Brooding pattern and larval production in wild stocks of the puelche oyster, Ostrea puelchana d'orbigny.

ABSTRACT Brooding pattern and fertility of the puelche oyster (Ostrea puelchana) were investigated in a native population from the San Matias Gulf (40[degrees]48'S; 65[degrees]05'W, Northern Patagonia, Argentina). Monthly samples of 100 oysters were randomly collected at the oyster ground during the period November 1999 to January 2000, and weekly in the 2000 to 2001 reproductive season. Complementary data were obtained from the literature (1976 to 1978) and samplings performed during the periods 1980 to 1984 and 1987 and 1998. The date at which oysters begin larval brooding may be placed somewhere within the period November 18 to January 11. In most seasons (77%), brooding starts within the period November 18 to December 5. The number of brooders is low at the debut (7.5% in 1999, 4% in 2000), peaking at the end of spring (20%), and gradually decreasing until reaching nearly 1% by middle February. Total number of larvae brooded on each sampling date showed a high correlation with brooding percentages ([r.sup.2] = 0.85). Mean fertility showed no correlation with the percentage of oysters brooding larvae ([r.sup.2] = 0.12). The highest value of mean fertility (2.7 million) was recorded at the beginning of the season when 4% of the oysters were brooding larvae. Mean fertility ranged from 900,000 to 2,700,000 larvae. Brood size ranges from 447,500 to 3,790,000 larvae (mean = 1,868,212; s = 813,808; n = 121). Individual fertility showed no correlation with neither size (total height) nor with internal volume of oysters within a size range of 60-115 mm. O. puelchana shows the highest fertility recorded in Ostrea species, a feature that seems consistent with its short incubation period, small egg size, long planktonic life, and small size of pediveligers.

KEY WORDS: oysters, Ostrea puelchana, brooding, larval production

INTRODUCTION

The puelche oyster, Ostrea puelchana, is a commercially valuable flat oyster species native to temperate waters of the San Matias Gulf (Northern Patagonia, Argentina). As with all other Ostrea species, O. puelchana broods the larvae and releases them as veligers, in this particular case after 6-7 days of incubation and at an initial size of 110-130 [micro]m (Pascual & Zampatti 1995).

This species has received a great amount of research effort clue to the biologic interest in its peculiar reproductive feature of "carriage" of epibiotic dwarf males (Calvo & Morriconi 1978, Fernandez Castro & Lucas 1987, Pascual et al. 1989, Pascual 1997, Pascual 2000). The commercial interest in its culture (Pascual & Bocca 1988, Pascual & Zampatti 1995) generated the oyster culture project that has been developed in Northern Patagonia since 1980.

Hatchery seed of this species was produced for the first time in France in 1988 (Pascual et al. 1991 ICES, Pascual & Zampatti 1995). Routine seed production in Argentina has been performed since 1997 in the state hatchery of Rio Negro Province.

From 1997 to 2003 research efforts were focused on reproductive studies and the application of the acquired knowledge on the improvement of broodstock conditioning to increase larval production.

As is the case with other flat oyster species (Perez Camacho 1987), hatchery seed production of the Puelche oyster has been constrained by the relatively low amount of larvae released by each individual oyster (fertility). Thus a large number of animals for broodstock conditioning in the hatchery is required. In consequence a detailed knowledge of brooding behavior in natural conditions is necessary to design a correct management plan for seed production.

In this study we present results gathered for several years, particularly in 2000 and 2001, regarding the annual pattern of brooding and larval production in Puelche oysters from one of the main natural oyster grounds of Northern Patagonia.

MATERIAL & METHODS

Condition Index of the Parental Stock

Monthly samples of 40 oysters of sizes ranging from 65-95 mm (total height) were randomly collected by scuba diving at the oyster ground of Las Grutas (40[degrees]48'S; 65[degrees]05'W; Fig. 1) during the period June 1997 to September 1998. Seawater temperature was recorded on each collection date.

