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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects
1988
Seasonal Changes in Fecundity of Oysters Crassostrea virginica (Gmelin): From Four Oyster Reefs in the James River, Virginia
Carrollyn Cox College of William and Mary - Virginia Institute of Marine Science
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Recommended Citation Cox, Carrollyn, "Seasonal Changes in Fecundity of Oysters Crassostrea virginica (Gmelin): From Four Oyster Reefs in the James River, Virginia" (1988). Dissertations, Theses, and Masters Projects. Paper 1539617594. https://dx.doi.org/doi:10.25773/v5-h6wd-y692
This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. SEASONAL CHANGES IN FECUNDITY OF OYSTERS CRASSOSTREA VIRGINICA (GMELIN)
FROM FOUR OYSTER REEFS IN THE JAMES RIVER, VIRGINIA.
A Thesis
Presented to
The Faculty of the School of Marine Science
The College of William and Mary in Virginia
In Partial Fulfillment
Of the Requirements for the Degree of
Master of Arts
by /
Carrollyn Cox APPROVAL SHEET
This thesis is submitted in partial fulfillment of the requirements
for the degree of
Master of Arts
Oij, Carrollyn^ Cox
Approved: May, 1988
Roger L.^Mann, Ph.D. Committee Chairman/Advisor
Mark W. Luckenbach, Ph.D.
Morris H. Roberts, Jr., ^h.D
Michael Castagna, M.
Evon P. Ruzecki, Ph.D DEDICATION
To Kevin J . McCarthy and Bernardita Campos for the times shared
during the summer of 1986 TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... v
LIST OF TABLES ...... vi
LIST OF FIGURES...... vii
ABSTRACT ...... viii
INTRODUCTION ...... 2
LITERATURE REVIEW...... 5 Historical D a t a ...... 5 Life History ...... 6 F e c u n d i t y ...... 12 Condition Index ...... 15
OBJECTIVES ...... 18
MATERIALS AND METHODS...... 19 Sample Collection ...... 19 Temperature, Salinity, and Dissolved Oxygen ...... 19 Fecundity ...... 21 Condition Index ...... 22 Sex Ratios...... 24 Abundance and Size-Frequency ...... 24 Total Egg Production...... 25 Spawning and Larval Viability ...... 25
RESULTS...... 28 Temperature, Salinity, and Dissolved Oxygen ...... 28 F e c u n d i t y ...... 31 Condition Index ...... 39 Sex Ratios...... 45 Abundance and Size-Frequency...... 49 Total Egg Production...... 49 Spawning and Larval Viability ...... 52
DISCUSSION ...... 57
LITERATURE CITED ...... 65
VITA ...... 74
iv ACKNOWLEDGMENTS
I wish to express my gratitude to all of those who have helped to make this thesis a reality. My major professor, Roger Mann, and my
thesis committee, Michael Castagna, Mark Luckenbach, Morris Roberts, and
Evon Ruzecki, supported and encouraged me. Mark Luckenbach spent countless hours making sense of the statistics. Jean Watkinson gave me the benefit of her expertise in bivalve spawning techniques. Bernardita
Campos listened. Kevin McCarthy, James Whitcomb, David Eggleston, Julia
Wilcox, Curtis Roegner, and especially Kenneth Walker assisted in the field. Jan Hodges digitized reef areas. Diane Walker proof read sections of the manuscript. The VIMS Computer Center Staff, especially
Gary Anderson and Kevin Kiley, and many others assisted me with my computer work. Distilled water was provided by Betty Salley and the
VIMS Nutrient Analysis Laboratory. Kenneth Kurkowski and the VIMS
Oyster Hatchery Staff provided oyster larvae, filtered water and advice.
This work was funded in part by a grant from the Virginia General
Assembly, and in part by two minor research grants to me from the
Virginia Institute of Marine Science, School of Marine Science of the
College of William and Mary.
v LIST OF TABLES
Table Page
1. Multiple Comparisons of Fecundity Among Stations. . . . . 34
2. Multiple Comparisons of Egg Number Among Dog Shoal Collections ...... 36
3. Multiple Comparisons of Egg Number Among Thomas Rock Collections ...... 37
4. Multiple Comparisons of Egg Number Among Wreck Shoal Collections ...... 38
5. Multiple Comparisons of Egg Number Among Horsehead Collections ...... 40
6. Sex Ratios of James River Oysters ...... 48
7. Number of Oysters per 0.25 square m e te r ...... 50
8. Estimate of Total Egg Production per R e e f ...... 53
9. Results of Spawning Trials...... 54
10. Results of Successful Spawning of Horsehead Oysters..... 55
vi LIST OF FIGURES
Figure Page
1. The James River, Virginia ...... 3
2. Study sites in the James River Seed A r e a ...... 20
3. Temperature and salinity at each study site from June to October, 1986 ...... 29
4. Percent Oxygen Saturation ...... 30
5. Regression of Egg Number and Shell Length ...... 32
6. Mean Egg Number in James River Oysters...... 33
7. Regression of Cl(grav) and Shell Length ...... 41
8. Mean Cl(grav) of James River Oysters...... 42
9. Regression of Cl(afdw) and Shell Length ...... 43
10. Mean Cl(afdw) of James River Oysters...... 44
11. Correlation of Mean Egg Number and Mean Cl(grav)...... 46
12. Correlation of Mean Egg Number and Mean Cl(afdw)...... 47
13. Mean Abundance of James River Oysters ...... 51
vii ABSTRACT
Oysters Crassostrea virginica (Gmelin) were examined during the reproductive season of 1986, to determine variation in fecundity among individuals from four different reefs, Dog Shoal, Thomas Rock, Wreck
Shoal, and Horsehead, in the seed area of the James River, Virginia.
Factors potentially affecting reproductive potential, sex ratio, oyster abundance, mean shell length, and larval viability, were also examined.
Additionally, fecundity was compared with gravimetric and ash-free dry weight condition indices to determine if fecundity and condition index were correlated.
Fecundity of female oysters was found to be highly variable, both within and among locations. The large variation was attributed to a combination of factors including time since last spawning, oyster size, disease, parasitism, and temperature and salinity regimes. As a group, oysters from Thomas Rock produced 3-4 times as many eggs as those from the other 3 reefs. The production of eggs on Horsehead was greater than that on Wreck Shoal due to the great abundance of oysters on Horsehead, and a more nearly equal sex ratio. In sum, Dog Shoal oysters produced the fewest eggs.
Results indicate that seed area oysters are potentially capable of providing for the large settlement of larvae in that area. Theories to account for the settlement failure of 1986 are discussed. SEASONAL CHANGES IN FECUNDITY OF OYSTERS CRASSOSTREA VIRGINICA (GMELIN)
FROM FOUR OYSTER REEFS IN THE JAMES RIVER, VIRGINIA INTRODUCTION
The Chesapeake Bay estuary (Fig. 1) supports one of the largest
Crassostrea virginica (Gmelin, 1791) fisheries in North America
(Thompson, 1986). Located centrally within the geographic range of the
American oyster, the Chesapeake offers a favorable climate for oyster growth (Andrews, 1970). In addition, the subestuaries of the Chesapeake contain large areas of low salinity water which provide oysters with a refuge from predators and diseases (Andrews, 1982). The James River
(Fig. 1) is the third largest and southernmost major tributary of the
Chesapeake Bay (Wolman, 1968). The James is an important oyster producing area because recruitment there is high and mortality of oysters older than one year is low (Haven and Fritz, 1985).
The James River has traditionally been used as an oyster seed area because, despite high recruitment and low mortality, growth rates are retarded by low salinity and meat quality of the oysters is poor (Haven and Fritz, 1985). Oysters less than 76 mm in length (seed) are harvested from the James and replanted elsewhere in the bay where growth rate is higher (Wood and Hargis, 1971; Haven and Fritz, 1985). The low salinity sector of the James (Fig. 1) historically has been the primary source of seed oysters for planters throughout the bay (Andrews, 1951,
1970). In the 50 years between 1930 and 1980, an estimated 75% or more of the seed oysters planted in Virginia came from the James River seed area (Haven et al., 1981). This seed area contains approximately 5,658
2 FIGURE 1. The James River, Virginia 0££P WATER SHOAL
w RCC k Sh o a l
6URWELL- 8AY
* NANSEMONO r id g e 4 hectares of public oyster reefs extending from near the James River Bridge upriver to Deepwater Shoal Lighthouse (Andrews, 1951, 1983; Haven and Whitcomb, 1983). Despite its importance, little is known about the broodstock which provides the larvae which recruit into the James River seed area. Andrews (1951, 1968) argued that the primary broodstock was located in the deep waters of Hampton Roads (Fig. 1). Larvae produced downstream were thought to be transported upstream to the seed area by gravitational circulation (Andrews, 1951). Alternatively, the primary broodstock may lie within the low salinity seed area north of the James River Bridge (Andrews, 1951). Results of dye studies in the James River Hydraulic Scale Model support this alternative, suggesting that larvae released from oysters within the seed area would provide maximum coverage of reefs in the seed area (Ruzecki and Hargis, in press). The potential contribution of oysters in the seed area to local recruitment is primarily dependent on their fecundities; presently data concerning the temporal and spatial variability in fecundity are lacking. This study estimates the reproductive potential of oysters from the seed area, comparing fecundities of oysters from different reefs over time. Condition index, a parameter used as an indicator of meat quality, is compared with fecundity to determine if a correlation between meat condition and egg production exists. Other parameters such as abundance, sex ratios, and larval viability which affect total reproductive output of oysters are also examined. Observed values of abundance, sex ratio, and fecundity are used to estimate total egg production per oyster reef. An attempt is made to relate findings of this study to observed recruitment of larvae in the seed area. LITERATURE REVIEW Historical Data Extensive historical data concerning the timing and quantity of spat settlement in the James River seed beds exists (Andrews, 1951, 1954, 1982; Whitcomb, 1983-1985, 1986b). The terms spatfall, strike, set, setting and settlement are used interchangeably to describe the metamorphosis of oysters from pelagic larvae to sedentary juveniles or spat (Galtsoff, 1964). Settlement in the James usually begins by late June and ends in early October (Andrews, 1951; Haven and Fritz, 1985). Setting was continuous throughout each spawning season before the onset of the disease known as MSX, caused by Haplosporidium nelsoni, in the late 1950's (Andrews, 1951; 1954). Since 1963 however, spatfall in the James has occurred in 1-2 week pulses throughout the spawning season (Haven and Fritz, 1985). In pre-MSX years, peak setting usually occurred on or about the first of September (Andrews, 1951). More recently, peak settlement has occurred in mid-July to mid-August (Whitcomb, 1986b). The heaviest settlement in the river is near the mouth and to the north of the shipping channel (Fig. 1), but survival increases upriver where salinities are low (Andrews, 1954; 1982; Haven and Fritz, 1985). The lower James River was virtually depleted of oysters by the pathogen MSX in the early 1960's (Andrews and Wood, 1967). Since that time, recruitment in the seed area has diminished to less than half of 6 that in earlier years (Andrews, 1970; Haven and Fritz, 1985). Light spatfall in the