, blotted on "Kimwipes," weighed and placed in 100 ml of 80% ethanol (ETOH)• After 48 hours of extraction in ethanol, two 1-ml aliquots were analyzed for radioactivity with a Beckman 200-LS liquid scintil- 14 lation counter and the C activity was expressed as counts per minute/mg ash-free dry weight. A long term experiment was conducted using Rangia acclimated to salinity of 15 ppt (15°/oo S) and a flowing system. Six liters of 15°/oo S water was filtered through a 0.45y Millipore filter and glycine was added to give a final concentration of 5.2 X 10~6 molar C12 + C14-glycine (16.7 yc of C14/liter). This solution was pumped at a rate of 1 ml/min by a 4-channel peristaltic pump past 4 Rangia in separate chambers and the effluent collected in a fraction collector. The medium entered at the bottom of the chambers and was drawn off at the top. The source solution and each chamber was stirred by magnetic air-powered stirrers to prevent heating. Four rows of 15 ml test tubes in the fraction collector received the effluent and the drop counting mechanism caused the machine to advance every 12 minutes or 12 ml of effluent. The system was run for a period of 14 hours and at termination the radioactivity of one 1-ml sample from each tube was counted. In addition, the 4 animals were analyzed for alcohol-soluble radioactivity as discussed above. The radioactivity of each effluent sample from zero time to 14 hours was plotted against time. Rangia gill tissue was also utilized in an uptake experiment. These lamellibranch molluscs possess two pairs (demibranchs) of gills, and since each pair was treated separately, this demi- branch will be referred to below as a gill. The gills were removed from animals which had been acclimated to salinities of 1, 2, 5, 10, 15, 20, 25, and 32°/oo (ppt). They were placed in beakers containing aerated water of the corresponding salinity until all gills had been dissected. Twenty gills from each of the salinities were placed in separate small disposable plastic 76 dishes with 5 ml of water of the proper salinity, containing 3.75 x 10 ^ molar C ^ + C^-glycine (33.2 yc of C^/liter) . At time intervals of 10, 20, 30, 60, and 90 minutes, four gills from each salinity were removed, rinsed in three changes of 75 ml of water (5, 10 and 5 minutes, respectively), blotted, weighed and placed in 10 ml of 80% ethanol for 72 hours. These extracts were then counted for radioactivity together with the initial and final samples of the exposure media. After appropriate calculations, the radioactivity of the gill in cpm per mg ash-free dry weight was plotted against time for each salinity. Experiments were conducted to determine the rate of glycine uptake by Rangia at several salinities. In each separate experiment for each salinity, five one liter containers were prepared which held 5 animals; all 25 had previously been adapted to the particular salinity for at least 7 days. After approximately one hour or when all animals were actively siphoning, a small volume of solution was added to each liter of water. Mixing occurred quite rapidly as a result of the aeration supplied to each container. The final solutions contained the same amount of 14 14 radioactivity in the form of C -glycine (20 yc of C liter) 14 12 but the total concentration of glycine (C plus C ) and each -3 -4 -4 -5 -5 container varied (10 , 5 x 10 , 1 0 , 5 x 10 , and 10 molar). The Rangia were exposed for one hour in these concentration gradient experiments and after the usual extraction procedure, the radio­ activity of each group was determined and the results expressed as follows:

cpm/gm/hr(animals) concentration _ Velocity cpm/ml (initial medium) (moles/ml) (moles/gm/hr)

It was then possible to prepare Lineweaver-Burke curves of the data, where the reciprocal of the velocity is plotted against the reciprocal of the substrate concentration. As noted by other workers (Ste­ phens, 1967), the use of this means of analysis is not a suggestion that uptake is an enzyme dependent process but this type of plot

77 illustrates the differences in uptake with concentration and with salinity. To examine the use of amino acids in the osmoregulatory 12 activities of the animals, they were first exposed to C + glycine at a concentration of 2 x 10 ^ molar (20 pc of C / liter) for four hours at a salinity of 22 ppt. All animals had been previously acclimated to this salinity for four days. After the exposure, three were treated as described for extraction, while the remainder were divided into groups of three and placed in separate 250 ml containers at salinities of 2, 4, 6, 10, 16, or 22 ppt for 24 hours. Samples of the medium from each of the 18 containers and six salinities were taken at 0.5, 1, 5.5, 16, and 24 hours and counted. The data are expressed as the amount of radioactivity released by each salinity group in cpm per mg ash-free dry weight. In order to determine the fate of the glycine after uptake with regard to both salinity and time, the following experiment was conducted. Rangia acclimated to salinities of 1, 2, 5, 10, and 15 ppt were exposed for 2 hours in a 1.02 x 10 ^ molar solution of C"^ + -glycine (16.7 yc of C^/liter). After exposure the clams were washed free of any surrounding activity by moving them through 6 successive changes of water of the appro­ priate salinity. The initial incubation medium had an activity of 17,000 cpm/ml and the 6th wash had an activity of approximately 20 cpm/ml, so it appears that the cleansing procedure was quite effective. The clams were then placed in glycine-free water of the appropriate salinity for periods up to 99 hours. Alcohol- 14 soluble and alcohol-insoluble C -labeled components were analysed at 3, 24, 48, and 99 hours after exposure. After extraction of the alcohol—soluble or free pool compounds the total activity of the incorporated or ETOH-insoluble components was measured using the following procedure. Tissue was removed from the alcohol, washed with alcohol, then blotted with firm pressure on "Kirawipes." It was then rinsed in tap water and homogenized in a micro Waring blendor with enough 80% ethanol to bring the total volume of the

78 homogenate to 50 ml. One ml of this homogenate was counted after being solubilized with 1 ml of Protosol. Alcohol-soluble and insoluble activities were corrected by the use of quenching curves. The results of this experiment were expressed as the relative percentages of the total activity in the free pool and alcohol-insoluble portions. This method facilitates comparisons between salinities and compensates for individual differences in total activity. To determine the fate of the glycine, the alcohol-insoluble fraction was quantitatively separated into protein, nucleic acids, lipids and polysaccarides (glycogen). The basic methodology (outlined in Figure 9) used was that of Shibko et al. (1967) with modifications as suggested by Graff (1970). After their quali­ tative separation the four tissue fractions were prepared for counting as follows: First, the dried components were put into solution with 5 ml of 10% KOH, using heat where necessary. Then 1 ml of each fraction was neutralized with glacial acetic acid and counted using the Beckman LS-20QB liquid scintillation system with an Aquasol cocktail. This uniform preparation for counting greatly reduced any differences in the quenching properties of the different fractions.

3. Results a. Uptake of glycine by whole animals In one of the preliminary experiments, 20 Rangia acclimated to 12°/oo S (ppt) were exposed to a 2.6 x 10 ^ molar solution of 14 12 14 C and C glycine (20 yc of C /liter). This concentration gave a specific activity of 14,268 counts per minute per ml (cpm/ml). After one hour of exposure to 2 liters of water the —8 clams had reduced the activity to 629 cpm/ml (1.15 x 10 molar). During the next 4 one-hour intervals the activity of the media was 503, 498, 500, and 470 cpm/ml, respectively. One could conclude from these data that while Rangia rapidly takes up glycine at concentrations of 1 x 10 ^ molar and above it is not —8 effective at removing the material at or below a 1 x 10 molar concentration. 79 1. Extract 0.8 gm wet weight of homo­ genized tissue in 10 ml of 80% ETOH for 24 hours.

2. Centrifuge* and pour off supernatant, repeat with 5 ml wash of 80% ETOH.

3. Extract lipids from pellet with TO ml of ether - 70% ETOH (3:1 v/v) for 18 hours with frequent mixing, centrifuge, re­ peat with 5 ml of ether - ETOH for 2 hours. '

f ------T Combined Supernates (lipids) Pellet

1. Evaporate in vacuum desiccator 1. Add 5 ml TCA (trichloroacetic (insert a small beaker of paraffin acid) and mix well. oil in desiccator).

2. Weigh when dry. 2. Heat to 80°C for 30 minutes, then hold at 50°C for 2 hours

3. Cool and centrifuge, repeat with 5 ml of cold TCA.

f------T Combined Supernates (glycogen and nucleic acids) Pellet (proteins)

1. Make the supernate 52% ETOH. 1. Wash twice with 10 ml of absolute ETOH.

2. Allow to stand at 0°C for 18 hours. 2. Dry to constant weight at 95°C and weigh.

3. Centrifuge * 'Jr Supernate (nucleic acids) Precipitate (glycogen)

1. Remove TCA by extraction with 1. Wash twice with 5 ml of 30 ml of ether in separatory absolute ETOH. funnel. Repeat 4 times.

2. Evaporate to dryness at 95°C 2. Dry to constant weight at and weigh. 95°C and weigh.

* All centrifugation at 3,000 rpm for ten minutes.

FIG. 9. - Flow sheet for biochemical separation of the major tissue components .

8o Uptake of glycine versus salinity is plotted in Figure 10. The plotted values represent the ethanol-soluble activity after -.5 14 5 hours of incubation in a 1 x 10 molar solution of C + 12 -3 C glycine (700 cpm = 1 x 10 micromoles). The resulting activity of the tissue after 5 hours remained relatively constant (450-550 cpm/mg at or about 0.7 y moles per gram) at salinities from 20 to 6°/oo, but decreased rapidly at lower salinities. The uptake at 6°/oo S was shown to be 3 times that at 2°/oo S. The uptake levels at the higher salinities represent a 75-fold accumulation of glycine over that in the ambient medium. The nature of the uptake process over time is shown in Figure 11. From these data it appears that the process is a continuous one, at least at the concentrations and time intervals tested. This experiment was conducted at 15°/oo S with the glycine being introduced via a flow-through system. The 4.4 micromole (y mole) differential, between source and outflow, presented on the graph, represents a rate of glycine uptake equal to 0.264 y moles per hour (calculated using the flow rate of 0.06 L/hr and the differential of 4.4 y moles/L between the glycine concentration of the source and the outflow water). Using the average ash-free dry weight of the clam tissue (1.58 gm) the uptake was 0.161 y moles per gram per hour. This constant rate of removal was maintained for 15 hours, after which the experiment caused the oxygen concentration to be reduced to about 1.5 ml 0^/L; however, the rate of glycine uptake remained uniform as the oxygen concen­ tration decreased indicating that the uptake process is independent of the oxygen concentration down to a relatively low level. Stephens (1963) supports this conclusion stating that the rate of accumulation of amino acids is independent of the oxygen tension of the medium for periods up to 24 hours. His data were obtained from experimental work with the intertidal polychaete Clymenella torquata.

8l CPM MG" FIG. 10.Ethanolsolubleradioactivityof - Rangia whole 100 0 1 2

------

where n=6. where oa lcn fr5hus S.D. shownas +, forglycine5molar hours. as a function of salinity. Exposed to110' Exposedx afunctionasofsalinity. 1 ------4

1------1 6

. SALINITY %. 8 ------

82 1 1------1 0

12 ------7 4 ± A

14 1 ------

1------r 16 I

W f 18

I “ ■A±n 20 5 6-,

5- f r B-g -SOURCE CONCENTRATION-— — —

(0 a> I 4- a.

< 3_1 • a Q Hi 2 D . 2-1 ° o #0 ° s o a o° ; 8 Ui z 1 ° □ ! « I -° o ° O %

> • □ 0 1 -I o ~ I ----- 1------1------1------1 1------1------1------1 1------1------1 1 1------1 J% A. 20 60 100 140 180 220 260 300 960 TIME (minutes)

FIG. 11. - Glycine uptake by 4 individuals in separate flow-through chambers.

83 b. Uptake of glycine by isolated gill tissue An experiment was conducted where after dissection the gills were placed in the acclimation salinity of the whole clams (Figure 12). The utilization of gill tissue made it possible to use much shorter incubation intervals than was possible with whole animals• An incubation interval of less than 2 hours was not feasible with intact Rangia because of the variability in the clams1 response to slight disturbances such as the addition of the labeled glycine. After 90 minutes of incubation in a 3.75 x 10 —6 molar solution of plus C^-glycine the gills at 1 and 2°/oo S (1 and 2 ppt) had taken up 0.56 and 0.75 y moles of glycine per gram of tissue, respectively. The data were converted from cpm to y moles using a ratio of 1000 cpm/mg = 0.74 y moles/g. The gills at 5°/oo S were intermediate in their uptake capacities as were whole clams' uptake at 5°/oo S (Figure 10). The reduction in uptake at 32°/oo S is probably a function of salinity stress which will be discussed later. The uptake process in the gills exhibits a slight leveling off after 60 minutes which implies that saturation is occurring. The leveling off in uptake is pronounced with the gills in the lower salinities and those in 32°/oo S. This phenomenon is not a function of the glycine concentration in the media as in no instance was _6 the glycine in the incubation media reduced below a 1 x 10 molar concentration, which is well above the minimum concentration for effective uptake.

c. Velocity of glycine uptake The relation between the ambient glycine concentration and the rate of uptake at different salinities by whole animals is shown in Table 5. From these data it is possible to plot the reciprocal of the rate of uptake against the reciprocal of the concentration in the ambient medium. The regression lines for this double reciprocal plot are shown in Figure 13. The high correlation coefficients (above 0.98).demonstrate a very good fit to the Lineweaver-Burke plot. The concentration at which the uptake of glycine was half maximal (Km) was approximately the same Table 5. The relationship between the ambient glycine concentration(s) and rate of uptake (V) at different salinities. Each V value represents the mean of 5 individuals. Rm represents that concentration of glycine at which uptake is half maximal (V max). Slope Correlation s V Of V max Km Coefficient —4 U moles/ 1/v p moles/ X10 o/oo X10 ^ Molar gm/hr versus gm/hr Molar 1/s

2 10.0 0.900 0.491 0.851 0.418 0.993 5.0 1.200 1.0 0.457 0.5 0.445 0.1 0.166 2 10.0 1.253 0.464 1.025 0.476 0.987 5.0 0.819 1.0 0.633 0.5 0.545 0.1 0.178 4 10.0 2.638 0.342 2.849 0.974 Q .989 5.0 1.905 1.0 1.478 0.5 1.078 0.1 0.264 6 10.0 2.859 0.159 2.079 0.331 0.987 5.0 2.398 1.0 1.289 0.5 1.054 0.1 0.492 10 10.0 - 0.172 2.703 0.462 0.992 L n O • 2.839 1.0 1.468 0.5 1.608 0.1 0.478 1 85 Table 5 (cont.)

Slope Correlation S V of V mâx Kiû Coefficient y moles/ 1/v y moles/ 4 X1(T o/oo Xio-4 Molar gm/hr versus gm/hr Molar 1/s

11 10.0 2.710 0.137 2.288 0.314 0.986 5.0 2.263 1.0 1.772 0.5 1.171 0.1 0.561

86 F G 12.soluble -radioactivitytissueEthanolgillfunctionofas aIG. CPM MG" f aiiyadtm. S.D. 7% valuesapproximatelyof were time.of salinityand theplottedpoints, n=4. where 87

o ®

FIG. 13. ~ Straight-line regression analysis of concentration (S) and velocity (V) relationships at various salinities.

88 (0.3 to 0.9 x 10 molar) within the range of salinities tested (2 to ll°/oo). However, the velocity at which uptake was maximal (V max) was significantly lower at 2°/oo S (less than one half) than those velocities exhibited by clams at higher salinities. Although these data are in general agreement with those presented in other sections above, the suppression of uptake at 4 and 6°/oo S is not as evident. Since considerable earlier data have shown the continuous decrease in uptake from 10°/oo S down to 2°/oo S, perhaps these concentration gradient experiments are not as applicable to the determination of salinity effects. The response of Rangia to a reduction of the ambient 14 salinity after being incubated with C -labeled glycine for 5 hours at 22°/oo S is represented in Figure 14. With decreasing salinity there is a corresponding increase in the radioactivity being released into the media. The greatest release of radio­ activity which occurred after 24 hours was at 2 and 4°/oo S (762 and 440 cpm/mg, respectively). The release at 6°/oo S was similar to that at 10°/oo S (249 versus 255 cpm/mg) and only slight amounts of radioactivity appeared at the higher salinity of 16°/oo. The 42 cpm/mg obtained at 22°/oo S was due not to release but to small amounts of "hot" exposure medium carried over when the clams were transferred to fresh medium. The low levels of released activity at the 5.5 hour interval for the clams at 2 and 4°/oo S were probably due to the extreme salinity inhibiting the siphoning activity of the clams. d. Fate of accumulated glycine The first phase of a study to determine the fate of the glycine after uptake was to examine the percentage of activity that remains as free pool compounds and the percentage that is incorporated into the tissue components. These percentages are plotted versus time for salinities 1 through 15°/oo (1-15 ppt) (Figure 15). The components that are soluble in 80% ethanol have been defined as free pool components and the insoluble

89 900-

oi s

Q. o

5J5 16 24 TIME (hours)

14 Fig. 14. - Release of C -activity as a function of salinity and time. Rangia previously exposed for 5 hours to cl4-glycine at 22°/<>o S. Vertical bars are standard errors, where n=3.

90 FIG. 15. - Percentage of total activity in the ETOH-insoluble fraction as a function of salinity and time. Vertical bars indicate S.D., where n=4.

91 components as incorporated compounds. Only the incorporated or ETOH-insoluble fraction is plotted, as the pool or ETOH-soluble fraction is the reciprocal of Figure 15. The most rapid and the greatest incorporation occurs at the lower salinities of 1, 2, and 5°/oo, resulting in approximately 75% of the total activity being converted to ETOH-insoluble components 49 hours after exposure to the labeled glycine. During the same time interval the clams in 10 and 15°/oo S had only incorporated 54 and 32%, respectively, of their total glycine accumulation. At 99 hours the figures for these clams at 1, 2 and 5°/oo S had not changed appreciably. However, incorporated radioactivity had increased for the Rangia at 10 and 15°/oo S, being 58 and 44%, respectively. The total (free pool and incorporated) activity, for the 99 hour interval, is plotted against salinity in Figure 16. The total 14 C activity due to accumulation of labeled glycine was 3.5 times greater at 10 and 15°/oo S than at l°/oo S and the total activity taken up by the animals decreases sharply below 10°/oo S. A comparison was made between the total activity present in the clams at 24 hours after exposure and that at 99 hours. The results of the comparison are presented in Table 6 as the percent decrease in the total tissue activity and as the equivalent decrease in moles of glycine per mg of tissue. It appears that with increasing salinity there is an increase in the utilization as well as uptake of glycine.

Table 6. The percentage decrease in radioactivity and moles of glycine in Rangia tissue that occurred at various salinities between the 24th and the 99th hour after 14 exposure to C -labeled glycine.

Decrease in Glycine Percent Decrease in Total Radio- (moles x 10-9 of Glycine/ Salinity ______activity______mg Tissue)______1 9.2 0.015 2 21.3 0.067 5 45.5 0.260 10 53.8 0.419 15 69.2 0.108

92 %oSALINITY

FIG. 16. - Total C-^-activity 99 hours after exposure to glycine at various salinities. Vertical bars = S.D., where n=4.

93 In Figures 17 and 18 the percentages of the incorporated activity which were found in the protein, nucleic acid, glycogen, and lipid fractions are presented for the 3 and 99 hour intervals, 14 respectively, after exposure to C -labeled glycine. These fractions made up the following percentages by weight of the total tissue components: protein, 59.4 +4.9; nucleic acid, 15.8 +1.3; glycogen, 19.0 + 5.3; and lipid, 5.7 + 1.8. Thé + refers to the standard deviation, where n = 39. These percentages total very close to 100% which was expected, since the free pool compounds were extracted before analysis and the inorganic matter was subtracted from the total weight. There was no significant difference in the percentages of the tissue components at the different salinities. There was, however, a significant difference in the fate of the labeled glycine with regard to both salinity 14 and time. After three hours of exposure to C -glycine those clams at 1, 2 and 5°/oo S had used approximately 90% of the incorporated glycine in protein synthesis, while the clams at 10 and 15°/oo S had used only 75%. This difference was accounted for by a higher percentage of labeled nucleic acids in animals from the 10 o 14 and 15 /oo salinities. After 99 hours the percentage of C in the protein of the low salinity clams had dropped to 87% and the percentage in the clams at 10 and 15°/oo S had increased to 93%. The nucleic acid fractions at 10 and 15°/oo S had de­ creased to a level where there was no significant difference with 14 salinity at 99 hours. The percentage of C activity in the glycogen fractions had increased from 1 or 2% to 5 or 6% at the low salinities. As pointed out in Table 6 the loss in the total tissue activity after 75 hours ranged from 9.2 to 69.2%, depending on salinity. This loss of activity was not accounted for by an increase in the media activity, thus the C^ must have been expelled as C“^ 0^, indicating metabolism of labeled compounds. FIG. 17.the -PercentageofactivityinETOH-insoluble X ETOH—INSOLUBLE C IN TISSUE FRACTIONS 9 5 aiu tsu fractions3 tissue aftervariousexposurehours to C-^-glycine. Bars = S.D., Bars=n=4. where toC-^-glycine. XoSALINIT Y XoSALINIT 95

95

90

85

80

75

25.

20

15

10

5

0

%» SALINITY

ig. 18. - Percentage of ETOH-insoluble activity in the various tissue fractions 99 hours after exposure to Cl^-glycine. Bars = S.D., where n=4.