[FIGURE 1 OMITTED]

Total height and length was recorded for each oyster. Internal volume was estimated in the following way: each live oyster was hang tied by a thread and sunk into a beaker filled with seawater and placed on a balance. Total live weight and shells weight were registered by this method, and internal volume (IV) was calculated as the difference between both weights. Condition index was calculated as: CI = IV/DMW x 100. Dry meat weight (DMW) was recorded a posteriori.

Brooding Pattern

A hundred oysters of size ranging from 60-100 mm (total height) were haphazardly collected on each sampling date from the population of Las Grutas (Fig. 1). Sampling was performed monthly during the 1999 to 2000 season (November, December, and January). In the 2000 to 2001 season, sampling frequency was increased and the collections were performed weekly (November to February) to assure an accurate coverage of brooding dynamics (brooding period lasts 5-7 days in this species, Pascual & Zampatti 1995).

Shell height and live weight were recorded before the oysters were opened. For brooding oysters, larvae were removed by gently washing the oysters with a spray bottle over a 1 L graduated beaker filled with seawater. Total number of larvae per oyster (fertility) was estimated by counting larvae--under a stereomicroscope--on 6 samples (100 [micro]L) collected with an Epphendorf pipette from the previously homogenized larval solution.

Internal volume of each sampled oyster was estimated using the method described earlier.

Complementary data on incubation dates and fertility were obtained from the literature (1976 to 1978, Morriconi & Calvo 1980) and from samplings performed during the periods 1980 to 1984 (Parma & Pascual, unpubl, data), 1987 (Zampatti, unpubl, data), and 1998 (Castanos, unpubl.).

RESULTS

Reproductive Behavior of Puelche Oysters in the Natural Ground

Condition Index of Parental Stock

Seawater temperature and condition index (CI) are inversely correlated throughout the year (Fig. 2). Minimal temperatures are recorded in July to August (6[degrees]C to 8[degrees]C), and maximal temperatures are recorded in December to January (19[degrees]C to 20[degrees]C). CI starts decreasing in December and reaches its minimal value in February (7.88). During autumn and winter CI gradually increases until reaching its maximal values during the period August to November (14.5-14.7).

[FIGURE 2 OMITTED]

Brooding Pattern

The beginning of larval brooding in natural oyster populations of the San Matias Gulf, registered over 13 y, occurs within the period November 18 to January 11 (Fig. 3). In most seasons (77%), brooding starts within the period November 18 to December 5. Even when there were some gaps in seawater temperature records, the available data suggest that brooding has a threshold temperature of 16[degrees]C, even when interannual fluctuations occur within a range of 16[degrees]C to 20[degrees]C.

[FIGURE 3 OMITTED]

Sampling design carried out in 1999 and, the more detailed one carried out in the 2000 to 2001 season, enabled the study of brooding behavior at two different seasons.

In 1999, brooding started early in the season, on November 18, with a low percentage of brooders in the population (7.5%). The three samplings carried out in 1999 showed a peak in the number of brooders in the population and in the total larval production by early December (12/5: 12% of brooders and 31.9 millions larvae). This value decreased at the beginning of the summer (January 5: 12 million larvae and 6% of brooding).

In 2000 brooding was delayed compared with the previous season, starting on December 4 with only a 4% of the oysters brooding larvae (Fig. 4). The percentage of brooders steadily increased afterwards, peaking on December 21 (20%). One week later, on December 28, the percentage of brooders abruptly fell to 6.7%, increasing again afterwards to 15%. Brooding numbers gradually decreased from January 12 until reaching a minimum of 1% by middle February (Fig. 4a).

[FIGURE 4 OMITTED]

Mean fertility (mean number of larvae incubated per oyster), all dates considered, ranged from 900,000 to 2,700,000 larvae (Fig. 4a). Mean fertility showed no correlation with the percentage of oysters brooding larvae at each date ([r.sup.2] = 0.12). In 2000, however, the highest values of mean fertility (2.7 and 2.5 million larvae) were recorded at the beginning of the season (December 4 and 12) when 4% and 13% of the oysters were brooding larvae (Fig. 4a).

