Factors influencing recruitment of hatchery reared pearl oyster (Pinctada Mazatlanica; Hanley 1856) spat.

ABSTRACT We evaluated experimental collection of Pinctada mazatlanica spat on substrates of different textures and colors placed at different depths within settlement tanks. When larvae reached the pediveliger stage (day 25), black-colored spat collectors ("envelope" type) composed of the following materials were offered as settlement substrates: onion bag, mosquito net, fishing net, and 63% shade-cloth. Spat collectors measured 30 x 30 cm and were composed of an outer bag and inner substrate made of the same material. The influence of the color of spat collector material on recruitment was investigated using a second type of spat collector composed of onion bags as the outer bag and inner substrate ("bag" type). Bags were prepared in 3 different outer/inner (O/I) color combinations of green/green, green/black, and red/black (O/I). Both types of collectors were deployed at different depths within settlement tanks. Approximately 2.7% of the initial larval population survived, resulting in 35,583 spat. Of these, 71.3% recruited to bag type collectors, 21.9% to envelope type collectors, and 6.8% to the surfaces of the culture tanks. Spat recruitment was significantly affected by collector material (P < 0.01), with fishing net and shade-cloth yielding highest and lowest density of spat, respectively. Similarly, spat collector color combination significantly influenced spat collection (P < 0.01), with higher recruitment to red and green substrates. For both spat collector types, there was significantly greater (P < 0.01) recruitment of spat to collectors in the middle of the water column (60-90-cm depth). This study showed that both the type and color of material used for spat collectors, as well as depth of deployment, influenced recruitment of P. mazatlanica spat. These results will help fine-tune current hatchery techniques for this species.

KEY WORDS: pearl oyster, Pinctada mazatlanica, spat, recruitment, hatchery culture

INTRODUCTION

Settlement and metamorphosis in marine bivalves is a complex process influenced by physical and chemical characteristics of available substrates and environmental factors such as water temperature, light intensity, and currents (e.g., Crisp 1974, Gray 1974, Hadfield 1978). Under hatchery culture conditions, several natural and artificial materials have been investigated as substrates for collection of bivalve spat, including monofilament gillnet, PVC, glass, plastic, nylon rope, onion bag, asbestos natural shrubs and leaves, and bivalve cultch (e.g., Pearce & Bourget 1996). In general, concave or convex substrates seem to be more attractive for larval settlement than flat or even substrates; similarly, poorly-illuminated areas are preferred to clear or illuminated surfaces (Ajana 1979, Phelger & Cary 1983, Alagarswami et al. 1983, Rose & Baker 1994, Taylor et al. 1998, Doroudi & Southgate 2002).

Despite the high commercial value of pearl oysters (Fassler 1991), there have been few studies assessing settlement substrates for hatchery production of the 3 main pearl producing species, Pinctada fucata (Alagarswami et al. 1983, Alagarswami et al. 1987), P. margaritifera (Alagarswami et al. 1989, Southgate & Beer 1997) and P. maxima (Minaur 1969, Rose & Baker 1994, Taylor et al. 1998). These studies have generally attempted to define physical and chemical stimuli for larval settlement to optimize hatchery production protocols. This goal has been achieved for P. fucata in India (Alagarswami et al. 1987, Dharmaraj et al. 1991, Victor et al. 1995) and more recently in Australia for P. maxima (Rose 1994) and P. margaritifera (Southgate & Beer 1997, Doroudi & Southgate 2003). In these countries, hatchery-reared spat are now used for commercial pearl production.

In Baja California, Mexico, over-exploitation and depletion of natural beds of pearl oysters prompted research into cultivation of the 2 native species, P. mazatlanica (Hanley) and Pteria sterna (Gould). Because cultivation based on wild spatfall has been successful (Monteforte 1996), research into hatchery culture of these species has been scarce and discontinuous (McAnally & Valenzuela 1990, Serrano & Salinas 1993, Araya et al. 1995, Martinez-Fernandez et al. 2003) and reliable methods for large-scale hatchery culture have not yet been developed. On this basis, many aspects of larval behavior during settlement, metamorphosis, and recruitment are still unknown for these species, particularly P. mazatlanica. In an effort to generate information that will allow more reliable hatchery culture of this species, this study evaluated the influence of spat collector material and color and their depth on recruitment of hatchery-reared P. mazatlanica spat.

MATERIALS AND METHODS

Larval Rearing

Gametes were obtained from ripe P. mazadanica in July (summer) and larvae were reared according to methods described by Robles et al. (2000). Larvae were stocked at an initial density of 2 m[L.sup.-1] in duplicate 5,000-L, white, conical fiberglass tanks containing 1 -[micro]m filtered, gently aerated seawater (~37 [per thousand]). They were fed a 1:1:1 mixture (by cell number) of Isochrysis galbana, Chaetoceros muelleri, and C. calcitrans at total densities of 30 x [10.sup.3] cell m[L.sup.-1] from day 1-10 and 60 x [10.sup.3] cell m[L.sup.-1] from day 11 to settlement. Culture tanks were drained, washed, and filled with clean seawater every 3 days and water temperature was maintained at 26 [+ or -] 1.5[degrees]C under a 12h:12h (light:dark) photoperiod.