96 4. Discussion In the present study, a consistent pattern emerges when the relationship between the uptake of glycine and the salinity of the medium is studied. A high and rather consistent level of glycine uptake was exhibited by those Rangia in salinities at or above 10°/oo, but a rapid decrease in uptake occurred at salinities below 10°/oo (uptake at 10°/oo S was 3.5 times that at 2°/oo S). This pattern very closely coincides with the previously established pattern of osmoregulation versus salinity, that is, uptake of glycine decreases sharply at salinities below 10°/oo, which is approximately the point at which osmoregulation begins. This observation is in support of Stephen's (1967) statement that possibly the processes which underlie osmotic regulation are incompatible with the accumulation of amino acids from the ambient medium. This incompatibility is apparently demonstrated by the inability of Raiigia to accumulate large amounts of glycine from the ambient medium when in salinities below 10°/oo and when working against rather extreme concentration gradients. The reason for this dif­ ference in uptake is still hypothetical; it may be due to energy competition between the osmoregulatory and uptake processes, or the reduction in specific ions as the salinity is decreased may play a significant role. Uptake was shown to be essentially zero at the concentration —8 of approximately 1 x 10 moles of glycine per liter. Estimates of glycine concentration in coastal waters are in the range of from 1 x 10 ^ to 1 x 10 ^ molar, while Stephens (1963) has reported a concentration of 1 x 10 ^ molar glycine from shallow mud flats. It would appear that an excess of glycine and other amino acids is available for uptake by Rangia and other soft-bodied marine invertebrates inhabiting the coastal and estuarine waters. The results of uptake experiments with gill tissue indicate that even short term uptake (less than one hour) is affected by salinity. The gills demonstrated an immediate uptake response, which was relatively linear over a period of 90 minutes. Again,

97 the glycine uptake at salinities below 10°/oo S was suppressed. It seems likely that gill tissue is the active site for uptake of amino acids by Rangia. It is interesting to note that there was some decrease in uptake at 32°/oo S, indicating that this salinity is somewhat stressful. This result compares with the critical temperature data, discussed later under respiration, since oxygen consumption at 32°/oo was less than that at 25°/oo at 35 and 40 C. The release of accumulated glycine back into the surrounding media after exposure to hyposmotic stress is in further support of the theory that osmoregulation and amino acid uptake are incompatible. The maximum release of glycine occurred at salinities below 5°/oo which corresponds to the salinity range over which Rangia exhibits the maximum osmotic differential between blood and external medium. Allen’s (1961) listing of the amino acids occurring in Rangia is unusual in its conspicuous absence of taurine which has been consistently reported as a major component of the amino acid pool in a long series of marine invertebrates including many lamellibranchs (Simpson, 35 et al., 1959; Lange, 1963; Bricteux-Gregoire et al., 1964; Lynch and Wood, 1966). Gilles (1972) has stated that !,The important part played by taurine as a cellular osmotic effector appears to be of general occurrence in the phylum Mollusca.” In an experiment involving the tracing of Rangia’s metabolic pathways Allen and Awapara (1960) demonstrated that taurine was formed, but was rapidly metabolized and excreted as an unknown compound. It is perhaps significant here to note that taurine has not been found in extracts of either fresh water or terrestrial molluscs (Simpson, 1959), which also demonstrate osmoregulation and indeed depend upon it for survival. Using the data on respiratory rates versus salinity and the data on rate of glycine uptake it is possible to calculate the contri­ bution of dissolved glycine to the energy budget of Rangia. At a glycine concentration of 4.6 x 10 5 moles/liter (the concentration at which uptake was half maximal or Km) the rate of glycine uptake at 10°/oo S was 101 yg of glycine/gm tissue/hr. As 1 mg of glycine requires roughly 1 ml of 0^ for complete metabolism (Stephens and Virkar, 1966) this amount of glycine represents 8.43% of the total 0^ consumption by Rangia. Thus, glycine taken up from the ambient medium could represent as much as 8.43% of RangiaTs energy needs. At 2°/oo S the glycine taken up was equivalent to 3.38% of the total 0 -5 ^ consumption. The concentration of 4.6 x 10 moles/liter is above the reported value of 2.5 x 10 ^ moles/liter in mud flats; however, -5 -4 it is below the 6 x 10 to 10 moles/liter concentration that has been reported for total free amino acids in the estuarine environment (Stephens, 1963). If one assumes that other amino acids as well as glycine may be taken up simultaneously and utilized, the above values for the contribution of amino acids to the energy budget of Rangia may be increased. This assumption has been shown to be valid with the polychete Clymenella (Stephens, 1963). The evidence presented in this portion of the study demonstrates that the glycine is rapidly synthesized into protein and there is some indication that at least at the higher salinities the formation of nucleic acids may be an intermediate step. In order to explain the differences in the distribution of radioactive material derived from glycine as they pertain to salinity, much more sophisticated biochemical analyses would be required. It is interesting to note these variations in relation to salinity, but a thorough investigation of all metabolic pathways is beyond the scope of this project. Another interesting and unexpected result of this work is the fact that the rate at which the accumulated glycine (alcohol-soluble and insoluble fractions) is metabolized was found to be related to salinity. This metabolic or turnover rate increased as salinity increased. The glycine-derived radioactivity was metabolized by Rangia at 15°/oo S at a rate 7.5 times faster than those maintained at l°/oo S. It is not possible to explain why this occurs, but the slower turnover rate at low salinities may be one factor contributing to the larger size of individuals from Old River Lake. However, as stated above, the uptake of glycine was much reduced at low salinities, thus negating the advantage of slower turnover rate.

99 Effects of Salinity and Temperature on Respiration

1. Introduction Many studies on effects of salinity and temperature variation on the respiration of bivalves have been conducted (Percy et al., 1971; Giese, 1969 (review); Van Winkle, 1968; Read, 1962; Bielawski, 1961; and Hopkins, 1949). However, to date no respiratory work has been conducted utilizing Rangia. Van Winkle (1968) has shown that the effect of salinity on the oxygen consumption of bivalve gill tissue varies from one species to another. His work demonstrated that respiration in Crassostrea virginica and Mytilus edulis remained relatively constant over a salinity range of 5-30°/oo, whereas Mercenaria mercenaria and Modiolus démissus responded to low salinities by an increase in respiration. The data of Percy et al. (1971) are in conflict with those of Van Winkle as Percy demonstrated that a dilution of the sea water medium stimulated gill respiration in Crassostrea virginica. In this study, the respiratory rates of both isolated gill tissue and whole Rangia under a variety of environmental conditions and acclimation periods were investigated. The results should provide a firm basis for future studies utilizing respiration as an indicator of physiological condition.

2. Materials and Methods a. Whole animal respiration A system built around the Yellow Springs Instrument Oxygen Meter (Model 154) was used to measure the 0^ consumption of whole Rangia. One unit of the basic system consists of a 700 ml chamber containing a Teflon stir bar, a glass pedestal, and a YSI 02 and temperature sensing electrode (probes)• The electrode was inserted through a tapered rubber stopper which served to seal the respirator chamber. Five identical respiratory chambers were set up and connected to the Model 54 YSI Oxygen Meter via a switching box. The system makes possible continuous analysis of the 0^ consumption in the 5 chambers while using one oxygen meter. The meter supplies power continuously to the individual probes while

100 the optical readout was intermittently switched from one probe to another. The readout was recorded manually in the present study, but the system could easily be automated. The electrodes are compen­ sated internally for temperature effects on both membrane permeability and oxygen solubility in water. Calibration of the oxygen electrodes was done using water saturated by aeration as recommended by the Yellow Springs Instrument Company. This procedure was checked, using the Winkler method (Standard Methods, 1960), and was found to be satis­ factory. The polarographic type of oxygen electrode requires agitation of the sample in the vicinity of the electrode as dissolved oxygen is being reduced at the cathode (Bielawski, 1961). Under quiescent conditions a gradient in the dissolved oxygen content would be established on the sample side of the membrane, resulting in an atypical response. Mixing in the respiratory chamber was provided through the use of air-powered magnetic stirrers. These stirrers were connected in series from the same air source and proved to be very satisfactory in both maintaining a uniform stirring rate and preventing any heat buildup, as they could be submerged in a water bath. The electrodes exhibited a very slight drift over time, but this was found to be constant and was corrected for in all experiments. A comparison between Millipore-filtered and unfiltered freshly mixed water was made and in neither case was there any measurable oxygen consumption. Thus unfiltered freshly mixed water was used in all experiments. Tests were made on the oxygen consump­ tion of clean empty Rangia shells and no measurable oxygen consump­ tion occurred even over a period of 7 hours. Temperature regulation was achieved by immersing the chambers in a constant temperature water bath (+ 0.05° C). The clams were scrubbed free of all attached algae or other growth and were then placed in the respiratory chambers on a glass pedestal. They were supplied with filtered aeration until they had been siphoning steadily for one hour; at this time the cork containing the oxygen electrode was inserted with care to avoid trapping any air bubbles. The oxygen concentration in each chamber was measured at 15 minute intervals for a 2 or 3 hour period. The

101 respiratory rate was then expressed in ml of 0^ per gram ash-free dry weight per hour (Q02)• Calculation of 0^ Consumption: Appm 0^ X Vol of media in liters * no. hours - 0.047* ash free = mg 0o/gm/hr X 0.698 = m/09/gm/hr dry wt. >^2 ' ^ ~ 2

*0.047 = correction factor for probe drift

The clams used were selected for uniform size, as mentioned under amino acid uptake methods. However, to prevent the possibility of error due to weight differences all respiratory rates were corrected to a standard weight following the method of Davies (1966). A logarithmic regression curve describing the relation­ ship between oxygen consumption and weight was constructed using data from 46 individuals. The b value (slope of the regression line) obtained was -0.83546; (the correlation coefficient = 0.838) and was used to correct all respiratory rates to a standard weight of 1.313. This b value is in good agreement with those values obtained for a variety of other invertebrates (Newell, 1970)• Kennedy and Mihursky (1972) working with 3 estuarine bivalves, ecologically similar to Rangia, have shown that the variation of b with temperature is not significant within the temperature range used in this study. Therefore the b value obtained at 22° C in the present study has been used to correct all rates. The data of Roberts (1957), Davies (1966), Sassaman and Mangum (1970), and Green and Hobson (1970) also indicate that b is relatively independent of temperature. b . Gill respiration The respiratory response of the gill tissue was measured utilizing a Gilson Medical Respirometer. The gills were placed in 7 ml Warburg flasks containing 3 ml of medium plus a C02 absorbant in the center well (0.5 ml of 10% KOH). A filter paper wick was added to the center well of the flask to facilitate C02 absorption. A shaking rate of 112 cycles per minute was used throughout the study.

102 Gills were dissected from clams of a uniform size that had previously been acclimated to the required salinity for at least 7 days. The gills were removed by peeling back the mantle and then snipping the thin septa that connect the gill to the body of the clam. Using this procedure it was possible to remove the entire pair of gills from one side of the clam with almost no tissue damage. Early attempts at using fragments of a gill proved unsuccessful because of the high variability in the respiratory response. After removal the gills were placed in dishes of clean water of the ex­ perimental salinity, usually the same as the acclimation salinity. When all the gills were dissected (a process requiring no more than 30 minutes) they were placed in the Warburg flasks together with 3 ml of Millipore-filtered water. The system was allowed to adjust for 30 minutes and then readings were taken every 30 minutes for 3 to 5 hours. The results were corrected to STP and expressed as ml 0^ per gram ash-free dry weight per hour ( C ^ ) •

3. Results a. Whole animal respiration As no base line respiratory information on Rangia was available it was necessary to answer certain basic questions before the effect of salinity on respiration was examined. First it was es­ sential to look for the presence of diurnal variation in the res­ piratory rates. For a group of ten individuals the midnight through 2:00 a.m. respiration (QO^) was 0.78 + 0.12 (S.D.) and the noon through 2:00 p.m. was 0.71 + 0.11. In addition, the Q0^ of 5 clams was examined over an 18 hour period from noon through 8:00 p.m. and the Q0^ remained constant over the entire period. A flow-through system maintaining a constant 0^ concentration in the medium was used in this experiment. From these data it appears that Rangia do not exhibit the diurnal or rhythmic activity characteristic of many littoral invertebrates. Next the response to starvation was examined (Figure 19). There is a slight drop in the Q0^ between 2 and 10 days of starvation, but between 10 and 20 days after collection the Q0^ remained constant.

103 .0 -, _-c O) 1.8- <$ _ 1.6 - E ^ 14 - Z O 12- |,o. 0 .8 -

3 0. 6 - g 0 . 4 - ^0.2-

8 T I I” 2 5 10 DAYS AFTER COLLECTION

FIG. 19. “ Respiratory rate as a function of days of starvation. Bars = S.D., where n=5. (1) Respiration at various oxygen tensions As Rangia lives in an environment where wide variations in the oxygen concentration are the rule, it was desirable to determine the respiratory response at various levels of oxygen tension. As shown in Figure 20 the Q0^ remained relatively constant over a broad range of oxygen levels. These data indicate that the of Rangia is relatively independent of the external 0^ tension between approximately 2.5 and 4.5 ml of O^/L and imply that Rangia has some degree of control over its respiratory mechanisms (Nicol, 1967) . In all other respiratory experiments utilizing whole organisms, the respiratory rates were computed from data taken at between 4.50 and 2.75 ml 0^ per liter, since this procedure served to reduce individual variation.

(2) Respiration after anaerobiosis The respiratory response of Rangia after being held under anaerobic conditions is plotted in Figure 21. After being held for 2 hours under nitrogen the clams were placed in the respiratory chambers and the Q0^ measured at intervals after the clams had begun siphoning. The Q0^ rapidly decreased from an initial 2.16 to constant level of 1.10 within 100 minutes. This transitory increase in the rate of oxygen utilization after oxygen became available indicates the repayment of an oxygen debt incurred through the storage of incompletely oxidized compounds. This in­ complete oxidation is a result of temporary anaerobic metabolism.

(3) Effects of salinity on whole animals. The effect of salinity on the respiratory response of Rangia after acclimation to all experimental salinities for 10 days is shown in Figure 22. In view of the extreme salinity range the Q0^ remains quite constant. However, if the respiratory rates at the various salinities are compared to an average value (1.00) there are significant decreases in the Q0^ at 1 and 15°/oo S (0.78 and 0.78, respectively) and significant increases at 5 and 10°/oo S (1.32 and 1.21, respectively). This same pattern was repeated in

105 FIG. 20. - Respiratory rate at 15°/oo S as a function of dissolved oxygen concentration. Bars = S.D., where n=5. OXYGEN CONSUMPTION (ml 02 g-1 hr“1) FIG. 21. - Respiratory rate-Respiratoryat where n=5. where aerated water from 2 hours without oxygen. Bars = S.D., Bars= from2aerated oxygen.hours water without T M (minutes) IME 15°/oo S 107 and 23°C upon return toreturnupon

F G 2. eprtr aea 2° s ucino aiiy Dotted function 22.rate 23°C -aofsalinity.atRespiratoryas IG. OXYGEN CONSUMPTION (ml 02 g -1 hr-1) where n=5.where ie ma frt falslnte. as S.D., = Bars allsalinities.ofrate mean line: 108

35

0.595 0.047 + 0.595 0.657 + 0.100 + 0.657 0.455 + 0.129 + 0.455 30 1.283 + 0.103 + 1.283 1.499 + 0.240 + 1.499 1.194 + 0.124 + 1.194 25 1.000 + 0.150 + 1.000 1.039 0.084 + 1.039 0.825 0.069 + 0.825 20 0.787 0.329 + 0.787 0.685 + 0.071 + 0.685 0.059 + 0.822 15 0.579 + 0.175 + 0.579 0.428 + 0.086 + 0.428 0.097 + 0.618 10 at different salinity and temperature combinations. standard +indicates combinations. The temperature and salinity different at deviations, where n = 5. = n where deviations, 0.074 + 0.171 + 0.074 0.412 + 0.064 + 0.412 0.090 + 0.286 ity 2 able 7. Respiratory rate of Rangia in ml of of ml in Rangia hour of per rate 7. weight able Respiratory ash-free gram per 15 32 T Salin­

109 several replicate experiments. A low QC^ at 15°/oo S was consistently demonstrated when the synergistic effects of different temperature and salinity combinations were examined (Figure 23). The clams were transferred from 22 + 1° C and held at each experimental temperature for 2 hours while their respiration was measured. The standard deviations for these data are presented in Table 7. Again, even in the case of rather extreme variations in external conditions the respiratory response was quite uniform at the three salinities tested. However, there were several significant departures from this uniformity. The clams at 2°/oo S demonstrated an inhibition of their siphoning activity at 10° C. This is reflected in the very low QC^ (0.074) and also in the high Q 10 (60.55) shown in Table 8 for the 10-15 C interval. Several repetitions at the 2 /oo S and 10 C combination proved that this response was consistent. Since there was a quite linear increase in QO2 for the animals at 2 /oo S from 15 to 30 C, it would appear that they were perhaps experiencing less stress than those at 15 and 32°/oo S. The Q1q values for clams at 15 and 32°/oo S were relatively high for the 25 to 30° C interval (2.42 and 2.08, respectively). It should be noted that regardless of salinity the critical temperature was found to be in the range between 30 and 35° C. The extremely low QO2 values exhibited by Fang-ia at 35° C can only be the result of damage to the metabolic systems. Another interesting aspect of the results was the lack of any response by the clams at 15°/oo S to a 5 C increase in temperature. This same temperature increase (10 to 15° C) caused

a relatively large increase in QO2 by Rangia at 2 and 32 / 0 0 S .

Table 8. Q 10 of the respiratory rate for Rangia over various thermal intervals.

Salinity 10-15° C 15-20° C 20-25° C 25-30° C 1.42 2°/ 0 0 60.55 1.84 1.61 15°/oo 1.08 2.55 1.45 2.42 32°/oo 4.68 1.76 1.60 2.08

110 10 15 20 25 30 35 TEMPERATURE (°C)

FIG. 23. - Respiratory rate as a function of tem­ perature and salinity. S.D. listed in Table 7.

Ill b. Gill respiration In Figure 24 the respiratory response of the isolated gill is plotted against the time of exposure to a given experimental tem­ perature. After two .hours of acclimation to the apparatus and the new temperature, a relatively stable rate of respiration was ex­ hibited by gills at all temperatures except 40° C. The small amount of variation at 35° C and the extreme variation shown by the gills at 40° C indicate that the critical temperature for the gill tissue is between 35 and 40° C. It is interesting to note that whole clams demonstrated a similar response at a lower temperature (between 30 and 35° C). It would appear that gill tissue is more tolerant than muscle and other Rangia tissues. In preliminary studies, the Q0^ of gills from animals acclimated to 10, 15 and 25°/oo S for six and fourteen days showed no significant variation in relation to the length of acclimation. Therefore, the effect of temperature and salinity on gill respiration was examined using gills from clams acclimated for 9 to 10 days at the appropriate salinity. Respiratory rates were taken for 2 hours after a 2 hour acclimation interval at the experimental temperature. The respiratory rates for the different temperature and salinity combinations are plotted in Figure 25, and the standard deviations for these data are listed in Table 9. The gills were apparently damaged by a temperature of 40° C, as shown by the negative Q10 values listed in Table 10. The average respiratory rate for the gills at the lower tem­ peratures remained quite uniform from 5 to 25 /oo ST However, as the temperature was increased to 25° C and above, the variability of the QO^ with regard to salinity increased. The greatest effect of temperature was seen with gills at 2 and 5 /oo S. They exhibited a very marked increase in respiration at 35 C; the QO2 of the gills at 2°/oo S increased from 2.76 to 5.05 when moved from 30° C to 35° C. It should also be noted that at 35 and 40° C the Q02 of the gills at 32° / 0 0 S was lower than the Q02 of those gills at 25°/oo, while at lower temperatures this differential was not

112 FIG. 24. - Respiratory rate of gill tissue at 15°/oo S as a function of time and temperature. One pair of gills per temperature. Animals and gills acclimated to 22°C.

113 FIG. 25. - Respiratory rate of gill tissue as a function of temperature and salinity. Animals acclimated to various salinities and 22°C for 9 days. S.D. listed in Table 9.

Ilk Table 9. Oxygen consumption of isolated gill tissue in ml O^/gm ash free dry weight/hour at different salinity and temperature combinations. The + values indicate standard deviations where n = 6.

Salinity o/oo

c° 2 5 10 15 25 32

10 0.516 + 0.035 0.583 + 0.031 0.545 + 0.053 0.468 + 0.013 .0.534 + 0.017 0.509 + 0.002 15 0.887 + 0.100 0.977 + 0.036 0.931 + 0.053 0.891 + 0.078 0.753 + 0.059 1.028 + 0.238 20 1.654 + 0.135 1.412 + 0.224 1.381 + 0.039 1.486 + 0.257 1.330 + 0.144 1.465+0.026 25 2.181 + 0.109 2.138 + 0.213 1.831 + 0.060 2.180 + 0.142 1.781 + 0.211 1.736 + 0.140 30 2.765 + 0.294 3.149 + 0.179 2.639 + 0.101 2.874 + 0.312 2.'443 + 0.170 2.719 + 0.573 35 5.048 + 0.152 5.144 + 0.137 3.882 + 0.203 3.748+0.292 3.892 + 0.050 3.213 + 0.052 40 2.292 + 0.051 3.149 + 0.130 2.960 + 0.190 3.435 + 0.152 3.512 + 0.350 2.952 + 0.121 — — — Table 10. Q10 for Rangia gill at different salinity and temperature combinations. The negative values indicate a decrease in the respiratory rate.

Salinity °/oo Temperature Interval C° .2 5 10 15 25 32 10-15 2.92 2.78 2.89 3.61 1.98 4.04 15-20 3.45 2.07 2.19 2.75 3.09 2.01 20-25 1.71 2.28 1.74 2.13 1.76 1.39 25-30 1.58 2.16 2.07 1.71 1.87 2.43 30-35 3.31 2.65 2.16 1.69 2.52 1.39 35-40 -4.84 2.65 -1.71 -1.18 -1.21 -1.16 significant. It would appear that temperature has more drastically affected respiration at the low salinities (2 and 5°/oo), where 35° C has resulted in an extreme increase, and 40° C has apparently damaged the tissue, causing a sharp decrease. Stress of salinity was again shown at 32°/oo S, since at both 35 and 40° C respiration was depressed below that of gills in the intermediate salinities. The fact that 40° C respresents the critical temperature is shown by the decrease in Q0^ from the 35° C values at all salinities, with the decrease becoming extreme at 2, 5 and 10°/oo S. In a study on the effects of salinity shock, clams were acclimated to 28.3° C and 13°/oo S for 10 days prior to the analysis of gill respiration which was also done at 28.3° C. The data plotted in Figure 26 represent the response of the gills to a rapid change in salinity. The respiratory rates were measured for a four hour period beginning one hour after the transfer to a new salinity. The gills taken from 13°/oo S and placed in 2 and 5°/oo S demonstrated an increase in their QO^, from 2.45 ml 02/g/hr at 13°/oo S to 4.59 at 2°/oo S and 3.98 at 5°/oo S. However, those gills moved to 20 and 25°/oo S exhibited no significant change in their Q02 .

4. Discussion The effect of salinity on the respiration of bivalves has been the subject of several investigations on both intact organisms and isolated tissues. If any increase is noted in the respiratory rate as the salinity is lowered the explanation is given that this is a response to increased energy requirements to maintain an osmotic gradient between the interior and exterior of the cells. Hopkins (1949) observed an increase in gill and mantle tissue respiration and a decrease in the adductor tissue respiration of Mercenaria mercenaria. He attributed this to the fact that the gill and mantle, being primarily epithelial tissues, are functionally adapted to resist water entrance, presumably using metabolic energy in the process. This explanation is in some question, as Van Winkle (1968) working with four euryhaline bivalves demonstrated that the gill tissue of two species, Crassostrea virgiriica and

117 FIG. 26. - Respiratory response of gill tissue at 28°C acclimated to 13°/oo S (28°C) and transferred rapidly to other salinities. Bars = S.D., where n=6.

118 Mytilus edulis, exhibited an essentially constant respiratory rate from 5 to 30°/oo S. The respiration of two other species, Mérceiiaria mercenària and .'Modiolus demissus, was increased at low salinities. As Crassostrea and Modiolus have the same lower salinity boundaries for activity of the adult bivalve this effect of salinity on res­ piration does not seem to be related to lower salinity limits, nor does the argument for increased 0^ consumption due to osmotic stress apply equally. The recent work of Percy et al. (1971) has presented results in conflict with the data of Van Winkle (1968). This additional study on the respiration of £. virginica gill tissue demonstrated an increase in the respiratory rate with a decrease in salinity. Percy did not consider the data of Van Winkle in his discussion, but does suggest that the stimulating effect of dilution of the medium on the respiration rate is a relatively transitory phenomenon. The present evidence indicates that in Mytilus edulis the rise in gill respiration with lowered salinities is only temporary, the rate gradually returning to normal, and the earlier work of Schlieper (1929) supports this theory. In addition, Galtsoff (1964) has shown that intact oysters exhibit no respiratory stimulation in dilute seawater if allowed to adjust for three days. A review of the methods used in the above studies indicates that the increased respiration, where it occurs, is probably due to the rapid introduction of the gill tissue into a medium of lower salinity. At least this has been shown to be the case with Rangia gill tissue. As the discussion of Figure 26 points out there is a twofold increase in 0^ consumption when gill tissue is shocked by moving it from 13°/oo to 2°/oo S. However, when acclimated gills are used there is no significant difference in the respiration at 2 and 13°/oo S, even at a wide variety of temperatures, until lethal temperatures are approached. It appears then that the osmotic load after acclimation does not appreciably affect the respiration of Rangia and other euryhaline bivalves .

119 The synergistic effects of near lethal temperatures and low salinity produced a marked increase in the gill respiration over

that at higher salinities. The Q^q data indicate a lower critical temperature at salinities below 10 /oo. Other data presented on temperature tolerance of intact Rangia support this assumption. As this increase in respiration coincides with the point at which osmoregulation begins there is the suggestion that with high temperatures and very low salinities the osmoregulatory process may compete with other metabolic processes for energy. It is interesting that Rangia tolerates the highest temperature in a medium of 25°/oo S, which in nature represents its maximum limit of distribution.

