Reproductive cycle and energy content of Tawera gayi (Hupe 1854) (Bivalvia: Veneridae) at the southernmost limit of their distribution range.

ABSTRACT The reproductive cycle and the energy content of soft parts of Tawera gayi were studied monthly from August 2001 to July 2002 in Ushuaia Bay, Beagle Channel. The degree of sexual maturity was determined histologically. The sex ratio was significantly different of 1:1 (896 females, 796 males, [chi square]-test, P < 0.05). First sexual maturity occurred at 12 mm shell height in both sexes. In males the reproductive cycle was determined analyzing the monthly percentage of gonadal stages. High percentages (85%-100%) were mature (ripe stage) during a large part of the year (March to September) and in October the 100% of individuals were partially spawned with recovery. An image analyzer was used to determine quantitatively female reproductive cycle. The percentage of gonadal area covered by oocytes was lower in December. The mean number of mature oocytes (diameter >40 [micro]m) by gonadal area and the mean diameter of oocytes reached the lower values during October, November, and December, indicating a spawning peak. This more intensive spawning activity coincides with the phytoplankton bloom in spring. The energy content of soft parts was measured using a microbomb calorimeter in monthly samples during a year. The mean energy content in both sexes was significantly different reaching 20.95 kJ/g AFDW for females and 21.39 kJ/g AFDW for males. The relative condition index attained the lower values in August, September, and October. The monthly values of the energy content did not show significant differences in both sexes. Results of this study indicate that notwithstanding T. gayi lives in a markedly seasonal environment as Subantarctic waters of Beagle Channel shows a protracted gamete production with a principal spawning episode and small pulses of evacuation during the rest of the year.

KEY WORDS: reproductive cycle, energetics, clams, Tawera gayi, Beagle Channel

INTRODUCTION

Periodic changes in temperature and food availability could be reflected in seasonal variations of reproductive activities of marine bivalves. The energy required to carry out the gametogenic process could be supplied directly by the ingested food (Jayabal & Kalyani 1986), or by the reserves previously stored in the gonad or in other tissues (Sastry 1979, Gabbot 1983). The simultaneous study of the annual reproductive cycle and the variation of energy content could give information about the specific pattern of energetic storage and utilization.

Though the reproductive cycles of bivalve venerids have been well studied (Lozada & Bustos 1984, Borzone 1989, Rodriguez-Moscoso et al. 1992, Stead et al. 1997, Morriconi et al. 2002, Delgado & Perez Camacho 2005), little information is available about the variation in the energy contents of their tissues, which could reflect the spatial and temporal energy distribution within the organism (but see Beninger & Lucas 1984, Lomovasky et al. 2001).

The venerid Tawera gayi (Hupe, 1864) is a species with a wide geographical distribution from 33[degrees]S in the Pacific littoral zone (Soot-Ryen 1959, Dell 1964) and 36[degrees]S in the Atlantic littoral zone to 54[degrees]50'S in the Beagle Channel (Carcelles 1944, 1950). This is the southern limit of the T. gayi distribution where water temperatures never overcome 11[degrees]C (Schroeder, unpubl. data) therefore growth and reproduction could be subject to physiological restrictions related to the low temperatures of this area.

T. gayi is commercially exploited in the Chilean coast (see http://webmail.sernapesca.cl, Osorio et al. 1979, Urban & Tesch 1996), whereas there is occasional fishing along the Argentinean Patagonian coast.

Few studies have been carried out on this species; the reproductive cycle was analyzed only during a short period of fall season in the northern Argentinean Patagonia (Punta Loma, Golfo Nuevo, Chubut province 42[degrees]S, 47[degrees]W; Schuldt 1975). In this study a high percentage of ripe individuals was found in March with recovering in June. In the southern limit of it geographical distribution (Ushuaia Bay, 54[degrees]50'S) the population dynamics was studied, and a maximum age of 15 y with a mean abundance of 1,091 individual [m.sup.-2] was founded (Lomovasky et al. 2005).

