A simple transport procedure for juvenile calico scallops, Argopecten gibbus (Linnaeus, 1758).

ABSTRACT A simple procedure for transporting live calico scallop, Argopecten gibbus (Linnaeus, 1758), juveniles for grow-out culture is described. The effects of 4 simulated transport periods (9, 16, 20 and 24 h), and subsequent recovery were examined. Success was assessed by survival rate and growth during 2-wk recovery. Survival rate was of 100% for all tested transport periods immediately following transport and following 2-wk recovery. Shell growth averaged 1.76-2.31 mm over the 2-wk period, yielding a growth rate 0.88-1.2 mm.[week.sup.-1]; comparable to average growth rate at same site. There was no significant difference in growth among transport exposure time. Monitored levels of heat shock protein 70 (HSP 70) during manipulation did not indicate any physiologic stress. Successful application of this procedure to a real life 14 h airway transport of another subtropical/tropical scallop species, demonstrates its use for transport of two scallop species juveniles ranging in size from 8-40 mm, without detriment to subsequent survival or growth.

KEY WORDS: Argopecten gibbus, calico scallop, heat shock protein, transport, recovery

INTRODUCTION

Scallops produced in hatcheries or collected from natural settlements normally require transportation to other sites for continued culture. Scallops are particularly difficult to transport because they are usually sublittoral organisms, unlike oysters or mussels, and seldom encounter aerial exposure; they have been shown to display progressive respiratory acidosis during emersion (Duncan et al. 1994, Maguire et al. 1999). In part for this reason, stress associated with transport often causes mortalities. Several methods of transport have been attempted; two of the most common are the use of insulated boxes with material soaked in seawater, referred to as dry transport, (Ventilla 1982, Maguire et al. 1999, Maeda-Martinez et al. 2000), and vivier lorry, where animals are transported in seawater with a continuous oxygen flow (Maguire et al. 1999). Although some success is obtained, survival reported in the literature does not exceed 61% immediately following 19 h of simulated dry transport (Maeda-Martinez et al. 2000), and increased mortality (up to 51%) is often seen in subsequent months during grow-out (Maguire et al. 1999). As for transporting in seawater, high densities cause a rapid deterioration in quality of holding seawater by reducing dissolved oxygen and increasing ammonia and bacterial levels; leading to gill tissue damage (Maguire et al. 1999).

This article describes a simple procedure for transport of juvenile scallops. This procedure is based on ornamental fish transport system, where fish are sealed in polyethylene bags filled with pretreated water over-saturated with pure oxygen and transported in insulated boxes (Lim et al. 2003). The procedure described here gives consideration to conditioning of scallops prior to simulated shipment, through temperature and starvation; and to loading density (g scallops. [L.sup.-1] of transport water) during transport. Four simulated transport time periods were tested; success was assessed by survival immediately following transport and by survival rate and growth during a subsequent grow-out period. To identify any stress-induced phase during this procedure, levels of heat shock protein 70 (HSP 70), used as biomarkers for environmental stress exposure in a range of organisms were also monitored (Feder 1999, Feder & Hofman 1999, Lewis et al. 1999).

MATERIALS & METHODS

Transport Procedure

In July 2003, 3-too-old calico scallop, Argopecten gibbus (Linnaeus, 1758), spat, originating from a hatchery spawn, were pooled from the nursery system at Bermuda Biologic Station for Research, reared at 26 [+ or -] 1[degrees]C. A subsample of 50 spat was collected from the pool and shell height was measured using vernier calipers. To lower the metabolic rate, and thus reduce oxygen consumption and accumulation of acidity, carbon dioxide and ammonia during transport, scallops were subjected to a cold water acclimation period (14[degrees]C for 6 h). The acclimation temperature, which is also the packaging temperature, selected is one that calico scallops are subjected to during winter months in Bermuda, and can thus tolerate (pets. obs.). An acclimation period of 4-6 h is also used for fish (Lim et al. 2003). During the acclimation period, scallops were not fed to reduce the amount of excreta during transport. Following acclimation, spat were weighed and 160 g subsamples were distributed into 1-L double zippered plastic bags filled with 600 mL of 1-[micro]m filtered cold seawater (14[degrees]C), This equated to 260 g.[L.sup.-1] or approximately 53 juvenile scallops.[L.sup.-1], a high loading density compared with other studies on pectinids (9.2 juvenile scallops. [L.sup.-1]) as reported by Maguire et al. (1999) and on ornamental fish (22-272 g.[L.sup.-1], Lim et al. 2003). Loading density and required volume of transport water for scallops were assessed during preliminary trials. Double ziplock bags were used to avoid any potential tearing or opening, especially critical during transport situations. Oxygen was bubbled into each bag using a Pasteur pipette, until the bag was full. It was assumed that supplied oxygen would be sufficient so as to not be a limiting factor. The bag was further secured with duct tape to avoid leakage. A polystyrene box (60 cm x 30 cm x 40 cm) was cooled to 14[degrees]C using four gel packs, and a false polystyrene bottom drilled with holes, was placed on top of the gel packs to avoid direct contact of scallops with the gel packs.