Effect of Salinity on Glycogen Utilization by Rangia

1. Introduction The use of glycogen as a storage product for vertebrates and invertebrates is well documented. Although some data are available on the rate of glycogen utilization by various organisms, few have examined the relationship to salinity. In a limited fashion, Allen (1961) noted a decrease in glycogen as he moved Rangia into higher salinities. He suggested this phenomenon could be interpreted as an indication of work being done in order to maintain osmotic equilibrium.

2. Materials and Methods The use of stored glycogen under aerobic and anaerobic conditions was determined by comparing the percentage of glycogen in the tissue of Rangia held under nitrogen with those held in aerated water. The clams under nitrogen were held in sealed flasks containing water previously bubbled with nitrogen gas to remove all oxygen. The water was changed every two days and bubbled with nitrogen every day to prevent the buildup of hydrogen sulfide. The control clams were held under identical conditions except that their water was constantly aerated. Six clams from each group were analyzed at 0, 4 , 8 and 11 day intervals. The glycogen analysis followed the method previously described except that in this case the six clams

120 at each interval were homogenized together and four samples of the homogenate were taken for analysis. One of the samples was used to obtain an ash-free dry weight and the other three were used for glycogen analysis. In addition, the glycogen utilization by Rangia at different salinities was examined. A large group of clams was collected and held at the environmental salinity (10 ppt) for 4 days to allow the clams to rid themselves of any food. Six of these clams were then analyzed for glycogen content as described above. The remaining clams were divided into groups of six and moved in steps of 5 ppt per two days to the final salinities ranging from 1 to 32 ppt (°/oo). After 16 days all the clams were analyzed for their glycogen content. The results were expressed as the percentage of the original glycogen content present in the tissue at different salinities.

3. Results In Figure 27 the percentage glycogen in the tissue of Rangia held under aerobic and anaerobic conditions at 10°/oo S and 22° C is plotted versus time. After 11 days the clams under anaerobic conditions had utilized 2.4 times as much glycogen as those clams under aerobic conditions. In both cases the clams continued siphoning throughout the experiment, but the clams under nitrogen seemed to siphon less than those under aerobic conditions. After 13 days under nitrogen, deaths began to occur at the rate of 1 or 2 clams per day. The utilization of glycogen at different salinities is plotted in Figure 28. After 16 days starvation the clams at 20°/oo S had utilized 37 per cent of their initial glycogen. The utilization was reduced at both lower and higher salinities and was almost negligible at 2 and 5°/oo S.

4. Discussion Rangia reacts to a short period of anaerobiosis by simply storing the incompletely metabolized acid end products in its tissues and thus accumulates an oxygen debt. This debt is repaid rapidly via a dramatic increase in the respiratory rate when oxygenated

121 FIG. 27. - Per cent utilization of glycogen under aerobic (dark circles) and anaerobic (open circles) conditions as a function of time. Bars = S.D., where n=6.

122 water becomes available. Under long term anaerobic conditions Rangia shifts its metabolic pathways to those of a typical facultative anaerobe as described by Newell (1964). Instead of a complete reduction of 6-carbon sugars to CO^ and H^O, an incomplete reduction to relatively large, high energy compounds occurs and these com­ pounds are excreted. Lactic acid is the common end product of such metabolism, but Rangia excretes primarily succinate (Stokes and Awapara, 1968) . Aerobic metabolism is a much more economical means of burning metabolic fuel (glycogen in this instance) and indeed this is the primary pathway for Rangia. As the discussion of Figure 27 indicates, 2.4 times as much glycogen is used in anaero- biosis as compared with clams under aerobic conditions. This ability of Rangia to become a facultative anaerobe is of considerable adaptive significance. On several occasions during the present study winds forced the water out of upper Trinity Bay to the extent that the Rangia beds were dry for periods up to five days. During such periods, clams must keep shells closed, conserving water but also excluding oxygen. Thus, while anaerobiosis is inefficient in terms of energy conversion, it is highly effective in terms of survival value. The results of these experiments correspond closely to the assimilation rate of accumulated glycine at various salinities (Table 6). Although the highest salinity recorded on Table 6 was 15°/oo, there was a steady increase in the per cent of glycine lost from a low of 9.2 per cent at 1 /oo S to a 69.2 per cent decrease at 15°/oo S. This trend would indicate that Rangia are not only utilizing stored glycogen but also accumulating glycine at a much more rapid rate when maintained at intermediate and high salinities. As noted in the introduction, Allen (1961) also found that Rangia contained less glycogen as he moved them to higher salinities The results of osmoregulatory research have shown that this increased utilization of glycogen at intermediate salinities cannot be merely the reflection of osmoregulatory work. Since Figure 28 shows a greater decrease in the glycogen content at 10, 15 and 20°/oo S, osmotic work alone cannot be responsible.

123 FIG. 28. Percentage of glycogen present after 16 days of starvation as a function of salinity. Bars = S.D., where n=6.

12k From the results of the osmoregulatory, respiratory and glycogen studies it should be possible to construct a picture of the metabolic responses and adaptations of Rangia in relation to the stress of salinity. However, only the glycine uptake and osmoregulatory datu correspond in their relationship to salinity. The point at which glycine uptake decreases (below 10°/oo S) is also the concentration at which osmoregulation becomes significant. When the data on glycogen use, respiration and osmoregulation are compared, no apparent correlation is seen. It seems that the energy required for osmoregu­ lation would be maximal at low salinities (l-5°/oo S), when glycogen utilization was found to be minimal. In addition, the lowest rates for respiration were at the intermediate salinities (low at 15°/oo S), which was the region of maximal glycogen utilization. As is often the case, the research results presented above have answered some questions, but brought to mind a number of additional questions. More research is required to attempt to explain the seemingly contradictory results regarding glycogen utilization and other metabolic parameters in relation to salinity.

Effect of Salinity on Feeding

1. Introduction Apparently, few investigations have been conducted to determine the effect of salinity on the feeding rate of bivalves. As might be assumed, most feeding studies concerning bivalves have been conducted with the commercially important American oyster (Crassostrea virginica) . Some workers have utilized artificial food sources as the test suspension (Mironov, 1948; Lund, 1957; Fox et al., 1937). Other investigations of feeding rate utilized various species of algae which were first labeled with a radioactive isotope (Chipman and Hopkins, 1954; Chipman, 1959; Smith, 1959). The rate of water propulsion by the bay scallop, Aquipecten irradians was found by Chipman and Hopkins (1954) to be dependent on the size of the individual. Rao (1953) found the weight of the soft parts of Mytilus to be related to the rate of water transport. After conducting experiments

125 with great numbers of oysters, over several years, Galtsoff (1964) concluded that there are considerable variations between the pumping rates of individual oysters, and between the rates of individuals at different times of the day or on different days. Experimental studies on the effects of salinity on the pumping rate of oysters were first conducted by Hopkins (1936) on CrasSQStrea gigas of the Pacific coast. However, as Galtsoff (1964) reported, the technique used was not reliable. Loosanoff (1952) exposed C. vlrginica, which had been acclimated to 27 ppt, directly to salinities of 20, 15, 10 and 5 ppt. The rate of water transport by these oysters was reduced by from 1 to 76% of the original rate. Collier et al. (1953) studied the effects of dissolved organic substances on the pumping rate of oysters. They showed that salinity was related to the amount of dissolved organic substance in the water, which in turn influenced the pumping rate. From the experimental evidence presented, it would appear that the organic material was directly responsible for alterations in pumping rate, and salinity had an indirect effect, if any. Extensive studies have recently been conducted with £. virginica (Haven and Morales—Alamo, 1970) and the mussel, Mytilus edulis (Widdows and Bayne, 1971) which are related to the efficiency of feeding or particle removal. Both of these studies utilized a constant-flowing sea water system and the bivalves were allowed to feed over a period of days or months. Haven and Morales-Alamo (1970) found that oysters removed particles in the 3 to 12 micron range with equal efficiency, but only removed particles in the 1-3 p range with about one-third the efficiency. Widdows and Bayne (1971) found no significant difference in the filtration rate over the 3 range of concentrations used (l-5»5 x 10 cells per ml).

2. Materials and Methods Rangia used in feeding studies were collected from McCollum Park and acclimated to the specific experimental salinity, without food, for at least one week. Animals used in any single experiment were all from the same collection and were approximately the same size. Before being placed in the experimental chambers, clams were 126 washed and scrubbed free of any debris, barnacles, or algae which might have been attached to the shell. The food source for nearly all feeding studies was the green phytoflagellate, Dunaliella peircei. This organism was shown to be a suitable food for Rangia larvae and has been used by other workers in similar experiments. The most important reason for utilizing this particular single-celled alga was that it was capable of growth and reproduction in salinities from 5 to 32 ppt. In order to maintain a constant number of cells per unit volume in the source container, the cells must remain viable during the course of the experiment (4-5 hours). Since Dunaliella could be grown at each experimental salinity, there was a greater probability that large numbers of cells would not be killed by osmotic stress during the experiment. Other food sources, which were tested during the project, included the green phytoflagellates Brachiomonas submarina and Isochrysis galbana, a finely powdered alfalfa, and the aquatic seed plant Batis maritima (salt wort). The salt wort was collected 14 from a marsh on Galveston Island, exposed to C 09 , dried, powdered 14 Z and used as C -labeled detritus. All feeding experiments were conducted in a flowing system, where a source algal culture was pumped, via a peristaltic pump, through tubing, in and out of chambers containing the Rangia. The concentration of algae was determined with a Coulter counter before and after the animals had been exposed to the solution. In most cases the solution was passed through small containers, each holding one clam, but in some instances large containers with five or six animals were used. All source containers and experimental chambers were stirred constantly with air-powered stirrers, and the source was aerated so that oxygen content would be high in the chambers. It was believed that this type of system closely approximates the natural environment. The use of a static system in which animals are merely placed in a container with a known number of algae is much less complex, but has several flaws. Two major problems with a static system are that excretory products

127 are building up, and that the animals are never acting on the same concentration of algae at any two points in time.

3. Results

Numerous feeding experiments were conducted from June 1971 through April 1972 and the results are presented in Tables 11, 12 and 13. All data listed are from experiments utilizing Dunaliella as a food source, but factors such as cell concentration, salinity, and flow rate vary. The two-hour time period, which was selected for comparisons, was found to be sufficient for all animals to reach an equilibrium in their rate of removal and not so long that irregular bursts in feeding occurred. All data are expressed as per cent removal of algae, regardless of the algal concentration. Table 11 lists the results of feeding studies as segregated according to the salinity of the test medium (5, 10, 15, 20, 25 and 32°/oo S). No attempt was made to determine the means and standard deviations at each salinity, since the values were generally widely spaced within a given salinity. It would not have been wise or valid to compare means when individual points were so widely scattered. Regardless of salinity, slightly more than one- half of the values were in the 20-40% range. Within one specific salinity (20°/oo) the range was 8 to 78% removal. Often within a specific salinity the range between high and low values was on the order of 50% removal. Even when removal rates are compared as to the flow rate (Figure 12) or the concentration of algae (Figure 13), there was no consistent relationship to salinity. It was possible to control for temperature, time of day and period of acclimation, but un­ fortunately individual variation between clams cannot be controlled. These results show that any effect of salinity on the rate of removal of algae must have been masked by individual variation. Of course, it is possible that there is no effect from salinity; otherwise, during the course of these numerous experiments a significant effect on feeding should have been exhibited. The lack of a salinity effect is likely, since it has been shown in other sections

128 Table 11. Rangia feeding on Dunaliella with data

segregated according to salinity.

Number of Salinity Concentration Flow Rate % Removal in (Cells x 103/0.5 ml) (mls/min) 2-hour Period Animals (°/oo)

4 5 113 1 34 1 5 49 3 45 4 10 84 1 33 4 10 21 2 53 1 10 49 3 62 4 15 133 1 29 4 15 94 1 35 1 15 43 1 12 2 15 58 1 59 1 15 52 3 34 1 15 49 3 58 4 15 21 3 37 4 15 31 5 24 5 15 4 5 27 6 15 13 10 26 4 20 117 1 23 7 20 64 1 78 1 20 40 1 8 2 20 60 1 33 1 20 53 3 45 1 20 48 3 31 4 20 5 5 56 6 20 26 10 69 6 20 16 10 20 4 25 95 1 35 8 25 110 1 70 1 25 47 1 24 2 25 59 1 35 1 25 53 3 30

129 Table 11. (cont.)

Number of Salinity Concentration Flow Rate % Removal in Animals (°/oo) (Cells x lO'Vo. 5ml) (mis/min) 2-hour Period

6 25 14 10 56 4 32 56 1 47 1 32 46 .1 8 1 32 51 1 17 4 32 15 2 52 1 32 52 3 30 1 32 47 3 26 4 32 35 3 61 3 32 5 5 30 6 32 17 10 24

130 Table 12. Rangia feeding on Dunaliella with data segregated according to flow rate.

Salinity Concentration Flow Rate % Removal in (°/oo) (Cells x 10^/0.5 ml) (mis/min) 2-hour Period

5 113 1 34 10 84 1 33 15 133 1 29 15 94 1 35 15 43 1 12 15 58 1 59

20 117 1 . 23 20 64 1 78 20 40 1 8 20 60 1 33

25 95 1 35 25 110 1 70 25 47 1 24 25 59 1 35

32 56 1 47 32 46 1 8 32 51 1 17

10 21 2 53 32 15 2 52 5 49 3 45 10 49 3 62 15 52 3 34 15 49 3 58 15 21 3 37 20 53 3 45 20 48 3 31

25 53 3 30

131 Table 12. (cont.)

Salinity Concentration Flow Rate % Removal in (°/od) (Cells x 103/0.5 ml) (mis /min) 2-hour Period

32 52 3 30 32 47 3 26 32 35 3 61

15 31 5 24 15 4 5 27

20 5 5 56

32 5 5 30

15 13 10 26

20 26 10 69 20 26 10 20 20 16 10 20

25 28 10 46 25 14 10 56 32 17 10 24

132 Table 13. Rangia feeding on Dunaliella with data segregated according to cell concentration.

Salinity Concentration Flow Rate % Removal in (Cells x 103/0.5 ml) (°/oo) (mis/min) 2-hour Period Range; 84-133

5 113 i 34 10 84 i 33 15 133 i 29 15 94 i 35 20 117 i 23* 25 95 i 35 25 110 i 70

Range; 40-64 5 49 3 45 10 49 3 62 15 43 1 12 15 58 1 59 15 52 3 34 15 49 3 58 20 64 1 78 20 40 1 8 20 60 1 33 20 53 3 45 20 48 3 31 25 47 1 24 25 59 1 35 25 53 3 30 32 56 1 47 32 46 1 8 32 51 1 17 32 52 3 30 32 47 3 26

133 Table 13. (cont.)

Concentration Salinity Flow Rate % Removal in (Cells x 10^/0.5 ml) (°/oo) Range: 3-35 (mis/min) 2-hr Period

5 10 21 2 53 15 21 3 37 15 31 5 24 15 4 5 27 15 13 10 26

20 5 5 56 20 26 10 69 20 16 10 20

25 28 10 46 25 14 10 56

32 15 2 52 32 35 3 61 32 5 5 30 32 17 10 24

13^ of this report that respiration is not affected after acclimation, which according to water and blood regulation data occurs within 24 hours. As it was concluded in the spring of 1972, from the data presented above, that salinity was not responsible for a consistent effect on feeding, further experiments utilizing other food sources did not seem likely to be productive. It was shown that Rangia would remove other phytoplankters and laboratory-prepared detritus, but no extensive attempt was made to quantitate removal in relation to salinity. No other alga tested could tolerate the wide range of salinities necessary for valid comparative tests. Although ar­ tificial detritus was prepared and preliminary tests were conducted, a number of problems were involved in the quantitation of removal rates. Certainly, if salinity were to affect feeding this should have been demonstrated in the many experiments with Duiialiella,

4. Discussion The results of experiments in which Rangia were fed Dunaliella have shown that within the salinity range tested (5 to 32°/oo) no dominant effect of salinity was exhibited. If subtle effects were present, these were masked by the extensive differences between individual Rangia. While controlling temperature, time of day and acclimation period, no consistent or significant effect from salinity, cell concentration or flow rate was found. As noted in the intro­ duction, other bivalves have also been found to be quite variable in their rate of water transport (Galtsoff, 1964), which in turn affects respiration and feeding. In certain bivalves, such as the bay scallop (Aquipecten irradians), water may be continuously pumped, but the food sorting activity of the cilia on the gills is not constant (Chipman and Hopkins, 1954). Unfortunately, the functional morphology of Rangia's feeding apparatus has not been closely examined. Observations made during feeding experiments showed that the siphons were extended, but once water was taken in it must have been acted upon differentially by individual

135 Rangia. Since the pumping rates of the clams were not measured, it is also possible that the flow rates through the animals varied between individuals and with time. Although few comprehensive studies have been conducted regarding the effects of salinity on the feeding of bivalves, workers have been successful at measuring particle discrimination (Haven and Morales-Alamo, 1970) and feeding efficiency (Widdows and Bayne, 1971). These studies and others that have provided reliable data utilized a constant-flowing natural sea water system. The availability of a constant supply of sea water and food is a tremendous advantage in feeding studies. By examining the rate of removal of food over a period of several days or months, it is possible to avoid problems related to short-term, irregular feeding activities. Since logistically it was impossible to conduct long-term experiments, it was hoped that a period of four to five hours would be sufficient to measure the effects of salinity. From the results presented above, which are highly irregular, it would appear that neither salinity nor any of the other variables tested have a consistent effect on feeding. Since the respiration of acclimated animals was not dependent on salinity and was relatively constant with time, it is quite possible that water is pumped at a constant rate, but the removal of particles is dependent on some intrinsic factor.

Effects of Salinity on Reproduction, Larval Development and Growth

1. Introduction A very common breeding habit in marine invertebrates is broadcast fertilization, in which eggs and sperms are released into the environ­ ment and fertilization is external. Giese (1959) has shown that most marine invertebrates are seasonal in their reproduction and exhibit synchrony in the release of gametes. Various environmental factors may contribute to the control of synchrony, and thus increase the efficiency of the fertilization process.

136 Fairbanks (1963) described the spawning cycle of Rangia in Louisiana, but differences between isolated populations may occur and salinity factors were not thoroughly examined. Populations of Crassostrea virginica from different regions were shown to require different temperature regimes for completion of gametogenesis and spawning (Loosanoff, 1969) , Ropes and Stickney (1965) have shown that My a arenaria in New England has only one annual spawn, while south of Cape Cod it releases gametes at two separate periods (Shaw, 1965) • The larval development of Rangia was described by Chanley (1965). In this study clams were induced to spawn by a temperature shock treatment. Thus, it had been shown that temperature exerts an effect on the spawning and presumably the maturation of gametes, but the effect of salinity had not been examined, until Cain (1972) in an unpublished Ph.D. dissertation reported data from detailed studies of the effects of salinity and temperature on survival and growth of Rangia larvae, and on reproduction, in Virginia.

2. Materials and Methods Rangia were obtained from either McCollum Park or Lake Anahuac, Chambers County, Texas. On arrival at the laboratory, they were placed in 15 gallon aquaria at salinities approximating that of the collection site at that point in time. They were gradually (2°/oo per day) taken through a series of salinities and held at the final salinity for at least 2 days before experimentation. Since it was found that even a slight temperature increase would often prematurely trigger spawning, all aquaria were maintained at 22+1° C. The ability of Rangia to release gametes was tested by placing clams from the various salinities, at a temperature of 22+1° C, into separate small aquaria at the same salinity, but at a temperature of 34° C. They were held at this temperature until spawning occurred or for a maximum of 24 hours. When gametes were released, the data regarding salinity, sex and the approximate number of spawning indivi­ duals were recorded. When it appeared that all animals capable of releasing gametes had done so, the clams were removed and the aquaria containing the gametes were returned to room temperature and allowed

137 to cool. Either by dilution with the same salinity water or by trans­ fer, the gametes were diluted and maintained overnight. Depending upon the particular experiment, fertilized eggs, trochophore larvae or veliger larvae were transferred to smaller vessels and tested for survival and growth under various conditions. Three different food sources were used in various experiments with larvae and young clams. Two of the three were unicellular green, flagellated algae (Dunaliella peircei, Brachiomonas submarina) and the last was a finely powdered alfalfa, which should be comparable to natural detritus. Larval growth and survival data were obtained with the use of various count­ ing chambers and a compound microscope. In a separate study, 161 small Raiigia (4.5 to 15.2 mm) were placed in individual compartments within aquaria at 2, 4, 5, 15 and 25°/oo salinity. These clams were initially measured with a vernier caliper (+ 0.1 mm) and continued to be measured periodically for 2 months, during which time they were fed phytoplankton and powdered alfalfa.