To determinate if the gonad development in T.gayi depends on the energy directly acquired from the food, or the energy stored in the body, we analyzed the energy content variation and the reproductive cycle, size of first maturation, and the sex ratio in a population at the southernmost distributional limit.

MATERIALS AND METHODS

Study Site and Sampling Procedure

Monthly samples of the hard clam Tawera gayi were collected in Ushuaia Bay (Beagle Channel, 54[degrees]50'18"S, 68[degrees]16'25"W), by SCUBA diving at 3-5 m depth between August 2001 and July 2002. Each month four randomly selected cores of 30 x 30 cm surface area and 15 cm depth were dug out. Specimens were sorted from the unsieved content of the cores. For each individual (n = 1,692) the shell height (umbo to ventral axis, H), and length (anterior to posterior axis, L) were measured to the nearest 0.01 mm. The shells were removed and the soft parts of body were weighted ([+ or -] 0.01 g).

During the study period the monthly mean seawater temperature ranged between 8.2[degrees]C in summer and 3.4[degrees]C in winter (Fig. 1; Schroeder unpubl.).

[FIGURE 1 OMITTED]

Histological Determinations

Monthly subsamples of approximately 20 clams for each sex were randomly selected for histological analysis; the shells were removed and the soft parts (n = 180 females; 272 males) were fixed in Bouin fluid, water washed, and transferred to 70% alcohol. A cross-sectional block was dissected from each clam, dehydrated in an alcohol series, cleared in benzene, embedded in Paraplast, sectioned at 5 [micro]m, and stained with Groat hematoxylin and eosin.

The sex ratio was determined by examining gonadal smears or histological slides, and it was tested against a 1:1 ratio with Chi-square test (Zar 1999).

Qualitative Reproductive Analysis

Male Gonadal Stages

The following male gonadal stages in Tawera gayi were established using the maturity scale described by Morriconi et al. (2002) for the clam Eurhomalea exalbida:

Inactive

This stage was only observed in juveniles. The gonads contain small isolated alveoli, separated by a great quantity of interalveolar tissue. The alveoli contain isolated spermatozoa in the center and few spermatogonia are located near alveoli walls. Scarce spermatocytes into the alveoli are observed in some individuals (Fig. 2A).

[FIGURE 2 OMITTED]

Early Active

The gonads contain abundant alveoli with reduced interalveolar space.

Spermatogonia are located at the periphery of the alveoli forming a row, whereas a wide band of spermatocytes and spermatids is observed toward the alveoli center. Into the central alveoli lumen scant spermatozoa are arranged radially with the tails pointing toward the center of alveoli (Fig. 2B).

Late Active

The band of immature germinal cells is narrower than the previous stage. It is detached from the alveolar walls and there are vacuities among the cell mass. The number of spermatozoa increases and they occupy the center of alveoli (Fig. 2C).

Ripe

The large distended alveoli are touching each other. Spermatocytes and spermatids are restricted to a layer close to the alveoli wall and the central lumen is wide and full of spermatozoa. In some alveoli the spermatozoa are disorganized, and ready to be expelled to the ducts suggesting the existence of reduced sperm evacuation (Fig. 2D).

Partially Spawned With Recovery Stage

Partially empty alveoli with a decrease in the number of spermatozoa filling the lumen are observed. The presence of a band of spermatocytes and spermatids indicates an imminent gonadal recovery. The microscopic aspect of this stage is similar to the early active stage. The greatest differences lie in the somewhat disorganized spermatocytes and spermatids band and the irregular shape of alveoli because of their partial contraction (Fig. 2E).

Quantitative Reproductive Analysis

The proportion among sexual cell types in Tawera gayi females varies not so markedly along the year as to determine gonadal stages. For this reason a quantitative analysis of the histological preparations of female specimens was performed using images captured by a video camera (Panasonic KR222) and viewed using the Image-Pro Plus 3.0 software. Three microscopic fields (x100) per cross section were analyzed for each specimen. In each field the non-gonadal area was disregarded and the remaining area was expressed as the gonadal area in [micro][m.sup.2]. The mean number of oocytes (divided into three size classes: <25 [micro]m, 25-40 [micro]m, and >45 [micro]m minor axis) per 100 [micro][m.sup.2] gonadal area, the mean diameter (minor axis) of oocytes, and the percentage of gonadal area occupied by oocytes were determined. To evaluate the null hypothesis of no difference in these variables throughout the year we performed a one-way ANOVA with subsequent Tukey-Kramer Multiple Comparisons test when differences were found. The assumptions of normality and homogeneity of variances were tested using the Kolmogorov-Smirnov test and the Levene test, respectively (Zar 1999).

