BULLETIN OF MARINE SCIENCE, 45(1): 130-138, 1989

GAMETOGENIC CYCLE OF RANGIA CUNEATA (MACTRIDAE, MOLLUSCA) IN MOBILE BAY, ALABAMA, WITH COMMENTS ON GEOGRAPHIC VARIATION

M. C. Jovanovich and K. R. Marion

ABSTRACT Stages of the gametogenic cycle of the brackish water clam Rangia cuneata were investigated in Mobile Bay, Alabama, by histological examination of the gonads. Water temperature and salinity measurements were related to the gametogenic cycle and compared to those made in other studies on the same species in different locations. Clams in the early active phase were found from February through April, and those in the late active phase predominated in May. Gonads were ripe from July through September and were partially spawned from September through October. Clams became spent between November and January. Changes in meat, gonad, and total wet weight reflected the stages of the gametogenic cycle, while length of the shells remained nearly constant year round. The gametogenic cycle of R. cuneata in Mobile Bay followed a similar pattern to that observed in clams studied in other locations, except that gametogenesis began earlier, during late winter, and clams became spent earlier in the fall. It is suggested that the interactive effects of temperature, salinity, and nutrients can account for the differences in the timing and length of the stages of the gametogenic cycle between locations.

Rangia cuneata (Gray) is a brackish water clam (Family Mactridae) abundant in the estuaries of the Gulf of Mexico. It is also found throughout the coastal areas of the eastern United States as far north as the upper Chesapeake Bay (Woodburn, 1962; Pfitzenmeyer, 1970). R. cuneata inhabits the upper layer of sediment in areas where the salinity ranges between 0 and 150/00and where water temperatures vary seasonally between 0.5 and 35°C (Hopkins et aI., 1973). In the James River, Virginia, Cain (1975) reported that this species made up 95% of the total benthic biomass. As a result of such high population densities and because R. cuneata converts detritus to biomass that can then be used as a food source for many bottom-dwelling and bottom-feeding animals (Odum and Copeland, 1969), the species is considered to be an important link in the food chains of estuaries. This clam has also been used in recent years as a biological indicator of pollution in the upper reaches of estuaries, because of its ability to accumulate organic pollutants, particularly the polycyclic aromatic hydrocarbons (PAH) (Fu- cik et aI., 1977; Neffand Anderson, 1981). During the course of a study conducted by the authors on the biological and physical factors influencing the uptake and depuration of the PAH anthracene by this clam, it became apparent that infor- mation on the reproductive cycle of R. cuneata in the Gulf of Mexico is limited. Fairbanks (1963) observed that R. cuneata in Louisiana had two "incompletely definitive" spawning periods per year. In February, clams developed a pre-spawn- ing condition, followed by the production of ripe gametes and spawning in March through May. According to Fairbanks, there was a short period of recovery in early June, followed by a more prolonged period of gamete production beginning at the end of the same month and lasting until November. Cain (1975) investigated the gametogenic cycle of R. cuneata in the James River, Virginia, and indicated that gametogenesis started in early April and continued throughout the summer. Ripe gametes were present from May to late November, with spawning beginning in early and mid-summer, but the major spawning period occurred in the fall.

130 JOVANOVICH AND MARION: GAMETOGENIC CYCLE OF RANGIA CUNEATA 131

30 I 12.0 25 0 0- en 10.0 r-» -W 20 a: Z ;:) 8.0 ~ =i c( 15 -< 6.0 a: Cl W - Q. 8- :E 10 4.0 - W ~ I 5 2.0

JJ ASONDJ F MAMJ JASON OJ F 1983 1984 1985 Figure 1. Rangia cunea/a. Seasonal changes in water temperature and salinity at Dog River, Mobile County, Alabama between June 1983 and January 1985.

The purpose of this study was to determine the gonadal development of R. cuneata under the temperature and salinity conditions prevailing in Mobile Bay, Alabama over a 16-month period. Since the gametogenic cycle of lamellibranchs can vary considerably between localities (Pfitzenmeyer, 1962; Ropes and Stickney, 1962; Shaw, 1964), knowledge of the cycle at additional locations can provide useful insights to factors controlling geographic variation in the reproduction of lamel- libranchs.