3. Results Studies conducted in the fall of 1971 (Oct. 25 to Nov. 12) utilized clams from both McCollum Park and Lake Anahuac. After ac­ climation to the various salinities and a temperature of 22 + 1° C, they were given a temperature shock (34° C) and in some cases a salinity shock. Lake Anahuac clams did not spawn at 0 /oo S, but did at 1 and 3°/oo, as well as 5°/oo when transferred from 3°/oo S. Rangia from McCollum Park which were maintained at 7, 8, 27 and 30°/oo S did not spawn, even when they were salinity shocked by a 2 to 5 /oo S increase. These preliminary experiments were conducted in our laboratory by Glenn Michael Hightower for a student project in an invertebrate zoology course. They were followed up by C. A. Bedinger who made a careful microscopic study of the embryos and larvae as they developed. Some of Bedinger’s drawings are presented as Figures 29, 30, 31 and 32. Later studies were made with the assistance of Susan Baldwin and Thomas Dillon. Bedinger and Hightower found larvae settling to the bottom after 6 days, so from day 7 on they should be called juvenile clams rather than larvae. 138 KEY TO FIGURES

1 Anterior adductor muscle 11 Mesoderm 2 Anus 12 Mouth 3 Apical tuft 13 Nephridium 4 Cerebral ganglion 14 Polar body 5 Crystalline style 15 Posterior adductor muscle 6 Digestive gland 16 Retractor muscle 7 Foot 17 Shell 8 Blastocoel 18 Statocyst 9 Gut 19 Velum

3

14

8

12

9

2

18

139 FIG, 30, - Straight-hinged larva.

FIG. 31. - Straight-hinged larva, dorsal view.

iko (

Note: The apparent position of the adductor, dorsal to the anus, is a graphic artifact. When relaxed the muscle is U-shaped; the middle part or bend of the U is shown, dorsal to rectum and to attached ends. In later stages the muscle is clearly ventral to the rectum. — C. A. Bedinger

FIG. 32. - Veliger larva, settling stage.

ibl Growth studies were conducted with-the larvae which were produced from the spawned Anahuac clams. The results of these experiments are presented in Figures 33 and 34. Although not shown in this report, similar growth was exhibited by larvae which were only fed once with Duriàlièlla, but it could be that the alga was also growing or multiplying during the investigation. The initial supply of algae seems to be very important, since the larvae show a very rapid growth rate in the first ten days (Figures 33 and 34). The figures also illustrate a relatively small effect of a doubling in the number of larvae present (14 to 28 larvae per ml). The larvae at the lower concentration, Figure 33, reached a maximum size of approximately 165 microns (y), while those at twice this concentration, Figure 34, leveled off at about 150 y. The only apparent difference between the growth of larvae at 3 and 5°/oo S was the fact that growth was slightly faster at 5°/oo S, so that they reached a maximum 1 to 2 days sooner. Some aspects of the above experiments were interesting to note. First, larvae did not survive at 0 or l°/oo S, although adults have been collected from these salinities. Second, some larvae were fed on powdered alfalfa and did not grow well or survive for long under these conditions. It seemed that this material was not a suitable food for larvae, or the population of bacteria and ciliates associated with the material were deleterious to the larvae. During May and June of 1972, collections were made from Lake Anahuac and McCollum Park. Very limited success occurred when attempts were made to spawn those Rangia from McCollum Park. Although clams were temperature and salinity shocked after being maintained at 22° C and a range of salinities between tap water and 30°/oo S, the only successful spawn of these Rangia occurred at 10°/oo S. The single exception was the release of a small amount of sperm from one clam at 20°/oo S. The animals from Lake Anahuac were also acclimated to a series of salinities before being tested for spawning. These clams, which were collected from a low- salinity region, were found to more readily release gametes and to

lk2 LENGTH IN MICRONS 1 2 5 . 9 6 1 73.80—r I. 33.FIG. -larvaefunctionofLengthasaofagesalinity. and 4 8 0 2 4 6 18 16 14 12 10 8 6 ----- 1 ----- AGE OF LARVAE (DAYS) LARVAE OF AGE 1 ----- 1 ----- 1 ----- ib3 1 ---- -1 1------1 ------1 ----- 1 ----- 1 ----- 1 ----- 1 1 LENGTH IN MICRONS FIG. 34. - Length of larvae as a function of age and salinity. and age of function a as larvae of Length - 34. FIG. avewr X h cnetaino i. 33. Fig.of concentration the 2X were Larvae AG O LRA (DAYS) LARVAE OF E Ikk do so at a wider range of salinities. Spawning occurred at 2, 5 and 10°/oo S and in one case some sperm were released at 2Q°/oo S. In most cases where spawning of both sperm and eggs occurred, fertilized eggs and cleavage stages were observed. However, the highest and lowest salinities at which the trochophore stage was reached were 10 and 2°/oo S, respectively, for Anahuac clams, and 10°/oo was the only salinity for McCollum Park clams. The tro- chophores from the above animals successfully developed to the veliger stage. Larvae from some of the spawns were fed Brachiomonas and maintained at the same salinity for 21 days. By this time the foot had developed and was observed to be highly ciliated. These juvenile clams were found to use the ciliated foot to move about, often to an area of dense algae, where the current from the cilia was used to bring in food. At this stage in development, the juveniles were slowly acclimated to a series of salinities ranging from 2 to 30°/oo. Approximately the same number of clams (15 to 25) were placed in small Petri dishes with 5 ml of 2, 4, 6, 8, 15, 20, 25 or 30°/oo S water. At intervals of between 1 and 3 days, they were fed, salinity was checked and readjusted, and length- width measurements were taken on six randomly selected clams from each salinity. It was found that width was directly correlated with length, so in the calculation of growth versus salinity, only length data were used. The results of these growth studies are shown in Figure 35, in which age of "larvae," actually juvenile clams, has been plotted against length in microns. The vertical bars shown at each point are not the standard deviations (SD) of the measurements for one salinity, but the SD for the distribution of lengths at all salinities around the mean. The SD for the six individual measure­ ments at each salinity were quite small (approximately 10% of the length). Figure 35 was prepared in this fashion since there was no consistent effect of salinity on the growth of "larvae" (juvenile clams). Growth was often in steps, thus masking the effects of salinity. Even at termination, it was found that there

1^5 320 -

CO 2 3 0 0 - O o: - cj — 2 8 0 - 2

2 2 6 0 - X i— CO 2 4 0 - 2 LU _ _J 2 2 0 -

2 0 0 - n ------1------1------1------1------1------1------1------1------i------1 21 23 25 27 29 31 33 35 37 39 41 AGE OF LARVAE (DAYS)

FIG. 35. - Mean length of larvae as a function of age. Bars for day 23 to day 41 are S.D. for length at all test salinities (2-30°/oo)•

lk6 was no direct correlation of size with salinity, since the larger individuals were in salinities of 4, 8, 15 and 20°/oo. From the results obtained it would appear that after the early stages of development (21 days) the juvenile Rangia can survive and grow equally well at salinities between 2 and 30°/oo. The research concerning the growth of young Rangia was un­ fortunately not productive. Even though an attempt was made to feed the clams in excess there may not have been enough food available to support growth. After approximately two months of periodic measurements, no significant growth was exhibited by Rangia in any test salinity. In addition, survival under test conditions was generally less than 50%. It was evident that the laboratory conditions were not suitable, but it is not clear which environmental factors limited growth and survival. Since survival did not seem to be correlated with salinity, apparently temperature (22 + 1 ° C) and insufficient food were the important factors. It could be that a soft, organic-rich substrate is required for significant growth.

4. Discussion These studies on the reproduction of adults and the growth of juvenile Rangia, although limited in scope, have provided some very interesting information. It has been shown that clams from McCollum Park, Trinity Bay did not spawn when tested in October 1971 and only exhibited a limited spawn at 10°/oo when examined in June 1972. Apparently, clams collected from the lower salinity Lake Anahuac site were more mature at these times, and therefore exhibited significant gamete release in response to temperature and/or salinity increases. Although salinities are characteristi­ cally higher at McCollum Park, probably temperature values were quite similar to those of Lake Anahuac. Food type and availability, substrate and organic content of the substrate are other factors which could produce differences between the two populations. Tenore, Horton and Duke (1968) have studied the effects of substrate on the growth of Rangia and found sand with high organic content to

1U7 be favorable to growth, Gooch (1971) also showed that high organic content of the substrate was advantageous for growth. Successful spawning of Rarigia from either population occurred only at salinities between 2 and 10°/oo. Recently, Cain (1972) reported on an extensive study of Rarigia reproduction conducted during 1970 and 1971 in the James River, Virginia. He compiled field and laboratory data concerning the seasonal reproductive state of gonads, the relationships between temperature, salinity and spawning, and the tolerance of larvae to temperature and salinity. This report on clams from various locations along the James River showed that animals inhabiting the region of highest salinities (5 to 10°/oo, with a maximum of 20°/oo) were the largest and were ripe from spring through fall. It was found that clams from these intermediate salinities required a decrease in salinity to spawn, while those at low salinity were stimulated to release gametes by an increase in both temperature and salinity. Judging from the above information, perhaps McCollum Park Rangia would have more readily released gametes if immediately placed at lower salinity and higher temperature. In this study, they were first acclimated to lower salinities and later given a temperature increase. Cain (1972) also tested the survival of Rangia embryos to the straight-hinge stage (48 hours) and to the 7th day at different combinations of temperature and salinity. He found that embryos could not develop at any temperature in salinities less than 0.3 ppt (fresh water). Survival was low for embryos and larvae at 35° C, which correlates with the stress effects of 35° C on adults reported above under Respiration. The combinations of 5 and 10 ppt salinity and 20, 25 and 30° C were found by Cain to give approximately the same high survival rates for larvae. A salinity of 8 ppt appears to be optimal for larvae, since he reported 100% survival at 24 C and the next highest survival was at 8 ppt and 16 C. No embryos or early larvae survived at any temperature in salinity of 20 ppt. Cain transferred larvae which survived to the straight-hinge stage at 28° C and 8 ppt to containers with salinities of 2, 8, 14 and 20 ppt and temperatures of 8, 16, 24 and 32° C. At this stage l48 of development, more than 75% of the larvae survived all combinations, and optimum growth occurred in 14 ppt salinity and at 32° C. At each temperature 14 ppt was optimal salinity and at each salinity 32° C was the optimal temperature. The results of this report combine nicely with those of Cain (1972), since we have measured growth rates at early and late stages of development, and he has studied the survival of larvae during the first seven days of development. From the results of both studies it is apparent that embryos and early larvae do not survive nor grow well at salinities below 3°/oo or above approximately 10°/oo. From the point at which the straight-hinge stage is reached (48 hours), Rangia are less sensitive to temperature and salinity, and as shown above, grow at approximately the same rate up to at least 41 days (Figure 35). Data presented in both studies indicate that at least within the densities examined, the number of larvae per unit volume had little effect on growth (Figures 33 and 34).

Phytoplankton Studies

1. Introduction Rangia cuneata has been found to feed on both living and dead suspended matter (Darnell, 1958, 1961, 1962). When considering the possible food sources of a population of clams, it is important to know the types of phytoplankters available at different seasons of the year. Due to difficulties regarding collecting techniques, analytical methods and the heterogeneous nature of phytoplankton populations, it was only possible to determine the relative per­ centage of phytoplankton species present in water samples.

2. Materials and Methods Samples were taken in the vicinity of the pier at McCollum Park, Trinity Bay, Chambers County, Texas. The collection dates were October 18, 1971, February 11, 1972, and April 8, 1972. Samples were taken on the first collection date by use of a plankton net (35 y opening), but as this method did not provide a large enough sample size, continuous centrifugation of a one

149 liter water sample was used on the February and April collections. Immediately after collection the samples were preserved with 50 ml of 100% formalin per liter of water. Temperature and salinity data were taken at the time of collection. Organisms were identi­ fied and counted with the aid of a compound microscope and the data were expressed as percentages of the total one-liter sample.

3. Results The salinities and temperatures at the time of collection are listed below: Date Temperature (°C) Salinity (°/oo)

11/18/71 31 15 2/11/72 18 12 4/8/72 25 12

Table 14 shows the composition of phytoplankton samples on the three dates as percentages, by species. The values were determined from the mean of three separate subsamples of the total phytoplankton. Unfortunately, there were several species of certain genera which could not be identified to the species level. A well-trained professional phycologist using sophisticated microscopic techniques, and more time, would have been required for more exact species identification.

4. Discussion It was shown that many species of phytoplankters are available as a food source for Rangia. In general, diatoms were the most prominent phytoplankters and Nitzschia was the dom­ inant genus. From these data, it would seem that in the fall and spring, when temperatures were relatively high (31 and 25° C, respectively), the distribution of phytoplankters was quite similar• However, in February, when the temperature was 18° C, there was a sharp increase in Ankistrodesmus sp., which lowered the percent­ age of other organisms. It might be assumed that low temperature was responsible for the increase in the concentration of this species, but neither the cause nor the duration of the abundance is known as yet. This phase of the project provided information 150 regarding the number of phytoplankton species over the Rangia site and the percentage composition, but it is not possible to directly relate this information to the feeding activity of Rangia until more is known about the ability of this clam to discriminate between different food particles. The composition of the phytoplankton was studied by Henry Elfstrom, a student, under the supervision of Dr. Elenor R. Cox, Associate Professor of Biology, Texas A&M University.

151 Table 14. Composition of phytoplankton from McCollum Park Trinity Bay, Chambers County, Texas.

Average Percentages Composition Phy toplankton 10/18/71 2/11/72 4/8/72

Navicula sp. a 5.0 1.2 4.8 Navicula sp. b 6.0 1.5 5.7 Nitzschia peregrina 6.1 2.5 7.1 Nitzschia acuminata 0.3 1.6 2.4 Nitzschia longissima 0 11.6 0 Nitzschia closterium 0 4.7 0 Nitzschia sp. a 10.3 4.4 12.5 Nitzschia sp. b 4.3 2.9 5.1 Nitzschia sp. c 2.8 1.2 2.8 Amphora sp. a 3.8 4.5 5.1 Gymnodinium sp. a 1.3 2.2 1.9 Gymnodinium sp. b 0 10.5 0 Peridinium sp. 0 4.1 0 Cerataulina pelagica 17.8 1.3 13.2 Skeletonema costatum 8.5 2.7 10.0 Coscinodiscus centralis 2.2 0.02 2.8 Surirella ovata 2.1 0.7 1.1 Synedra sp. 2.2 1.6 2.4 Ankistrodesmus sp. 7.9 24.5 9.1 Asterionella japonica 0.7 0 0 Gyrosigma sp. 0.7 0 0.6

152 SOME INDICES OF SEASONAL AND ENVIRONMENTAL EFFECTS

Effects of Salinity arid Temperature on Ciliary Activity

Introduction The structure and the function of cilia have been long studied and are well documented (Sleigh, 1962). In molluscs, they are important in ventilation and feeding processes. Cilia of clam gills have been studied extensively in order to define some of the factors that influence their activity and efficiency. The effects of different ions according to their concentration and balance have been examined in a number of molluscs, especially in Mytilus (Gray, 1920, 1923, 1926, 1930). Since our survey did not reveal any published information on the effects of ions on ciliary activity of Rangia, a preliminary study was undertaken. In using the data from this study, we concluded that we must allow complete adaptation/acclimatization of Rangia to the test salinities if we are to measure steady rates of ciliary activity. We noted that both ionic species concentrations and ratios of ions affect the rate of ciliary beat in Rangia, thus when the test organisms are acclimated to various salinities, they will show different base level activity at any set temperature. It was our intent to examine the effects of temperature on cilia of Rangia acclimated to various salinities in order to be able to estimate the tolerances of these clams to various temperature - salinity conditions, as measured by their ciliary activity. Similar studies have been performed on other bi­ valves (Galtsoff, 1928; Hopkins, 1935; Vemberg et al., 1963).

Methods Rangia were acclimated to the test salinities at least one week prior to the experiments. The clams were opened by transection of the adductor muscles, then the gills were excised and placed in sea water of the appropriate salinity until used. The experiments at each temperature were performed in temperature-controlled attach­ ments holding the experimental chambers to the stage of the microscope. For temperature below ambient a cold room was used.

153 The light source of the microscope was replaced by a strobo­ scope, Calibration of this instrument was checked against the electric tachometer of a Sorvall RC2B centrifuge and a manually operated tachometer. The speed of the strobe was increased or decreased until the cilia appeared motionless - thus in synchrony with the pulse of the strobe. Three to seven replicates of each measurement were made. The data were recorded and graphed, prior to analysis. Larval Rangia were reared as described in another section of this report. Larval ciliary activity was measured as above, except that cilia were studied in situ in intact living larvae, since the larvae themselves were of microscopic size.

Results and Discussion The response curves of frontal gill cilia of adult Rangia in various salinity/temperature conditions are shown in Figure 36. The highest rate of ciliary activity occurred in clams acclimated to 10°/oo (ppt) salinity, followed by those at 15°/oo; those at 20°/oo were third highest followed by the response of cilia at 25°/oo. Cilia of gills of clams kept at 5°/oo gave the slowest temperature-related response. It is interesting to observe that the curves at 5°/oo and at 25°/oo are quite similar. Both of these salinities represent the short-term extremes to which Rangia is exposed under natural conditions in Trinity Bay, with the ex­ ception of very short time freshwater conditions following major precipitation in the area. Examining the data from the point of view of temperature effects, the frontal cilia of Rangia gills appear to reach peak activity at approximately 22° C. By the time 28° C is reached the activity is considerably diminished, followed by virtual cessation of ciliary beat (at least organized beating) at 35° C and above. Below ambient temperature, the activity is considerably decreased by exposure to 16° C. Further cooling results in the loss of coordination of ciliary beat and reduction of rate to very low levels.

15U FIG. 36. - Plot of ciliary beat frequency as a function of temperature of excised gills of Rangia adapted to various salinities.

155 It is significant that at low temperatures Rarigia probably requires less 0 ^ 9 thus the loss of effectiveness of ciliary activity would not place the organisms into jeopardy due to lack of venti­ lation. High temperature on the other hand is quite severe in its effects, since not only is the ciliary effectiveness reduced, but also the amount of dissolved in the surrounding water is lower, thus placing the organisms into double jeopardy physiologically. It is interesting to note that our observations in the field support this view; when collecting in Trinity Bay there seemed to be a higher number of empty shells in relation to live clams during summer months when water was warm than in winter when the temperature was near freezing on the partly exposed clam beds. The analysis of the frequency of ciliary beat vs. salinity at specific temperatures indicates that the response is not predictable; probably it is influenced by other physiological parameters which were beyond the scope of this study. It would be of interest to attempt to define some of these parameters in a future study. Ciliary activity of larval Rangia was measured under the same conditions as that of gill cilia from adult clams, except that intact animals were studied under the microscope instead of excised gills. We were able to get reproducible results only at 10 and 25 ppt salinity at three temperatures. At other temperatures and salinities, the results could not be reproduced; individual replicates were irregularly different and unpredictable. It is our tentative conclusion that the effects of salinity/temperature conditions on larval ciliary activity cannot be used as an indicator. Even at the two salinities where we were successful, the profiles of the response curves are different (Fig. 37). It cannot be determined if these differences are indeed significant or perhaps are the result of experimental conditions beyond our control.

156 F G 37. frequency -ofciliaryfunctionPlotas aof beatIG. CYCLES SEC" at salinities.various temperature 22-to24-hour-oldoflarvalRangia 157

Dissolved Carbohydrate Uptake by Rangia

Introduction In examining the literature, we have seen no published evidence on the uptake of carbohydrate by bivalves as a function of salinity condition. It was evident from preliminary work, that the major carbohydrate in Rangia is glycogen. (Also, see Allen, 1959.) Glycogen is synthesized from glucose in all organisms which possess it: glucose derived from dietary sources, synthesized from gluconeo­ genic amino acids, or taken up through the body surfaces. As far as published information indicates, the methods of glycogen synthesis in molluscs have not been described. Investigation of the effects of altered salinity on the uptake and assimilation of glucose was undertaken in our study.

Methods Rangia were collected and transported to the laboratory as described elsewhere. Following adaptation of the clams to laboratory conditions for at least 3 days, they were acclimated to different salinities from 5 to 25 ppt by 5 ppt increments every three days. For uptake and glycogen synthesis studies, whole clams, shucked clams and excised mantle pieces were used. Incubations for various lengths of time at specified glucose concentrations were 14 performed at the acclimated salinities, using glucose- C U.L. solutions in artificial sea water. Following incubations, the radioactivity remaining in the incubation medium, extracted by 70% ETOH from whole, shucked clam or mantle pieces, and the radioactivity in glycogen isolated by the previously described procedure were quantitated. Based on these data, the glucose-equivalent radioactivity removed from the medium, the glucose equivalent radio­ activity recovered by ethanol extraction from the clam meat and that from glycogen were calculated. The data were analyzed by standard kinetic analyses to determine the role of salinity in affecting uptake of glucose by the clam and synthesis of glycogen within the clam.

158 Results Initial experiments with whole clams and shucked clams indicated that individual variations of clams even at a single salinity were so great as to make meaningful analysis of glucose uptake and gly­ cogen synthesis impossible. To circumvent this problem we have resorted to attempting standardization of excised mantle pieces for the remainder of experiments. Using mantle pieces, we were able to determine that an incubation period of 5 minutes will give us reproducible data based on the recovery of glucose equivalent radioactivity from clam mantles in all salinity conditions tested (Fig. 38). The following experiments established the relationship of concentration of glucose in the medium to the velocity of the uptake at various salinities (Figure 39). The analysis of these data by Michaelis- Menten kinetics (Fig. 40) and other methods led us to the conclusion, that there is no direct correlation of uptake by the clam mantles with increasing or decreasing salinities, thus suggesting that this parameter will not serve as an indicator of salinity effects on Rangia even in the laboratory, much less under field conditions. As for glycogen synthesis or exchange of glucose taken up from the medium into glycogen, again the data do not give positive correlation. We have been unable to show net synthesis of glycogen by Rangia preparations in the laboratory. Even to show exchange reactions, our incubation times and glucose concentrations had to be increased beyond limits where individual variability of clams was overriding any effect of salinity which may have been evidenced. Based on this, we must reject the possibility of using this method of examining salinity effects on Rangia physiology. Our data confirm the apparent lack of correlation of salinity and glycogen content under laboratory conditions reported by Allen (loc. cit.).

159 FIG. 38. - Plot of glucose equivalent radioactivity in ETOH extracts of mantle pieces of Rangia as a function of time. Clams were adapted to test salinities prior to incubations.

160 FIG. 39. - Plot of velocity of glucose uptake of Rangia mantle pieces as a function of glucose concentration in the incubation media. Clams were adapted to the various salinities prior to incubations.

161 FIG. 40. - Plot of glucose uptake analyzed by Michaelis-Menten kinetics indicating no direct correlation between salinity change and uptake of dissolved glucose by Rangia.

162 Seasonal Physical - Chemical Parameters of the Composition of Rangia

Introduction There are very few comprehensive studies on the seasonal changes in composition of marine invertebrates. Some of the more recent papers are Blackmore (1969) reporting on Patella vülgáta L., Ansell et al. (1964) on Venus mercenaria, Barnes et al. (1963) on two species of Balanus , Heath and Barnes (1970) on Cárcinus maenas L ., Reeve et al. (1970) on Ságitta hispida. Stone (1970) on Hymeniácidon perléve and Williams (1970) on Littorina littorea. In these studies, attempts were made to correlate seasonal changes with parameters of growth, reproduction and biochemical composition of the inverte­ brates under field conditions. Such studies were not published on the brackish water clam, Rangia cuneata, even though Allen (1959) did report its chemical composition with respect to effects of salinity on total carbohydrate, protein and amino acid content under laboratory conditions. To attempt to determine the changes in chemical composition due to seasonal variations, the present study undertook the examination of Rangia chemical composition from samples collected from natural habitats through the year. In setting up the parameters to be examined, the suggestions and reviews of Giese (1967, 1969) were used to great advantage.

Methods Rangia were collected at McCollum Park on Trinity Bay. They were transported to the laboratory in styrofoam or plastic ice chests, cooled with crushed ice. The physical measurements, weight, volume of shell and meat were recorded prior to placing the meat into 25 ml of 95% ethanol. Following this step, the clams were homogenized in a cooled Waring Blendor and the homogenates were stored until analysis at 2° C. Aliquots of the homogenate were centrifuged to pellet the non-ethanol-soluble portion. The pellets were dried to a constant weight at 100° C. The total dry weight of the meat was calculated and recorded.

163 Glycogen was isolated from pellets by digestion for 1-1.5 hours in 30 - 60% KOH. The addition of 1.2 volumes of 95% ethanol to the digest flocculated the glycogen; washing of the glycogen was also done with 95% ethanol. Quantitation of glycogen was made on aliquots of water solutions of the washed glycogen by the phenol- sulfuric acid method of Dubois et al. (1956). The glycogen concentration of the whole meat portion of the clam was calculated and was expressed as percentage of dry weight. Protein content of the dry pellets was measured by the method of Lowry et al. (1951) following dissolution of aliquots (wet or dry) in IN NaOH. Total protein concentration of the total clam meat was calculated. Based on these measurements, a condition index was estimated.

Results The physical characteristics of Rangia, based on a sample of 12 clams per collection date, are summarized in Tables 15, 16, 17 and 18. Table 15 defines the range, mean size and standard deviation of length, breadth and depth of the shell of the clams. Table 15 lists the range, means and standard deviation of volume of whole (closed) shell, open shell and internal cavity of the same group. Table 17 lists the range of weights of the whole clams, that of the shells and by difference the weight of the meat and water contained in the shell. Based on analyses of these sets of numbers it is evident that the organisms collected during this time interval were approximately the same physical size; thus, compositional analysis may show variations of seasonally dependent nature in total glycogen, protein and in "condition index" based on wet weight/ shell cavity volume. The wet weight, dry weight, tissue water content and condition index of each collection sample are shown in Table 18.*

*The "condition index" used by oyster biologists is a percentage figure, i.e., our ratio index multiplied by 100. To convert to the "condition index" based on dry weight, divide by 6.25 - SHH.

1 6k Table 15. External measurements

Collection Length (mm) Depth (mm) Breadth (mm) Date Range Meant Range Meant Range Meant

5/1/71 54-61 57.2 ± 3.2 48-56 50.6 ± 3.4 36-41 38.8 ± 2.2 6/1/71 52-63 57.1 ± 3.2 47-55 50.9 ± 2.1 33-41 38.3 ± 1.9 7/1/71 52-58 55.1 ± 1.8 48-55 50.8 ± 2.4 34-41 37.7 ± 2.4 8/1/71 51-66 57.1 ± 4.5 45-60 51.6 ± 3.8 34-43 39.0 ± 2.9 9/1/71 49-60 54.9 ± 2.9 43-53 49.3 ± 2.4 33-40 36.1 ±2.0 11/1/71 53-61 57.3 ± 2.9 47-56 51.4 ± 2.8 35-43 39.0 ±2.6 1/1/72 53-59 55.3 ± 1.9 47-54 49.9 ± 2.3 34-39 37.0 ± 1.6 2/1/72 54-60 56.3 ± 1.8 45-51 47.9 ± 1.4 35-41 37.8 ± 1.5 Table 16. Volumes

3 3 Collection Total Volume (cm ) Shell Vo]Lume (cm^) Internal Volume (cm ) Date

Range Mean ± Range Mean± Range Mean!