low salinity area in the years subsequent to the appearance of MSX suggests that the few oysters remaining in the lower river produce too few larvae to sustain high recruitment in the seed area, and that local spawning in the seed area does not contribute significantly larval recruitment there (Andrews, 1970). In the late 1970's to mid 1980's however, spat settlement in the seed area was high (Whitcomb, 1985), similar to pre-MSX levels (Whitcomb, personal communication). It is apparent from these data that recruits were produced by seed area oysters, since oyster abundance has not recovered in the lower river. If settling larvae are produced by adults within the seed area, then the broodstock may consist of large numbers of relatively small oysters, each of which produce few gametes in a season (Andrews, 1951). Oyster beds in the middle of the seed area contain large numbers of oysters and are suitably located for settlement and survival of spat (Andrews, 1982). Low recruitment in the post-MSX years may be due to a reduction in broodstock fecundity (number of gametes produced in a season) or to reduced larval survival (e.g., Berg, 1969). Fecundity reduction may be caused by poor water quality (Galtsoff, 1964; Haven and Fritz, 1985) or inadequate food (Kennedy, 1986); larval survival may be related to water quality, food availability or predation (Loosanoff, 1965; Kennedy, 1986). These possibilities have not been examined. Life History The genus Crassostrea Sacco (1897) is comprised of estuarine oysters which can tolerate turbid, low salinity waters (Nelson, 1938) 7 "where hot summers and cold winters prevail" (Andrews, 1979). Crassostrea virginica (Gmelin, 1791), the American, Virginia, or Eastern oyster, is found from the Gulf of St. Lawrence to Rio de Janeiro, Brazil (McLean, 1941; Galtsoff, 1964). The oviparous Crassostrea virginica has been described as a primarily protandric, consecutive hermaphrodite (Burkenroad, 1931; Needier, 1932a), though individuals can mature first as females (Coe, 1936). C. virginica can change sex from male to female or vice versa (Needier, 1932a, 1942; Burkenroad, 1937; Galtsoff, 1937). In the terminology of Hoagland (1984), C. virginica possesses "alternating sexuality" since the species is neither protandric nor protogynic. The change from male to female is purported to be hastened by good growing conditions or retarded by proximity of females (Needier, 1932a; Coe, 1936; but see Galtsoff, 1964). Rarely, C. virginica are reported to contain both eggs and sperm at the same time, making self-fertilization possible (Kellog, 1892; Burkenroad, 1931; Needier, 1932a, 1932b; Coe, 1936, 1938; Galtsoff, 1964). Females tend to be larger than males of the same age until they reach their fourth summer (Needier, 1932a; Coe, 1936) . There is usually a 1:1 sex ratio (males:females) in groups of adult oysters, or an excess of older females (Needier, 1932a; Coe, 1938). In the James River, the sex ratio is close to 1:1 (Andrews, 1984 unpublished; Morales-Alamo, 1986). Individuals from Wreck Shoal (Fig. 1) under 50 mm in length are predominately males, while those over 50 mm in length are mostly females (Andrews, 1951 unpublished, 1984 unpublished). Lacking secondary sex characteristics, the sex of oysters can be determined by examining gonadal material for the presence of eggs or sperm (Churchill, 1920). After the end of the spawning season, oysters resorb remaining gametes (Loosanoff, 1965); during this period no discrete gonads exist and sex is recognizable only by careful cytological examination (Galtsoff, 1964; Andrews, 1979). American oysters become sexually mature at an early age. In the mid-Atlantic region from Cape Cod to the Chesapeake Bay, most one year old oysters can spawn (Coe, 1938; Hayes and Menzel, 1981). South of Virginia, functional maturity is reached within 10-12 weeks (Coe, 1936) In the warm southern waters of Louisiana, young oysters may produce functional gametes in as few as 28 days after attachment (Menzel, 1951) Males tend to produce mature sex products earlier in the season than do females (Coe, 1938; Loosanoff and Davis, 1952), and females probably do not spawn in nature until sperm enter the water and provide a stimulus (Andrews, 1979). Eggs of Crassostrea virginica vary in size between 45 um and 62 um in diameter, independent of female size (Loosanoff and Davis, 1963); other authors (e.g., Kennedy and Battle, 1964; Galtsoff, 1964) report that eggs are about 40-50 um in diameter. In the ovary, eggs are compressed and "rendered polygonal by mutual pressure" (Brooks, 1880). The long axis varies from 55 um to 75 um, the width is 35 um to 55 um, depending on the shape (Galtsoff, 1964). Eggs retain this form until sometime after discharge; after coming in contact with seawater, they become round and turgid. Eggs and sperm are shed directly into the water where fertilization occurs and planktotrophic larvae develop (Gunter, 1950; Loosanoff, 1965). Oysters rarely release all eggs or sperm at a single spawning, but continue to expel gametes at intervals over extended periods (Andrews, 1954; Davis and Chanley, 1955; 1956). 9 Crassostrea virginica develop through several stages on a continuum from fertilized egg through metamorphosis. Development time depends upon several factors including temperature, salinity, food quality and availability, and presence of toxins. Spiral cleavage begins within 15- 20 minutes after fertilization at 22-24°C (Galtsoff, 1964). At the same temperature, gastrulation occurs within 4-6 hours (Galtsoff, 1964). The gastrula possesses a few large cilia at the vegetal pole; the rest of the body is finely ciliated (Galtsoff, 1964). After a few minutes, a ciliated girdle is formed, polar bodies are lost, and the larva swims upward. From the time it begins to swim, the larva is called a trochophore (Galtsoff, 1964). The trochophore appears in 6-8 hours at normal spawning temperatures (Andrews, 1979). The next stage, the veliger, is so named because the larva possesses a velum, a ciliated organ with which it moves, feeds, and respires. From the time that the larva acquires a velum until metamorphosis, it is called a veliger larva. Veliger stages are differentiated from each other by shell morphology. Larvae begin to produce shells as trochophores (Galtsoff, 1964), but the first recognizable shelled larva is a veliger, often termed straight-hinge or D-hinge larva because of its characteristic shape. Straight-hinge oyster larvae are formed within 24-30 hours of fertilization (Andrews, 1979), and measure 70-75 um in length (Loosanoff and Nomejko, 1951a) where larval shell length is the greatest shell distance parallel to the hinge line (Loosanoff and Davis, 1963; Jablonski and Lutz, 1980). The next stage is the umbo larva which has a hinge line less than half the total shell length and a well developed umbo (Chanley and Andrews, 1971). Crassostrea virginica larvae have a 10 skewed umbo which is peculiar to the genus Crassostrea (Chanley and Andrews, 1971). The most advanced larval stage is the pediveliger (sensu Carriker, 1961). This veliger differs from earlier stages in that it possesses a foot. The pediveliger can swim with its velum or crawl with its foot protruding (Andrews, 1979). The pediveliger larva settles to the bottom for a period of crawling to find an attachment site within a limited area. The larva attaches to a suitable site by exuding a droplet of liquid cement through a pore at the heel of the foot, and pressing the left valve into it (Carriker, 1961). After the larva attaches to the substrate, it metamorphoses, a process involving the absorbtion of the velum and foot and internal reorganization, thus assuming a sedentary mode of life (Galtsoff, 1964). Attachment and metamorphosis usually occur when the larva is between 310 and 350 um in length (Chanley and Andrews, 1971), but free swimming larvae 355 um in length have been reported (Loosanoff and Davis, 1963). Length of the larval period depends upon environmental conditions (Nelson, 1908; Medcof, 1939; Ingle, 1951). Larvae of Crassostrea virginica metamorphosed in approximately 18 days when cultured in the laboratory at 23°C and fed a diet of Isochrvsis galbana Parke (Loosanoff and Davis, 1963). Larval life is shorter at higher temperatures. Metamorphosis occurred in just 9-11 days when larvae were cultured at 27°C and fed a combination of 3 algal species (Dupuy et al., 1977). Salinity also influences growth rates; larvae at low salinities grow slower than do sibling larvae in high salinities (Calabrese and Davis, 1970) . 11 The species Crassostrea virginica from Canada to South America consists of several local races which lives under widely different environmental conditions and differ in some aspects of their inheritance (Coe, 1938; Loosanoff and Engle, 1942; Stauber, 1950; Ingle, 1951; Loosanoff and Nomejko, 1951b). As it is used here, race describes a group of organisms which fit a described species on morphological grounds, "but which are functionally different in certain respects from one another" (Stauber, 1950). These groups have been alternatively termed physiological species (Stauber, 1950) and geographic or local races (Mayr, 1964). The existence of local races of Crassostrea virginica is evidenced by great differences in maturation rates between populations, as well as differences in stimuli and duration of those stimuli which are required to initiate spawning (Coe, 1938; Loosanoff and Engle, 1942; Stauber, 1950; Ingle, 1951; Loosanoff and Nomejko, 1951b). The temperatures required for gonadal development and spawning of Northern races are lower than those of Southern races (Stauber, 1950; Loosanoff and Nomejko, 1951b). Overwintering oysters from Long Island Sound, containing small undeveloped follicles typical of hibernating oysters, developed ripe spermatozoa and fertilizable eggs in 5 days when held in the laboratory at 25°C (Loosanoff and Davis, 1952), These oysters spawned naturally on the 11th day in the laboratory (Loosanoff and Davis, 1952). In contrast, average time required for oysters from Delaware Bay to ripen from winter conditions to spawning at 25°C was 35 days (Price and Maurer, 1971). Chesapeake Bay oysters also require high temperature for 4-6 weeks to induce spawning (Andrews, 1979). In a more 12 recent study by Roberts et aJL. (1987), however, sexually indeterminate oysters from the Chesapeake region maintained at 25°C produced mature gametes in less than 2 weeks. Fecundity The term fecundity is used to describe the number of mature gametes produced by an organism over a specified time interval (Scott, 1962). This time interval may be the lifespan of the animal, one spawning season, or one reproductive bout within a spawning season. Variable useage of this term is a source of confusion in the literature. In this study, fecundity is defined as the total number of eggs contained in from a female oyster at any one time. Fecundity of bivalves has been estimated by many authors employing various techniques. One method involving the difference between weight of gonad before and after spawning (e.g.. Fox and Coe, 1943), is not applicable to Crassostrea virginica because outlines of the gonad are indistinct (Galtsoff, 1964). A similar index, the change in soft tissue weight before and after spawning has also been used (Griffiths, 1977; Bayne