MATERIALS AND METHODS

Clams were collected by hand at the mouth of Dog River, Mobile County, Alabama, USA (30°34' 10"N; 88°05'OO"W) approximately every 6 weeks from September 1983 to January 1985. The salinity and temperature of the water were mcasured at every collection and, whenever possible, between collec- tions, using a YSI salinity-temperature meter. Samples for histological examination consisted of 16 mature individuals per collection. Each clam was measured by taking the total length from the anterior to the posterior of the valve. Total wet weight was recorded, after which each clam was shucked and the tissue wet weight determined. Gonads were then dissected, weighed, and fixed in Bouin's fixative for 24 h (Pantin, 1969). The tissues were embedded in paraffin, sectioned at 6 jlm thickness, stained with Mayer's Acid Hema]um as modified by Lillie (Clark, 1981) and counterstained with Eosin Y. Each slide was examined at 63 x and 250 x magnification to determine the status of gonadal deve]- opment. Since the gametogenic cycle of this clam is a nearly continuous process without sharp demarcations, five artificial stages of gonadal development, as employed by Cain (1972), were used for reproductive assessment. These were: early active, late active, ripe, partially spawned and spent. The average number of eggs in five follicles for each female collected (total number of females = 50) was also determined, and the vertical and horizontal axis of five eggs selected at random were measured, together with the diameter of the nucleus of each of these eggs.

RESULTS Water Temperature and Salinity. - Water temperatures at the sampling site (Fig. 1) showed a clear seasonal cycle, with maximum values in September (29°C in 1983 and 32°C in 1984), and minimum values in February 1983 (11°C) and 132 BULLETIN Of MARINE SCIENCE, VOL. 45, NO. I, 1989

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December 1984 (10°C). Changes of more than 10°C were observed between April and May 1984, and again between September and October of the same year. Salinity also followed a seasonal pattern (Fig. 1) with a maximum value in November 1983 (14%0) and another peak value in October 1984 (100/00).Minimum salinity was observed in March 1984 (00/00).Salinity values reflected the discharge from the nearby rivers, being high during extended periods of low river influx and very low under flooding conditions, approaching 00/00(Schroeder and Lysinger, 1979). Seasonal Variation in Size. - The length of the shells remained nearly constant throughout the 16-month period, averaging 47.0 ± 0.6 mm (Fig. 2); however, the total weight of individual clams and the weight of their meat showed a seasonal cycle (Fig. 2). Both values were minimal between October 1983 and January 1984, while maximum weights were observed in August (58.1 ± 2.4 and 8.3 ± 0.3 g, respectively). Gonad weight also reflected this pattern (Fig. 2), reaching minimum values during October 1983 through March 1984, followed by increased weights from May 1984 through September 1984, with a maximum in August (2.7 ± 0.2 g). Histology: Female Gonadal Development. - Using the system of Cain (1972), fe- males judged to be in the early active stage had few oogonia at the periphery of the follicle (Fig. 3a, b). Oogonia appeared partially embedded in the membrane or attached by a short stalk. In the late active phase, larger oocytes (Fig. 3c), measuring between 29 and 38 !lm, with a conspicuous basophilic nucleolus present in the nucleus became prevalent. The nuclei measured between 20 and 25 !lm. Gonads of ripe females (Fig. 3d, e) contained the largest number offollicles, with densely packed oocytes in their lumina. The mature oocytes measured between Figure 3. Rangia cuneata. Sections of gonad tissue of clams from Mobile Bay, Alabama. a and b) female in early active phase of oogenesis (x 63 and x 250); c) female in late active phase (x 250); d and e) ripe female (x 63 and x 250); f and g) partially spawned ovary (x 63 and x 250); h) spent female (x 63). 134 BULLETIN OF MARINE SCIENCE, VOL. 45, NO. I, 1989