Total number of larvae brooded (total larval production) on each sampling date along the season showed a high correlation with brooding percentages ([r.sup.2] = 0.85) being the highest value found on December 21 (Fig. 4b).

Individual fertility estimates show that Puelche oysters may brood from 447,500 to 3,790,000 larvae (mean = 1,868,212; std = 813,808; n = 121). Individual fertility did not show any correlation neither with size (total height) nor with internal volume of oysters within a size range of 60-115 mm.

DISCUSSION

Phylogenetic relations among brooding oysters have been the matter of controversial discussions. Harry (1985) published a polemical paper presenting a morphologically based reclassification of flat oysters. Perhaps the most controversial point was the case of the Southern Hemisphere species. Harry (1985) synonymized several species as Ostrea puelchana: O. chilensis Philippi (Chile and New Zealand), O. angassi Sowerby (Australia), and O. algoensis Sowerby (South Africa). Jozefowicz and O'Foighil (1998) performed a comprehensive molecular phylogenetic analysis on ostreinid taxa. Their main goal was to determine if flat oysters occurring between latitudes 35[degrees]S and 50[degrees]S constituted a single circumglobal species or distinct regional taxa. Among other very conclusive remarks, the authors point out that the molecular data together with other distinctions undermine Harry' s (1985) assumption that O. puelchana has a circumglobal distribution beneath 35[degrees]S. They recommend retaining the original names for Southern Hemisphere taxa: O. puelchana, O. angasi, O. chilensis, and O. algoensis.

Jozefowicz and O'Foighil (1998) also pointed out that if it were true, the fact that genomic mutation rates are proportional to the relative frequency of germline cell divisions (Shimmin et al. 1993, Liu et al. 1996), the low fecundity of Ostrea species could explain the low genetic divergence levels found among them, compared with those of highly fecund cupped oysters from America and Asia. This same conclusion was reached earlier by Buroker (1985) who, additionally compared several Ostrea species and suggested that Tiostrea chilensis Philippi, the species that best characterizes the "Ostrea type;" has lower genetic variation than other Ostrea like O. permollis or O. lurida Carpenter, which tend to be more closely aligned to nonbrooding species.

In this regard, the fertility of O. puelchana is strikingly high compared with the available information recorded for other Ostrea species (Table 1), its mean value doubling the highest value recorded for Ostreas corresponding to the European oyster; Ostrea edulis Linnaeus releases 9.57 x [10.sup.5] larvae (Cole 1941, Walne 1964).

There seems to be a gradient in several life history traits such as incubation period, egg size, duration of planktonic life, and size of pediveligers. In this gradient O. puelchana can be placed in one extreme with the highest fertility recorded, a feature that seems consistent with its short incubation period, small egg size, long planktonic life, and small size of pediveligers. O. chilensis may be placed in the opposite extreme of the gradient regarding these same traits (Table 1).

Mean fertility in Puelche oysters decreases from its initial and highest value (2.7 million larvae per oyster) recorded in the end of spring to its lowest value (1.1 million larvae) registered at the end of the reproductive season.

Detailed research on the reproductive biology of the Puelche oyster (Castanos, unpublished) gives support to this behavior because the majority of oysters spawning as females at the debut of the season show a much more intense gonad development than those spawning as females late in the season, following a first and intense male spawning.

This data match those of Cerruti (1941) who estimated that late spawners of O. edulis spawned one-third to one-sixth the number of eggs produced by those oysters spawning early in the season. The same behavior and explanation was offered by Cole (1941) and Korringa (1952) for O. edulis, even when Walne (1964) studying the same species could not find evidence of this behavior.

Cranfield and Allen (1977) for O. lutaria Hutton, from New Zealand found that even when fertility varied widely in the different sampling periods; there was no consistent seasonal trend in this variation as was previously suggested by Hollis (1963).