Initial Survival

Assessment of initial spat survival and subsequent survival and growth was conducted after each of four transport periods: 9, 16, 20 and 24 h. Care was taken to avoid any fluctuations in temperature during sampling. A visual assessment of scallop behavior was done at this time, and bag temperature recorded. On sampling, scallops were transferred into a cold water bath (14[degrees]C), and seawater temperature was gradually increased to 26[degrees] [+ or -] [degrees]C over a 12 h period. After a 3-day acclimation to ambient temperature, spat were transferred to the field in 3-mm Japanese pearl nets. Initial shell height of spat was of 8.9 [+ or -] 1.7 mm, as measured with vernier calipers ([+ or -] 0.1 mm). Spat were maintained on longlines for a period of 2 wk, after which growth and long-term survival were assessed.

Recovery--Long Term Survival and Shell Growth

A subsample of 50 spat was taken from each treatment for shell height measurements after 2-wk grow-out period. Survival was assessed by counting the number of dead spat seen in each treatment. Statistical differences in shell growth rate after the recovery period for each transport period was evaluated by Model 1 ANOVA (STATVIEW program, P = 0.05).

HSP 70 Analysis

Aliquots of spat for heat shock protein expression analysis were taken prior to acclimation, post acclimation, after each mock transport time period (9, 16, 20 and 24 h), and 2 wk after transfer in the field (long term survival, recovery). For each time point, five aliquots of 3 g wet weight were taken, placed in plastic vials on ice, and transferred to a -70[degrees]C freezer, until analysis.

For HSP 70 analyses of each sample, ~1.0 g of scallops (shells included) was homogenized in 8 mL of 65[degrees]C sample buffer (125 mM Tris pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS)). Samples were boiled for 3 min and spun at low speed for 10 min. The supernatant was removed and spun a second time for 10 min at low speed. The resulting supernatant was then diluted with sample buffer at a 1:2 ratio (sample to buffer) for total protein and HSP 70 analyses. Total protein concentrations of diluted samples were determined using the Lowry-based BioRad DC protein assay (BioRad Laboratories, Hercules, California) with a bovine serum albumin standard.

Samples of equal total protein content (17 [micro]g) were prepared with the addition of the reducing agent, dithiothreitol. Samples were boiled for 3 min prior to separation by SDS-polyacrylamide gel electrophoresis (8% resolving gel). Gels were stained with SYPRO Tangerine protein gel stain (Molecular Probes, Eugene, Oregon) and scanned for protein load analyses. Proteins were then transferred to nitrocellulose (Hybond, Amersham Pharmacia Biotech), analyzed by immunoblotting with monoclonal antiHSP 70 clone BRM-22 antibodies (Sigma, St. Louis, Missouri), and visualized by chemiluminescence (Lumiglo, Cell Signaling Technology, Beverly, Massachusetts) using Kodak X-Omat film.

Band intensity on all gels and blots were quantified by densitometric analysis. Two sets of analyses were conducted. The first compared HSP 70 expression among preacclimated, acclimated and mock transport (9, 16, 20 and 24 h) samples. The second compared mock transport (9, 16, 20 and 24 h) samples with recovery samples for each individual transport time. Five replicates of each sample type for each set of analyses were run. Relative intensity measurements of protein bands on gels were used to correct for uneven protein loads caused by loading error. A ratio of intensity measurements for the first set of analyses were based on preacclimation sample load. Those for the recovery analyses were based on load for each individual mock transport time. Converted ratios of relative band intensities were compared across mock transport times and acclimation samples using a Model I ANOVA and between mock transport and recovery samples using a Student t-test with each time point analyzed independently (Zar 1984). A value of 0.05 was used for all analyses (Zar 1984).

RESULTS

Transport and Initial Survival

During the mock transport experiment, temperature remained constant within all the transport bags at 14[degrees]C for 20 h; in the last 4 h, temperature in the bag and in the box increased to 16[degrees]C. At each sampling time, scallops showed normal behavior by swimming and clapping when disturbed; no mortality was recorded at the time of sampling following each transport period, resulting in a survival rate of 100% immediately following all simulated transport periods.