34 and 44 /lm, with a nucleus averaging 25 /lm in diameter. The number of oocytes in the follicles decreased and appeared free in the lumina when females became partially spawned (Fig. 3f, g). Oocyte diameter remained the same, averaging 44 /lm. As spawning continued, follicles were no longer well-defined, and increased numbers of leucocytes or phagocytes were observed in the lumina. The lumina of the follicles of spent females were generally void of oocytes, or a few oocytes were still scattered over the surrounding tissue (Fig. 3h). The presence of phago- cytic cells was common in these follicles. Histology: Male Gonadal Development. -In the early active phase, darkly stained spermatogonia and spermatocytes appeared in the thickened walls of the follicles (Fig. 4a), corresponding to what appeared to be the first meiotic divisions (Cain, 1972). Some spermatocytes were observed scattered in the lumina. Further de- velopment in the late active stage (Fig. 4b, c) resulted in dense masses of sper- matocytes and sperm at ids filling most of the follicles, with a few spermatozoa appearing in their center. Follicles of ripe males contained dense masses of mature, strongly basophilic spermatozoa which, occasionally, were arranged in swirls (Fig. 4d, e). As spawning continued, the follicles were not as densely packed with spermatozoa as in the ripe phase (Fig. 4f). At times, earlier stages of spermato- genesis could still be detected on the periphery of the follicles. Males were termed spent when a few spermatozoa were observed in the nearly-empty follicles (Fig. 4g, h). An increased number of phagocytic cells was common, as in spent females. Seasonal Frequency in Developmental Stages. -Concurrent gametogenic devel- opment was observed throughout the cycle for both sexes. Approximately 80% of the clams in the early active phase were observed between February and April, with only a few in January and May (Fig. 5). Further development ofthe gametes in the late active phase occurred in May only; however, clams in three other stages of the cycle (e.g., early active, ripe and partially spawned) were also observed during the same period. From July through September, approximately 80% of the clams were ripe and the other 20% were in the partially spawned phase. More ripe clams (81%) were found in September 1984, as compared with September of the previous year (45%). The percent ripe clams decreased after September, and clams in the partially spawned stage predominated in October. Gonads were spent beginning in November and continued in this phase through January before tissues in the early active phase were observed again. The sex of the majority of clams in the spent phase could no longer be determined from December until February due to the absence of gametes in later stages of oogenesis and sper- matogenesis.

DISCUSSION A direct relationship exists between water temperature and the initiation of the reproductive cycle in bivalves (Brown, 1984). Gametogenesis in R. cuneata from Mobile Bay began in late winter-early spring, coinciding with an increase in water temperature from 11°C in February to 15°C in March (Fig. 1). From his study on the reproductive cycle of R. cuneata in the James River, Va., Cain (1975) reported that a temperature of 15°C appeared to be important in initiating gametogenesis in spring and summer. The first maturation of gametes in R. cuneata from Lake Pontchartrain, La., also began when water temperatures were rising (Fairbanks, 1963). Cain (1975) observed that gametogenesis began in early April and contin- ued throughout the summer months. In Mobile Bay, clams were observed in the late active stage of gametogenesis at the end of May, when water temperatures Figure 4. Rangia cuneata. Sections of male gonad tissue of clams from Mobile Bay, Alabama. a) male in early active phase of spermatogenesis (x 250); b and c) male in late active phase (x 63 and x 250); d and e) ripe male (x63 and x 250); f) partially spawned testis (x63); g and h) spent male with few spermatozoa retained (x 63 and x 250). 136 BULLETIN OF MARINE SCIENCE, VOL. 45, NO. I, 1989

100 - ~ r- r- r- r- r- r-

80 f-

-; 60 f- e G) I a.. 40 - t: 'r )

20 - i

I "" ~ '" I T§ 1~ 1983 SON 0 J F M A M J J A SON 0 TJ 1985 o Spent .Early Active EmJLate Active [EJRipe ~ Partially Spawned