When pediveliger larvae were seen on day 25, larvae were transferred to duplicate 5,000-L settlement tanks identical to larval culture tanks. Conditions within the settlement tanks were as described for larval culture tanks. Tank water was again replaced every 3 days and a microalgal diet of the same composition as that fed to larvae was provided at a density of 80 x [10.sup.3] cell m[L.sup.-1]. Settlement tanks contained spat collectors of the compositions outlined below.

Spat Collectors

Materials chosen to make spat collectors, as well as their color and design, were based on materials used successfully to collect wild spat of P. mazatlanica and P. sterna in Bahia de La Paz, Baja California Sur, Mexico (Monteforte & Wright 1994, Monteforte et al. 1995). To assess the effect of type of substrate on recruitment of spat, black-colored onion bag, mosquito net, fishing net, and 63% shade-cloth were tested as collector materials. Physical properties of each of these materials are shown in Table 1. Spat collectors measured 30 x 30 cm and were composed of an outer bag and inner substrate made of the same material. The weight of the inner substrate was 35 g, and this type of spat collector was called "envelope." For each material, four spat collectors were uniformly distributed along a 120-cm rope. Triplicate ropes were made for each material type to provide 12 spat collectors per material per tank. Ropes were hung from 30-120 cm below the surface of the water in settlement tanks to determine any bathymetric variation in the spat density.

The influence of color of spat collector material on recruitment was investigated using a second type of spat collector called "bag." These were composed of onion bags as the outer bag and inner substrate. Each "bag" was filled with 68 g of substrate. Bags were prepared in 3 different outer/inner (O/I) color combinations of green/green, green/black and red/black (O/I). Colors closely match those used in the Windows Standard Color Palette Classification (black = 000000, red = FF0000, and green or teal = 008080). For each color combination, two spat collectors were tied at 60-cm and 120-cm to a 120-cm rope, and triplicate ropes were used to provide six collectors per combination per tank.

Spat Management and Data Treatment

Spat collectors were deployed in settlement tanks for 1 mo and maintenance of settlement tanks followed the methods described by Pit & Southgate (2000). Spat were carefully recovered from spat collectors and counted to determine mean density per spat collector type at each depth. Given the l-mo period between deployment of spat collectors and assessment of spat numbers, the mobility of pearl oyster spat following settlement (Gervis & Sims 1992) and their ready ability to detach from settlement substrates (Taylor et al. 1997), spat numbers were reported as recruitment (not settlement) throughout this article. Differences in physical structure of spat collectors, whereby "bags" had volumetric arrangement and offered a much larger mass of substrate for spat collection than "envelopes," required that statistical matrixes for each treatment were run independently. Two-way ANOVA, followed by the Tukey test for posthoc mean comparisons, were applied to study the effect of substrate type and depth on mean density of spat (Sokal & Rohlf 1981). Another two-way ANOVA was performed to examine differences in spat density as a function of color combination and depth of spat collectors.

RESULTS

D-stage veliger larvae had a mean antero-posterior shell length and dorso-ventral shell height of 85 [micro]m and 66 [micro]m, respectively. Umbone veligers first appeared on day 13 and had mean shell length and shell height measurements of 142 [micro]m and 129 [micro]m, respectively. First "eyed" larvae (184 x 169 [micro]m) were seen on day 21 and pediveliger larvae (230 x 210 [micro]m) were seen on day 25. From an initial larval population of 1.3 x [10.sup.6] larvae, survival to the end of the experiment was low (2.7%) with a yield of 35,583 spat. Of these, 71.3% were associated with the "bag" type spat collectors, 21.9% with the "envelope" type spat collectors, and 6.8% were attached to tank surfaces.

Mean density of spat on each type of spat collector is shown in Figure 1. There were significant differences in density of spat in relation to substrate type (F = 27.9; P < 0.01), depth of collectors (F = 11.4; P < 0.01), and the interaction of factors (F = 15.7; P < 0.01). Spat were found at greater densities on spat collectors containing fishing net placed at 90-cm depth and significantly less on collectors containing shade-cloth immersed at 120 cm.

[FIGURE 1 OMITTED]

Spat collection was significantly influenced by the color of collectors (F = 84.6; P < 0.01), depth (F = 9.22; P < 0.01), and interaction of factors (F = 38.6; P < 0.01) (Fig. 2). On the basis of spat numbers, there were 3 significantly different groups showing higher (red/black at 60 cm), intermediate (green/black at 60 cm and red/black and green/black at 120 cm) and lower (green/green at 60 and 120 cm) recruitment.