and Worrall, 1980). Historically, oyster biologists estimated "quantity of spawn" produced by measuring gonad thickness (Loosanoff and Engle, 1942; Loosanoff, 1965). Loosanoff (1965) stated that the method was reliable only if oysters of the same size were compared and the gonad thickness was measured at the same place on every oyster. The above methods are of little use in quantitative studies of reproduction. Several quantitative methods of estimating fecundity are reported in the literature. Yankson (1986) found that the difference in gamete 13 volume fraction (proportion of the gonad which contained gametes in various stages of development) immediately before and after spawning was a good, quantitative estimate of fecundity. Kennedy (1986) took this method one step further and determined the percentage volume of gonad devoted to different tissue types (e.g.. developing gametes, mature gametes, spawning/spent tissue, and non-reproductive tissue). Gamete volume fraction and percentage volume fraction are especially useful fecundity estimators because both males and females can be assessed by these methods (Yankson, 1986; Kennedy, 1986). Drawbacks of these methods include the high cost of preparing the histological sections and the length of time required to examine them. A less costly, yet quantitative, method for estimating fecundity of female bivalves is to count the number of eggs actually produced by an individual. The number of eggs produced by Crassostrea virginica has been estimated by several authors. Brooks (1880) estimated that the number of eggs contained in a female at one time was between 18,750,000 and 125,000,000. This estimate was based on the volume of eggs removed from a ripe female divided by the volume of a single egg. Since much foreign matter and water was included in initial volumes, his final estimate was approximately 9 million eggs per "medium sized" female; an unusually large oyster contained an estimated 60 million eggs. Churchill (1920) estimated that a female oyster would produce 16 million eggs (presumably in one season), but he did not state how he arrived at that value. Nelson (1921) found 16-60 million eggs present in females at the onset of the breeding season, depending upon the size and condition of the oyster. According to Nelson, a large 5 or 6 year old female, if fat the preceeding spring, would produce 50-60 million eggs 14 during the spawning season. His rough estimate of eggs released at a single spawning was 100,000 per female. Nelson did not explain how seasonal fecundity values were obtained, but single spawning estimates were made by dip netting eggs from the field immediately after spawning. Galtsoff (1930) estimated a maximum of 500 million eggs per female per season for C. virginica as compared to 92 million for C. gigas. For a single spawning, individual female C. virginica were observed to release 15 to 114.5 million eggs (Galtsoff, 1930). Eggs were obtained by subjecting ripe females to elevated temperature and adding sperm to the water. Each female was spawned separately in a 20 liter tank from which 5 samples of 100 ml each were taken from the agitated water. Burkenroad (1947) states that Galtsoff's estimate is about ten times too high on the basis that computed egg volumes exceed the total body volume. Galtsoff (1964) countered that his counts were correct +/- 10%, with non-uniform distribution of eggs being the principal source of error. Galtsoff claimed that egg volume in the ovary is compressed because the eggs are tightly packed, and that upon release the volume of the eggs increases as they become more rounded (Galtsoff, 1964). Davis and Chanley (1955, 1956) induced oysters to spawn at 3, 5, and 7 day intervals and found that the more often they were induced, the more frequently oysters spawned. As spawning frequency decreased, the number of eggs discharged at each spawning increased. Davis and Chanley (1955) reported that up to 70,000,000 eggs were released by a single female in a single spawning. Mean fecundity of all oysters tested was found to range between 27-31.4 million eggs per individual per season depending upon spawning interval (Davis and Chanley, 1956) . 15 Egg counts for oysters traditionally yield widely varying totals (Andrews, 1979). Large variation in egg numbers may be due, in part, to such factors as age, size, nutritional condition, disease, parasitism, and spawning season length (Andrews, 1979). Differences in egg numbers of two similar females may also be due to sample timing; one oyster may be ready to spawn, while the other has just spawned. Condition Index The physiological state of an oyster is known as its condition. An oyster that is in good condition is generally fat, glycogen rich, and fills its shell cavity completely (Medcof and Needier, 1941; Gunter, 1941); an oyster in poor condition is thin, clear, and watery (Gunter, 1941; Engle, 1950). This state is approached after spawning, but recovery is usually rapid (Chipman, 1947). Condition index determination has often been employed in oyster research because of the economic importance of detecting good quality oyster meats (Westley, 1959; Lawrence and Scott, 1982). Many methods of determining oyster condition have been used since the concept of condition was introduced. In 1912, Grave described a method for evaluating the "fatness" of oysters by determining the percentage of the shell cavity which is occupied by the meat: Condition Factor Index = Vol. Meat x 100 Vol. Shell Cavity. This Index of Condition was also reported by Baird (1958) who found it to objectively measure the "fatness" of oyster meats, irrespective of shell thickness or size. If fresh water is used as the displacement medium, however, oyster meats may become swollen, causing an increase in 16 meat volume (Engle, 1950). Westley (1959) used Grave's method and found it to "lack reproducibility in volumetric measurement and estimation of meat quality". The method was also found to be of limited value since it should not be used in cases where spawning is possible (Baird, 1958; Mann, 1978) . Hopkins (in Higgins, 1938) proposed the ratio of dry meat weight to shell-cavity volume as a condition index. Index of Condition — Drv weight meat (g) x 100. Shell-cavity volume (ml) Meats were dried to constant weight at 100°C. The Hopkins method has been used with various modifications by many authors. Medcof and Needier (1941) modified the Index by substituting 1000 for 100 as the constant in the equation. They found the condition of oysters to vary with size, shell shape, and the average depth of water from which they were fished. Drying methods have been changed by Galtsoff et al. (1947) to initial drying at 50°C for 86 hours and final drying at 100°C for 24 hours; Lawrence and Scott (1982) dried meats at 68°C for 48 hours; and Korringa (1955) suggested drying meats by toluene distillation. This index is supposedly valid when spawning is possible because it accomodates the increase in water content of the meat and the decrease in dry meat weight which occur after spawning (Masumoto, et al., 1934, in Mann, 1978; Quayle, 1969). Galtsoff (1964) found fault with this method of determining oyster condition because some oysters such as young or over crowded individuals are flat, having little space between the valves. These individuals may have an artificially high condition 17 index since the soft tissues fill most of the shell-cavity volume (Galtsoff, 1964). Galtsoff offers no data to support this statement. Several other methods have recently been employed to determine condition. Walne and Mann (1975) and Mann and Ryther (1977) used a totally gravimetric index: Index of Condition — Dry meat weight (g) x 1000. Dry shell weight (g) This method is easy to use when processing a large number of samples since both the meat and the shell are analyzed using the same method (Mann, 1978). The method cannot be used, however, when shells are damaged for example by boring sponges or worms. de Wilde (1975) used an ash-free dry weight index : Index of Condition = Ash-free dry meat weight (mg') x 1000. 3 Shell length (mm) This method can be used for specimens which have shell damage since 3 shell weight is not determined. Using the shell length term assumes isodiametric shell growth, but neglects differences in shell thickness (Mann, 1978). OBJECTIVES It is the purpose of this thesis to compare temporal and spatial variability of broodstock fecundity among different reefs in the James River seed area. Several factors potentially affecting reproductive potential, specifically sex ratio, abundance, size-frequency, and larval viability are determined for oysters from the seed area. Additionally, fecundity and condition indices are compared to determine if a correlation between them exists. 18 MATERIALS AND METHODS Sample Collection Oysters were collected from 4 oyster reefs in the James River during the spawning season of 1986. Collection sites were Horsehead (HH), Wreck Shoal (WS), Thomas Rock (TR), and Dog Shoal (DS) (Fig. 2). In an average year, the reproductive season for oysters in the James River begins during June and lasts into October (Haven and Fritz, 1985). Ten collections were made at intervals of approximately 14-15 days beginning June 2, 1986 and ending October 16, 1986. Samples taken from June 17 through September 9 were obtained by dredging. On June 2, 2 August 25, and October 16, 4 quantitative 0.25 m samples were collected haphazardly from each reef by SCUBA divers. Half of the oysters collected at Horsehead and Dog Shoal from June 17, 1986 through September 9, 1986 were randomly selected for use in spawning and larval viability experiments. All remaining oysters were frozen for subsequent analysis. Temperature. Salinity, and Dissolved Oxygen Temperature and salinity was recorded for all stations on each collection date. Surface and bottom water temperatures were measured to the nearest 0.5°C. Bottom water obtained with a modified Van Dorn bottle was placed in glass bottles and returned to the laboratory for FIGURE 2. Study Sites in the James River Seed Area. HOG ISLAND HORSEHEAO .WRECK SHOAL THOMAS /ROCK / River OOG SHOAL- •»*oTlC*l. MttCS « analysis of salinity and dissolved oxygen. Salinity was determined by the VIMS Nutrient Analysis Laboratory using a portable Beckman Induction Salinometer Model RS-10. Water samples for dissolved oxygen analysis were fixed in the field using manganous sulfate solution and alkali- iodide-azide reagent (APHA, 1980). Dissolved oxygen was determined by the VIMS Nutrient Analysis Laboratory using the azide modification of the Winkler titration method following acidification with concentrated I^SO^ (APHA, 1980). Percent oxygen saturation was calculated for each station on all sample dates. Data were analyzed using a 2-way analysis of variance (ANOVA) to determine if temperature, salinity, and oxygen saturation differed significantly among stations or collection dates. Where differences were found, Student-Neuman-Keuls multiple range tests were employed to determine which stations or dates were different. In this and all subsequent statistical tests, significance was tested at anlevel of 0.050. Fecundity Ten females were randomly selected from each collection for fecundity determination. Shell length was measured to the nearest millimeter using dial