Jeffs et al. (1996) studied the brooding behavior of Tiostrea chilensis Philippi in Northern New Zealand and suggested that in this year-round brooder, larger broods seem to be associated with increasing levels of brooding activity and lower water temperatures. Despite year-round brooding, the proportion of the population brooding during peak periods remained comparable to the highest proportions reported for populations elsewhere with shorter brooding seasons.

Hopkins (1936) suggested that, for O. lurida Carpenter (Northeast Pacific), the higher number of brooders appear within a period of about 6 wk at the beginning of the spawning season, though occasionally gravid individuals may be found during the following 5 or 6 mo. A secondary wave of spawning may occur later in the season.

Cole (1941) found a positive correlation between parent size and brood size in O. edulis.

This same relationship was found by Hopkins (1937) in O. lurida. However, oysters of the same length show a great variation in brood size suggesting that length is probably not a good criterion and that perhaps volume of the meat or fatness, on which reproductive capacity probably depends, may vary considerably and could better explain the relation.

In the case of O. edulis, an oyster almost doubles its capacity (fertility) each year, until the fourth year of life when fertility stabilizes in its definitive value (Cole 1941). In several incubating oysters, a relation can be found between size, weight, meat volume, and brood size (Walne 1964, Jeffs et al. 1997a, Jeffs et al. 1997b, Cranfield & Allen 1977).

Morriconi and Calvo (1980) suggested that in O. puelchana fertility (specially if it is measured as number of embryos per brooder) increases with size and dry meat weight. Our data could not confirm this result suggesting that fertility increases neither with size nor with the internal volume of the oyster. A more detailed study should be performed to make this point clear taking special care on the fact that we are looking at a sequential hermaphrodite that alternates sexual phases and shows differences in the intensity of female gonad development depending not only on size but also on the time of the season this sexual phase becomes functional. This same observation was made by Cole (1941) who suggested that the relation between fertility and size might be obscured by the time in the season.

The compiled experience suggests that conclusions can only be driven once a deep knowledge is acquired on the reproduction of each particular species.

Chaparro et al. (1993) describes the behavior of O. chilensis larvae within the mother's brooding chamber. They also discussed the energy costs of brooding suggesting that, because larvae actually feed during their long incubation period (8 wk), the female may increase its clearance rate so as to compensate for the retention of particles by the larvae. This cost, summed to the cost in generating the currents that transport the larvae through the pallial cavity should be possibly measured. Our own unpublished observations, however, suggest that brooding Puelche oysters decrease their filtration rate by reducing the degree to which they open their valves, perhaps a behavior meant to prevent larvae loss.

In fact, brooding oysters show extremely low filtration rates compared with nonbrooding oysters (Castanos, unpublished). This preliminary observation suggests that at least in the case of O. puelchana, brooding probably does not imply an extra energetic cost. This may be consistent with the short brooding period in this species (5-7 days, Pascual & Zampatti 1995) during which the small larvae may grow fueled only by egg reserves. This fact could be backed by the behavior of the condition index, which decreases abruptly from November to December, coincidently with male and female spawning.

Jeffs et al. (1996) conclude from their study on the annual cycle of brooding and fertility in T. chilensis that the shortage of spat in hatcheries is related to the brooding dynamics of this species. There have been continuous difficulties in developing a hatchery technique for conditioning and synchronizing larval production in Tiostrea broodstock. They recommend water temperature manipulation as the most fruitful area for research aimed to improve larvae hatchery production.

The Puelche oyster seems to be an exception in this regard due to its high fertility that enables an easy hatchery manipulation and a high larval production based on a relatively small broodstock. The biologic knowledge acquired, related to the reproductive behavior of this species, has been the key for a successful seed production program of this species.

TABLE 1.
Life history traits recorded for several Ostrea species.