[FIGURE 2 OMITTED]

DISCUSSION

In general, development of P. mazatlanica larvae to settlement was similar to that reported for other members of the family Pteriidae, such as P. fucata (Alagarswami et al. 1983), P. margaritifera (Alagarswami et al. 1989, Doroudi & Southgate 2003) and P. maxima (Rose & Baker 1994), and to that previously reported for P. mazatlanica (Martinez-Fernandez et al. 2003) (Table 2). However, the low survival recorded in this study, regardless of substrate type, color, or depth tested, indicates that some aspects of the husbandry conditions were inadequate and require improvement. Similarly, low survival has been reported in other studies with Pinctada larvae. For example, Alagarswami et al. (1989) reported that only 6.3% of hatchery cultured P. margaritifera larvae survived to become spat, whereas Southgate & Beer (1997) reported survival of approximately 4.3% to 5.7% for the same species during larval development.

Our results show that P. mazatlanica larvae recruited more heavily to spat collectors with particular textures, colors, and depths. Significant differences in the distribution of spat at the end of the experiment indicated preferential recruitment to fishing net as a substrate, to the red/black (outer/inner) spat collector color combination, and to tank depths from 60-90 cm below the water's surface. Fishing net had a rough fibrous texture composed of thick knotted cords (Table 1) and these characteristics were clearly more attractive for recruitment of spat when compared with shade-cloth, which recorded poor recruitment. Fishing net presumably provided better tactile stimuli to settling larvae or crawling spat or possibly favored stronger byssal attachment to either cords or knots. A similar hypothesis was offered by Taylor et al. (1998) to explain preferential settlement of P. maxima spat on filamentous polypropylene rope when compared with plain PVC and glass substrates. Interestingly in this study, mosquito net ranked second to fishing net in terms of spat density but is not a rough fibrous material (Table 1). This smooth fiberglass material is flat, with a 1-[mm.sup.2] pore size, and offered a relatively large area (per gram substrate) for attachment of spat. Although onion bags and shade-cloth are also flat substrates, it is likely that either the wide mesh size/small area for settlement of onion bags or the smooth vinyl surface of shade-cloth did not offer attractive tactile stimuli for P. mazatlanica spat. It is notable that shade-cloth has been widely used as a settlement medium for the closely related P. margaritifera (Southgate & Beer 1997, Pit & Southgate 2000, Doroudi & Southgate 2002).

Great contrast has been shown in larval settlement studies with bivalves when flat and 3-dimensional substrates and smooth versus rough surfaces have been compared (e.g., Phelger & Cary 1983, Pearce & Bourget 1996). This is well illustrated in prior studies with pearl oysters. Alagarswami et al. (1983), for example, reported that P. fucata showed a preference for materials with a smooth surface such as fiberglass. Similarly, P. sterna showed good settlement on plastic plates (Serrano & Salinas 1993). In contrast, recruitment of P. maxima spat has been shown to be more abundant on 3-dimensional materials such as monofilament gillnet (Rose & Baker 1994) or fibrous substrates like rope (Taylor et al. 1998). While the present study did not compare flat versus 3-dimensional substrates, our results indicate that P. mazatlanica shows a similar preference for a more textured 3-dimensional substrate.

Numerous studies have reported that bivalve larvae show negative phototrophic behavior when recruiting to substrates (e.g., Ritchie & Menzel 1969, Ajana 1979, Baker 1997, O'Foighil, et al. 1990). Similar behavior has been reported for pearl oysters (Alagarswami et al. 1983, Alagarswami et al. 1989, Monteforte & Wright 1994, Rose & Baker 1994, Monteforte et al. 1995, Taylor et al. 1998). On this basis, all collectors in the substrate experiment were offered only in black. When spat collectors were offered in various color combinations, however, each was composed only of onion bags. Given the same substrate in each type of "colored" spat collector, differences in substrate selectivity must be ascribed to spat collector color. The red/black (outer/inner) color combination supported significantly greater recruitment of spat than collectors with the green/green color combination. This is likely to result from the preference of spat for darker, less reflective substrates, which perhaps offer greater cryptic advantage (Ajana 1979, Baker 1997, Taylor et al. 1998).

P. mazatlanica also recruited in greater density to spat collectors in the middle of the water column. This behavior may also represent preference for lower light intensity and avoidance of brighter light at the water's surface and light reflected from the bottom of settlement tanks (white in color). However, our results may simply reflect hydrodynamic conditions within settlement tanks that favored recruitment to collectors in the midwater column. Pearce et al. (1998) and Taylor et al. (1998) similarly reported that spat of Placopecten magellanicus and P. maxima, respectively, preferentially recruited to spat collectors in the middle of the water column of settlement tanks.