Size at First Maturity

Height at first maturity was based on 133 specimens between 7 and 18 mm H. Specimens were classified histologically as sexually active (with oocytes or sperm) and inactive (with scant and small alveoli containing few immature sexual cells). The median of the cumulative frequency distribution of sexually active specimens was considered to represent the height at which 50% of animals are sexually mature (Masello & Defeo 1986).

Relative Condition Index and Soft Mass Energy Content

Relative Condition Index and the energy contents of approximately 15 individuals per month (186 females; 179 males) with shell heights >18 mm (this size corresponds to adults specimens) were determined. After removing valves, the sex of each individual was established using gonad smears. The soft parts were identified and stored at -20[degrees]C until processing. The soft parts were dried at 80[degrees]C to constant weight.

The relationship between shell height and dry mass was represented by:

log (dry mass) = a + b x (log H), (1)

Monthly condition values were analyzing using

condition index (CI) = Dry mass/Shell height (b), (2)

where b is the slope in (1), and were normalized using

relative condition index (RCI) = (CI - mean CI)/SD of CI. (3)

The monthly differences were analyzed using an analysis of Variance (ANOVA).

The caloric contents were obtained by burning pellets (25-200 mg) in a Parr model 1425 microbomb calorimeter following Beukema and De Bruin (1977) and Lucas (1996). The values obtained were corrected for ash and acid contents and were expressed as kJ/g ash free dry weight (AFDW).

To evaluate the null hypothesis of no difference in the energy content between sex and across the year we performed a t-test and a Kruskall-Wallis test respectively, the last-one given the assumptions of normality and homogeneity of variances were rejected. When differences found a Dunn multiple comparison post-test was used (Zar 1999).

RESULTS

Tawera gayi is a gonochoristic species. The gonad is made up of interconnected gonadal alveoli, infiltrating the muscular tissue of the foot and surrounding the digestive gland and gut.

The alveoli are surrounded by connective tissue and contain different types of germinal cells. The sexes were identified histologically in individuals larger than 7 mm and 8 mm H in males and females respectively.

The sex ratio differed significantly of 1:1 (896 females, 796 males, [chi square] test, P < 0.05), the differences were only significant in January (51 females, 25 males) and April (171 females, 121 males).

Male Reproductive Cycle

A high percentage of male individuals (85% to 100%) were mature (ripe stage) during a large part of the year (March to September) (Fig. 3). In September only few individuals (13%) and all individuals in October were in partially spawned with recovery stage. In November and December the 100% of the male gonads resumed the maturation process because they were in the early active stage. During January and February the 100% and the 76% of the clams respectively were in late active stage.

[FIGURE 3 OMITTED]

Female Reproductive Cycle

During the annual reproductive cycle, female gonads contained a variable number of the different types of germinal cell simultaneously. Oogoniae (5-7 [micro]m) and small spherical oocytes ([less than or equal to] 25 [micro]m) had basophilic cytoplasms and nuclei with several nucleoli. Medium size pedunculated oocytes (25-40 [micro]m) and free spherical oocytes (40 65 gin) showed acidophilic cytoplasms. The latter were considered mature (Fig. 4A). In fresh smears mature oocytes were observed surrounded by a transparent jelly coat, they had a diameter of 100 [micro]m proximately.

[FIGURE 4 OMITTED]

The higher values of monthly mean number of mature oocytes (diameter >40 [micro]m) by gonadal area (Fig. 5) were reached during September, February, June, and July. In October, November, and December the values decreased significantly indicating spawning had occurred (one-way ANOVA, F = 16.28, P < 0.0001; Tukey-Kramer Multiple Comparisons test P < 0.05). The smaller (<25 [micro]m) and medium (25-40 [micro]m) size oocytes fell in number after the spawning, except in March, when the medium size oocytes increased. Histological images during these months showed the presence of a moderate number of ripe oocytes suggesting partial spawning (Fig. 4B).