5/1/71 40-58 46.3 ± 5.4 21-34 26.0 ± 3.8 16-25 20.2 ± 2.8 6/1/71 35-53 45.8 ± 4.4 16-32 24.2 ,± 4.0 13-30 21.7 ± 4.3 7/1/71 37-55 44.8 ± 6.7 17-29 22.2 ± 3.6 14-33 22.6 ± 5.1 8/1/71 30-63 46.3 ±10.1 13-35 23.1 ± 6.5 16-27 23.3 ± 4.1 9/1/71 30-53 38.6 ±5.9 13-27 19.9 ± 3.8 7-26 18.7 ± 4.7 11/1/71 35-56 46.8 ± 6.9 15-32 26.0 ±4.9 11-27 20.8 ±4.0 1/1/72 38-52 43.6 ± 3.9 17-28 23.1 ± 3.2 16-26 20.5 ± 3.1 2/1/72 39-49 44.3 ± 3.0 21-31 24.3 ± 2.7 15-26 20.3 ±3.1 Table 17. External weights

Internal Weight (g)

Total Weight (g) Shell Weight (g) (Meat + Free Water) Collection Date Range Meant Range Meant Range Meant

5/1/71 75.4 - 116.7 91.0 ± 11.5 56.5 - 91.0 69.9 ± 9.8 18.1 - 25.8 21.0 ± 2.5

6/1/71 62.1 - 112.4 89.6 ± 12.4 43.8 - 86.3 68.3 ±10.7 18.3 - 26.1 21.4 ±2.4

7/1/71 62.2 - 105.9 87.0 ± 13.1 44.5 - 84.1 65.2 ±11.8 14.9 - 42.0 21.9 ± 6.8

8/1/71 58.4 - 130.7 92.4 ± 20.7 40.4 - 99.7 68.3 ±16.7 18.0 - 32.7 24.1 ±4.3

9/1/71 55.2 - 98.6 78.4 ± 10.7 41.8 - 71.8 59.4 ± 8.2 13.4 - 27.4 19.0 ±3.2

11/1/71 64.9 - 110.0 89.9 ± 14.4 48.8 - 82.8 69.0 ±10.9 15.2 - 29.5 21.0 ±4.4

1/1/72 67.3 - 94.8 81.3 ± 7.7 49.5 - 71.3 61.1 ± 6.4 17.7 - 23.5 20.3 ± 1.7

2/1/72 : 74.2 - 93.5 82.9 ± 5.5 56.7 - 74.3 65.7 ±5.0 8.8 - 25.3 17.1 ± 3.6 Table 18. Internal weights and condition (meat) index

Blotted Meat Dry Meat Tissue Wat er Condition (Meat) Weight (g) Weight(g) Collection Weight (g) Index Date

Range Meant Range Meant Range Meant Hange Meant

5/1/71 7.3-12.1 10.0 ± 1.1 ;0.39-2.13 1.00 t 0.45 6.9-10.3 9.0 t 1.0 0.38-0.56 ;0.50 t 0.04 6/1/71 9.3-14.4 11.2 ± 1.6 1.04-3.07 1.83 t 0.54 6.9-12.3 9.4 t 1.5 0.32-1.10* 0.55 t 0.18 7/1/71 7.9-13.2 10.1 ± 1.8 1.15-2.39 1.74 t 0.36 6.4-11.1 8.4 t 1.5 0.30-0.55 0.47 t 0.13 8/1/71 6.0-12.7 9.5 i 2.0 0.80-2.29 1.44 t 0.40 5.2-10.5 8.0 t 1.6 0.30-0.55 0.41 t 0.06 9/1/71 6.7-11.7 9.2 t 1.3 1.29-2.07 1.70 t 0.21 5.4- 9.7 7.5 t 1.1 0.36-1.36* 0.55 t 0.26 11/1/71 5.2-9.2 6.7 ± 1.2 0.72-1.22 0.92 t 0.16 4.5- 8,3 5.8 t 1.0 0.24-0.48 0.33 t 0.07 1/1/72 6.2-9.2 7.6 ± 1.0 0.83-1.52 1.12 t 0.19 5.3- 7.8 6.4 t 0.8 0.30-0.43 0.37 t 0.03 2/1/72 6.6-9.5 7.9 ± 0.9 0.93-1.64 1.23 t 0.18 5.4- 7.9 6.7 t 0.7 0.29-0.59 0.40 t 0.09

C°nditi°n index above 1.0 in these samples represents that of two individual clams only. These individuals had very high shell volume so that the internal volume calculated was unusually low. Whether this was an abnormality of the clam or a measurement error could not be determined when FEB- O PROTEIN 0 ± 8.8 •± 1 2 .0 • g ly c o g e n

JAN- o ± 4 ' i ± 7 .5 + 1

• CO NOV_ *6 5 O

SEPT- 0±16.9 •±24.2 (/) I zI- o AUG- 0 ± 3.8 • ± 1 1 0

JULY- 0 ± 8.9 • ±12.9

H

JUNE- • ±i o P ± , , °

±56 MAY- t D ± 6.6

t------1------1------1------1------1------1------r 5 10 15 20 25 30 35 40 45 50 MG C C -1 OF INTERNAL SHELL VOLUME

FIG. 41. - Plot of glycogen and protein in meat of Rangia expressed as mgcc~l of shell cavity volume as a function of time of collection.

169 FEB. A GLYCOGEN #±6.3 A + 10.2 • PROTEIN

± 5.2 JAN. • A±11.3

NOV. A±n.2 # ± 1C.0

+4.0

AUG. #—7.4 A ±10.8

LU 2 h - JULY. • ±3.3 A ±9.1

A± l l 5 JUNE. A # ± 10.5

± 5.1 MAY. • A ± 9.5

0 10 20 30 40 50 60 70 80 90 % OF DRY WEIGHT

FIG. 42. - Plot of glycogen and protein in the meat of Rangia expressed as per cent dry meat weight as a function of time of collection.

170 The condition index appears to be lowest in the November - February time period, rising to high level by May and June. Following a slight dip in the July - August period, it rises again in September. It would seem that for good harvesting conditions most of the year is suitable, except perhaps the October - January period. The levels of glycogen and protein in the meat of the clam 3 expressed as mg/available cm of shell cavity volume are shown in 3 Figure 41. Lowest level of glycogen/cm of shell cavity is during the November - January period. It rises by February then drops again to a low by May. It is highest during the June - September 3 period. The protein level/cm of shell cavity volume follows essentially the same pattern. Glycogen and protein, expressed as per cent dry meat weight of the clams, are shown by Figure 42. Glycogen levels based on this parameter reach a peak in February, drop to intermediate level by June, rise from June to August. Drop in level begins by September and reaches the low for the year by November. Protein content analysis is not so clear; while the level appears to be the same almost year around, there is a peak around February and November. There appears to be no direct correlation to salinity conditions that can be drawn from this set of data.

171 COMPARATIVE STUDIES OF CRYSTALLINE STYLE ENZYME ACTIVITY IN POPULATIONS FROM WATERS OF DIFFERENT SALINITY

Introduction arid Literature Review

The crystalline style of molluscs is a flexible gelatinous rod found in a blind diverticulum of the stomach called the style sac. Cilia of the sac epithelium rotate the style and move it for­ ward so that the style material is released into the gut lumen by rotation of the style against the gastric shield, a horny platelike structure in the stomach. The crystalline style occurs in most if not all lamelli- branchia (Yonge, 1931). Anton de Heide (1686) presented the first written notice of discovery of the crystalline style in molluscs, and guessed it was an intestinal ferment or performed some role in the act of generation. Since that time the styles of numerous organisms have been extensively investigated and many varied theories have been advanced as to their origin, nature and function (Nelson, 1918). The enzymatic role of this organ was first demonstrated in 1900 by Coupin who found amylolytic enzymes in the crystalline style of Cardium edule. Since then, a great number and variety of enzymes have been reported to be components of the style. In addition to the amylase originally reported by Coupin and later confirmed by others (Mitra, 1901; Nelson, 1918, 1925; Yonge, 1926; Graham, 1931; Horiuchi & Lane, 1966), numerous enzymes such as cellulase (Purchon, 1941; Lavine, 1946; Newell, 1953; Fish, 1955; Horiuchi & Lane, 1965, 1966), lipase (George, 1952, Hozumi, 1959), alginase (Franssen & Jeuniaux, 1965), chitobiase (Jeuniaux, 1963) and others have also been reported. It is generally agreed that the only extracellular digestion of carbohydrates in herbivorous molluscs occurs in the lumen of the stomach through the action of enzymes liberated by dissolution of the crystalline style.

172 The composition of the styles of various molluscs (Mya arenaria, Yonge, 1923; Ostrea edulis, Nelson, 1918; Crepidula fornicata, Mackintosh, 1925; Mactra solidissima (now called Spisula solidissima), Lavine, 1946) has consistently been reported to be 87% water, 12% organic matter, and 1% inorganic matter. The organic matter is largely protein, and the presence of a globulin (Mackintosh, 1925), an albumen (Nelson, 1918) and a mucin (Mackintosh, 1925; Nelson, 1918; Berkeley, 1935) has been reported. Bailey & Worboys (1960) have shown the style mucoprotein of Pinna nobilis to belong to the hexosamine- galactose-fucose group of mucoproteins, and found the amino acids threonine, serine, proline, and tyrosine to be present in relatively large amounts. The organism used in this study, the fresh-to-brackish-water mactrid clam Rangia cuneata (Gray, in Sowerby, 1831) is quite common in Gulf coastal estuaries from northwest Florida to Texas (Abbott, 1954) and reportedly ranges from the Potomac River in Maryland (Pfitzenmeyer, 1964) to Alvarado, Mexico (Pulley, 1952a). Rangia is a filter feeder on detritus and phytoplankton. It is usually buried 1-6 inches in the bottom substrate. Along the Gulf coast, Rangia spp. have been reported from brackish or low- salinity water by Strecker (1935), Ladd (1951), Gunter (1961), Pulley (1952a & b), Hedgpeth (1953, 1954), Gunter & Shell (1958), and Parker (1960); and from fresh water as well by Maury (1920). It has been reported that no living Rangia were found where the average salinity was over 18 ppt (Parker, 1966). Rangia is highly variable in size, adults usually ranging from about 20 mm in length and depth to about 70 mm in length and 60 mm in depth. The variations in size among different Rangia populations have often been attributed to differences in the environmental salinity. Ladd (1951) observed that these clams attain their largest size at lower salinities. The observations that low-salinity environments seem to be optimum for this species suggested to Hedgpeth (1953) that Rangia may be an excellent organism for use as an indicator of estuarine salinity conditions. When conditions with­ in a bay or lake are drastically changed, the Rangia population

173 appears to be affected quite rapidly. For example, according to Parker (1955), up to 1951 a large San Antonio Bay population of Rangia occurred during a period of low salinity, but it practically disappeared when the salinity level rose to about 20 ppt. Its possible use as a biological monitor of estuarine conditions is by no means the only value of Raiigia cuneata. Darnell (1958) describes Rangia as the most Important bottom invertebrate in Lake Pontchartrain, Louisiana, and lists small Rangia as a primary com­ ponent of fish food. Rangia is also a valuable source of shell. Vast quantities of Rangia shell have been dredged and used extensively in road construction along the Gulf coast. In addition, the shells have numerous industrial uses. There is also a limited market for the meats as human food. Of importance to this study is the fact that Rangia cuneata possesses a crystalline style which is approximately 20-40 mm in length, depending upon the clam’s size. The anterior end of the style enters the posterior dorsal side of the stomach and terminates against the gastric shield in the anterior dorsal portion of the stomach. The styles of Rangia lend themselves to study since they are relatively stable when compared with the styles of many other bivalves; they have not been shown to appreciably diminish or de­ compose even under conditions of starvation or lack of oxygen. In an extensive review of the literature, no reports have been found concerning the carbohydrases of the crystalline style of Rangia cuneata. It was believed, therefore, that an investigation of the enzymology of the carbohydrases of the Rangia crystalline style would contribute to a better understanding of the physiology of this important estuarine invertebrate.

Materials and Methods

Specimens of Rangia cuneata used in this study were collected from three ecologically different locations on the Texas Gulf coast: the McCollum Park area of upper Trinity Bay, the northwestern shore of Lake Anahuac near Anahuac, Texas, and the Neches River above Beaumont, Texas. The McCollum Park region has a mean yearly salinity of approximately 11 ppt (Parker, 1966), while Lake Anahuac is essen­ tially a freshwater lake (salinity less than 1 ppt). The salinity of the Neches River at our collecting site varies widely with flow rate. It ranges from 0.3 ppt during periods of high flow rate up to 13 ppt during periods of low river flow (Hopkins & Andrews, 1970). The Beaumont population is separated from the nearest other popula­ tion of Rangia, in Sabine Lake, by 20 miles of polluted, nearly sterile river. The largest specimens of Rangia from the Trinity and Anahuac locations were found to be approximately 60-70 mm in length and depth, while the Neches Rangia were only 30-40 mm in length and depth. After collection, the clams were brought to Texas A&M University and stored in a cold room at 0-4° C. The styles were extracted, usually within 12 hours, the time depending upon the number of specimens collected. Following extraction, the styles were washed several times in cold 0.2 M phosphate buffer (pH 6.6), made isotonic to the water of each respective collection site by the addition of NaCl solution. They were then homogenized in phosphate buffer using ground glass homogenizers and stored for 6-8 hours at 0-4° C to completely solubilize the protein. The viscous homogenate was centrifuged at 0-4° C for thirty minutes at 20,200 G, the supernatant retained and either frozen immediately or shell frozen, lyophilized and stored at -10° C for later assay. Preliminary work revealed no appreciable difference in enzymatic activity between freshly prepared, frozen, or lyophilized preparations. When used for assay, the lyophilized homogenate was suspended in either phosphate buffer or glass-distilled water, depending upon the type of assay to be performed. When allowed to stand at room temperature for several hours, the lyophilized powder dissolved readily in both glass-distilled water and phosphate buf­ fer (Horiuchi & Lane, 1966). The suspended homogenate was dialyzed against glass distilled water at 2-4° C for at least 48 hours, or until free of glucose and chloride ions as shown by testing aliquot samples with Glucostat reagent (Worthington Biochemical Corporation) and AgNO^, respectively. Precipitates which appeared in the solution

175 during dialysis were shown to be enzymatically inactive and therefore were separated by low-speed centrifugation and discarded. Only the clear supernatant was used for analysis. For the purposes of this report, detailed accounts of analytical procedures and several discussions are omitted. These will be found in the 1972 Ph.D. dissertation of Melvin Frei entitled "Carbohydrases of the crystalline style of the brackish water clam Rarigia cuneata."

Results and Discussion

The results of assays to determine substrate specificity are shown in Table 19; the specific activity values are expressed either as jig glucose/hour/mg Lowry protein or as yg glucose equivalents/ hour/mg Lowry protein, depending upon the reagent used for product analysis. The values are shown as a range of specific activity since the values tend to fluctuate with only minor alterations in the reaction conditions. By the use of Glucostat as the indicator of enzymatic activity, cleavage of soluble starch, glycogen, dextrin and maltose was demonstrated, with specific activity values ranging from 8 to 20 yg glucose liberated/hour/mg Lowry protein. When the reducing sugar test was employed, it indicated that in addition to the above- mentioned substrates, carboxymethyl cellulose-fine was cleaved to smaller units. The values obtained using the reducing sugar test were consistently higher than those obtained using the Glucostat reagent; however, the reducing sugar values represent not only the glucose liberated but all units with a free reducing end which are capable of yielding a positive reducing sugar test. After considering the results of the substrate specificity assays, it was decided to explore and compare the style enzyme activity of the three populations of clams on only two of the carbohydrates, soluble starch and maltose (both of which are a 1, 4 glucosides). The remainder of this study therefore deals with a comparison and partial characterization of the amylase and maltase activity of the crystalline style enzymes of the Trinity, Anahuac, and Neches populations of Rangia cuneata. Since the presence of

176 Table 19. Carbohydrate digestion in Rangia cuneata

Substrate Concentration Enzyme Activity 0 y moles/reaction Glucostat Reducing Sugar

Soluble Starch 50 16-20 35-40 Glycogen 15-18 35-40 Dextrin 50 8-10 20-25

Maltose 10 8-10 —

C M Cellulose(fine) 50 — 20-25 CMC (med.) 0 0 Cellulose Phosphoric Acid Swollen 0 0 Sulfuric Acid Digested 0 0 Cellobiose 10 0 0 Raffinose 50 0 0 a-Lactose 10 0 0 3-Lactose 10 0 0 Melibiose 10 0 0 Melizitose 50 0 0 Sucrose 10 0 0 a-Cellulose 50 0 0 3-Cellulose 50 0 0 Trehalose 50 0 0 Pectin 0 0 Salicin 0 0 Sodium Alginate 0 0

1) Specific Activity expressed as yg Glucose/hr/mg Lowry Protein 2) Specific Activity expressed as yg Glucose equivalents/hr/mg Lowry Protein Assays conducted for 2 hrs at 37° C and pH 6.6

177 both amylase and maltase was established by preliminary studies, the end products of amylase activity (i.e. maltose) would be broken down to glucose; thus Glucostat may be used to measure both amylase and maltase activities. Since the original work by Coupin (1900) in which the presence of amylolytic enzymes in the crystalline style of Cardium edule was established, numerous reports have demonstrated the presence of not only amylase, but other carbohydrases as well, in the molluscan crystalline style. It is difficult to make valid comparisons with the results of other investigators since the species tested and the reaction con­ ditions employed are different. However, the results are in agree­ ment with the majority of other reports in that the same carbohy­ drases appear to be cleaved by the crystalline style enzymes of Rangia as are cleaved by those of other molluscs.

Chlorinity In view of the fact that the style amylases were found to be Cl dependent, assays were conducted to determine the relationships between chlorinity and amylase activity of style extracts from Trinity, Anahuac, and Neches Rangia. The reaction mixtures, at a total volume of 4.0 ml, contained 50 y moles soluble starch, 200 y moles phosphate buffer (pH 6.6), 2.0 mg lyophilized style extract and the various concentrations of NaCl, as shown in Fig. 43. Prior to assaying for the effects of chlorinity upon style amylase activity, particular attention was given to complete removal of Cl . All extracts were dialyzed against glass-distilled water for at least 48 hours and/or until free of Cl as evidenced by a negative test with AgNO^. From Fig. 43 it may be observed that in all three Rangia populations, amylase activity was definitely Cl dependent. This is not surprising since all known animal amylases have been shown to be Cl dependent. No amylase activity was observed in the absence of NaCl (after dialysis); however, amylolytic activity was detectable after adjusting the NaCl concentration of the reaction mixture to as little as 1.8 x 10 M. Other investigators (Yonge, 1923; Yonge,

178 FIG. 43. - Plot of effect of chloride concentration on glucose released from soluble starch by amylase of Trinity, Anahuac, and Neches Rangia crystal­ line style extracts.

179 1926; Lavine, 1946; etc.) have similarly shown the amylases of the molluscan crystalline style to be Cl dependent, but have mentioned no specific concentrations. Although with all three populations there was detectable amylase -4 activity at 1.81 x 10 M NaCl, each appeared to respond differently to NaCl concentration increases, and furthermore, each appeared to require a different Cl concentration for maximum activity (Fig. 43). Table 20 shows a comparison of the effects of chlorinity upon style amylase activity for the three populations of Rangia. The specific activity at each NaCl concentration is expressed in terms of per cent of maximum activity. Although no statistical analysis can be performed on this type of data, it perhaps more dramatically emphasizes the differences in Cl concentration required for maximum activity by each population. It may be noted from Table 20 that with the minimum addition of NaCl, equivalent to making the reaction -4 mixture 1.81 x 10 M, the style amylases of the Trinity, Anahuac, and Neches Rangia had already reached 23.13%, 12.74% and 4.41% of maximum activity, respectively. Results therefore indicate that the Cl requirement for initiation of activity is probably much lower although assays at lower concentrations were not conducted. At -4 NaCl concentrations between 10.87 and 36.24 x 10 M, the Trinity and Anahuac style preparations almost parallel one another in per cent maximum activity, while being quite different from the Neches —4 preparation. At concentrations greater than 36.24 x 10 M, the Anahuac clam style enzyme begins to exhibit a decrease in activity not noticeable in the preparation from the other two populations. When considering Fig. 43 and Table 20, it is interesting to note that the Trinity Rangia which inhabits the most saline of the three environments required the least amount of NaCl to exhibit maximum activity, in spite of the rigorous dialysis to remove the Cl . It should also be noted that the style enzymes of the Trinity and Anahuac populations reach maximum activity with NaCl addition to a concentration of 18.12 - 36.24 x 10-V , while that of the Neches population did not reach maximum activity until the concentration had been raised to

l8o Table 20. Comparison of effects of chlorinity variation upon amylolytic activity of the crystalline styles of Trinity, Neches, and Anahuac Rangia.

NaCl ^ % of Maximum Activity Molarity x 10 Trinity Anahuac Neches

1.81 23.13 12.74 4.41 3.62 37.22 26.50 17.64 5.43 50.71 36.33 27.20 7.24 59.27 46.43 31.60 9.06 64.01 57.79 38.98 10.87 69.76 65.36 51.45 12.68 74.48 69.79 54.40 14.49 78.60 74.72 63.23 16.30 88.57 80.29 64.76 18.12 100.00 89.44 73.52 36.24 99.68 100.00 73.57 54.36 96.51 99.19 74.81 72.48 93.34 95.05 79.37 90.60 93.18 92.66 91.13 108.72 94.77 84.09 94.30 126.84 94.61 83.28 96 .83 144.96 97.31 84.09 100.00 163.08 97.45 85.67 100.00 181.20 94.71 85.67 100.00

Substrate used was 50 y moles soluble starch Reactants incubated for 2 hours @ at pH 6.6

l8l approximately three to five times that of the other two populations. Assays were conducted in this study to determine the relation­ ship between pH and amylase activity of style extracts from Trinity, Anahuac, and Neches Rangia. The reaction mixtures, at a total volume of 4.0 ml contained 50 y moles soluble starch, 1812 y moles NaCl, 2.0 mg of lyophilized style extract, and 400 y moles of either phos­ phate or citrate-phosphate buffer at the various pH levels indicated in Fig. 44 and Table 21. When phosphate and citrate-phosphate buffers were used the style amylase activity was noted to be unaffected by a change from citrate-phosphate to phosphate at the same acidity. It should be mentioned that buffers other than the two above were tested. When buffers such as citrate, acetate, phthalate-NaOH, and succinate were used, the activity values were depressed when com­ pared with either of the two above-mentioned systems at the same pH levels. A tabular comparison of the pH effects upon style amylase ac­ tivity of the three populations of Rangia is presented in Table 21. The values shown are the specific activities at the specified pH?s expressed in terms of a percentage of the maximum specific activity. The rationale behind expression of the data in this manner is twofold; it emphasizes more dramatically the relationships between preparations as affected by pH and, most importantly, the data are presented in such a way that the inherent differences in specific activities which occur at any pH, and the differences imposed by experimental design, are not statistically perpetuated. It may be noted from Table 21 that at pH 5.2 the Trinity style amylase had reached approximately 20% of maximum while that of the Anahuac and Neches populations did not reach this level until pH 5.6 and 5.8, respectively. In all three cases, however, the activity rose sharply and reached maximum at pH 6.6, which represents the value upon which the other percentages were calculated. The style amylase activity of the Trinity Rangia was lost between pH 7.6 and 7.8, while for the Anahuac and Neches Rangia the activity was lost between pH 7.8 and 8.0.