This study provides valuable new information relating to hatchery culture of P. mazatlanica. The major findings are that: (1) recruitment of spat was influenced more by substrate texture and structure than by surface area offered; (2) spat recruited in greater numbers to collectors in the middle of the water column (60-90 cm depth); and (3) black-colored fishing and mosquito net supported greater recruitment as spat collector materials for P. mazatlanica and shade-cloth and clear or light-colored materials should be avoided. The low survival of spat at the end of the study, however, indicates that further research is required to determine optimal conditions for maintaining early P. mazatlanica spat in the hatchery.

TABLE 1.
Physical properties of the different materials used as
spat collectors to evaluate recruitment of Pinctada mazatlanica spat

                                 Materials

Properties          Fishing net                 Onion bag

Common use        Large-scale commercial      Agriculture
                    fishing activities in       application (e.g.
                    tuna/sardine fisheries      vegetable storage)

Composition       Polyester fibers            Polypropylene fibers

Surface texture   Rough and fibrous;          Moderately smooth
                    woven cords with
                    grooves

Mesh/pore size    2 [cm.sup.2]                3-5 [mm.sup.2]

Area for          59.3 [cm.sup.3]             27.5 [cm.sup.2]
attachment          [g.sup.-1] substrate        [g.sup.-1] substrate
                    (cords) 60.5
                    [cm.sup.3] [g.sup.-1]
                    substrate (knots)

Structure         3-D net formed by 4-mm      Flat and flexible
                    diam. knotted cords

Example           [ILLUSTRATION OMITTED]      [ILLUSTRATION OMITTED]

                                   Materials

Properties          Mosquito net                Shade-cloth

Common use        Window, door, cradle        Aquaculture (e.g. fishing
                    screening protection        cages, pond protection)
                    against insects             and industry usage
                                                (shades, canopies)

Composition       Vinyl-coated fiberglass     Extruded high-density
                                                polyethylene

Surface texture   Moderately smooth           Smooth and slippery

Mesh/pore size    1 [mm.sup.2]                0.5-[mm.sup.2] offering
                                                63% shade

Area for          88.5 [cm.sup.2]             103 [cm.sup.2]
attachment          [g.sup.-1] substrate        [g.sup.-1] substrate

Structure         Flat and flexible           Flat and semi-rigid

Example           [ILLUSTRATION OMITTED]      [ILLUSTRATION OMITTED]

TABLE 2.
Time series of larval developmental stages for different Pinctada
species reared under hatchery conditions.

                                       Larval development

                          Set     SL x SH      Met      SL x SH
Species                  (day)   ([micro]m)   (day)    ([micro]m)

Pinctada fucata           22     250 x 240      24     330 x 300
                          20     220 x 200      24        300
Pinctada margaritifera    23     260 x 240      28     350 x 300
                          20     214 x 217      NS         NS
                         -22        230         NS         NS
Pinctada maxima           24     268 x 222      28     349 x 289
Pinctada mazatlanica      24     223 x 206      NS         NS
                          25     230 x 210      30     268 x 240

Species                  Surv (%)   Reference

Pinctada fucata             NS      Alagarswami et al. (1983)
                           4.1      Alagarswami et al. (1987);
                                      Dharmaraj et al. (1991),
                                      Victor et al. (1995)
Pinctada margaritifera     6.3      Alagarswami et al. (1980)
                         1.9-9.6    Southgate and Beer (1997)
                            NS      Doroudi and Southgate (2003)
Pinctada maxima             23      Rose and Baker (1994)
Pinctada mazatlanica       3.1      Martinez-Fernandez et al. (2003)
                         2.3-3.0    Present study

Set, settlement of plantigrade larvae; SL x SH = shell length x shell
height; Met, metamorphosis into earl spat; Surv, percentage larval
survival and spat production at the end of the study; NS, not
specified.

ACKNOWLEDGMENTS

The authors thank the following staff of CIBNOR: M. Robles, G. Gurrola, J. J. Ramirez, and I. Fogel for assistance with this study; also H. Acosta-Salmon (James Cook University, Australia) and anonymous reviewers for valuable comments and suggestions. Additional support was provided by International Foundation for Science (IFS-Sweden), the Comision Nacional de la Biodiversidad of Mexico (CONABIO), and the Australian Academy of Science. This work was supported as part of an institutional project of CIBNOR.

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PEDRO E. SAUCEDO, (1,) * HORACIO BERVERA-LEON, (1) MARIO MONTEFORTE, (1) PAUL C. SOUTHGATE (2) AND PABLO MONSALVO-SPENCER (1)

(1) Centro de Investigaciones Biologicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo Santa Rita, La Paz, Baja California Sur 23090, Mexico and (2) pearl Oyster Research Group, School of Marine Biology & Aquaculture, James Cook University, Townsville, Queensland 4811, Australia

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