calipers. Adult oyster shell length was defined as the maximum distance between the umbo and the ventral shell margin. Oysters were thawed at room temperature (ca. 24°C) and shucked. Individual oyster meats were placed in a blender containing 150 ml of York River water diluted to 20°/oo salinity with distilled water (hereafter termed estuarine water) and blended on medium setting for 30 22 seconds. The resultant mixture was filtered through stacked 90 um and 53 um mesh Nitex nylon sieves, and the liquid containing eggs was retained in a 1-1 graduated cylinder. Blender, sieves and funnel were rinsed with 750 ml estuarine water. Additional water was added to bring the volume in the cylinder up to 1-1. Eggs were sampled using the plunger suspension method described in Loosanoff and Davis (1963). The sample was plunged for 30 seconds, then 10-1000 ul depending on egg concentration, was withdrawn using a variable volume microliter pipette, and placed in a Sedgwick-Rafter cell for counting. The cell was then filled to capacity with estuarine water. Egg number was calculated from the mean of 3 aliquots withdrawn from the egg suspension. The number of eggs per sample was multiplied by the appropriate factor to determine total number of eggs per female; total egg number hereafter is termed fecundity. Fecundity was regressed on oyster shell length for all stations and sample dates combined to determine if change in shell length explained a significant portion of the observed variation in egg number. Mean egg number was plotted for each station over time. A non- parametric Kruskal-Wallis 1-way analysis of variance test was used to test for variation in egg number. Multiple comparisons were performed using Dunn's Approximation (1964) with an experiment-wise error rate of 0.050 when the Kruskal-Wallis test showed significant variation in mean egg number. Condition Index Forty individuals were chosen randomly from each sample for condition index (Cl) analysis. This large sample size was chosen 23 because of the "large inherent variability in the condition of oysters...within a sample" (Baird, 1958). Oysters were scraped and scrubbed to remove fouling organisms and mud. Shell length was measured to the nearest millimeter using dial calipers. Gonadal material was collected with a Pasteur pipette and examined microscopically. Sex was determined by the presence of eggs or sperm. Meats and shells were placed in separate ashed and tared aluminum weighing pans. Dry weight was obtained by drying meat and shell to constant weight at 100°C for 24 hours. Pans were weighed after cooling in a desiccator, dry weights determined, and gravimetric condition index, Cl(grav), was calculated using the method of Walne and Mann (1975). An additional condition index based on ash-free dry weight, Cl(afdw), was calculated for each oyster analyzed for Cl(grav). Pans containing dried oyster meat were ashed in a muffle furnace at 500°C for 4 hours. After cooling in a desiccator, pans were weighed to the nearest milligram and Cl(afdw) was calculated using the method described by de Wilde (1975). Regressions between condition index and shell length were computed for both condition indices to determine if they varied with shell length. Correlations between fecundity and each condition index (including values obtained for females only) were performed to determine if either Cl(grav) or Cl(afdw) was a good indicator of female spawning potential. Sex Ratios Sex ratios (males:females) were calculated for oysters collected from each of the 4 oyster reefs on the 10 sample dates employing the same animals used for Cl determination. Calculated ratios were compared to the expected 1:1 ratio using chi-square analysis. A 2-way ANOVA was used to determine whether sex ratios varied among reefs and through the spawning season. Total sex ratio over the entire season was calculated for each station. Total sex ratio was compared with the expected ratio, again employing chi-square analysis. Abundance and Size-Frequency 2 Oysters collected from 0.25 m plots were used to determine approximate abundance and size-frequency of oysters on each reef. Oysters from each of the 4 replicate samples from each station on the 3 sample dates were counted and shell length was measured to the nearest millimeter. Counts included all oysters except the young of the year as they were too small to be seen with the naked eye for part of the season, and even when larger, they were too difficult to remove from the culch to assess sex. A 2-way ANOVA was performed on natural log transformed abundance values to determine if mean abundance varied among stations or over time. Where differences were found, multiple comparisons were performed using the Student-Neuman-Keuls test. Mean oyster shell length was compared among stations using the Kruskal-Wallis nonparametric 1-way analysis of variance test. Multiple comparisons were made with Dunn's Approximation. 25 Total Egg Production Seasonal fecundity for each station was estimated as the mean egg number from all dates if dates were not significantly different from each other. If a significant date effect was found, seasonal fecundity was calculated as mean egg number on dates when oysters contained significantly more eggs than the background level. Mean abundance was 2 multiplied by mean sex ratio to calculate number of females per m for the 4 stations. Boundaries of Dog Shoal, Thomas Rock, and Horsehead were estimated and area within the boundaries obtained by digitizing NOAA chart 12248. Boundaries of Wreck Shoal were unclear, so area reported for Wreck Shoal by Moore (1910) was used. Areas reported are representative of area sampled, and do not necessarily include the entire reef. Estimates of total spawning potential for the broodstock at each station were obtained by multiplying mean estimated fecundities, number 2 of females per m , and reef area. Spawning and Larval Viability Seawater from Wachapreague, Virginia was used in all spawning experiments instead of local York River or James River water because it was considered to be cleaner than the river waters which sometimes contain high levels of TBT, copper, and other pollutants. Seawater was diluted to 15°/oo salinity with distilled water, 1-um filtered, and chemically disinfected using sodium hypochlorite followed by dechlorination with sodium thiosulfate (Castagna and Kraeuter, 1981). 26 Approximately 75 randomly selected oysters from each Horsehead and Dog Shoal collection made from June 17 through September 9 were brought into the laboratory for spawning. Protocol for spawning was as follows: 1. Oysters were washed on the date of collection. 2. Oysters were placed in spawning tank of filtered seawater at 16-19° C overnight. 3. The following morning, water temperature was raised to 23°C for 1 hour. 4. Oysters were fed several liters of the naked flagellate Isochrvsis galbana Parke (approx. 5,000,000 cells/ml). 5. After the water was cleared, the tank was partially drained. 6. The tank was refilled with heated water from a head tank and water temperature in the spawning tank was quickly raised to 30°C to induce spawning. 7. Live sperm from several sacrificed males was added to the tank (Calabrese and Davis, 1970). 8. The first male to spawn was allowed to remain in the tank. 9. Subsequent spawners were transferred to individual 2-1 beakers as soon as they began to spawn. 10. Gametes were collected. 11. After spawning, oysters were removed from their beakers, sexed and measured. 12. Sperm from the first 3 male spawners was pooled. 13. Several milliliters of sperm was added to the eggs of the first 5 female spawners. 14. After 5 minutes, the eggs were filtered through a series of stacked sieves (200 um and 90 um) and collected on a 20u nitex nylon mesh. 15. Eggs were rinsed with 15°/oo water and washed into a 1-1 graduated cylinder for counting. 16. The volume was brought up to 1 liter. 17. Eggs were suspended by stirring with a plunger. 18. 3 1-ml aliquots were removed and placed in Sedgwick- Rafter Counting Cells. 19. Eggs, both fertilized and unfertilized, were counted. 20. If oysters did not spawn, they were cooled to 16-19°C over night. 21. The next day, the above procedure was repeated. 22. If spawning was still unsuccessful, eggs were stripped from 5 randomly selected females. 23. Sperm stripped from 3 randomly selected males was combined and added to the stripped eggs. Larval Culture Protocol was as follows: 1. 1.5-1 cultures were started with embryo densities of 15/ml (22,500/beaker). 27 2. Two replicate cultures were prepared with embryos from each of the 5 females (10 cultures total). 3. Cultures were maintained in a water bath at 25°C. 4. After 4 hours, the percentage of cleaved eggs was determined and recorded. 5. Cultures were examined again after 24 and 48 hours. Percentage of live straight-hinge larvae was recorded. RESULTS Temperature. Salinity, and Dissolved Oxygen Bottom water temperature and salinity values recorded at the study sites are depicted in Figure 3. Horsehead had the largest range of temperature and salinity, 19-31°C and 6.62-18.38°/oo, respectively. Dog Shoal had the smallest temperature and salinity ranges, 19-29°C and 14.89-20.76°/oo. Temperature peaked at all stations on July 29, then decreased to the lowest recorded on the final collection date. The temperature varied significantly over the course of the study (ANOVA: F=63.98, df=9, P<0.00005), but there was no difference in temperature among the stations (ANOVA: F=1.36, df=3, P=0.277). Salinity generally increased throughout the summer (ANOVA: F=13.68, df=9, P<0.00005). At each station, salinity recorded on the final date was higher than the salinity on June 2. Salinity varied significantly among stations (ANOVA: F=65.51, df=3, P<0.00005) with salinity at Horsehead significantly lower than that at the other stations (SNK: P<0.050). There was no difference in salinity among the other stations. Percent oxygen saturation of the bottom water ranged from 77% to 123% (Fig. 4). Since it was impossible to tell if saturation values greater than 100% were the result of supersaturation of the water, calculation rounding error, or error in sample collection or processing, Figure 3. Bottom water Temperature and Salinity at Study Sites June to October 1986 TEMPERATURE Figure 4. Percent Oxygen Saturation c m \ r j iiinmiiiimimiiiiimmmiiimitiiim irn iiinmiiiimimiiiiimmmiiimitiiim _LN30d3d 3 d 0 3 N L _ S V i n d V l I O N 11 o o 1II M ...... m uiiniiiinu 1 1 M N 1111 m m 1 1 111 m Ml 11 Mh 1111M 11 1111 n 1 1 1 1 n 1 1 1 i um im m o O ~3 3 —3 3 Z Z CM ~~3 3 o o I— CD Q < LU 31 all values were included in the analysis. Oxygen saturation varied significantly over time (ANOVA: F=9.23, df=9, P<0.0005), but more importantly, variation among stations was not significant (ANOVA: F-2.96, df=3, P-0.052). Fecundity Egg numbers were to have been standardized for shell length in order to obtain size-specific fecundity. Although the regression between egg number and shell length appeared to be significant (b=0.32, N=330, P<0.0005), it was not, because the assumption of homogeneity of variance was violated (Sokal and Rholf, 1981). Values of the dependent variable, egg number, were not normally distributed for each value of shell length, the independent variable (Fig. 5). Since a valid regression was not