182 pH

FIG, 44. - Plot of effect of pH on crystalline style amylase activity of Trinity, Anahuac, and Neches Rangia, indicating pH optimum of starch hydrolysis.

183 Table 21. Comparison of effects of pH variation upon amylolytic activity of the crystalline styles of Trinity, Neches, and Anahuac Rangia.

% of Maximum Activity pH Trinity Néchés Anàhuac

5.0 0.00 0.00 0.00 5.2 17.28 0.00 0.00 5.4 35.39 0.00 0.00 5.6 45.00 0.00 19.54 5.8 60.80 19.44 36.25 6.0 74.52 33.30 52.76 6.2 84.34 61.11 74.72 6.4 88.24 77.76 76.90 6 .6 100.00 100.00 100.00 6.8 92.18 86.14 82.41 7.0 52.92 55.53 65.92 7.2 33.32 41.67 47.26 7.4 25.47 22.23 37.36 7.6 9.78 16.65 26.35 7.8 0.00 4.94 10.09 8.0 0.00 0.00 0.00 Results of pH assay presented above are in partial agreement with results of several other investigators working with crystalline style amylases of marine molluscs (Spisula solidissima, Crassostrea virgiriica, Ostrea edulis, etc.). In addition to species differences, assay conditions used by these investigators varied greatly, making a valid comparison impossible. In order to ensure that substrate was not a limiting factor in determinations of specific activity, assays were conducted to determine the relationship between substrate concentration and amylase activity of style extracts from Trinity, Anahuac, and Neches Rangia. The reaction mixtures, in a total volume of 4.0 ml, contained 200 y moles phosphate buffer (pH 6.6), 1812 y moles NaCl, 2.0 mg lyophilized style extract, and various concentrations of soluble starch between 5 and 80 x 10 M, as indicated in Fig. 45. Fig. 45 shows the relationship between substrate concentration and specific style amylase activity for the Trinity Rangia. Data for the Anahuac and Neches style preparations are not presented since the results were essentially identical. Specific activity increased almost directly with increasing substrate concentration up to approximately 45 x 10 ^ M. Further increases in substrate concentration did not appreciably increase the specific activity, indicating saturation of active sites of the enzyme. The enzyme concentration of approximately 1.5 mg Lowry protein, the pH, the amount of NaCl, the incubation period, etc., used in these assays relating substrate concentration to specific activity closely parallels that used in the majority of experiments in this study. It is therefore reasonable to assume that as long as soluble —6 starch is in excess of 45 x 10 M, the substrate will not be a limiting factor in the style amylase reaction. When maltose was used as substrate, at a concentration of 50 x 10~^M, the contaminating glucose or the maltose itself (depending upon the type of analysis used) made the colorimetric readings with even substrate alone too high to read reliably. It was found, after setting up several series of maltose dilutions, that at a maltose

185 FIG. 45. - Plot of effect of substrate (starch) concentration on amylase activity of Trinity Rangia showing substrate saturation of the active site between 40-50 x 10""^M.

186 _6 concentration of 10 x 10 M, valid results were obtainable and reliably reproducible. Therefore the assays in which maltose was used as substrate were conducted at a substrate concentration of —6 10 x 10 M more out of necessity than by choice.

Incubation Time It has been reviewed previously that various parameters such as pH, temperature optima, etc., may be affected by length of incubation. It became of interest therefore to determine if the length of incubation (under the specified conditions) had any effect upon the specific activity of the crystalline style amylase of Rangia. Fig. 46 illustrates the relationship between length of incuba­ tion and the amount of glucose liberated/mg Lowry protein. The re­ action mixtures, at a total volume of 4.0 ml, contained 50 y moles soluble starch, 200 y moles phosphate buffer (pH 6.6), 1812 y moles NaCl, and 2.0 mg lyophilized style extract. The reaction mixtures were incubated at 37° C for various periods of time between 2.5 and 180 minutes, as indicated in Fig. 46. The reactions were stopped at the end of each interval by placing the assay tubes in an ice bath until such time as Glucostat was added for the colorimetric test. Amylase activity shown in Fig. 46 was very slight for the first 20 minutes of incubation and began to rise sharply thereafter. Previous experimentation pointed to the presence of either two distinct enzymes, an amylase and a maltase, or an amylo-maltase, as described by Bernfield 1955. On the basis of this, it is possible that this time (5-10 minutes) might be required to convert the starch to smaller components which are acted upon by the maltase to liberate measurable glucose. The specific activity of amylase after each of the incubation periods is presented in Table 22. The specific activity is more meaningful because it takes into consideration the amount of product liberated/unit time/mg protein and not simply the amount of product liberated. The data reveal that the specific activity was lower for the first hour of incubation; however, after about 80 minutes, the specific activity at this specified pH, temperature, etc., did not appreciably change. Therefore the incubation time 187 pG GLUCOSE MG"' PROTEIN 3 5 . FIG. 46. - Plot of relationship between incubation timeincubation 46.ofrelationshipFIG.andamylase -Plot between activityofcrystallinestyle.Trinity Rangia 188

Table 22. Change in incubation time - pg Glucose/mg Lowry Protein.

Time ______Specific Activity

60 min - 6.0 yg Glucose/hr/mg Lowry Protein 80 min 7.85 100 min - 7.97 120 min - 8.60 140 min - 9.55 160 min 9.46 180 min 8.92 yg; Glucose/hr/mg Lowry Protein

Trinity Rangia - 37° C Temp. Incubation Rx. Mixture - 1.0 ml 50 mM Sol. Starch (50 p moles) 1.0 ml Phos. Buffer pH 6.6 0.1 ml NaCl (1.7M) 1.0 ml FDS (2 mg/ml GDH) - 1.25 mg L.P. 0.9 ml Glass-distilled Water

I89 of two hours used in the majority of the assays in this study was considered to give an accurate representation of the specific activity as measured by the Glucostat reagent. In order to determine the significance of variations in the in­ cubation temperature, assays were conducted to find the relationship between temperature variation and the specific amylase and maltase activity for style extracts of the Trinity, Anahuac, and Neches Rangia. The assay mixtures for the amylase experiments, at a total volume of 4.0 ml, contained 50 y moles soluble starch, 200 y moles phosphate buffer (pH 6.6), 1812 y moles NaCl, and 2.0 mg lyophilized style extract. The reactions were allowed to proceed for two hours at various incubation temperatures ranging from 2 to 60° C, as indicated in Fig. 47. At incubation temperatures of 4 and 10° C there was detectable amylase activity with the Anahuac clams, and between 10 and 16° C all three preparations exhibited significant activity. Specific activity for all preparations increased sharply with increasing temperature until the apparent optima were reached, at approximately 30° C for enzyme of the Neches population and at 37° C for that of the Trinity population. Although the optimum was not as sharply defined for the Anahuac clam style enzyme it is reasonable to assume that the temperature which resulted in the highest specific activity, 37° C, closely approximates the optimum. Activity began to decrease with the enzyme of the Neches population between 30 and 37° C; with the other two populations, the decrease began between 40 and 45° C. Specific activity for all preparations dropped sharply between 45 and 50° C, and at 60° C the activity had been completely lost in all. A tabular comparison of the effects of incubation temperature variation upon style amylase activity for the three previously mentioned populations is presented in Table 23. The values shown are the specific activities at the specified incubation temperatures expressed in terms of percentage maximum specific activity. No experiments were conducted between the temperatures of 10 and 16° C; however, it was indicated that the actual temperature required

190 FIG. 47. - Plot of temperature optimum determination for the amylase of Trinity, Anahuac, and Neches Rangia crystalline style.

191 Table 23. Comparison of effects of temperature variation upon amylolytic activity of the crystalline

styles of Trinity, Neches, and Anahuac Rangia.

Reaction % of Maximum Activity Temperature (° c) Trinity Neches Anahuac

0 0.00 0.00 0.0 4 0.00 0.00 2.74 10 0.00 0.00 7.69 16 16.75 24.57 34.61 20 39.25 50.55 61.53 25 66.98 88.26 96.70

30 88.45 ____ 100.00 97.86 37 100.00 91.05 100.00 45 81.43 56.93 76.92 50 19.16 0.00 8.78 55 11.39 0.00 0.00 60 0.00 0.00 0.00

192 for activation of the amylase of the Trinity and Neches populations is considerably below 16° c. As previously mentioned, the style amylase of the Neches clams reached maximum activity at about 30° C while that of the Anahuac and Trinity clams did not exhibit maximum activity until about 37° C. Assay mixtures for maltase contained, in a total volume of 4.0 mg, 10 p moles maltose, 200 p moles phosphate buffer (pH -6.6), 1812 u moles NaCl, and 2.0 mg lyophilized style extract. The reactions were allowed to proceed for two hours at various incubation temperatures ranging from 2 to 60° C, as indicated in Fig. 48. Incubation temperatures were maintained and product analysis was performed as previously described for the amylase assays. Fig. 48 illustrates the relationship between incubation temperature and specific maltase activity for the Trinity Rangia; results for the other two populations were quite similar. Maltase activity was still present at 60° C, a temperature at which amylase activity had been completely lost. Maltase activity was not shown to be Cl dependent, in contrast to amylase activity; however, NaCl was added to the maltose reaction mixtures to preserve uniformity. The level of NaCl added was found to have no inhibitory effect on maltase activity. It is evident that differences do exist between the temperature optima for maltase and for amylase activity even though reaction conditions were the same for both assays. In the previously described temperature studies the entire reaction mixtures were incubated for two hours at the specified temperatures in order to estimate the optimum temperature for optimum activity. Another temperature-related assay useful in characterizing and comparing enzyme systems is the heat denaturation study. In denaturation studies, the enzyme alone is incubated at various temperatures for specified periods of time prior to conducting the actual assay. The denaturation curves, which show the relationships between length of enzyme exposure to specified temperatures and style amylase

193 FIG. 48. - Plot of temperature optimum determination of Trinity Rangia crystalline style maltase.

19^ activity, for the Trinity, Anahuac and Neches Rangia are shown in Figs. 49A, 50A, and 51A, respectively. The results are expressed as the percentage of control values obtained after incubation at 37° C. The exposure of Trinity clam style extracts (Fig. 49A) to 45° C for 2 and 4 minutes resulted in approximately 44% loss of style amylase activity, and approximately 80% of the activity is lost after heating for 16 minutes at this temperature. By comparison, heating the enzyme solutions for only 2 minutes at 50° C resulted in approximately 80% activity loss, while after 16 minutes at 50° C approximately 90% of the activity had been lost. Heating at higher temperatures for varying lengths of time showed a progressive decrease in activity so that after 16 minutes at 65° C no activity was detected. In studying the Anahuac style preparation, given in Fig. 50A, it may be noted that at 45° C, exposure for 2 and 4 minutes resulted in approximately 20% loss of activity which decreased sharply to approximately 75% loss after 16 minutes at 45° C. By comparison, heating at 50° C for only 2 minutes showed approximately 80% activity loss while 16 minutes at 50° C resulted in 95% loss of activity. After heating the enzyme solutions for 4 minutes at 65° C, no activity was detected. Results of experiments with Neches clam style preparations given in Fig. 51A indicate that exposure for 2 minutes at 45° C resulted in approximately 20% loss of style amylase activity; this loss sharply increased to approximately 80% after 16 minutes at 45° C. Heating at 50° C for only 2 minutes resulted in approximately 80% activity loss, and approximately 95% loss after 16 minutes o at 50 C. No amylase activity was noted after heating the enzyme solutions for 16 minutes at 65° C. The relationships between length of enzyme exposure to specified temperatures and style maltase activity for the Trinity, Anahuac, and Neches Rangia are presented in Figs. 49B, 50B, and 51B, respectively. The results are again expressed as a percentage of control values obtained by incubation at 37° C.

195 FIG. 49. - Plot of temperature dénaturation of Trinity Rangia crystalline style amylase and maltase. Style extract was heated to specified time intervals and temperatures prior to assay.

196 100 A. ANAHUAC-AMYLASE

FIG. 50. - Plot of temperature dénaturation of Anahuac Rangia crystalline style amylase and maltase. Style extract was heated to specified time intervals and temperatures prior to assay.

197 F IG. 51. - Plot of temperature dénaturationtemperatureofof -Plot 51.Neches Rangia IG. % OF CONTROL X OF CONTROL extract was heated totimeintervalsextractspecified heated was Style andcrystallinestyleamylase maltase. and temperatures prior totemperaturesandassay.prior 198

In assays conducted with Trinity clam style preparation, given in Fig. 49B, after heating the enzyme solutions for 2 minutes at 45° C there was a decrease in maltase activity of approximately 30%; however, heating at 45° C for 8 and 16 minutes resulted in only 25 and 20% decreases, respectively. Incubation for 2 minutes at both 50 and 55° C resulted in about a 25% decrease, while 16 minutes at 50 and 55° C showed decreases of 60 and 80%, respectively* Heat treatment of the enzyme solutions at higher temperatures for varying lengths of time resulted in a progressive decrease in activity so that after 2 minutes at 75° C all style maltase activity for the Trinity Rangia was abolished. Assays conducted with Anahuac clam style preparations given in Fig. 50B indicated that heating the enzyme solutions for 2 minutes at 45° C reduced maltase activity about 35% and after 16 minutes at 45° C the activity was reduced to about 55% of control. Heating for 16 minutes at 50, 55 and 60° C caused approximately an 80% decrease in activity as in the Trinity Rangia; heating for 2 minutes at 75° C resulted in complete loss of activity for the Anahuac Rangia. Results of the denaturation experiments with maltase of the Neches population, given in Fig. 51B, are quite different from those of the other populations. When enzyme solutions were incubated for 2 minutes at 45° C, rather than noting a decrease in activity there was an increase of approximately 15% when compared with con­ trols. Heating for longer periods of time at 45° C showed progres­ sively higher activities until after 16 minutes at 45° C the maltase activity was approximately 25% above controls assayed at 37° C. Heating of the enzyme solutions for 2 minutes at 50 and 55 C likewise resulted in increased activity; approximately 25% at 50° C and 35% at 55° C. However, after 16 minutes at both 50 and 55° C, the style maltase activity for the Neches Rangia had decreased to approximately 60% of control and when the enzyme was exposed to 70° C for 4 minutes or 75° C for 2 minutes all style maltase activity was lost.

199 The results of other studies indicate that most molluscan style amylases are denatured by exposure to temperatures of 50 to 70° C for a period of 15 minutes. Results of the denaturation assays of the present study show that heating of the enzyme solution for 16 minutes at 65° C results in a complete loss of the style amylase activity in Rangia cuneata. The style maltase activity was found to be lost when the enzyme solutions were heated for 15 minutes at 70 C. Figs. 52A & B and 53A & B are summaries of the amylase and maltase activity changes in response to heating the enzyme solutions at certain temperatures for various periods of time. The data presented in this manner illustrate more clearly that the style amylases of all three populations responded similarly to enzyme heating for certain periods of time, but that the maltase response was quite different from that shown for the amylase. The maltase activity in response to enzyme heating was quite similar for the Trinity and Anahuac clams but the maltase response of the Neches population was found to be quite different from that of the other two populations. It is probable that there are in all prep­ arations separate amylase and maltase enzymes, and that there is not an amylo-maltase to perform the hydrolyses of amylose and maltose. It is further probable that the maltase of the Neches population is different from that of the Trinity and Anahuac clams. This would lend support to the possibility that the Neches clams are a distinct and separate population.

Ionic Effects It is well established that molluscan, as well as other animal amylases, require ions such as Cl for activity (Yonge, 1923, 1926; Lavine, 1946; Horiuchi & Lane, 1966; etc.). In addition to NaCl, Yonge (1926) noted activation of the style amylase of Ostrea edulis _l_ j | -j | by chlorides of K , Ba , Mg , and others. Lavine (1946) likewise infers that the presence of salts other than NaCl in sea water might increase the rate of starch digestion by style amylases of Spisula solidissima.

200 F % OF CONTROL ACTIVITY % OF CONTROL ACTIVITY IG. 52. - Plot of temperature dénaturation of Trinity, of dénaturation temperature of Plot - 52. IG. Anahuac, and Neches Rangia crystalline style crystalline Rangia Neches and Anahuac, amylase and maltase. Comparison of relative of Comparison maltase. and amylase ae o ls o atvt t 5 n 0 C. 50° and 45 at activity of loss of rates 201

FIG. 53. - Plot of temperature dénaturation of Trinity, of dénaturation temperature of Plot - 53. FIG. % OF CONTROL ACTIVITY % OF CONTROL ACTIVITY 14 90-1 0 . Anahuac, and Neches Rangia crystalline style crystalline Rangia Neches and Anahuac, ae o ls o ciiya 5 ad 0 C. 70° and 55 at activity of loss of rates amylase and maltase. Comparison of relative of Comparison maltase. and amylase 202

It is also well established that various ions such as the heavy metals are powerful inhibitors of amylase activity. Such reports as that of Englard, Sorof, & Singer (1951) show that reagents such as HgCl^ and CuSO^, typically thought of as selective for -SH groups, exert inhibitory effects upon amylase activity. It has also been recently noted that chlorides of Fe++, Hg”^, and Cu added to aquarium water in which Raiigia were being retained resulted in high mortality; no explanation was offered for these findings. In view of the above-mentioned reports and since various ionic species are known to be environmental pollutants common to most estuarine regions, it became of interest to determine the effect of a series of ions and divalent cations upon style amylase activity °f Rangia. In these studies, as in the determination of the effect of chlorinity upon amylase activity, particular attention was given to complete ion removal prior to beginning the assays. As previously described, it has been established that Cl" is required for style amylase activity in Rangia cuneata. In order to establish whether the Ca ionic species had any effect other than that exerted by NaCl alone, NaCl was added to all reaction mixtures to a molar -4 concentration of 18.12 x 10 . This amount had previously been shown to approximate the concentration required for maximum style amylase activity. Each reaction mixture, in a total volume of 4.0 ml, contained 50 y moles soluble starch, 200 y moles phosphate buffer (pH 6.6), 1812 y moles NaCl, and 10 y moles of one of the chloride salts indicated in Table 24. The results illustrate the effects of various ionic species upon style amylolytic activity of the three popula- I i tions of Rangia. As may be seen, the chloride salts of Fe , [ | j j _|_ |_ | j Hg , Sn , Ni , and Zn completely inactivated the style amylase of all three populations of Rangia. It should be pointed out that this effect could not be due to the additional Cl alone, since 10 y moles of NaCl added to controls in addition to the 1812 y moles present in the reaction mixture did not show any appreciable effect.

203 0.00 0.00 0.00 0.00 0.00 75.00 89.00 31.03 12.06 100.00 203.45 129.31 181.21 106.03 100.00 100.00 100.00 Neches 3.57 0.00 0.00 0.00 0.00 0.00 71.43 74.99 74.99 78.57 57.14 85.71 12.50 144.64 453.56 489.20 1Q0.00 Anahuac % % of Control 0.00 0.00 0.00 0.00 0.00 74.41 77.47 90.21 84.78 89.16 24.19 26.84 204.76 243.20 104.78 120.63 100.00 Trinity crystalline styles of Trinity, Anahuac, and Neches Rangia ciineata. 2 2 2 CI (Molarity x 10 ) 0 Ionic Species +MgCl2 +CsCl2 +BaCl2 +CaCl +SrCl +LiCl +ZnCl +CdCl2 +KC1 +RbCl +MnCl2 +SnCl2 +NiCl2 +C +FeCl2 +HgCl2 able 24. Effect of various ionic species upon amylolytic activity of the Reactants incubated for 2 hours at 37° C. Values expressed are means of 5-10 determination. 10 y moles of each ionic species added to reactionmixtures. NaCl Only T

20k I I I I The salts of Co and Cd when added to the reaction mixtures also showed a high degree of inhibition; between 70 and 95% for all three populations. For the Trinity and Anahuac populations K+ I j and Cs resulted in approximately 25% inhibition while no inhibi­ tory effect was noted with the Neches population. Rb+ resulted in approximately 15% inhibition in the Trinity and Anahuac clams while I j the Neches clams showed no appreciable change. Mn showed slight inhibition with all three populations and Li+ exerted some inhibitory action upon the amylases of the Trinity and Anahuac I j populations. A slight excitatory effect by Mg was seen with the Trinity Rangia; however there was distinct inhibition of enzymes in the other populations. Barium salts caused an increase of 20 to 45% of the amylase in all three preparations when compared with controls. Strikingly, I | | | when the chloride salts of Ca and Sr were added to the reaction mixtures, activity was at least doubled for the Trinity and Neches preparations and approximately quadrupled for that of the Anahuac clams. The complete inhibition of amylase activity by ions such as I | j | Fe , Hg , etc., indicate that even extremely small amounts of these ions in the estuarine habitat could severely decrease the digestive efficiency of Rangia and possibly lead to its elimination. On the other hand, the stimulatory or excitatory effects noted with I | | | | | j | the salts of Mg , Ba , Ca , and Sr tend to infer that the pres­ ence of salts other than NaCl in sea water might also increase starch hydrolysis, as first noted by Lavine (1946).

Conclusions

Digestive enzymes are present in the crystalline style of molluscs and are released into the stomach when the style dissolves. Crystalline styles of the mactrid Rangia cuneata, from three eco­ logically different locations on the Texas Gulf coast, were assayed for carbohydrase activity. In common with other previously

205 mentioned investigations of molluscan styles, this study has failed to show any trace of enzymatic activity on substrates of sucrose, raffinose, melibiose, and several other glucosides. It is concluded that substrates such as those mentioned above are probably digested intracellularly by cells of the digestive divertic­ ulum, as was first mentioned by Yonge (1926). Enzyme activity was noted in all three populations with substrates of soluble starch, glycogen, dextrin, maltose and CMC-fine, as evidenced by the liberation of glucose or an increase in reducing sugar. Based on results of this and numerous other studies there is little doubt that the molluscan crystalline style contains enzyme systems capable of degrading the a-glycosidic linkages of starch, glycogen, etc. Although it appears that the style of Rangia is incapable of digesting pure cellulose, the 8-glycosidic linkage which occurs in cellulose was shown to be susceptible to cleavage, since CMC- fine was at least partially degraded by the style extract. The presence of a cellulase, together with a cellobiase as shown in Strombus gigas by Horiuchi & Lane (1965), would logically be of great advantage to Rangia since organic debris, which is largely cellulose, probably constitutes the bulk of ingested material. On the other hand, it is highly probable that much of the ingested material has already been partially degraded by microbial action to a form utilizable by the clam. The amylases of all three populations of Rangia studied were found to be Cl"" dependent, as are other animal amylases, while Cl was shown not to be required for the other carbohydrases found. Amylase activity was detected in all three populations at NaCl con- -4 centrations of as low as 1.8 x 10 M; however, as mentioned, each population responded differently to increasing NaCl concentra­ tions and each required a different concentration for maximum activi- ty: 18.12 x 10-Si, 36.24 x 10_^M, and 144 x 10_^M, for the Trinity, Anahuac, and Neches Rangia, respectively. The Trinity Rangia which inhabits the most saline of the three environments required the least amount of Cl for maximum activity.