Figure 5. Rangia cuneata. Stages in the reproductive cycle of clams from Mobile Bay, Alabama. The length of each shaded area represents the percentage frequency of clams in each stage of development for both sexes during each collection. Refer to text for description of stages. had increased to 28°C. Fairbanks (1963), on the other hand, observed a three- month spawning period of R. cuneata between March and May. During this same period, clams in the present study were still in the early and active stages of gametogenic development. Gonads of clams from Mobile Bay were ripe by the end of July through the months of September, when temperatures in the Bay ranged between 28° and 32°C; however, mature gametes were found from late May to October in the late active, ripe, and partially spawned phases (Figs. 3c, d, e, and 4b, d, f). The major period of spawning began sometime in August and continued through September, although some spawning could have also occurred during early summer. Although temperature is an important factor controlling gametogenesis, changes in salinity from 0 to 5 or from 10 to 150/00 were found to induce spawning in R. cuneata in the laboratory (Cain, 1973). In the present study, salinity measurements from the sampling site were too variable and infrequent to determine if spawning was indeed induced by these changes. By November, the gonads contained few, if any, mature gametes (Figs. 3h, 4g). This is in contrast to the works of Cain (1975) and Fairbanks (1963), both of whom found ripe clams through late November. These differences are probably due to the varied effect of environmental conditions prevailing in different geo- graphical locations (e.g., temperature, salinity, food availability) (Sastry, 1979). It is also possible that an increased rate of spawning occurred in Mobile Bay in September when maximum temperatures (32°C) were recorded, resulting in the appearance of the spent phase earlier than found by either Cain or Fairbanks. In the James River, a maximum of 29°C was measured in early August (Cain, 1975); JOVANOVICH AND MARION: GAMETOGENIC CYCLE OF RANGIA CUNEATA 137 however, the clams continued to spawn later in the fall when temperatures dropped between 20 and 10°e. Weight changes of clams collected during the 16-month period (Fig. 2) indicated a relationship to the gametogenic cycle. The whole weight, meat weight and gonadal weights paralleled each other and were maximal in August, during the ripe phase preceding major spawning. Ansell and Trevallion (1967) reported that the body weight of Tellina tenuis increased greatly as the gonads proliferated. Cain (1975) found that spawning patterns were related to clam total weight, and Fairbanks (1963) suggested that gonadal condition of R. cuneata was related to size, growth rate, and shell weight. In contrast, the lengths of the shells measured in this study remained constant year round, indicating that the observed tissue growth in this organism is closely associated with reproduction. Constancy in shell length during annual collections was also observed in R. cuneata in Maryland (Andersen and Bilger, 1976). According to Sastry (1979), the key factors that influence growth rates, and in turn reproduction, are temperature and food con- centration. Several factors are probably responsible for differences in the gametogenic cycle of marine invertebrates over a geographic range (Seed and Brown, 1977). Tem- perature has been implicated as the main factor. Physiologically different races, each adapted to different temperature regimes, have been shown to exist (Loo- sanoff and Nomejko, 1951). Latitudinal differences were observed in Macoma nasuta, which spawn year-round in milder southern portions of their range but have a unimodal gametogenic pattern in northern areas (Rae, 1978). Another factor that could be equally important, however, is food availability (GaItsoff, 1964; Bayne et aI., 1975). This parameter was not measured in the present study since the primary objective was to relate the gametogenic cycle of R. cuneata to changes in biochemical composition and to seasonal uptake and release of an- thracene (Jovanovich and Marion, 1987). Differences in initiation of gametogen- esis and in spawning time of approximately one month were observed between the present study and works conducted by Cain (1972) and Fairbanks (1963). The interactive effects of temperature, salinity and nutrients could account for this variation. In conclusion, seasonal gonadal changes in R. cuneata follow a pattern similar to that observed by Cain (1975) and Fairbanks (1963) in different geographic areas except that gametogenesis and the spent phase occur earlier in the cycle. Gonadal changes observed in this study were also typical of those found in other pelecypods (Sastry, 1979). Triggered by changes in environmental factors such as temperature, salinity, and food availability, gametogenesis proceeds through the summer, followed by spawning. After spawning, there is a recovery phase in which unspawned gametes are resorbed by autolytic processes and phagocytosis. Mitosis and gametogenic proliferation then begin the next spawning season in late winter.

ACKNOWLEDGMENTS

The authors are grateful to Ms. D. Davis for her assistance in the field and to Dr. L. Chiarappa for his helpful suggestions with the manuscript. This project was partially sponsored by the NOAA Office of Sea Grant under Grant #NA81 AA-D-00050 and by a fellowship from the Mississippi-Alabama Sea Grant Consortium to the senior author.

LITERATURE CITED

Andersen, A. M. and M. D. Bilger. 1976. Growth of marked Rangia clams in the Potomac River. Nat. Shellf. Assoc. Abstract 66: 120. 138 BULLETINOFMARINESCIENCE,VOL.45, NO.1, 1989

