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Improvement of Water Chemistry with Bacillus Probiotics Inclusion during Simulated Transport of Yellowfin Tuna Yolk Sac Larvae

Ian C. Zinka; Daniel D. Benettia; Philippe A. Douilletb; Daniel Marguliesc; Vernon P. Scholeyd a Aquaculture Division, Marine Affairs Department, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida, USA b EcoMicrobials LLC, Miami, Florida 33137, USA c Inter-American Tropical Tuna Commission, La Jolla, California, USA d Achotines Laboratory, InterAmerican Tropical Tuna Commission, Las Tablas, Provincia Los Santos, Republic of Panana First published on: 02 February 2011 To cite this Article Zink, Ian C. , Benetti, Daniel D. , Douillet, Philippe A. , Margulies, Daniel and Scholey, Vernon P.(2011)

'Improvement of Water Chemistry with Bacillus Probiotics Inclusion during Simulated Transport of Yellowfin Tuna Yolk Sac Larvae', North American Journal of Aquaculture, 73: 1, 42 48, First published on: 02 February 2011 (iFirst) To link to this Article: DOI: 10.1080/15222055.2011.544622 URL: http://dx.doi.org/10.1080/15222055.2011.544622

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North American Journal of Aquaculture 73:4248, 2011 C American Fisheries Society 2011 ISSN: 1522-2055 print / 1548-8454 online DOI: 10.1080/15222055.2011.544622

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Improvement of Water Chemistry with Bacillus Probiotics Inclusion during Simulated Transport of Yellown Tuna Yolk Sac Larvae
Ian C. Zink* and Daniel D. Benetti
Aquaculture Division, Marine Affairs Department, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, Florida 33145, USA

Philippe A. Douillet
EcoMicrobials LLC, 2000 Bayshore Drive, Suite 205, Miami, Florida 33137, USA

Daniel Margulies
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Inter-American Tropical Tuna Commission, 8604 La Jolla Shores Drive, La Jolla, California 92037, USA

Vernon P. Scholey
Inter-American Tropical Tuna Commission, Achotines Laboratory, Las Tablas, Provincia Los Santos, Republic of Panana

Abstract
The effects of adding a probiotic Bacillus spp. blend on shipping bag water quality and survival of yolk sac larvae of yellown tuna Thunnus albacares during a 24-h mock shipment were investigated. To better detect effects on water quality, the trial was designed without the utilization of available chemical water quality or temperature modulators. Shipping water salinity (30.731.0 ) and temperature (24.026.7 C) reected conditions utilized during larval rearing. Probiotic incorporation (15 mL/L, about 1.5 106 colony-forming units/mL) resulted in signicantly lower nal concentrations of total ammonia nitrogen and un-ionized ammonia in comparison with the control. Signicantly higher nal mean dissolved oxygen concentration observed in the probiotic treatment could have resulted from stress reduction. Although no statistical difference was detected in larval survival upon termination of the trial, improvements in water quality (reduced total ammonia nitrogen and increased dissolved oxygen) resulting from incorporation of Bacillus probiotics would yield added levels of safety during shipping and would reduce the chances of negative results while incurring minimal increases in shipping costs.

Due to high market demand and value, aquaculture of tuna species has been a subject of interest for the past few decades. Recent criticism of current ranching practices have intensied
*Corresponding author: izink@rsmas.miami.edu Received January 28, 2010; accepted May 1, 2010

research efforts to consistently produce juveniles for growout operations. Japanese researchers have been studying tuna spawning and larviculture since the late 1970s (Kaji et al. 1996; Margulies et al. 2007; Masuma et al. 2008). Recent advances, such as completion of the life cycle of Pacic bluen tuna Thunnus orientalis under aquaculture conditions (Sawada et al. 2005), have provided further inspiration. Similarly, considerable interest in spawning, larval production, and larval growth of yellown tuna T. albacares has grown, with broodstock facilities in Bali, Japan, and Panama contributing to the knowledge of these processes (Margulies et al. 2005). Shipments of larvae from one of these facilities to another laboratory for experimental larviculture trials may be desirable, and thus successful techniques for closed-system shipments of yellown tuna yolk-sac larvae need to be developed. Concerns during transport of live shes include maintenance of high water quality and minimization of stress. Stress and degrading water quality can act synergistically to cause mortality either during or after shipment (Harmon 2009). Water quality issues focus upon the maintenance of high dissolved oxygen (DO) concentrations while reducing toxic ammonia accumulation and buffering water from pH shifts. As bacterial probiotic technology develops, continued realization of its