[FIGURE 5 OMITTED]

The percentage of gonadal area covered by oocytes (Fig. 6) reached a maximum in September and a minimum in December. The values of the last month were different from August, September, February, March, April, June, and July (one-way ANOVA, F = 16.90, P < 0.0001; Tukey-Kramer Multiple Comparisons test, P < 0.05).

[FIGURE 6 OMITTED]

The mean diameter (minor axis) of oocytes (Fig. 7) reached the lower values during October, November, and December. They were different in August, September, and February to July (one-way ANOVA, F- 18.85, P < 0.0001; Tukey-Kramer Multiple Comparisons test, P < 0.05).

[FIGURE 7 OMITTED]

Height at First Maturity

In males and females the heights at first maturity (Fig. 8) were approximately 12 mm H, corresponding to individuals of one or two years old (Lomovasky et al. 2005). The smallest males and females with active gonads measure 10 and 11 mm of height respectively.

[FIGURE 8 OMITTED]

Relative Condition Index and Soft Mass Energy Content

The monthly relative condition index (RCI) in both sexes showed the lower values in August, September, and October (Fig. 9; females: ANOVA F = 9.85, P < 0.001; males: ANOVA F = 12.07, P < 0.001).

[FIGURE 9 OMITTED]

The mean energy content in both sexes was significantly different reaching 20.95 kJ/g AFDW for females and 21.39 kJ/g AFDW for males (Student t = 4.056, P < 0.0001). The monthly values of the energy content (Fig. 10) showed no significant differences in males (Kruskal Wallis H = 16.84; P = 0.1124). In females the monthly values of the energy content (Fig. 10) showed differences (Kruskal Wallis H = 34.45; P < 0.05). There were no differences among months (Dunn multiple comparison post test P > 0.05).

[FIGURE 10 OMITTED]

DISCUSSION

During the greater part of the year T. gayi showed a high percentage of ripe individuals and no variations of the energy contents, nevertheless there were large seasonal variations of the temperature (Fig. 1) and food availability (Hernando 2006) in the studied zone.

Both sexes perform a similar reproductive pattern: Males (Fig. 3) showed a higher percentage of evacuated individuals (partially spawned with recovery stage) in October (mid-spring), and a new maturation (early active stage) in late spring (November and December). Also, in this season (October, November, and December) females showed a decrease in the values of the percentage of gonadal area covered by oocytes, the mean number of mature oocytes by gonadal area, and the mean diameter of oocytes (Fig. 5, 6, 7). These results indicate that T. gayi undergoes the main spawning throughout this period; in coincidence a maximum recruitment peak was found during summer (Lomovasky et al. 2005).

Besides, a small quantity of recruits was detected in the Beagle Channel year round (Lomovasky et al. 2005); these findings are in agreement with a high percentage of male ripe gonads showing a reduced sperm evacuation throughout a greater part of the year. Coincidently, in mature female gonads, the largest oocytes never totally occupied the alveoli suggesting a gradual expulsion of oocytes as was described in Eurhomalea exalbida (Morriconi et al. 2002).

An extended period with ripe gonads and without sexual quiescence described in this study was also observed in other Patagonian venerid clams such as Eurhomalea exalbida from the Beagle Channel (Morriconi et al. 2002) and from Golfo Nuevo (Argentine, 42[degrees]47'08"S; Schuldt 1975), Venus antiqua from Ancud Bay (Chile, 41[degrees]51'S, Lozada & Bustos 1984) and Golfo Nuevo (Schuldt 1975, Verdinelli & Schuldt 1976, Borzone 1989); and clams from other high latitudes such as the Antarctic clam Laternula elliptica (Urban & Mercuri 1998, Bigatti et al. 2001) and the Baltic clams Astarte borealis, A. elliptica and Cyprina islandica (von Oertzen 1972).