                                                           Maximal
                                                           No. of
                                          Brooding         Brooders
Species            Locality               Pattern            (%)

Ostrea edulis      England                May to Sept.     13-20.6
                   Ireland

Ostrea chilensis   Chile                  Nov. to Jan.     12-48

=0. lutaria        Northern New Zealand   Year-round        18.2

=T. chilensis      Central and Southern   Aug. to March    2.6-5.6
                     New Zealand

Ostrea lurida      Washington St.,        May to Aug.        55
                     USA/British
                     Columbia, Canada,
                     California, USA

Ostrea permollis   Florida, USA

Ostrea puelchana   San Matias Gulf        Nov. to Feb.       20
                   (Argentina)

Ostrea equestris   Georgia, USA           Apr. to Oct.       3.1

Ostrea spreta      San Matias Gulf        Dec. to Feb.      13.3
                     (Argentina)

Ostrea aupouria    Northern New Zealand   Dec. to May

Ostrea angasi      Tasmania, Australia    Nov. to Feb.     16 (in 2 mo)

                                    Days of
                    Days of       Planktonic                Egg Size
Species            Incubation        Life                  ([micro]m)

Ostrea edulis        7-10             10                       150

Ostrea chilensis    21-56         5 min-48 h                 220-323

=0. lutaria                                                  264-323

=T. chilensis       25-30         Some minutes                 280

Ostrea lurida         10            7(24[degrees]C)          100-110
                                 10-11(20-22[degrees]C)
                                 10-23(19-22[degrees]C)
Ostrea permollis     7-9            30-33                       70

Ostrea puelchana     5-7            17-20                     60-90

Ostrea equestris

Ostrea spreta                       22-30

Ostrea aupouria                     10-15

Ostrea angasi                       12-20

                     Size of        Larval
                     Released       Size at      Fertility
                     Larvae        Settlement    (Larvae
Species             ([micro]m)     ([micro]m)     x 105)

Ostrea edulis        180-190          300         4.1-9.57

Ostrea chilensis     390-541         470-556      0.5-0.6

=0. lutaria          448-556                      0.5-0.6

=T. chilensis        394-541                        0.5

Ostrea lurida        165-189         250-325      2.15-3

Ostrea permollis     108-127          290           2.21

Ostrea puelchana     110-130          284           19

Ostrea equestris

Ostrea spreta          123            320

Ostrea aupouria      125-140         270-320

Ostrea angasi        186-203         300-320       0.3-15.2

Species             Author(s)

Ostrea edulis       Orton 1927, Cole 1941
                    Korringa 1952, Walne
                      1964, Wilson & Simons
                      1985, Millican & Helm
                      1994

Ostrea chilensis    Walne 1963, Solis 1967
                    DiSalvo et al. 1983, Winter
                      et al. 1983, Toro &
                      Chaparro 1990, Chaparro
                      et al. 1993

=0. lutaria         Jeffs et al. 1996, 1997a,
                      1997b

=T. chilensis       Hollis 1963, Cranfield &
                      Allen 1977, Cranfield &
                      Michael 1989

Ostrea lurida       Hori 1933
                    Hopkins 1936, 1937
                    Loosanoff & Davis 1963
                    Coe 1932

Ostrea permollis    Forbes 1966

Ostrea puelchana    Calvo & Morriconi, 1980,
                      Pascual & Zampatti 1995
                    This work

Ostrea equestris    Gutsell 1926, Menzel 1955
                    Walker & Power 2001

Ostrea spreta       Zampatti (unpubl.)

Ostrea aupouria     Dinamani 1981

Ostrea angasi       Sumner 1972, Dix 1976

ACKNOWLEDGMENTS

The authors thank the International Foundation for Science (Grant A-704 to MP) and the Agencia Nacional de Promocion Cientifica y Tecnologica (PICT98#4221) for financial support.

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C. CASTANOS,* M. S. PASCUAL, I. AGULLEIRO, E. ZAMPATTI AND M. ELVIRA

Laboratorio y Criadero de Moluscos, Instituto de Biologia Marina y Pesquera Alte. Storni. Guemes 1030, 8520 San Antonio Oeste, Rio Negro, Argentina

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