Ansell, A, D, and A. Trevallion, 1967. Studies on Tellina tenuis Da Costa. L Seasonal growth and biochemical cycle. J. Exp. Mar. BioI. Ecol. I: 220-235, Bayne, B. L., P. A. Gabbott and J. Widdows. 1975. Some effects of stress in the adult on the eggs and larvae of M. edu/is. L. Mar. BioI. Assoc. U,K. 55: 675-689. Brown, R. A. 1984. Geographical variations in the reproduction of the horse mussel, Modiolus modiolus (Mollusca: Bivalvia). Mar. BioI. Assoc. U.K. 64: 751-770. Cain, T. D. 1972. The reproductive cycle and larval tolerances of Rangia cuneata in the James River, Virginia. Ph.D. Dissertation, University of Virginia, Charlottesville, Va. xii + 121 pp. --. 1973. The combined effects of temperature and salinity on embryos and larvae of the clam Rangia cuneata. Mar. BioI. 21: 1-6. ---. 1975. Reproduction and recruitment of the brackish water clam Rangia cuneata in the James River, Virginia. Fish. Bull. 78: 412-430. Clark, G. (ed.). 1981. Staining. Williams and Wilkins, Baltimore/London. 512 pp. Fairbanks, L. D. 1963. Biodemographic studies of the clam Rangia cuneata, Tulane Stud. Zool. 10(1): 3-47. Fucik, K. W., H. W. Armstrong and J. M. Neff. 1977. The uptake of naphthalenes by the clam, Rangia cuneata, in the vicinity of an oil-separator platform in Trinity Bay, Texas. Pages 637- 640 in Proceedings of the 1977 oil spill conference (prevention, behavior, control, cleanup). American Petroleum Institute, Washington, D.C. Galtsoff, P. S. 1964. The American oyster Crassoslrea virginica Gmelin. U.S. Fish. Wild\. Servo Fish. Bull. 64. 480 pp. Hopkins, S. H., J. W, Anderson and K. Horvath. 1973. The brackish water clam Rangia cuneata as indicator of ecological effects of salinity changes in coastal waters. U,S. Army Engineer Water- ways Experiment Sta., Vicksburg, Mississippi, Cont. Rep. H-73-1. 250 pp. Jovanovich, M. C. and K. R. Marion. 1987. Seasonal variation in uptake and depuration of an- thracene by the brackish water clam Rangia cuneata. Mar. BioI. 95: 395-403. Loosanoff, V. L. and C. A. Nomejko. 1951. Existence of physiologically-different races of oysters Crassostrea virginica. BioI. Bull. Mar. BioI. Lab., Woods Hole 101: 151-156, Neff, 1. M. and 1. W. Anderson. 1981. Response of marine animals to petroleum and specific petroleum hydrocarbons, London: Applied Science Publishers Ltd. 177 pp. Odum, H. T. and B. J. Copeland. 1969. A functional classification of the coastal ecological systems. Pages 9-86 in H. T. Odum, B. J. Copeland and E. A. McMahan, eds. Coastal systems of the United States. Rep. Fed. Water Pollut. Control Admin., Washington, D.C. Pantin, C. F. A. 1969. Notes on microscopical technique for zoologists. Cambridge University Press. 65 pp. Pfitzenmeyer, H. T. 1962. Periods of spawning and setting of the soft-shelled clam, Mya arenaria, at Solomons, Maryland. Chesapeake Sci. 3(2): 114-120, ---. 1970. Project C. Benthos. Pages 26-38 in L. E. Cronin, ed. Gross and biological effects of overboard spoil disposal in upper Chesapeake Bay. Maryland Natural Resources Inst., Spec. Rep. Rae III, J. G. 1978. Reproduction in two sympatric species of Macoma (Bivalvia). BioI. Bull Mar. BioI. Lab., Woods Hole 155: 207-219. Ropes, J. W. and A. P. Stickney. 1962. Gametogenesis in Mya arenaria from New England. Nat. Shellf. Assoc. Abstract 51: 123. Sastry, A. N. 1979. Pelecypoda (excluding Ostreidae). Pages 113-292 in A. C. Giese, ed. Reproduction of marine invertebrates. Academic Press, New York. Schroeder, W. W. and L. Lysinger. 1979. Hydrography and circulation of Mobile Bay. Pages 75-94 in H. A. Loyacano and J. P. Smith, eds. Symposium on the natural resources of the Mobile estuary, Alabama. U.S. Army Corps of Engineers, Mobile District. Seed, R. and R. A. Brown. 1977. A comparison of the reproductive cycles of Modiolus modiolus (L.) Cerastoderma (=Cardium) edule (L.) and Mytilus edu/is (L.) in Stranford Lough. Oecologia 30: 173-188. Shaw, W. N. 1964. Seasonal gonadal changes in female soft-shell clams, Mya arenaria, in the Tred Avon River, Maryland. Nat. Shcllf. Assoc. 53: 121-132. Woodburn, K. D. 1962. Clams and oysters in Charlotte County and vicinity. Florida State Bd. Conser. Mar. Lab. FSBCML No.: 62-12, CS No.: 62-1, 29 pp.

DATEACCEPTED: April 28, 1988.

ADDRESSES:(K.R.M,) Biology Department, University of Alabama at Birmingham, Birmingham, Alabama 35294; (M. c.J.) Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706.