obtained, egg number could not be standardized. Mean egg number per oyster differed significantly among the 2 stations (KW: X =11.97, n=330, P=0.0075). Mean egg numbers suggested that Thomas Rock oysters produced the most eggs followed by Wreck Shoal, Dog Shoal, and finally Horsehead (Fig. 6). The most fecund Thomas Rock female contained approximately 45.95 x 10^ eggs, while the most fecund g Horsehead female contained approximately 22.03 x 10 eggs. Because of unequal sample size and large variation in fecundity among individual females, however, the only statistical difference discernible was that Thomas Rock oysters were more fecund than Dog Shoal oysters (Table 1). Commencement of spawning was marked by a decrease in mean egg number, but large variation about the means suggest that spawning was not synchronous at any station except Horsehead (Figure 6). m e n on dob Nvan FIGURE 6. Mean Fecundity of James River Oysters. Histograms represent -3 mean egg number x 10 . Lines above represent upper limit of 95% confidence interval. * Denotes peaks significantly higher than background (estimated by Dunn’s Approx: P<0.050). MEAN EGG NUMBER / FEMALE 20000 28000' 24000' 32000' 36000 12000 40000 20000 16000’ 44000 28000 24000 32000 20000 26000 36000 40000 24000 32000 36000 44000 16000 40000 12000 12000 16000 44000 4000 8000 20000 24000 26000 8000 32000 36000 4000 4000 6000 40000 12000 44000 16000 4000 8000 O' 0’ O' 0- - - ' ' ’ ’ < ' ' U 0 JN 7 U 0 JL 4 U 2 AG2AG 6SP 9 E 2 OT 16 OCT 23 SEP 09 SEP 26 AUG AUG12 29 JUL 14 JUL 01 JUL 17 JUN 02 JUN ...... HMS ROCK THOMAS RC SHOAL WRECK HORSEHEAD DOG SHOALDOG Table 1. Multiple Comparison of Fecundity Among Stations DS HH WS TR MEAN EGG # (x 106) 4.54______4.23 5.37 9.80 Means not connected by a line are significantly different (Dunn's Approx: P<0.050) 35 It appears that Dog Shoal oysters had at least 3 periods of mass spawning. These periods are represented by high mean egg number and large variation about the mean on July 14, August 12, and September 9 followed 2 weeks later by low mean egg number. The decrease in egg number from June 17 to July 1 is not considered to be due to spawning * since DS oysters did not contain ripe gametes on June 17. Large variation about the mean which was indicative of spawning, also obscured differences among those means (Table 2), Large scale spawning at Thomas Rock appears to have begun between July 14 and July 29. Mean egg number on July 14 was significantly different from background level (Table 3). Large variation observed in mean egg number on July 17 and July 1 can be attributed to asynchronous gonad maturation of the oysters. As in the case of Dog Shoal, Thomas Rock oysters' did not contain fully mature eggs in June collections. There was no indication of a second spawning bout of Thomas Rock oysters. Oysters at Wreck Shoal and Horsehead began to produce mature eggs before those at the downriver stations, Thomas Rock and Dog Shoal. Although mean egg number does not differ significantly at Wreck Shoal (Table 4), the increase in mean and variation indicates that they began to spawn between July 1 and July 29. There was a small, non-significant (Dunn's Approx: P>0.050) increase in mean egg number in the beginning of September, indicating a second spawning bout. Spawning patterns at Horsehead were different than those seen at stations downriver. Horsehead oysters contained fewer eggs than those from other reefs and there was less variation in egg numbers between individuals at any given time. Lower variation relative to the other TABLE 2. Multiple Comparison of Egg Number Among Dog Shoal Samples VO VO vQ 1 < o CN r^ o CN ON VO 00 VO CN 00 vO o ON cn co CN ON ON o W I I I I I I I i VO 00 CO H o 00 00 oo O CN 1^* o 00 o o 00 LO VO ON ON 00 ON ON W o VO 00 CN CO 0 uo 1 • Denotes significantly different pairs (Dunn’s Approx: P<0.050) Table 3. Multiple Comparison of Egg Number Among Thomas Rock Samples vo oo cn ON CN CO o vO vO vO tH I"'. t-- CN 00 H N C n o o H t O CN I"- O ov n o I i I I I I I vO <3= n c CN o H O vO CN co CO uo CO r^. uo on CO CN N O CN in in in CN n o CO m o o in w OvO VO CO O CN CN O 1 1 1 1 n • • t S nin in CN 1^. H t H t H nCO in H n o • vO st CN ON r^. H t • H r H t H t CN ON 1 1 1 1 1 1 • • sf co CN CN H t 00 CO CN OON VO m m CN 00 • • • co m o NON ON CN o CO CN o H t VO ON H t H t O • •H in o •H •H T—1 •H "c o o W •ri « > O 00 CN vo cn 0 o\ iH oo OV oo CN cn hv 00 r-l CN O CN O V CN o CN rH 0 0 1 ■ VO •vf o cn H r cn ■ i • 0 0 cn 0 0 0 0 CN H r cn ■ I • VO vo H r o t o H 1 i • 39 stations resulted in more significant differences between collection dates (Table 5). There was a mass spawning at Horsehead in the beginning of July. A second, significant (Dunn's Approx: P<0.050) build-up of eggs in late July was followed by spawning which lasted into September. Horsehead oysters spawned out before those at other stations; no oysters containing eggs were found on October 16. Condition Index Gravimetric condition index values were not standardized for shell length, although a significant regression was obtained (b= -0.08, n-1574, P<0.0005). Variation in shell length accounted for only 3% of 2 the variation in Cl(grav) (r =0.032) (Fig. 7). Mean Cl(grav) values were highly variable as expected, and the variation increased with the mean (Fig. 8). Estimates of mean Cl(grav) were highest at Thomas Rock followed by Dog Shoal, Horsehead, and Wreck Shoal (KW: X^=56.94, n=1574, P<0.0005). Ash-free dry weight condition index values were not standardized for body size even though a significant regression was obtained (b=- 0.02, n=1572, P<0.0005). Shell length was incorporated in the calculation of Cl(afdw), so it had already been taken into account (Fig. 9), and dividing Cl(afdw) by shell length did not remove the size effect. Dog Shoal and Thomas Rock oysters possessed higher mean Cl(afdw) than did Wreck Shoal and Horsehead oysters (Fig. 10). It is interesting to note, for Horsehead oysters, both condition indices were low in late August and early September during late stages of spawning. In mid- Table 5. Multiple Comparison of Egg Number Among Horsehead Samples vo <}■ vO vO 0 0 00 CO <3 H t S C ON tH cs CS ON n o cN o cs O r^. tH o n o W Q H I I I I I vo vO vO vO CS O C CO 00 ON CO H t o o N O m ON ON m tH CO m uo co K O v H t co co sr*. o cs 1 1 • • • • sf vO in tH m 1 1 r-" 00 ON O H t ■ 1 11 CO H t m H t i 1 vO o ON r". N O cs ■ i • • • vO 00 vO cs H t 1 1 co O V cs 00 CS I 1 NON ON t N O m N O 0 * H 1 1 • CO CS N O O C o 1 1 • Denotes significantly different pairs (Dunn*s Approx: P<0.050) Figure 7. Regression of Cl(grav) and Shell Length o o O C o o o o o CO o ( abj B) io O M C o txx, xx o O C ■ in o cn o o o o CO SHELL LENGTH (MM) FIGURE 8 Mean Cl(grav) of James River Oysters. Histograms represent mean Cl(grav). Lines above represent upper limit of 95% confidence interval. MEAN GRAVIMETRIC CONDITION INDEX 0 2 25 30- 35 25 15- 30- 20 30 15- 40' - - - - U 0 JN 7 U 0 JL 4 U 2 AG 2AG 6SP 9 E 2 CT 16 CCT 23 SEP 09 SEP 26 12AUG AUG 29 JUL 14 JUL 01 JUL 17 JUN 02 JUN RC SHOAL WRECK HORSEHEAD THOMAS ROCK THOMAS O SHOAL DOG Figure 9. Regression of Cl(afdw) and Shell (MOdV)IO SHELL LENGTH FIGURE 10. Mean Cl(afdw) of James River Oysters. Histograms represent mean Cl(afdw). Line above represents upper limit of 95% confidence interval. MEAN ASH-FREE DRY WEIGHT CONDITION INDEX 2.2 2.6 1.0 30 34 2 2 2.6 14 0 3 2.2 34 2.6 0 3 34 22 6 2 0 3 34 JUN 02 JUN 17 JUL 01 JUL 14 JUL 29 AUG 12 AUG 26 SEP 09 SEP 23 OCT 16 OCT 23 SEP 09 SEP 26 12 AUG AUG 29 JUL 14 JUL 01 JUL 17 JUN 02 JUN RC SHOAL WRECK HMS ROCK THOMAS HORSEHEAD DOGSHOAL 45 September after spawning was complete, both condition indices increased (Figs. 8 and 10). Correlation between the 2 condition indices and egg number showed that while Cl(afdw) was only a poor indicator of female oyster fecundity (r=0.37, n=37, P=0.026)(Fig. 11), Cl(grav) and fecundity (r=0.19, n=37, P-=0.264) (Fig. 12) were not correlated. Since condition indices were not highly correlated with fecundity, detailed statistical analysis of condition indicies was not warranted. Sex Ratios The sex ratios of James River oysters are reported in Table 6. In most collections, males outnumbered females; the male to female ratio ranged from approximately 1 to 4. Values for June 2 were excluded because oysters did not possess mature gonads at that time. Since developing eggs were more readily identifiable than developing sperm, the sex ratios estimated from these data would be artificially low. Data for September 23 and October 16 were also excluded because few oysters from those collections contained any gametes, and sex determination was difficult. Most oysters containing residual gametes were males. Sex ratios were not found to differ significantly among stations (ANOVA: F=1.39, df=l, P=0.279) nor over time (ANOVA: F=0.76, df=l, P=0.607). Total sex ratio (Table 6) was greater than the expected 1.0 for each station (Chi-square: DS X2=40.32, df=l, P<0.001; TR X 2= 2 0 .9, df-1, PC0.001; WS X 2=24.5, df-1, PC0.001; HH X 2=4.3, df=l, P<0.05). Mean shell length of male oysters was significantly smaller than that of female oysters at all stations excluding Dog Shoal (K.-W: DS Figure 11. Correlation of Egg Number and Cl(grav) «“ o M C o O C o cn O C CL M C O C N- 3Wi 993 NV3W 3 9 9 d38WriN o ♦ o MEAN CI(GRAV) Figure 12. Correlation of Egg Number and CKafdw) cu MEAN CKafdw) * * # o vO CS r^ H CS o m m CM o m CS CO o m m o cs o VO o H vo o • •• •• •' • • • o 9 o o o o o o o o v CM § w CO 0} Pi r-- ON r H cs o vo 00 CO U O *• •••• • • ctf PC o o CS H co r H o t H 3 o* 09 A rd O r-l •fc ■fc * ** 00 t H cs vo ON vo t H t 3 m o cs m o o Mfr o <19 P m o o o r^ c s o o o p . 