206 The Neches population which lives in fresh water for 7 months of the year required the greatest amount of Cl . However, Bedford & Anderson (personal communication) have recently found Raiigia to be an osmoconformer at salinities above 10 ppt and an osmoregulator below this salinity, becoming significantly hyperosmotic to the environment at salinities below 5 ppt. It was also found that populations of Raiigia from two different areas of the Trinity estuary having different average salinities showed no difference in their osmoregulatory capabilities. Although no work was done specifically on the internal Cl concentration, it is possible that all three populations investigated in this study are capable of regulating their internal Cl concentration to that required for maximum amylase activity. Whether the differences in Cl requirements found in this study are environmental adaptations to the different environmental salinities or whether there is sufficient real difference to suggest different evolutionary history are difficult questions to answer. The Neches population is so drastically different in its response to Cl from the other two populations that on this and other evidence one is tempted to postulate that it is a genetically different race. In assays to determine the relationship between pH and style amylase activity, it was found that at pH’s below 5.0 none of the three populations exhibited detectable activity (Fig. 44). In the presence of 0.2 M citrate-phosphate buffer at pH 5.2, activity was noted for the Trinity style amylase while with either 0.2 M citrate-phosphate or 0.2 M phosphate buffer at pH 5.6 and 5.8 activity was noted with the Anahuac and Neches style amylases, respectively. The style amylases of all three populations showed an almost direct relationship to increasing pH; the optimum was very sharply defined and lies at pH 6.6, the efficiency of the en­ zyme being rapidly reduced on either side of this point. Increasing the pH above 6.6 resulted in a decrease in specific activity for

207 all three populations up to pH 8.0, when all amylase activity was los t, presumably due to enzyme dénaturation. Although the style amylase activity of each population was initiated at a different pH, all three populations exhibited maximum activity at pH 6.6; therefore it is doubtful that any significant conclusions can be drawn as to differences among the populations as far as response to pH is concerned. A result which should be pointed out is the decreased activity which was noted when citrate, acetate, or succinate buffers were used at the same pH levels as the citrate-phosphate and phosphate buffers. Data indicated that these buffers exerted an inhibitory effect upon style amylase activity. When assays were conducted to determine the effect of substrate concentration upon style amylase activity, it was found that all three populations responded quite similarly to substrate concentration changes (Fig. 45). Amylase activity was noted for all three populations with substrate concentrations of as little as 5 x 10 —6 M. With additions above this concentration the specific activity of all three populations increased almost directly with increasing substrate concentration up to approximately 45 x 10 —6 M at which point further increases did not appreciably increase the specific activity. When assays were conducted to determine the effect of enzyme concentration upon style amylase activity, it was again found that all three populations responded quite similarly. It was important that the response was linear over the range of concentrations used during the assays in this study. In most assays the style amylase of the Anahuac Rangia appeared to be higher than that of the other two populations; however, since all activity values changed quite drastically with only a slight alteration in reaction conditions, little emphasis may be placed on this apparent difference in specific activity. Results of experiments to determine the relationship between length of incubation time and style amylase activity showed that

208 activity was less for the first 20 minutes of incubation in spite of the fact that all reactants had been acclimated to the assay temperature for 30 minutes prior to beginning the assays. Several explanations for this apparent lag are possible; however, the most plausible one in view of other evidence presented is the presence of two distinct enzymes, an amylase which first cleaves the starch to smaller units and a maltase which in turn liberates the measur­ able glucose. Although the specific activity was somewhat low for the first hour of incubation, it began to rise shortly thereafter and did not appreciably change over the period between 80 and 180 minutes. In assays to approximate the optimum temperature for style amylase activity, no activity was noted at 0° C for enzymes from any of the three populations, due to enzyme inactivation, not destruction. At incubation temperatures of 4 and 10° C there was detectable amylase activity with the Anahuac Rarigia style and between 10 and 16° C all preparations showed significant activity. This activity increased sharply with increasing temperature until the optima were reached; approximately 30 C for the Neches Rangia and approximately 37° C for the Trinity and Anahuac populations. With temperatures above 37° C the activity for all populations began to decrease and dropped drastically between 45 and 50 C. At 60 C the style amylase activity had been destroyed in all three populations. It has been suggested that the difference in optimum temperature might be associated with a difference in environmental salinity; that the higher the environmental salinity, the higher the temperature optimum. This view is doubtful since the Anahuac population, which has a temperature optimum of 37° C, lives and has lived for many years in a nearly freshwater habitat. The temperature optima differences between the Trinity and Anahuac Rangia as compared with the Neches Rangia may simply reflect an adaptation to the differences in environmental temperature, but it must be remembered that in none of these cases does the optimum temperature represent anything approaching the temperature at which digestion must normally occur.

209 The denaturation studies, in addition to establishing the characteristic temperatures at which the activity of the amylase and maltase enzymes were lost, also revealed several points which bear repeating. First, the style amylases of all three populations responded similarly to enzyme heating for certain periods of time; secondly, the style maltase response was quite different from that shown for the style amylase; thirdly, the maltase activity in response to enzyme heating was quite similar for the Trinity and Anahuac clams; and fourthly, the response of the Neches Rangia was found to be drastically different from that of the other two populations. These findings, in conjunction with previously mentioned results, clearly indicate the presence of distinct maltase and amylase enzymes in the crystalline style of Rangia cuneata, each of which has a characteristic optimum temperature and a characteristic temperature of denaturation, and one of which (the amylase) has a definite Cl requirement. The apparent differences between the style enzymes of Trinity and Anahuac Rangia as compared with the Neches Rangia in Cl requirement, optimum temperature for activity, and the drastically different response to enzyme heating all strongly suggest that the Trinity and Anahuac populations are or at one time were members of the same population, while the Neches population is or was of a different heritage, although no speculation can be made as to the time or circumstances involved. It is possible that at a time in the past the Trinity and Anahuac populations were one and the same, but over an unknown period of time became separated as by natural or induced environmental change. Although the environmental salinity of the Trinity and Anahuac regions is drastically different, the clams are the same in size and show similar enzyme characteristics; therefore, they may be considered to be identical.

210 In the material presented here, perhaps more questions have been raised than were answered; however, it is believed that the knowledge gained during this study has significantly contributed to the knowledge of the physiology of this important estuarine organism. Further investigations will be needed to answer some of the questions raised by this study.

211 OVERALL SUMMARY AND DISCUSSION

From the literature and from our own earlier studies we knew at the beginning of this investigation that Rangia cuneata is a low-salinity estuarine species that seems incapable of main­ taining permanent populations outside the zone where salinity normally varies between 0 and 15 parts per thousand; that it is usually the dominant species within this zone, making up most of the benthic biomass; that it is important in the ecology of the low-salinity zone because it is the principal converter of detritus and phytoplankton into meat that is eaten by the principal fishes and crustaceans of this zone (and seasonally by wild ducks); that it is a commercially valuable natural resource now worth millions of dollars annually for its shells and potentially worth many times more for its meat; and that the adult clams can live in waters of salinity ranging from fresh to at least 25 ppt, apparently indefinitely, which made the reason for their restriction to the zone of 1-15 ppt an unsolved mystery. If this reason could be discovered, it seemed likely that Rangia cuneata, being a key species in the ecology, could serve as a useful indicator of the biological effects of any permanent change in salinity of the upper region of an estuary - raising it by cutting off inflow of fresh water, lowering it by diverting water from another drainage system into the estuary, or stabilizing it by building a dam upstream. Our findings during this investigation, and the findings of others who were working simultaneously in other laboratories, have given us a much better knowledge of how salinity differences affect Rangia cuneata, and most importantly, an understanding of its distribution in estuaries and of its population dynamics. Among these findings, supported by evidence presented in this report, are the following: 1. Rangia cuneata is an osmoconformer (internal salinity varies with external) at salinities above 10 ppt, but begins to regulate its internal salinity when the salinity of the sur­ rounding water drops below 10, and its internal salinity is kept

212 significantly higher than the external salinity at 5 ppt. This is a very unusual ability for a mollusc of marine affinities (Rangia belongs to a family of marine clams). R. cuneata adjusts to a decrease in external salinity much quicker than to a rise in salinity. When salinity is 3 ppt or less, osmoregulation takes 2.4% of the clam’s metabolic energy. 2. Rangia cuneata, thanks to its powers of adjustment (combining conformity with regulatory ability), can live for at least 8 months and probably much longer in salinities main­ tained at levels from almost 0 to 38 ppt. Cain (1972) reported that a population in James River, Virginia, survived from 1965 to 1970 and later in fresh water (salinity below 0.5 ppt), with no increase in salinity at any time during this 5-year period. 3. Rangia clams take up glycine from surrounding water rapidly in salinities above 10 ppt. Below 10 ppt the rate of uptake of glycine decreases rapidly, in the salinities in which osmoregulation occurs; there may be competition between regulation and uptake processes for sources of energy. Glycine uptake from water could represent 8.43% of energy needs of this clam. There is more release of glycine back into water at low salinities, especially at salinities lower than 5 ppt. Glycine taken up is rapidly synthesized into protein. The rate at which glycine is metabolized is also related to salinity, being 7.5 times faster at 15 than at 1 ppt. 4. Oxygen consumption is highest at salinities of 5 and 10 ppt, decreasing at 1 ppt and at 15 ppt. In all salinities, 30-35°C is a critical temperature range and there seems to be damage to the metabolic system at 35° C. Temperature affects respiration most drastically at low (2 and 5 ppt) and high (32 ppt) salinities. Gill tissues operate at higher temperatures than whole clams, oxygen use falling only in the 35-40° C range. The increase in oxygen use (respiratory rate) occurs in the same salinity range where osmoregulation becomes active; osmoregulation may compete with other metabolic processes for energy.

213 5. When starved, clams in aerated water used up 37% of their initial stored glycogen as a source of energy, reducing the amount from 23% of the dry weight of the clam to 20% in 11 days and to 14.5% in 16 days in a salinity of 20 ppt, the salinity at which maximum use occurred. Reduction in stored glycogen was less at salinities both above and below 20; after 16 days the remaining glycogen was 18% of dry weight at 25 and 32 ppt, and 20.5 to almost 23% at 1, 2 and 5 ppt. Under anaerobic conditions clams used glycogen 2.4 times as fast as under aerobic conditions, reducing the stored amount from 23% to 16% of dry weight in 11 days. After 13 days clams began to die at the rate of 1 or 2 per day. 6. Attempts to determine the effects of salinity on feeding rate were unsuccessful, that is, if there was any effect of salinity difference it could not be detected by the methods used. The percentage of algal cells, Dunaliella peircei, removed from a flowing stream of water in a 2-hour period varied erratically in all test salinities from 5 to 32 ppt. It was as low as 8% and as high as 78%; no pattern suggesting a possible correlation with salinity, flow rate, or concentration of algal cells could be noted. 7. Cilia on gills of adult Rangia were found to be most active in salinity of 10 ppt and least active in 5 and 25 ppt, the lowest and highest salinities tested. Temperature had much greater effect on rate of ciliary beat than salinity; cilia were most active at 22° C, slower at 16° C and 28° C, and almost stopped at 35° C. Measurements of rate of ciliary movement in trochophore larvae gave such irregular and irreproducible results that it was concluded ciliary activity of larvae could not be used as an index of the effect of salinity. 8. Tests of the rate of uptake of glucose from water of different salinities also showed so much variation between individual clams that it was decided rate of glucose uptake or glycogen synthesis would not serve as an index of salinity effects.

21b 9. Glycogen in Rangia clams just taken from McCollum Park (Trinity Bay) had percentages of glycogen in dry meat weight varying from 20% in November to over 60% in February, varying from 35 to 50% in other months. After the February peak there was a spring drop corresponding to the spawning period, and a partial recovery in summer (July and August). The later autumn drop may have resulted from an autumn spawning such as Cain (1972) reported from James River, Virginia. Protein in the same clams, as percentage of dry weight of meat, varied only from 35 to 50% during the year. Condition index, the ratio of the wet weight of meat in grams to the internal volume of the shell in milliliters, varied only from 0.33 to 0.55 during the year, being lowest in November and highest in spring and summer. When expressed as percentage of dry weight of meat in shell cavity volume, a condition index often used in shellfish biology, the low of 4.4% was in November; January and February showed 5.5 and 6.6% respectively. There was a drop to 4.95% in May, followed by a rise to 8.4, 7.7 and 6.2 in the summer months, and then a rise to the high for the year, 9.1% on September 1, possibly reflecting a buildup of gonadal tissue and gametes followed by spawning which caused the drop to 4.4% on November 1. These changes were evidently seasonal and were not correlated with changes in salinity. 10. Three populations of Rangia cuneata living in different salinity climates were used in studies of the carbohydrate- digesting enzymes amylase and maltase found in the crystalline style. The populations were those in Trinity Bay, Lake Anahuac in the Trinity River delta, and Neches River. The latter popula­ tion is isolated from any other Rangia population by 20 miles of polluted river. The enzymes from the three populations differed slightly in response to chloride concentration, in rate of activity at identical pH, in effect of different temperatures on activity, in rate of denaturation at high temperatures, and in response of activity to metallic ions. Frei was so impressed by the differences shown by the Neches River population that he

215 suggested that it might be a distinct race, or even subspecies. However, there are other possible explanations for his results, including possible artifacts due to minor defects in procedures. Even if the differences are truly genetic, they could well be caused by single gene differences, which would hardly be enough to make the populations different races, much less sub­ species. Considering the fact that the species Rangia cuneata is made up of many populations in the upper, or low-salinity, ends of separate estuaries, and thus all more or less isolated from each other, it is remarkable that all populations on the Atlantic and Gulf coasts, spotted over a stretch of some 2500 miles, have remained so nearly identical. For instance, Cain (1972) working in James River, Virginia, got almost exactly the same results in his work with the larvae of R. cuneata that we got with the Lake Anahuac population in Texas. Even the responses of R. cuneata to temperature seem quite similar over a spread of nearly 12 degrees difference in latitude. 11. As several field workers have reported (e.g., Fair­ banks, 1963 and Cain, 1972) Rangia cuneata has mature gonads producing gametes throughout more than half the year but does not spawn continuously. In fact, as we found and Cain found, it is quite difficult to get this clam to spawn in the labora­ tory. The clam, though loaded with gametes, will seldom release them until shocked by a sudden change in temperature, in salinity, or both; even then, the additional stimulus of a dose of sperm stripped from a male may be needed to make a female clam release eggs. Our field studies and those of others indicate that in the field, as in the laboratory, spawning seldom occurs until clams are stimulated by a change, usually a change in salinity. Cain (1972) concluded, after extensive field and laboratory studies, that ffa change in salinity either up from 0°/oo or down from 10°/oo or 15°/oo is necessary for spawning - a rise from near 0°/oo to 5°/oo was the best stimulus for spawning.11 This agrees with our experience with Lake Anahuac and Trinity Bay clams.

216 Larvae of R. cuneata are easy to rear in the laboratory, but they require salinity within a narrow range: 2 to 10 ppt for early larvae, 2 to 20 ppt for later larvae. After reach­ ing the setting stage 6-7 days after fertilization, juvenile clams are more tolerant. After the first 21 days, we reared them in 2 to 30 ppt; survival was only 50% but was not correlated with salinity. The slow growth rate as well as the 50% mortality indicated unfavorable laboratory conditions, perhaps including lack of the right food, lack of a soft substrate, and over­ crowding. There are many references in the literature to the occur­ rence of populations of R. cuneata made up of a single size class, evidently representing one year class and indicating that recruitment occurs only at intervals of several years. Cain (1972) studied one such population in the upper part of the James River estuary, in a zone where the water had been fresh (salinity below 0.5 ppt, the threshhold for tasting salt) continuously for years. From previous records, he found that in 1965, a year of low river flow, measurable salinity had reached this far upstream. The estimated age of the clams agreed with the theory that they all came from larvae that had been carried upriver in saline bottom currents and set here in 1965. Large size of individuals in one-class populations has also been reported, although not all such one-class populations contain large clams. Gooch (1971) has commented on this, and on the fact that in what he considered favorable environments the population consists of several, as many as six or seven, year-classes. Tarver (personal communication) reports that in Lake Pontchartrain one-class populations, some consisting of very large clams, occur in both the fresh and the high-salinity fringes of the Rangia zone. So far, there is little or no evidence to support the theory that salinity has a significant effect on the growth rate of Rangia.

217 From all of the information now accumulated from all sources, we visualize the following situation: the population of the Rangia zone in an estuary is made up of three sub­ populations. The central subpopulation, in the zone where salinity is usually between 2 and 10 ppt, is the reproducing one; spawning and recruitment occur nearly every year so that a wide range of sizes is found here most of the time. Inland from the breeding population, in salinities which are below 1 ppt most of the time, is a subpopulation made up of only one, two or three year-classes. If there are two or three classes they are likely to be of very different sizes. If there is a single class, it may be made up entirely of very large clams, entirely of small ones, or entirely of some intermediate size, depending on the lapse of time since the last recruitment occurred. These clams may go years without reproducing, but they produce gametes every year and if there is sufficient rise in salinity, for a long enough time, they can spawn and produce a new year class. Otherwise, they will get a new year class only when saltwater intrusion enables swimming larvae to be brought upstream (in saltier bottom currents, the "salt wedge17) from the breeding population in the central zone. Likewise, seaward of the breeding population there may be a subpopulation of non­ breeding clams which grew from larvae that were washed down­ stream from the breeding zone by freshets, at some time in the past, but are not able to reproduce in normal years because the salinity is too stable and too high. We started this investigation with the hope that we would find some type of measurement of condition or of activity, an index of health, so to speak, that would be better than survival or death as an indication of the effect of salinity change on Rangia cuneata. Hoese (1972) reported two cases of populations of Rangia in southwestern Louisiana being wiped out, apparently

218 by engineering works. White Lake, where Gunter and Shell (1958) had found many Rangia cuneata living in 1952, had no live Rangia that Hoese and his helpers could find in 1971-1972, though Gooch (1971) had found a few clams surviving in 1969. Hoese (1972) attributed the disappearance of the White Lake population to the control structures built in 1951 to prevent saltwater intrusion into the lake (so that the lake could be used as a source of water for irrigating rice and other crops). If Hoese is right, it took 19 years to prove by the final extinction of the population that the control structures had altered the environment enough to make it unsuitable for Rangia and therefore less suitable for all the fishes, crustaceans and birds that feed on Rangia. A quicker way of finding out what was happening to Rangia, and therefore to the brackish water community, might have made it possible to modify the water controlling system in such a way that the fauna could have been conserved without interfering with the use of the lake for irrigation. The Rangia population would have needed only a short period, probably less than a month, of enough saltwater intrusion to stimulate spawning (by raising salinity to 5 ppt) once every 4, 5 or 6 years. The other case cited by Hoese (1972) was Calcasieu Lake, where an abundance of shells showed that a large Rangia population had formerly lived, but where no live clams were found in 1971-1972. Kellogg (1905) had reported Rangia to be extremely common in upper Calcasieu Lake, accord­ ing to Hoese. Hoese (1972) attributed the apparent extermination of Rangia in Calcasieu Lake to the higher salinities (15.5 - 26.0 ppt at the time of his study) caused by saltwater intrusion through the Lake Charles Ship Channel. This hypothesis seems highly probable, though not proved. Our hopes that we would find some test that could be made on adult individuals, to measure the adverse or beneficial effects of salinity changes, were discouraged by the discovery that Rangia cuneata adults had a system of compensations that allowed them to adjust to almost any salinity. As individuals,

219 these clams seem almost invulnerable, except that they will not stand very much rough handling and exposure to the air, especially in hot weather. The key to the welfare of a Rangia population is not the physiology of the adult individual but reproduction and recruitment. Adults live on for years in habitats where reproduction is impossible. Spawning will not occur unless salinity changes, up from low salinity or down from high salinity. If spawning does occur, embryos and early larvae will not survive unless salinity is between 2 and 10 ppt. These are the two vulnerable points in the biology of Rangia cuneata. Once the larvae have developed past the swimming stage and settled to the bottom as juvenile clams, salinity is no longer a critical factor.

CONCLUSIONS The brackish water clam Rangia cuneata can be used as an indicator of the ecological effects of changes in salinity of estuarine waters such as might be caused by engineering works (channels, dams, diversions of rivers, etc.). The adult clams usually live in the zone where salinity ranges between 1 and 15 parts per thousand (ppt), but may be found in fresh water or in bay salinities as high as 25 ppt. The adults are highly adaptable to salinity changes, and can survive for years in salinities near 0 and at least for months in 32 ppt. The keys to the use of this clam as an indicator are the facts that (1) a change in salinity, either up from 0 or down from 15 ppt, is necessary to induce spawning, and (2) the embryos and early larvae survive only in salinities between 2 and 10 ppt. In places where changes in salinity, sufficient to stimulate spawning (changes of approximately 5 ppt), occur annually at some time during the long season (spring to fall) when the clams hold mature gametes, the Rangia population is made up of several

220 to many size classes each representing one year's crop of young. The juvenile clams, less than 25 mm (1 inch) long, less than a year old, and still sexually immature, make up a large part (ideally more than half) of such a regular breeding population. In waters where salinity is too stable for spawning or too low or too high for survival of early larval stages, the population consists of one or two size classes only, and usually has few or no young-of-the-year. Such uniform populations of old adults are in danger of extinction if the stable conditions that prevent recruitment continue. A population that receives a new supply of young clams only in unusual years, widely spaced in time, is not only living dangerously, but it is doing little for the ecosystem; few animals are able to eat adult clams until they die, and individual clams live on year after year for at least 10, sometimes 15, and very rarely perhaps as long as 20 years before they die of old age. The juvenile clams, young-of-the-year, from less than 1 mm up to 15-25 mm in length, are eaten in tremendous numbers by many fishes, crustaceans, and birds including several kinds of wild ducks. It is these young, easily eaten clams that make Rangia cuneata an important ecological asset and one of the main links in the estuarine food chain. Recruitment of another year class occurs only when the young are produced in such large numbers that they swamp the predators and still have numerous survivors at the end of the first year. Even in waters that are fresh most of the time and have saltwater intrusions that permit recruitment only at 4- or 5- year intervals, Rangia cuneata may be abundant enough to be of great economic value, worth as much as $775 per acre per year on a sustained yield basis, even though ecological value as a link in the food chain is limited because large size of adult clams prevents their use as food by moist animals.