Recovery--Long Term Survival and Shell Growth

Survival remained at 100% following acclimation of scallops exposed to all simulated transport periods. Further handling of spat to pearl nets, and transfer to the field on longlines for a 2-wk test recovery period did not incur any mortality. Shell height following 2 wk in the field ranged from 10.68 [+ or -] 1.99 mm to 11.23 [+ or -] 1.76 mm (Fig. 1). There was no significant difference in growth among transport exposure time (P < 0.05). Shell growth averaged 1.76-2.31 mm over the 2-wk period, yielding a growth rate 0.88-1.2 mm.[week.sup.-1]. This growth rate is within the range measured at this site on previous occasions (1-2.3 mm.[week.sup.-1], Sarkis unpublished).

HSP 70 Analysis

There was constitutive expression of HSP 70 in all samples analyzed (Fig. 2a and 2b). However, there were no significant differences in expression among preacclimated, acclimated and mock transport times (9, 16, 20 and 24 h) samples (P > 0.05). There were also no significant differences in HSP 70 expressions between mock transport and recovery at each individual transport time (P > 0.05).

DISCUSSION

The transport procedure yielded maximal survival (100%) for a mock transport of up to 24 h, in accordance with results obtained with fish using similar transport procedure (Lim et al. 2003). More promisingly, results of the current study demonstrate continued high survival throughout the recovery period, when scallops were manipulated for transfer into pearl nets, and to the field for a 2-wk grow-out period. Comparable postshipment shell growth seen in tested scallops implies that animals experimented upon were healthy and that transport conditions has negligible effect on their physiology. It is probable that conditioning of scallops prior to packaging, by food depravation and acclimation to low temperature, enabled scallops to tolerate any potential stressful conditions during transport and enhance subsequent survival, as was shown in guppies (Lira et al. 2000). In addition, gradual acclimation over a 24-h period to ambient temperature following transport may have further contributed to enhanced survival of scallops when transferred to the field (Fig. 1).

[FIGURE 1 OMITTED]

The described procedure was developed to optimize scallop survival to transport in the short-term and the long-term; for this reason, care was taken to minimize stress by maintaining conditions as close as possible to their tolerance range, namely to their natural temperature range. Results of the HSP analyses further suggest that scallops experienced little stress during acclimation and mock-transport and confirmed their tolerance to transport conditions.

Additional physiologic analyses would provide further insight into the stress experienced during transport processes. Although, as initially assumed the oxygen supply provided in the transport system described in this article, was sufficient for the biomass transported, monitoring of oxygen tension throughout the process, coupled with respiration rate analysis, would be useful in optimizing the biomass of scallops within such a transport system. The determination of oxygen-to-scallop ratio, expressed in volume of oxygen (mL) to the total weight of (g), is accepted as a good measure of the actual amount of oxygen used in relation to the animal biomass (Lim et al. 2003). In this way, the procedure may be modified accordingly for a range of scallop species.

It is worthwhile adding that this simple transport procedure has also proven successful for 14 h transport of another subtropical/ tropical scallop species, Euvola (Pecten) ziczac). Juvenile E. ziczac of 40 mm shell height, were transported from Bermuda to the Turks & Caicos Islands via airway on two occasions. In the first trial, some mortality ensued because of the lack of proper sealing of zippered bags, and exposure of scallops to air. Despite this, 10-day recovery survival rate for this first trial was of 90%. During the second trial, when transport bags were secured, 10-day recovery survival rate was of 100%.

In conclusion, this procedure has proven successful for juveniles of two scallop species ranging in size from 8-40 mm. With further insight into some of the physiologic processes undergone during transport, this procedure should become easily adaptable to various scallop species.

ACKNOWLEDGMENTS

This study would not have been possible without the dedication of the Bermuda Biological Station hatchery team, and without the generous support of the private and corporate donors encouraging scallop culture in Bermuda. The majority of the funds was obtained from the Centennial Trust, Bank of Bermuda, and the Christian Humann Foundation.

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SAMIA SARKIS, (1) * ANNE BOETTCHER, (2) NOBUO UEDA (2) AND CLAUDIA HOHN (3) (1) Department of Conservation Services, Flatt's, Bermuda; (2) University of South Alabama, Department of Biological Sciences, Mobile, Alabama; (3) Bermuda Biological Station for Research Inc., Ferry Reach, Bermuda 

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