1^ I"- t H t H Xl o i H t H cs CS CS t H O CM! o r H r H m m o O o • • • • • • • p o o o o o o O o T—1 § V o! § CO 09 o C! U •H o cs cs t H Hj- 0 0 Q) 44 o • • • •• » • • P •H PC CO t H t H t H r H cs CO t H CQ C H Xi 00 W j o •H W; 09 u a) O •H .a P Pi X2=0.31, n=268, P=0.5784; TR X2=5.12, n=306, P=0.0237; WS X2=8.50, n=305, P=0.0035; H H X 2=12.20, n=254, P=0.0005). Abundance and Size-Frequency Oyster distribution on the reefs was very patchy, ranging from 0 to 2 107 oysters per 0.25 m (Table 7). Oysters were more abundant at Horsehead than at other stations (SNK: PC0.050). Thomas Rock and Wreck Shoal had significantly more oysters than did Dog Shoal (SNK: P<0.050), but their abundances were not significantly different from each other 2 (SNK: P>0.050). Mean abundance per 0.25 m remained fairly constant through the study period at Dog Shoal and Wreck Shoal, while mean oyster abundance decreased at Thomas Rock and Horsehead (Table 7, Fig. 13). Oyster abundance declined at Thomas Rock from June 2 to October 16 (SNK: P<0.050) Reduction in abundance at Horsehead was not statistically significant. Mean shell length of oysters was significantly different among the 2 4 stations (KW: X =155.06, P<0.000005). Horsehead oysters were significantly smaller than oysters from all other stations (Dunn's Approx: P<0.050). Oyster shell length at Dog Shoal, Thomas Rock, and Wreck Shoal was not significantly different (Dunn's Approx: P>0.050). Total Egg Production Reef areas obtained by digitizing NOAA chart No. 12248 were used for Dog Shoal, Thomas Rock, and Horsehead. In the case of Wreck Shoal, boundaries are unclear on the chart, so area reported in Moore (1910) Table 7. Oyster Abundance in 0.25 square meter Collections CD o o 1— LD CM < CD C\J co ■m-o'M; co ■m-o'M; id CM CM CO -M- coin i— CM Q O < CD X CO O LU LU < Q O COo CT>CO "M" f'- oin co coo r± ) T C O ^ B C CM CO CM O CM (O CM (O CM * - t - CM - t CM - * f I CM CM X < X * o CO o o i O Ol in in co in o £ < co cn n- nc c oc Or- r O co co co in co ■M" CO CM ' cn - Lri m - r*— oo co "M- oi mooo in cm o r- T- r- T- CO 5 X LU o cn O < m c X MC oID o CM CM cm LU LU Q LU < co oo o co cn co co CM cn id ) O D C N m ■M-OOCM r~- CM CD N. CO CM’ CO ■sr r- co CD COin in CD m c x O x LU X LU < Q cn m c in MEAN 71.8 65.0 44.5 ST. DEV 40.7 17.4 22 2 ® f s 1 & 0 + + o& h m o> ID O E (0 ■ o < o 060 LU • 2 * % co ID TJ JO < CO S 16 a a £ 2 aoNVQNnsv Nvan 52 was employed in subsequent calculations. August 27 values were excluded from Dog Shoal mean fecundity calculations. Mean fecundity for Thomas Rock oysters was computed as the average of values for June 17 through July 29. All dates were included in Wreck Shoal mean fecundity since no dates were significantly different. Horsehead mean fecundity was calculated as the average of mean egg number from June 17 through August 12. Estimated seasonal egg production per reef, considering reef size, sex ratio, mean oysters abundance, and mean fecundity, revealed that there were more eggs produced at Thomas Rock than at the other sampling stations (Table 8). Total estimated egg production at Dog Shoal was an order of magnitude lower than that at other stations. Spawning and Larval Viability Spawning was attempted 9 times with Dog Shoal oysters and 6 times with oysters from Horsehead (Table 9). The only successful trial was that for oysters collected from Horsehead on August 14. Oysters began to spawn within 20 minutes of thermal shock. Spawning continued for 2.5 hours, and 55 out of 71 oysters in the tank spawned. Of the 55 spawners, 28 were male and 27 were female. Numbers of eggs obtained from the first five female female spawners ranged from 56,875 to 1,437,500 (Table 10). Fertilization was achieved in all cultures, yet no straight-hinge larvae were obtained in any trial, with spawned or stripped eggs. The same problem was experienced by two other researchers working in the same laboratory, one working with Crassostrea virginica. the other working with Mulinia lateralis (Say), Snisula solidissima (Dillwyn), and Rangia cuneata (Gray). Eggs of C. virginica which were spawned and cn 9 rH W 04 4 Os O' os • CN Z 00 Z < M w s *4 W W NJ a M M Eh a CO TABLE 9. Number of Oysters Spawning in 1986 Trials. Brackets connecting results of two trials indicate that spawning was attempted twice with the same batch of oysters. * Denotes oysters which were stripped after unsuccessful spawning trails. LU o _l < * * < o o o r^ O LU 2 C\J X LU LU U_ CO o o C D 0 0 (J) c r LU _l CM O X < UU _l * * * < o o o o o o 0 ^ 0 O X LU CO X CD LU O O O KT O h- C Q ? o O Q OOCDCMCOLOCOr^O'^-'M-LOCDCDO t- t-OO' t- t- t- cOCQ t- t- t- cM cO Z Z —J-J—I—i_J-J—iCDCDCDCDCD DDDDDDDDDDDDDD ““ D—D“D-3“D—5“ o o o o ^ c/> o o o o in © CD o m o co •4—• h-* in co cn a LO >N co c \i co c\j in o UJ : in in ■O CD o jr a) in o X s O) JO cz co JD “O oo o cn c i- ~Q> _d cn CD t~ Cl cn tn cn a> ZJ CO O O) O) c: o> (D © T3 CD CD jQ E u JD E C\i Nf CD T— CO JD E CD N CO ^ r^» £ co 56 fertilized in the VIMS Oyster Hatchery and subsequently transferred to the laboratory failed to develop into shelled larvae while sibling larvae which remained in the hatchery developed shells within 24 hours. DISCUSSION Fecundity of female oysters from four major reefs in the James River was observed to be highly variable, both within and among locations, in 1986. The large variation may be explained by several biotic and abiotic factors. Biotic influences include time since last spawning, food supply and resultant nutritional status of the individuals, size of females, and prevalence of disease and parasitism. Major abiotic factors potentially influencing fecundity include water temperature (which affects gonad maturation rate), and salinity regime. Variation in fecundity among females on a given reef occurred in part because not all oysters were at the same stage of reproductive maturity at the same time (e.g. Nelson, 1928; Loosanoff and Davis, 1952 Kennedy and Krantz, 1983). Despite various mechanisms which result in some synchronicity of spawning within oyster communities, not all oysters spawn at the same time. An oyster which spawned immediately prior to collection presumably contained fewer eggs than a similar oyster which was ready to spawn, but did not do so before collection. It was not possible to distinguish between those oysters which spawned recently and those which had few eggs because they were actually less fecund. Egg production is dependent upon the nutritional health of the oyster since accumulation of egg reserves takes place at the expense of stored glycogen (Engle, 1950; Galtsoff, 1964; Gabbott, 1975; Mann, 58 1979). It was not possible in this study to distinguish the variation in fecundity caused by differential food supply since condition index, the parameter chosen to represent nutritional status, was not highly correlated with egg production. Condition index usually decreases when spawning begins, and does not increase significantly until after the end of the spawning season when oysters begin fall fattening (Medcof and Needier, 1941; Chapman, 1947; Galtsoff, 1964). Lack of correlation between fecundity and condition index may be explained by the fact that oysters with a high Cl may possess large numbers of gametes and little glycogen, few gametes and moderate amounts of stored glycogen or no gametes and large glycogen stores as in the case of parasitic castration (e.g.. Quayle, 1969). A portion of the variation in fecundity among female oysters may be attributed to body size. Large oysters usually produce more eggs than do small oysters (e.g.. Davis and Chanley, 1956; Andrews, 1984 unpublished). Results reported herein showed that in the James River, small oysters generally contained a small number of eggs, but oysters over 60 mm in shell length possessed highly variable egg numbers (Fig. 5). Horsehead oysters, which were significantly smaller than those from the other stations, had the lowest mean egg number. It was not possible to quantify the effect of body size on fecundity, however, since the assumptions of regression analysis were violated because of heterogeneity of variance. Disease may have been the cause of some of the fecundity variation among female oysters. Record high salinities caused by consecutive droughts in 1985 and 1986 allowed oyster diseases to invade areas where they normally would not occur (Burreson, unpublished). Oysters from 59 Wreck Shoal, Dog Shoal, and Horsehead were infected by Haplosporidium nelsoni (MSX) with Wreck Shoal showing the highest infection rate in August, 1986 (Burreson, unpublished). Although Thomas Rock oysters were not plagued by MSX, at least 20% and perhaps as many as 96% were infected by the protozoan parasite Perkinsus (=Dermocvstidium) marinus (Burreson, unpublished; Burreson, personal communication) Oysters from the other 3 reefs were not tested for P. marinus. but Dog Shoal oysters were most likely also infected. Oysters from Wreck Shoal and Horsehead probably were free of the disease by virtue of their distance upriver and the somewhat lower salinity. Perkinsus caused high mortality during the fall of 1986 (Burreson, unpublished). The observed decline in abundance of oysters on Thomas Rock is clearly attributable to disease- related mortality. Some variation in fecundity among individuals was probably caused by parasitism. A substantial portion of oysters from all 4 reefs contained cercaria of the digenic trematode Bucephalus cuculus which occur in the gonads and digestive gland, eventually resulting in sterilization (Tennent, 1906; Menzel and Hopkins, 1955). Percentage of oysters parasitized by B. cuculus could not be estimated with any reliability since oysters were not examined for the occurrence of the parasite per se: it was merely noted if cercaria appeared in gonad smears. Cursory examination did not appear to indicate a greater infection of Bucephalus at one reef over another. Oysters of both sexes, as well as those of indeterminate sex, were observed to contain Bucephalus cercaria. 60 Egg maturation and peak spawning occurred earlier at the upriver stations, Horsehead and Wreck Shoal, than it did downriver at Dog Shoal and Thomas Rock (Table 6). Although bottom water temperature did not vary significantly among the stations during the study period, earlier differences in spring-time temperature may account for the timing of spawning. Data collected by Whitcomb (1986a) revealed that bottom water temperature at Horsehead on Hay 1 was 19.5°C. On May 6, temperature at Wreck Shoal was 19,0°C, while downriver at 2 shoals close to the Thomas Rock and Dog Shoal stations, temperatures were 17.1°C and 17.0°C, respectively (Whitcomb, 1986a). Egg maturation and subsequent spawning most likely was delayed by the extended period of cool water temperatures at Dog Shoal and Thomas Rock (e.g. Loosanoff and Davis, 1952). Salinity may be important in determining fecundity of oysters among different reefs in an estuary. Andrews (1984, unpublished) theorized that there was less spawning in the low salinity waters upriver than in the high salinity water downriver in the James. In this study, the salinity at Horsehead was significantly lower than at the other stations. Although salinity was measured only at a point in time, not over a tidal cycle, it is intuitively obvious that the salinity would be lowest since Horsehead is the farthest upstream station. Horsehead oysters were found to have the lowest mean fecundity of all the stations (Table 1). Water quality variation potentially influences fecundity variation among oysters from different reefs. Dissolved oxygen concentration, one measure of water quality, did not differ significantly among the study 61 sites (Fig. 4). Levels of pollutants were not monitored in this study, but their presence at one reef or another could constitute a stress on the oysters there. Stressed oysters presumably have less energy available for reproduction. Horsehead, for example, is adjacent to a anchorage for a large number of "mothballed" naval ships. Horsehead oysters are therefore potentially impacted by chemicals used in antifouling bottom paints such as copper and TBT (e.g. Roberts, et al.. 1987). Stress resulting from accumulation of these chemicals may partially account for the low mean fecundity of female Horsehead oysters (Table 1). Kennedy (1983) hypothesized that environmental stress may lead to increased numbers of males in an oyster population. In this study, male oysters were generally found to outnumber female oysters at all stations. This is inconsistent with the findings of Andrews (1951 unpublished; 1984 unpublished) and Morales-Alamo (1986) who found nearly equal numbers of both sexes on Wreck Shoal. One explanation of this inconsistency is that while all oysters over 1 year of age were included in the present study, Morales-Alamo (1986) examined only oysters approximately 60 mm in length or larger. Since a greater proportion of small oysters (i.e., <60 mm) are male (Burkenroad, 1931, Needier, 1932a; Andrews, 1984 