221 To usé Rangia cuneata as an ecological indicator, the biologist must collect a representative sample of the population, measure a large number (several hundred if possible), and con­ struct a histogram to show the modal peaks that indicate year classes. The collection must include large samples of the sub­ strate. The bottom sediments are strained through a set of sieves with graded mesh sizes to retain young—of-the-year clams, including those less than 1 mm in length. If several year classes are present, good conditions for sustained production of Rangia clams are indicated, especially if young- of-the-year are present. Such an area has not only the 2 to 10 ppt salinity, during the critical period, that is necessary for survival of the early larvae, but also the variable salinity that is necessary to induce spawning. If the population is made up of only one or two year classes, the area is marginal for Rangia from the ecological standpoint but may still be highly productive of the clams themselves, with a high commercial value per acre. If the population of a freshwater area con­ sists entirely of very old clams, Rangia is in danger of becoming locally extinct and can be saved only by entrance of enough salt water to raise the bottom salinity to 5 ppt for a few weeks. Rangia cuneata is one of the few permanent inhabitants of the low-salinity brackish water zone; most of the fauna of this zone is made up of swimming species (fishes and crustaceans) that move up and down the estuary as the salinity changes, but Rangia requires a changing salinity for its existence. Perfect stabilization of the salinity at any level would eventually wipe out a Rangia population. Disappearance of Rangia, which makes up as much as 99% of the benthic biomass of the low- salinity zone and is a principal source of food for many fishes (including both freshwater and saltwater species), would inevitably decrease the productivity of a low-salinity estuary,

222 9. Glycogen in Rangia clams just taken from McCollum Park (Trinity Bay) had percentages of glycogen in dry meat weight varying from 20% in November to over 60% in February, varying from 35 to 50% in other months. After the February peak there was a spring drop corresponding to the spawning period, and a partial recovery in summer (July and August). The later autumn drop may have resulted from an autumn spawning such as Cain (1972) reported from James River, Virginia. Protein in the same clams, as percentage of dry weight of meat, varied only from 35 to 50% during the year. Condition index, the ratio of the wet weight of meat in grams to the internal volume of the shell in milliliters, varied only from 0.33 to 0.55 during the year, being lowest in November and highest in spring and summer. When expressed as percentage of dry weight of meat in shell cavity volume, a condition index often used in shellfish biology, the low of 4.4% was in November; January and February showed 5.5 and 6.6% respectively. There was a drop to 4.95% in May, followed by a rise to 8.4, 7.7 and 6.2 in the summer months, and then a rise to the high for the year, 9.1% on September 1, possibly reflecting a buildup of gonadal tissue and gametes followed by spawning which caused the drop to 4.4% on November 1. These changes were evidently seasonal and were not correlated with changes in salinity. 10. Three populations of Rangia cuneata living in different salinity climates were used in studies of the carbohydrate- digesting enzymes amylase and maltase found in the crystalline style. The populations were those in Trinity Bay, Lake Anahuac in the Trinity River delta, and Neches River. The latter popula­ tion is isolated from any other Rangia population by 20 miles of polluted river. The enzymes from the three populations differed slightly in response to chloride concentration, in rate of activity at identical pH, in effect of different temperatures on activity, in rate of denaturation at high temperatures, and in response of activity to metallic ions. Frei was so impressed by the differences shown by the Neches River population that he

215 suggested that it might be a distinct race, or even subspecies. However, there are other possible explanations for his results, including possible artifacts due to minor defects in procedures. Even if the differences are truly genetic, they could well be caused by single gene differences, which would hardly be enough to make the populations different races, much less sub­ species. Considering the fact that the species Rangia cuneata is made up of many populations in the upper, or low-salinity, ends of separate estuaries, and thus all more or less isolated from each other, it is remarkable that all populations on the Atlantic and Gulf coasts, spotted over a stretch of some 2500 miles, have remained so nearly identical. For instance, Cain (1972) working in James River, Virginia, got almost exactly the same results in his work with the larvae of R. cuneata that we got with the Lake Anahuac population in Texas. Even the responses of R. cuneata to temperature seem quite similar over a spread of nearly 12 degrees difference in latitude. 11. As several field workers have reported (e.g., Fair­ banks, 1963 and Cain, 1972) Rangia cuneata has mature gonads producing gametes throughout more than half the year but does not spawn continuously. In fact, as we found and Cain found, it is quite difficult to get this clam to spawn in the labora­ tory. The clam, though loaded with gametes, will seldom release them until shocked by a sudden change in temperature, in salinity, or both; even then, the additional stimulus of a dose of sperm stripped from a male may be needed to make a female clam release eggs. Our field studies and those of others indicate that in the field, as in the laboratory, spawning seldom occurs until clams are stimulated by a change, usually a change in salinity. Cain (1972) concluded, after extensive field and laboratory studies, that "a change in salinity either up from 0°/oo or down from 10°/oo or 15°/oo is necessary for spawning - a rise from near 0°/oo to 5°/oo was the best stimulus for spawning.11 This agrees with our experience with Lake Anahuac and Trinity Bay clams.

216 Larvae of R. cuneata are easy to rear in the laboratory, but they require salinity within a narrow range: 2 to 10 ppt for early larvae, 2 to 20 ppt for later larvae. After reach­ ing the setting stage 6-7 days after fertilization, juvenile clams are more tolerant. After the first 21 days, we reared them in 2 to 30 ppt; survival was only 50% but was not correlated with salinity. The slow growth rate as well as the 50% mortality indicated unfavorable laboratory conditions, perhaps including lack of the right food, lack of a soft substrate, and over­ crowding . There are many references in the literature to the occur­ rence of populations of R. cuneata made up of a single size class, evidently representing one year class and indicating that recruitment occurs only at intervals of several years. Cain (1972) studied one such population in the upper part of the James River estuary, in a zone where the water had been fresh (salinity below 0.5 ppt, the threshhold for tasting salt) continuously for years. From previous records, he found that in 1965, a year of low river flow, measurable salinity had reached this far upstream. The estimated age of the clams agreed with the theory that they all came from larvae that had been carried upriver in saline bottom currents and set here in 1965. Large size of individuals in one-class populations has also been reported, although not all such one-class populations contain large clams. Gooch (1971) has commented on this, and on the fact that in what he considered favorable environments the population consists of several, as many as six or seven, year-classes. Tarver (personal communication) reports that in Lake Pontchartrain one-class populations, some consisting of very large clams, occur in both the fresh and the high-salinity fringes of the Rangia zone. So far, there is little or no evidence to support the theory that salinity has a significant effect on the growth rate of Rangia.

217 From all of the information now accumulated from all sources, we visualize the following situation: the population of the Rangia zone in an estuary is made up of three sub­ populations. The central subpopulation, in the zone where salinity is usually between 2 and 10 ppt, is the reproducing one; spawning and recruitment occur nearly every year so that a wide range of sizes is found here most of the time. Inland from the breeding population, in salinities which are below 1 ppt most of the time, is a subpopulation made up of only one, two or three year-classes. If there are two or three classes they are likely to be of very different sizes. If there is a single class, it may be made up entirely of very large clams, entirely of small ones, or entirely of some intermediate size, depending on the lapse of time since the last recruitment occurred. These clams may go years without reproducing, but they produce gametes every year and if there is sufficient rise in salinity, for a long enough time, they can spawn and produce a new year class. Otherwise, they will get a new year class only when saltwater intrusion enables swimming larvae to be brought upstream (in saltier bottom currents, the "salt wedge") from the breeding population in the central zone. Likewise, seaward of the breeding population there may be a subpopulation of non­ breeding clams which grew from larvae that were washed down­ stream from the breeding zone by freshets, at some time in the past, but are not able to reproduce in normal years because the salinity is too stable and too high. We started this investigation with the hope that we would find some type of measurement of condition or of activity, an index of health, so to speak, that would be better than survival or death as an indication of the effect of salinity change on Rangia cuneata. Hoese (1972) reported two cases of populations of Rangia in southwestern Louisiana being wiped out, apparently

218 by engineering works. White Lake, where Gunter and Shell (1958) had found many Rangia cuneata living in 1952, had no live Rangia that Hoese and his helpers could find in 1971-1972, though Gooch (1971) had found a few clams surviving in 1969. Hoese (1972) attributed the disappearance of the White Lake population to the control structures built in 1951 to prevent saltwater intrusion into the lake (so that the lake could be used as a source of water for irrigating rice and other crops). If Hoese is right, it took 19 years to prove by the final extinction of the population that the control structures had altered the environment enough to make it unsuitable for Rangia and therefore less suitable for all the fishes, crustaceans and birds that feed on Rangia. A quicker way of finding out what was happening to Rangia, and therefore to the brackish water community, might have made it possible to modify the water controlling system in such a way that the fauna could have been conserved without interfering with the use of the lake for irrigation. The Rangia population would have needed only a short period, probably less than a month, of enough saltwater intrusion to stimulate spawning (by raising salinity to 5 ppt) once every 4, 5 or 6 years. The other case cited by Hoese (1972) was Calcasieu Lake, where an abundance of shells showed that a large Rangia population had formerly lived, but where no live clams were found in 1971-1972. Kellogg (1905) had reported Rangia to be extremely common in upper Calcasieu Lake, accord­ ing to Hoese. Hoese (1972) attributed the apparent extermination of Rangia in Calcasieu Lake to the higher salinities (15.5 - 26.0 ppt at the time of his study) caused by saltwater intrusion through the Lake Charles Ship Channel. This hypothesis seems highly probable, though not proved. Our hopes that we would find some test that could be made on adult individuals, to measure the adverse or beneficial effects of salinity changes, were discouraged by the discovery that Rangia cuneata adults had a system of compensations that allowed them to adjust to almost any salinity. As individuals,

219 these clams seem almost invulnerable, except that they will not stand very much rough handling and exposure to the air, especially in hot weather. The key to the welfare of a Rangia population is not the physiology of the adult individual but reproduction and recruitment. Adults live on for years in habitats where reproduction is impossible. Spawning will not occur unless salinity changes, up from low salinity or down from high salinity. If spawning does occur, embryos and early larvae will not survive unless salinity is between 2 and 10 ppt• These are the two vulnerable points in the biology of Rangia cuneata. Once the larvae have developed past the swimming stage and settled to the bottom as juvenile clams, salinity is no longer a critical factor.

CONCLUSIONS The brackish water clam Rangia cuneata can be used as an indicator of the ecological effects of changes in salinity of estuarine waters such as might be caused by engineering works (channels, dams, diversions of rivers, etc.). The adult clams usually live in the zone where salinity ranges between 1 and 15 parts per thousand (ppt), but may be found in fresh water or in bay salinities as high as 25 ppt. The adults are highly adaptable to salinity changes, and can survive for years in salinities near 0 and at least for months in 32 ppt. The keys to the use of this clam as an indicator are the facts that (1) a change in salinity, either up from 0 or down from 15 ppt, is necessary to induce spawning, and (2) the embryos and early larvae survive only in salinities between 2 and 10 ppt. In places where changes in salinity, sufficient to stimulate spawning (changes of approximately 5 ppt), occur annually at some time during the long season (spring to fall) when the clams hold mature gametes, the Rangia population is made up of several

220 to many size classes each representing one year’s crop of young. The juvenile clams, less than 25 mm (1 inch) long, less than a year old, and still sexually immature, make up a large part (ideally more than half) of such a regular breeding population. In waters where salinity is too stable for spawning or too low or too high for survival of early larval stages, the population consists of one or two size classes only, and usually has few or no young-of-the-year. Such uniform populations of old adults are in danger of extinction if the stable conditions that prevent recruitment continue. A population that receives a new supply of young clams only in unusual years, widely spaced in time, is not only living dangerously, but it is doing little for the ecosystem; few animals are able to eat adult clams until they die, and individual clams live on year after year for at least 10, sometimes 15, and very rarely perhaps as long as 20 years before they die of old age. The juvenile clams, young-of-the-year, from less than 1 mm up to 15-25 mm in length, are eaten in tremendous numbers by many fishes, crustaceans, and birds including several kinds of wild ducks. It is these young, easily eaten clams that make Rangia cuneata an important ecological asset and one of the main links in the estuarine food chain. Recruitment of another year class occurs only when the young are produced in such large numbers that they swamp the predators and still have numerous survivors at the end of the first year. Even in waters that are fresh most of the time and have saltwater intrusions that permit recruitment only at 4- or 5- year intervals, Rangia cuneata may be abundant enough to be of great economic value, worth as much as $775 per acre per year on a sustained yield basis, even though ecological value as a link in the food chain is limited because large size of adult clams prevents their use as food by moist animals.

221 To use Rangia cuneata as an ecological indicator, the biologist must collect a representative sample of the population, measure a large number (several hundred if possible), and con­ struct a histogram to show the modal peaks that indicate year classes. The collection must include large samples of the sub­ strate. The bottom sediments are strained through a set of sieves with graded mesh sizes to retain young-of-the-year clams, including those less than 1 mm in length. If several year classes are present, good conditions for sustained production of Rangia clams are indicated, especially if young- of-the-year are present. Such an area has not only the 2 to 10 ppt salinity, during the critical period, that is necessary for survival of the early larvae, but also the variable salinity that is necessary to induce spawning. If the population is made up of only one or two year classes, the area is marginal for Rangia from the ecological standpoint but may still be highly productive of the clams themselves, with a high commercial value per acre. If the population of a freshwater area con­ sists entirely of very old clams, Rangia is in danger of becoming locally extinct and can be saved only by entrance of enough salt water to raise the bottom salinity to 5 ppt for a few weeks. Rangia cuneata is one of the few permanent inhabitants of the low-salinity brackish water zone; most of the fauna of this zone is made up of swimming species (fishes and crustaceans) that move up and down the estuary as the salinity changes, but Rangia requires a changing salinity for its existence. Perfect stabilization of the salinity at any level would eventually wipe out a Rangia population. Disappearance of Rangia, which makes up as much as 99% of the benthic biomass of the low- salinity zone and is a principal source of food for many fishes (including both freshwater and saltwater species), would inevitably decrease the productivity of a low-salinity estuary, its value for commercial and sport fisheries, and its value for other wildlife (including ducks). Shellfish biologists would like to have more knowledge of the biology of Rangia cuneata, not only for better management of wild populations but also for possible future cultivation of these clams for market in marshland ponds with controllable salinity. The phases most in need of study in order of priority, are: (1) the factors involved in the setting of larvae and the survival and growth of juvenile clams during their first year, and especially during their first few weeks; (2) all aspects of feeding including the use of nonliving material such as the detritus resulting from breakdown of seed plants and algae as well as the use of living plankton organisms; (3) the influence of various factors on growth rate, including salinity, temperature, substrate, currents, and food; and (4) the factors causing mortality of Rangia at all ages, including the adults .

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21+6 Hopkins, S. H. 1970. Ecology of the brackish-water clam, Rangia cuiieäta. Paper read at Texas Academy of Science Annual Meeting, March 7, 1970. Horvath, K. 1971. Hydrolytic enzymes of Rangia cuneata« Paper in Program of Texas Academy of Science Annual Meeting, March 13, 1971. (This paper was not presented.) Ingersoll, E. 1884. Part IV. Mollusks. W. Mollusks in general. Pp. 683-710. In: Goode, G. B., ed. The fisheries and fishery industries of the United States. Sect. I. Natural history of useful aquatic animals. U. S. Commission of Fish and Fisheries. Washington, D. C. xxxiv + 895 p p . (+ xx + 277 pis.), (p. 708 : "Gnathodon cuneatus of the Gulf of Mexico is already an article of diet, as well as useful in roadmaking.") Johnson, C. W. 1934. List of marine mollusca of the Atlantic coast from Labrador to Texas. Proc. Boston Soc. Nat. Hist., 40(l):l-203. (Rangia on p. 56.) Lamy, E. 1917. Revision des Mactridae vivants du museum d f histoire naturelle de Paris. J. de Conchyl., Ser. 4, 17(3): 173-275, pi. 6; (4):291-418, pi. 7. (Rangia, pp. 342-347). Lankford, R. R. and J. J. W. Rogers. 1969. Holocene geology of the Galveston Bay area. Houston Geological Soc., Houston, Tex. xiii + 141 p p . Menzel, R. W. 1968. Chromosome number in nine families of marine pelecypod mollusks. Nautilus, 82(2):45-58. (Rangia not included, but 3 other Mactridae have N=18, 2N=36 chrom.) Moore, R. C., C. G. Lalicher and A. G. Fischer. 1952. Inverte­ brate fossils. (1st ed.) McGraw-Hill Book Co., New York xiii + 766 pp. (Mactracea on p. 412, figs, on p. 445.) Morrison, J. P. E. 1958. Brackish water genera of Mactridae. Am. Malacol. Union Ann. Report 1958:26.

24-7 Newton, R. B. 1891. Systematic list of the Frederic E. Edwards collection of British Oligocene and Eocene Mollusca in the British Museum (Natural History) -— London. Brit. Mus. (Nat. Hist.). 365 pp. (dates of Sowerby’s publications, p. 321). Odum, H. T. 1972. A study of marine pond ecosystems developing with treated sewage inflow. (Abstract). Abstr. of Papers Submitted for 35th Ann. Meet., Am. Soc. Limnol. Ocean- ogr., Tallahassee, Fla., March 19-22, 1972. (Brackish ponds contained Rangia among other spp.) Olsen, L. A. 1972. Comparative functional morphology of feeding mechanisms in Rangia cuneata Gray and Polymesoda caroliniana Bose. Paper read at National Shellfisheries Ass. Meeting, June 26, 1972. (Abstract included in collection of abstracts given out at meeting.) Olson, K. R. and R. C. Harrel. 1972. Effect of salinity on the TL for mercury, copper and chromium for Rangia cuneata, a brackish water clam. Paper read at Texas Academy of Science Annual Meeting, March 10, 1972. Parker, R. H. 1956. Macro-invertebrate assemblages as indicators of sedimentary environments in East Mississippi Delta region. Bull. Am. Ass. Petrol. Geol. 40(2):295-376. (Rangia, pp. 309, 315, 321, 322, 323, 371, 372.) Patterson, L. and R. C. Harrel. 1972. Physico-chemical con­ ditions and community structure of macrobenthos in the vicinity of the Neches River salt water barrier. Paper read at Texas Academy of Science Annual Meeting, March 10, 1972. Pennak, R. W. 1953. Fresh-water invertebrates of the United States. The Ronald Press Co., New York, ix + 769 pp. (Rangia cuneata on p. 708.) Petrocelli, S. R., A. Hanks and J. Anderson. 1971. The uptake of dieldrin by Rangia cuiieata. (Abstract.) Amer. Zool.,

11(4):694. Plummer, F. B. 1933. Cenozoic systems in Texas. Univ. Texas Bull. 3232, pp. 519-817, figs. 28-54. (Abstract by H. B. Stenzel, p. 888 in Geol. Soc. America Mem. 67(1957), Vol. 2. Poirier, H. 1954. An up-to-date systematic list of.3200 sea- shells from Greenland to Texas: translation, explanation and gender of their names. Y. Villedieu, Hudson View Gardens, New York 33, N. Y. (Mimeo.) (Rangia on p. 177.) Reed, C. T. 1941. Marine life in Texas waters. Texas Academy of Science (Anson Jones Press, Houston), xii + 88 p p . (Rangia on pp. 54, 81.) Richards, H. G. 1939. Marine Pleistocene of Texas. Bull. Geol. Soc. America, 50 (no. 12, pt. 1):1885-1898, 3 pis. (Abstract by H. B. Stenzel, p. 888, Geol. Soc. America Mem. 67(Vol. 2), x + 1077 pp., 1957). Shidler, J. K. 1959. Key to shelled mollusks from lower salinity areas of Texas bays. 12 pp. incl. 1 p. biblio. & 2 pis. (Ditto copy). Simpson, J. W. and J. Awapara. 1964. Phosphoenolpyruvate carboxykinase activity in invertebrates. Comp. Biochem. Physiol., 12:457-464'. (Rangia cuneata on p. 459, 460, 461, 462.) Simpson, J. W. and J. Awapara. 1965. Biosynthesis of glucose from pyruvate-2-C^ and aspartate—3—C in a mollusc. Comp. Biochem. Physiol., 15:1-6. (The mollusc was Rangia

cuneata.) Smith, M. 1937. East Coast marine shells. Edwards Bros., Ann Arbor, Mich, vii + 308 pp., illus. (Rangia cuneata on p. 65.)

2b9 Sowerby, J. 1820-1834. The genera of recent and fossil shells, for the use of students in conchology and geology. Continued by George Brettingham (Sowerby) ("1st of the name”). London (private). 8 v o . not paged. Not dated. (For dates see Newton, 1891, p. 321.) (In the 1831 installment Sowerby published the name and description of Gray’s manuscript new genus and species, as !,Gnathodon cuneatus Gray1’.) Vokes, H. E. 1967. Genera of the Bivalvia: A systematic and bibliographic catalogue. Bull. Am. Paleontol., 51 (232):104-394. (p. 276, Rangia Des Moulins, 1832; p. 274, Columbia (Blainville MS) Rang, 1835, and Gnathodon Gray, 1832, non Oken, 1816 (Pisces), synonyms of Rangia.)

Ward, H. B. and G. C. Whipple. 1918. Fresh—water biology. Wiley, New York, ix + 1111 pp. (includes Rangia cuneata on p. 1020).

ADDENDA

(Cited References Inadvertently Omitted)

Chitwood, B. G. 1951. North American marine nematodes. Texas J. Sci., 3(4):617-672. Kellogg, J. L. 1905. Notes on marine food mollusks of Louisiana. Bull. Gulf Biol. Station, 3:1-43. Martinez, R. 1966. Coastal hydrographic and meteorological study. Project N - . MH-R-2 (Job No. 8). Texas Parks and Wildlife Department, Coastal Fisheries Project Reports, 1966:105-146. Timm, R. W. 1952. A survey of the marine nematodes of Chesapeake Bay, Maryland. Chesapeake Biological Laboratory, Solomons, Md; Publication No. 95. 70 pp.

25 0 Unclassified Securit^^lassificatioi^ DOCUMENT CONTROL DATA - R & D (Security classi fication^J^tUAe^body^of^jibstTacJ^and^indexjn^ännotaüj^

. originating a c t iv it y (Corporate author) r2a. REPORT SECURITY CLASSIFICATION Unclassified______Department of Biology, Research Foundation 2b. G R O U P Texas A&M University, College Station, Texas

3. REPORT TITLE

THE BRACKISH WATER CLAM RANGIA CUNEATA AS INDICATOR OF ECOLOGICAL EFFECTS OF SALINITY CHANGES IN COASTAL WATERS

4. DESCRIPTIVE n o t e s (Type oi report and inclusive dates) Final report 5- AUTHOR(S) (First name, middle initial, last name) S. H. Hopkins J. W. Anderson K. Horvath

6. REPORT DATE 7a. TOTAL NO. OF PAGES 7b. NO. OF REFS June 1973 257 273 8a. C O N T R A C T O R G R A N T NO. DACW 39-71-C-000 7 9a. ORIGINATOR'S REPORT NUMSER(S)

b. PROJEC T NO.

9b. OTHER REPORT NO(S) (Any other numbers that may be assigned this report) ^ Army Engineer Waterways Experiment Station Contract Report H-73-1

10. DISTRIBUTION STATEMENT Approved for public release; distribution unlimited.

11. SUPPLEMENTARY NOTES 12. SPONSORING MILITARY ACTIVITY Prepared under contract for U. S. Army Engineer Waterways Experiment Station, CE, Vicksburg, Miss. Office, Chief of Engineers, U. S. Army Washington, D. C.

13. ABSTRACT In the search for a laboratory test that could be used to determine when salinity changes were favorable or unfavorable, salinities in the range from 0 to 38 were tested on adult Rangia clams for effects on sur­ vival; regulation of internal salinity; intake, use and release of amino acids; respiration; glycogen use under aerobic and anaerobic conditions; feeding rate; ciliary activity; uptake of glucose; glycogen storage and "index of condition" in natural environments through a seasonal cycle; carbohydrate-digesting enzymes; and reproduction. It was determined that Rangia cuneata has a system of compensating reactions that allows it to adjust to changes in salinity over the range from 0 to 38 ppt and over the temperature range from 10 to 35° C without harm. It was concluded from these and further studies that the key to the welfare of a Rangia population is not the physiology of the adult individuals, but reproduction and recruitment. The keys to the use of Rangia cuneata as an indicator were found to be two facts: (1) a change in salinity, either up from near 0 or down from 15 ppt and above, is necessary to induce spawning; (2) the embryos and early larvae can survive only in salinities between 2 and 10 (or 15) ppt. On the basis of laboratory and field studies, the model proposed for Rangia in estuaries has the population consisting of: (1) a central subpopulation in the most favorable breeding zone where the salinities between 2 and 10 ppt and the changes in salinity necessary for reproduction occur in most years; (2) a low-salinity population upstream that is made up of one, two, or three year-classes resulting from larvae that were carried up the estuary, set, and survived in infrequent favorable (high-salinity) years; and (3) a similar subpopulation of one or a few year-classes downbay that set in years of freshets. Since a change in salinity, not just a favorable level, is required for reproduction, perfect stabilization of salinity at any level will result in dying out of the population in 15-20 years when old clams reach the limits of their life span. To use Rangia as an indicator of salinity climate, a large number of clams of all sizes are collected at random and measured for construction of a histogram that will reveal the number of modal peaks, which represent year-classes. If there are several such peaks, especially if there is a peak in the lengths below 30 mm, the population is in good shape and can feed many fishes, crustaceans, wild ducks, and other desirable clam-eaters. If there is only one size class, the population is in danger unless there is a nearby breeding population to replenish it whenever conditions permit. When Rangia dies out, there will inevitably be a decrease in the number of fish, crustaceans (crabs and shrimp), and birds that the brackish-water area can support.

FORM 1 ^ " 7 0 REPLACES DD FORM 1473, 1 JAN 64, m J m J 1 NOV 65 I OBSOLETE FOR ARMY USE. Unclassified Security Classification Unclassified Security Classification

14. LIN K A L IN K B LIN K C K EY WORDS ROLE WT ROLE WT ROLE W T

Aquatic ecosystems Clams Coastal environment Ecology Environmental effects Salinity

Unclassified Security Classification