The spawning in bivalves is controlled mainly by two factors: temperature and food availability (Giese & Pearse 1974). This appears to be what happened with T. gayi in the Beagle Channel. When the temperature (Fig. 1) and the phytoplankton concentration increase in late spring (Hernando 2006) the more intense spawning occurs. Thus larval development matched the spring algal bloom enhancing the prospects for larval survival (Sastry 1979). On the other hand the high percentage of ripe specimens during an extended period, expelling gametes in small pulses is corroborated by the presence of recruits (<12 mm H) year round (Lomovasky et al. 2005).

The populations of T. gayi (this study) and E. exalbida (Morriconi et al. 2002) studied in Beagle Channel showed the height of first maturity to be about half of their maximum height. In T. gayi the first maturity occurs around 12-mm height in both sexes, this size corresponds to one- or two-year-old individuals. The H[infinity] of this clam reaches 28.03 mm with a maximum age of 15 years (Lomovasky et al. 2005). In E. exalbida from the Beagle Channel, the first maturity was established at 39-40-mm height, corresponding to 4-y-old, with a H[infinity] : 74 mm. This species can reach 70 y old (Lomovasky et al. 2002). Unfortunately, in other veneroid species the first maturity size was determined using different methods (Ropes et al. 1984, Kanti et al. 1993, Sato 1995, Eversole 2001), making comparisons with this study very difficult.

The analysis of the relationship among the reproductive cycles, energy stored and utilization, allows understanding of the internal energy transfer process for a given species (Lucas 1996). The mean energy content (kJ/g AFDW) in T. gayi was higher in males than females. This result is unusual because in several species of molluscs higher values were found in females (Paine 1971, Dame 1976, Lucas et al. 1978, Heral & Deslous-Paoli 1983). The RCI showed lower values in spring. During this period sexual maturation ended and the spawning began, whereas the higher values are reached in winter when the food availability is reduced, (Hernando 2006) probably reflecting the increase in gonadal mass caused by sexual maturation. There were no differences among monthly values of energy content in any sex (Fig. 10). This pattern may indicate the nonexistence of a change in the biochemical composition of soft tissue in relation to reproductive output and reserve accumulation. However, it is difficult to analyze the monthly variations of RCI and the transfer of energy between different tissues and/or their capacity of reserve because of the small body size of this species and the impossibility to separate the gonad from the digestive organs.

In contrast, in the hard clam E. exalbida from Ushuaia Bay, it was possible to analyze the energy content of soft body in three compartments (foot-visceral mass, Gills-mantle-siphons, and adductor muscle) showing only an annual variation in the foot-visceral mass with the lower values in coincidence with the main spawning period (Lomovasky et al. 2001, Morriconi et al. 2002), which was attributed to a decrease in the lipid content related to gamete emission (Lomovasky et al. 2004).

In summary, our results show that notwithstanding T. gayi lives in a markedly seasonal environment such as the Subantarctic waters of Beagle Channel, they have ripe gonads during an extended period with a principal spawning episode and small pulses of evacuation during the rest of the year. Furthermore the body energy contents do not show temporal variation.

ACKNOWLEDGMENTS

The authors thank Daniel Aureliano and Adalberto Ferlito for field assistance, the first one collaborated in the histological and image analysis process. Dr. Oscar Iribarne provided valuable suggestions that largely improved the manuscript. This project was supported by Fundacion Antorchas (13817-4/2000) and PIP 02944 (CONICET).

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ELBA MORRICONI, (1,2) * BETINA J. LOMOVASKY (2,3) AND JORGE CALVO (1,2,4)

(1) Centro Austral de Investigaciones Cientificas (CADIC-CONICET), C.C. 92, V9410BFD Ushuaia, Tierra del Fuego, Argentina; (2) Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina; (3) Laboratorio de Ecologia, Dpto. Biologia, FCEyN, Universidad Nacional de Mar del Plata, Argentina; (4) Universidad Nacional de la Patagonia San Juan Bosco, Sede Ushuaia, Argentina

* Corresponding author. E-mail: [e-mail]