unpublished), the inclusion of these small oysters in the present study may account for the observed preponderance of males. Andrews (1951 unpublished, 1984 unpublished) examined oysters of all sizes and reported a sex ratio of approximately 1.0; subsequent re- analysis of Andrew's data revealed male to female ratios significantly 62 greater than 1.0 for the 1951 data (X^— 13.23, PC0.001) and for the 1984 data (X2= 19.36, P<0.001). Seasonal egg production was estimated from the highest mean fecundity for oysters from each station. The highest means were averaged with other means not significantly different from them. Since high variation prevented the largest means from differing significantly from those on most other dates, seasonal egg production estimates are very conservative. Additionally, it is difficult to accurately estimate seasonal production because Crassostrea vireinica spawn several times each season and exhibit successive periods of gonadal build-up during the season (Andrews, 1979). Thomas Rock oysters produced 3-4 times more eggs per female in the season than oysters from other stations. Horsehead oysters ranked second in terms of seasonal fecundity; however estimated seasonal fecundity of individual Wreck Shoal oysters may be artificially low due to the missing sample during the height of the spawning season. Dog Shoal oysters had the lowest seasonal fecundity. With the exception of Thomas Rock, estimated seasonal fecundities of individual oysters by station in this study were an order of magnitude lower than those found in the literature. The potential contribution of eggs to the seed area made by the different oyster groups was estimated and termed total egg production per reef. Estimates of total egg production by oysters on each reef revealed that Thomas Rock was the most productive reef in 1986 because of tremendous individual seasonal fecundity (Table 8). This is especially interesting since Wreck Shoal is usually considered to be the 63 most productive reef in the seed area (e.g.. Andrews, 1983). Surprisingly, Horsehead was the second most productive reef. Although Horsehead is a fairly small reef, the great abundance of oysters and a more nearly equal sex ratio resulted in the production of a large number of eggs. Wreck Shoal was third in order of seasonal gamete production. In reality, Wreck Shoal probably surpassed Horsehead in total egg production by virtue of its tremendous area, but this was not apparent in the data because of the underestimate of individual seasonal fecundity. Estimated production on Dog Shoal is an order of magnitude lower than that on Wreck Shoal and Horsehead owing to the combination of small reef size, low oyster abundance, and low individual seasonal fecundity. These results illustrate the importance of the combination of factors in determining reproductive potential of an area. Results of this study indicate that seed area oysters are certainly capable of producing sufficient larvae to account for a large set in the seed area, providing that the larvae are viable, and that they are not lost from the system because of the hydrography. It is not possible to draw any conclusions concerning the potential viability of larvae produced by Dog Shoal and Horsehead oysters from the results of laboratory experiments conducted in this study. It is believed that the observed developmental failure was most likely caused by the laboratory environment, not by some inherent problem with the larvae or mishandling since siblings of larvae which developed normally in the VIMS Hatchery were malformed in this laboratory. More experiments to determine the viability of larvae from various reefs within the seed area are sorely needed. 64 Spat settlement in the James River seed area in 1986 was the lowest recorded in 7 years, similar to the low level of the 1970's (Whitcomb, 1986a). Lack of settlement in 1986 could have been caused by a spawning failure, but results of this study do not support this theory. Oysters from all 4 reefs examined produced mature gametes, and observed reductions in mean egg number over time indicate that spawning did occur. An alternative hypothesis to explain reduced spatfall is the occurrence of disease at Thomas Rock. Thomas Rock oysters were the most fecund in this study. A significant portion of them died from infection with Perkinsus marinus by the end of the spawning season. It is reasonable to postulate that even though Thomas Rock oysters spawned, the resulting larvae were not viable, and so did not recruit into the seed area. LITERATURE CITED APHA. 1980. Standard methods for the examination of water and wastewater, 15th Edition. Washington, D.C. 1134 pp. Andrews, J. D. 1951 Unpublished. Sexuality in Crassostrea virginica. Available upon request: Virginia Institute of Marine Science, Gloucester Point, Virginia. Andrews, J. D. 1951. Seasonal patterns of oyster setting in the James River and Chesapeake Bay. Ecology 32:753-758. Andrews, J. D. 1954. Setting of oysters in Virginia. Proceedings of the National Shellfisheries Association 45:38-46. Andrews, J. D. 1968. Oyster mortality studies in Virginia. VII. Review of epizootiology and origin of Minchinia nelsoni. Proceedings of the National Shellfisheries Association 58:23-36. Andrews, J. D. 1970. The mollusc fisheries of the Chesapeake Bay (USA). In: Proceedings of the Symposium on Mollusca held at Cochlin Part III. pp. 847-856. Marine Biological Association of India. Andrews, J. D. 1979. Pelecypoda: Ostreidae. In: Reproduction of Marine Invertebrates. Volume 5. Molluscs: Pelecypoda and lesser classes, A. C. Giese and J. S. Pearse, eds. pp. 293-341. Academic Press, New York. Andrews, J. D. 1982. The James River public seed area in Virginia. Special Report in Applied Marine Science and Ocean Engineering. Number 261. 60 pp. Virginia Institute of Marine Science, Gloucester Point, Virginia. Andrews, J. D. 1983. Transport of bivalve larvae in James River, Virginia. Journal of Shellfish Research 3:29-40. Andrews, J. D. 1984 Unpublished. Sex ratios in Crassostrea virginica oysters in Virginia. Available upon request: Virginia Institute of Marine Science, Gloucester Point, Virginia. Andrews, J. D. and J. L. Wood. 1967. Oyster mortality studies in Virginia. VI. History and distribution of Minchinia nelsoni. a pathogen of oysters in Virginia. Chesapeake Science 8:1-13. Baird, R. H. 1958. Measurement of condition in mussels and oysters. Journal du Conseil 23:249-257. 66 Bayne, B. L. 1975. Aspects of physiological condition in Mvtilus edulis (L.), with special reference to the effects of oxygen tension and salinity. In: Proceedings of the Ninth European Marine Biological Symposium, H. Barnes, ed. pp. 213-238. Aberdeen University Press, Aberdeen, Scotland. Berg, C. J., Jr. 1969. Seasonal gonadal changes of adult oviparous oysters in Tomales Bay, California. Veliger 12:27-36. Brooks, W. K. 1880. Development of the American oyster. Report of the Maryland Fish Commission for 1879. Reprinted in: Studies from the Biological Laboratory of the Johns Hopkins University 1:1-81. Burkenroad, M. D. 1931. Sex in the Louisiana oyster, Ostrea virginica. Science 74:71. Burkenroad, M. D. 1937. The sex-ratio in alternational hermaphrodites, with especial reference to the determination of sexual phases in oviparous oysters. Journal of Marine Research 1:75-84. Burkenroad, M. D. 1947. Egg number is a matter of interest in fisheries biology. Science 106:290. Burreson, E. M. Unpublished. Prevalence of the major oyster diseases in Virginia waters-1986. A summary of the annual monitoring program. Available from the author upon request: Virginia Institute of Marine Science, Gloucester Point, Virginia. Calabrese A., and H. C. Davis. 1970. Tolerances and requirements of embryos and larvae of bivalve molluscs. Helgolander Wissenschaftliche Meeresuntersuchungen 20:553-564. Castagna, M. and J. N. Kraeuter. 1981. Manual for growing the hard clam Mercenaria. Special Report in Applied Marine Science and Ocean Engineering Number 249. 110 pp. Virginia Institute of Marine Science, Gloucester Point, Virginia. Carriker, M. R. 1961. Interrelation of functional morphology, behavior, and autecology in early stages of the bivalve Mercenaria mercenaria. Journal of the Elisha Mitchell Scientific Society 77:168-242. Chanley, P. and J. D. Andrews. 1971. Aids for identification of bivalve larvae of Virginia. Malacologia 11:45-119. Chipman, W. A. 1947. Seasonal changes in the fattening of oysters. Addresses delivered at the Convention of the National Shellfisheries Association, pp. 28-32. Churchill, E. P. Jr. 1920. The oyster and the oyster industry of the Atlantic and Gulf States. U. S. Bureau of Fisheries Document Number 890. Appendix VIII to the Report of the U.S. Commissioner of Fisheries for 1919. 51 pp. 67 Coe, W. R. 1936. Environment and the sex in the oviparous oyster Ostrea virginica. Biological Bulletin 71:353. Coe, W. R. 1938. Primary sexual phases in the oviparous oyster (Ostrea virginica). Biological Bulletin 74:64-75. Davis, H. C. and P. E. Chanley. 1955. Spawning and egg production of oysters and clams. Proceedings of the National Shellfisheries Association 46:40-51. Davis, H. C. and P. E. Chanley. 1956. Spawning and egg production of oysters and clams. Biological Bulletin 110:117-128. de Wilde, P. A. W. J. 1975. Influences of temperature on behavior, energy, metabolism, and growth of Macoma balthica (L.). In: Proceedings of the Ninth European Marine Biology Symposium, H. Barnes, ed. pp. 239-256. Aberdeen Press, Aberdeen, Scotland. Dunn, 0. J. 1964. Multiple comparisons using rank sums. Technometrics 6:241-252. Dupuy, J. L., N. T. Windsor, and C. E. Sutton. 1977. Manual for design and operation of an oyster seed hatchery. Special Report in Applied and Marine Science and Ocean Engineering. Number 142. 104 pp. Virginia Institute of Marine Science, Gloucester Point, Virginia. Engle, J. B. 1950. The condition of oysters as measured by the carbohydrate cycle, the condition factor and the per cent dry weight. Proceedings of the National Shellfisheries Association 1950:20-25. Fox, D. L. and W. R. Coe. 1943. Biology of the California sea mussel (Mvtilus californianus). II. Nutrition, metabolism, growth, and calcium deposition. Journal of Experimental Zoology 93:205-249. Gabbott, P. A. 1975. Storage cycles in marine bivalve molluscs: a hypothesis concerning the relationship between glycogen metabolism and gametogenesis. In: Proceedings of the Ninth European Marine Biological Symposium, H. Barnes, ed. pp. 191-211. Aberdeen Press, Aberdeen, Scotland. Galtsoff, P. S. 1930. The fecundity of the oyster. Science 72:97-98. Galtsoff, P. S. 1937. Observations and experiments on sex change in the adult American oyster, Ostrea virginica. Collecting Net 12:187. Galtsoff, P. S. 1964. The American oyster, Crassostrea virginica (Gmelin). U. S. Fish and Wildlife Service Bulletin 64. 480 pp. 68 Galtsoff, P. S., W. A. Chipman, J. B. Engle, and H. N. Calderwood. 1947. Ecological and physiological studies of the effect of sulfate pulp mill waste on oysters in the York River, Virginia. Bulletin No. 43. Fishery Bulletin of the U.S. Fish and Wildlife Service 51:59-186. Grave, C. 1912. A manual of oyster culture in Maryland. Fourth Report of the Shellfish Commission of Maryland, pp. 279-348. Reprinted in 1944 by King Brothers, Inc., Baltimore. Griffiths, R. J. 1977. Reproductive cycles in littoral populations of Choromvtilus meridionalis (Kr.) and Aulacomva ater (Molina) with a quantitative assessment of gamete production in the former. Journal of Experimental Marine Biology and Ecology 30:53-71. Gunter, G. 1941. Seasonal condition of Texas oysters. Texas Academy of Science, Proceedings and Transactions 25:89-93. Gunter, G. 1950. The status of living oysters and the scientific name of the common American species. American Midland Naturalist 43:439- 449. Haven, D. S. and L. W. Fritz. 1985. Setting of the American oyster Crassostrea virginica in the James River, Virginia, USA: temporal and spatial distribution. Marine Biology 86:271-282. Haven, D. S., W. J. Hargis, Jr., and P. Kendall. 1981. The oyster industry of Virginia: its status problems and promise. Special Papers in Marine Science No. 4. 1024 pp. Virginia Institute of Marine Science, Gloucester Point, Virginia. Haven, D. S. and J. P. Whitcomb. 1983. The origin and extent of oyster reefs in the James River, Virginia. Journal of Shellfish Research 3(2):141-151. Hayes, P. F. and R. W. Menzel. 1981. The reproductive cycle of early setting Crassostrea virginica (Gmelin) in the northern Gulf of Mexico, and its implications for population recruitment. Biological Bulletin 160:80-88. Higgins, E. 1938. Progress in biological inquiries, 1937. Report of the commissioner of fisheries for the fiscal year 1938, Appendix 1. Administrative report No. 30. pp. 1-70. U.S. Bureau of Fisheries, Washington, D. C. Hoagland, K. E. 1985. Use of the terms protandry, protogyny, and hermaphroditism in malacology. American Malacological Bulletin 3(1):85-88. Ingle, R. M. 1951. Spawning and setting of oysters in relation to seasonal environmental changes. Bulletin of Marine Science of the Gulf and Caribbean 1():111-135. 69 Jablonski, D. and R. A. Lutz. 1980. Molluscan larval shell morphology: ecological and paleontological applications. In: Skeletal Growth of Aquatic Organisms, D. C. Rhoads and R. A. Lutz, eds. pp. 323-363. Plenum Press, New York. Kellog, J. L. 1892. A contribution to our knowledge of the morphology of lamellabranchiate mollusks. Bulletin of the U. S. Fish Commission 10:389-436. Kennedy, A. V. and H. I. Battle. 1964. Cyclic changes in the gonad of the American oyster, Crassostrea virginica (Gmelin). Canadian Journal of Zoology 42:305-321. Kennedy, V. S. 1983. Sex ratios in oysters, emphasizing Crassostrea virginica from the Chesapeake Bay, Maryland. Veliger 25(4):329-338. Kennedy, V. S. 1986. Expected seasonal presence of Crassostrea virginica (Gmelin) larval populations emphasizing Chesapeake Bay. American Malacological Bulletin, Special Edition Number 3(1986):25-29. Kennedy, V. S. and L. B. Krantz. 1983. Comparative gametogenic and spawning patterns of the oyster Crassostrea virginica (Gmelin) in central Chesapeake Bay. Journal of Shellfish Research 2(2) : 133-140. Korringa, P. 1955. Quality estimation of mussels and oysters. Archiv fuer Fischereiwiss 6:189-193. Lawrence, D. R. and G. I. Scott. 1982. Determination and use of condition index of oysters. Estuaries 5:23-27. Loosanoff, V. L. 1965. Gonad development and discharge of spawn in oysters of Long Island Sound. Biological Bulletin 129:546-561. Loosanoff, V. L. and H. C. Davis 1952. Temperature requirements for maturation of gonads of northern oysters. Biological Bulletin 103:80-96. Loosanoff, V. L. and H. C. Davis. 1963. Rearing of bivalve molluscs. In: Advances in Marine Biology. Volume 1. pp. 1-136. Academic Press, London. Loosanoff, V. L. and J. B. Engle. 1942. Accumulation and discharge of spawn by oysters living at different depths. Biological Bulletin 82:413-422. Loosanoff, V. L. and C. A. Nomejko. 1951a. Spawning and setting of the American oyster 0. virginica in relation to lunar phases. Ecology 32:113-134. Loosanoff, V. L. and C. A. Nomejko. 1951b. Existence of physiologically different races of oysters, Crassostrea virginica. Biological Bulletin 101:151-156. 70 McLean, R. A. 1941. The oysters of the western Atlantic. Notulae Naturae 67:1-14. Mann, R. 1978. A comparison of morphometric, biochemical, physiological indexes of conditions in marine bivalve molluscs. In: Energy and Environmental Stresses in Aquatic Systems, J. H. Thorp and I. W. Gibbons, eds. Department of Energy, Symposium Series Number 48. pp. 484-497. Mann, R. 1979. Some biochemical and physiological aspects of growth and gametogenesis in Crassostrea gigas and Ostrea edulis grown at sustained elevated temperatures. Journal of the Marine Biological Association of the United Kingdom 59:95-110. Mann, R. and J. H. Ryther. 1977. Growth of six species of bivalve molluscs in a waste reeyeling-aquaculture system. Aquaculture 11:231-245. Masumoto, B., M. Masumoto, and M. Hibino. 1934. The biochemical studies of Magaki (Ostrea gigas Thunberg) II. The seasonal variation in the biochemical composition of Ostrea gigas Thunberg. Journal of Science of Hiroshima University, Series A 4:47-56. Mayr, E. 1964. Animal Species and Evolution. Belknap Press, Cambridge. 797 pp. Medcof, J. C. 1939. Larval life of the oyster (Ostrea virginica) in Bideford River. Journal of the Fisheries Research Board of Canada 4:287-301. Medcof, J. C. and W. H. Needier. 1941. The influence of temperature and salinity on the condition of oysters (Ostrea virginica). Journal of the Fisheries Research Board of Canada 5:253-257. Menzel, R. W. 1951. Early sexual development and growth of the American oyster in Louisiana waters. Science 113(2947):719-721. Menzel, R. W. and S. W. Hopkins. 1955. The growth of oysters parasitized by the fungus Dermosvstidium marinum and by the trematode Bucephalus cuculus. Journal of Parasitology 41:333-342. Moore, H. F. 1910. Condition and extent of the oyster beds of the James River, Virginia. U. S. Bureau of Fisheries Document No. 729. 83 pp. U.S. Fish and Wildlife Service. Morales-Alamo, R. 1986. Sex ratios and gonad condition of James River oysters. In: Chesapeake Bay Research Initiatives at the Virginia Institute of Marine Science of the College of William and Mary. Accomplishments for the 1984-1986 biennium and projected activities for the 1986-1988 biennium. Virginia Institute of Marine Science, Gloucester Point, Virginia. 71 Needier, A. B. 1932a. Sex reversal in Ostrea virginica. Contributions to Canadian Biology and Fisheries 7:285-294. Needier, A. B. 1932b. American Atlantic oysters change their sex. Progress Reports of the Atlantic Biological Station, St. Andrews, N. B., and Fisheries Experimental Station (Atlantic), Halifax, N. S., Number 5. pp. 3-4. Needier, A. B. 1942. Sex reversal in individual oysters. Journal of the Fisheries Research Board of Canada 5:361-364. Nelson, J. 1908. Report of the Biological Department of the New Jersey Agricultural Experiment Station for the year 1908. pp. 149-177. Nelson, T. C. 1921. Aids to successful oyster culture. New Jersey Agricultural Experimental Station Bulletin 351:7-59. Nelson, T. C. 1928. Report of the Department of Biology of the New Jersey Agricultural College Experimental Station for the year ending June 30, 1927. pp. 77-83. Nelson, T. C. 1938. The feeding mechanism of the oyster. I. On the pallium and branchial chambers of Ostrea virginica. 0. edulis, and 0. angulata, with comparisons with other species of the genus. Journal of Morphology 63:1-61. Price, K. S. Jr. and D. Maurer. 1971. Holding and spawning of Delaware Bay oysters (Crassostrea virginica) out of season. II. Temperature requirements for maturation of gonads. Proceedings of the National Shellfisheries Association 61:29-34. Quayle, D. B. 1969. Pacific oyster culture in British Columbia. Bulletin of the Fisheries Research Board of Canada. Number 169. 192 pp. Roberts, M. H. Jr., M. E. Bender, P. F. DeLisle, H. C. Sutton, and R. L. Williams. 1987. Sex ratio and gamete production in American oysters exposed to tributyltin in the laboratory. Proceedings of Oceans 87:1471-1476. Ruzecki, E. P. and J. H. Hargis, Jr. (In press). Interaction between circulation of the estuary of the James River and transport of bivalve larvae. Sacco, F. 1897. I molluschi dei terreni terzaiarii del Piedmonte e della Ligura. Part 23 (Ostreidae, Anomiidae e Dimyidiae). Bullettino deo mesei di zoologia e di anatomia comparata della R. Universita, Torino 12(298):99-100. Scott, D. P. 1962. Effect of food quality on fecundity of rainbow trout, Salmo gairdneri. Journal of the Fisheries Research Board of Canada 19(4):715-731. 72 Sokal, R. R. and F. J. Rholf. 1981. Biometry, 2nd Edition. W. H. Friedman and Co., San Francisco. 859 pp. Stauber, L. A. 1950. The problem of physiological species with reference to oysters and oyster drills. Ecology 31:109-118. Tennent, D. H. 1906. A study of the life history of Bucephalus haimeanus: a parasite of the oyster. Quarterly Review of Microscope Science 49:635-690. Thompson, B. G . , Chief. 1986. Fisheries of the United States, 1986. Current Fishery Statistics Number 8385. U.S. Department of Commerce. National Oceanographic and Atmospheric Administration. 119 pp. Walne, P. R. and R. Mann. 1975. Growth and biochemical composition in Ostrea edulis and Crassostrea gigas. In: Proceedings of the Ninth European Marine Biology Symposium, H. Barnes, ed. pp. 587-607. Aberdeen University Press, Aberdeen, Scotland. Westley, R. E. 1959. Selection and evaluation of a method for quantitative measurement of oyster condition. Proceedings of the National Shellfisheries Association 50:145-149. Whitcomb, J. P. 1983. Oyster spatfall in Virginia rivers. 1983 annual summary. Marine Resources Special Report. The Virginia Sea Grant Program. Virginia Institute of Marine Science, Gloucester Point, Virginia. Whitcomb, J. P. 1984. Oyster spatfall in Virginia rivers. 1984 annual summary. Marine Resources Special Report. The Virginia Sea Grant Program. Virginia Institute of Marine Science, Gloucester Point, Virginia. Whitcomb, J. P. 1985. Oyster spatfall in Virginia rivers. 1985 annual summary. Marine Resources Special Report. The Virginia Sea Grant Program. Virginia Institute of Marine Science, Gloucester Point, Virginia. Whitcomb, J. P. 1986a. Oyster shoal survey, spring 1986. Virginia Marine Resource Report No. 86-6. Virginia Institute of Marine Science, Gloucester Point, Virginia. Whitcomb, J. P. 1986b. Oyster spatfall in Virginia rivers. 1986 annual summary. Marine Resources Special Report. The Virginia Sea Grant Program. Virginia Institute of Marine Science, Gloucester Point, Virginia. Wolman, M. G. 1968. The Chesapeake Bay: geology and geography. In: Proceedings of the Governor's Conference on the Chesapeake Bay. pp. 7-48. Wye Institute, Maryland. 73 Wood, L. and W. L. Hargis, Jr. 1971. Transport of bivalve larvae in a tidal estuary. Proceedings of the Fourth European Marine Biological Symposium 4:24-44. Yankson, K. 1986. Reproductive cycles of Cerastoderma glaucum (Brugiere) and C. edule (L.) with special reference to the effects of the 1981-82 severe winter. Journal of Molluscan Studies 52:6-14. VITA Carrollvn Cox Born in Durham, North Carolina, March 30, 1961. Graduated from First Colonial High School, Virginia Beach, Virginia in 1979. Earned B.A. in Biology with a minor in Environmental Sciences from the University of Virginia in 1983. Entered the master's program at Virginia Institute of Marine Science, School of Marine Science, College of William and Mary in 1984.