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applicability to increasing numbers of aquaculture-related activities will occur. While protocols and readily available chemical treatments address water quality issues, bacterial probiotics utilization could improve protocols and provide further safeguards. However, studies investigating probiotic utilization during shipping have been sparse. Furthermore, as transport conditions continue to increase biomass loads in order to maximize protability (Estudillo and Duray 2003; Benetti et al. 2007; Colburn et al. 2008), increasing safeguards to better ensure transport success are warranted. Bacterial probiotics have been demonstrated to yield multiple benets for targeted hosts, including innate immune system stimulation, stress reduction, control of bacterial communities, water quality improvement, and reduction of opportunistic and potentially pathogenic bacteria (Gatesoupe 1999; Skjermo and Vadstein 1999; Verschuere et al. 2000; Vine et al. 2006). Some authors narrowly dene probiotics to include live microbial adjuncts that are administered orally and that colonize the gastrointestinal tract (Gatesoupe 1999; Vine et al. 2006), while others broaden the denition to include external colonization (Skjermo and Vadstien 1999), water quality remediators that improve the hosts environment (Verschuere et al. 2000), or both. The present study considers the denition of probiotics to incorporate these broader contexts. One study has investigated the efcacy of nitrifying bacteria during transport of shes (Turner and Bower 1982). Studies incorporating the more recently developed probiotic blends, which potentially provide more benets than simply ammonia removal, are lacking. Bacillus spp. are capable of direct ammonia uptake for use as a nitrogen source during amino acid synthesis (Stadtman and Ginsburg 1974; Donohue and Bernlohr 1981). Indeed, studies report that when utilized as probiotic inoculates, Bacillus spp. are able to reduce ammonia concentrations within open-container shipping protocols (Gomes et al. 2008, 2009) and recirculating aquaculture systems (Chen and Chen 2001; Lalloo et al. 2007). Bacillus spp. probiotic additions could exhibit other potential benecial effects on yolk sac larvae during shipment by reducing stress during transport (Gomes et al. 2008, 2009), stimulating the innate immune response (Rengpipat et al. 2000; Guillan et al. 2004; Ziaei-Nejad et al. 2006; Aly et al. 2008), reducing pathogenic bacterial loads (Moriarty 1998; Vaseeharan and Rasmasamy 2003; Guillan et al. 2004; Lalloo et al. 2007; Hill et al. 2009; Nakayama et al. 2009), and reducing mortality after hosts are challenged with pathogens (Rengpipat et al. 1998, 2003; Vaseeharan and Rasmasamy 2003; Aly et al. 2008; Far et al. 2009). This trial was designed without the utilization of available chemical water quality or temperature modulators to better detect water quality effects and to verify manufacturers claims of water quality improvements when incorporating a commercially available Bacillus spp. probiotic blend during shipment. The trial emulated shipping conditions that would arise if a shipment lasted longer than anticipated and chemical water quality modulators became compromised.

METHODS A mock transport trial was conducted at the Inter-American Tropical Tuna Commission (IATTC) Achotines Laboratory (Achotines Bay, Los Santos Province, Republic of Panama) utilizing yellown tuna yolk sac larvae. The larvae were hatched from eggs produced by the natural spawning of broodstock held at ambient-temperature conditions at the same facility. Fertilized eggs were collected during late-evening hours, and incubation was conducted in 300-L, conical berglass tanks held at ambient temperature (about 26 C) as described by Margulies et al. (2005). Incubation systems were of a ow-through design with a 200% water exchange daily; incoming seawater (mean salinity SE = 31.0 0.1 ) was previously ltered to 1 m and sterilized with ultraviolet (UV) light. Yolk sac larvae were assumed to be healthy; this assumption was based on observation rather than quantication of a high hatching rate. Similarly, in an unrelated larval rearing trial utilizing larvae from the same spawn, early survival to rst feeding was high; although not constituting a true negative control, these observations support the assumption that yolk sac larvae were healthy. On 1 d posthatch, yolk sac larvae were concentrated within incubation tanks and volumetric subsampling was utilized to determine larval concentration. Transport packing was initiated by adding 3 L of ltered (1 m), UV-sterilized seawater at ambient temperature to each experimental polyethylene shipping bag. Larvae were then transferred to each of six shipping bags via 3-L beakers; after larval additions were complete, ltered seawater at ambient temperature was added to attain a nal volume of 20 L/shipping bag. For the probiotic treatment, 300 mL of EcoAqua (108 colony-forming units/mL in a mix of Bacillus subtilis, B. licheniformis, B. megaterium, and B. laterosporous; EcoMicrobials LLC, Miami, Florida) were added to each of three shipping bags. This volume was assumed to produce an inoculate of 1.5 106 colony-forming units/mL. It was assumed that the high concentration of inoculate would more rapidly achieve a climax bacterial community during the short duration of the trial. The three remaining shipping bags served as the control (i.e., no probiotic addition). Final shipping density of yolk sac larvae was 871 larvae/L. Oxygen was added to shipping water until DO saturation was above 300%; temperature, salinity, DO saturation, and pH were then recorded. Care was taken to eliminate cross-contamination while determining water quality variables by obtaining measurements rst from control replicates and then from probiotic treatment replicates. Water quality probes were rinsed between each replicate. Water samples for initial ammonia chemistry were then collected. Air within the shipping bags was expelled and replaced with pure gaseous oxygen. Bags were then secured, placed within an additional shipping bag that was also secured, and packaged individually within styrofoam coolers, which were then packed within cardboard boxes. During the trial, transport boxes remained within an airconditioned room held at 24 C. Periodically, boxes were lightly shaken for 10 s to simulate physical disturbance during shipping. The trial was terminated 24 h after completion of packing.

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TABLE 1. Mean ( SD) temperature, salinity, and dissolved oxygen (DO) in shipping bags and survival of yellown tuna yolk sac larvae during a mock shipping trial. Within a column, values sharing the same letter are signicantly different (p < 0.05).

Period Initial Final

Treatment Control Probiotic Control Probiotic

Temperature ( C) 26.7 0.1 z 26.6 0.2 y 24.5 0.1 zx 24.0 0.1 yx

Salinity ( 31.0 0 31.0 0 30.3 1.1 30.7 0.6

DO (mg/L) 20.3 0.1 z 20.4 0.2 y 16.0 0.7 zx 21.9 1.2 yx

pH 7.93 0.00 z 7.93 0.01 y 7.48 0.35 zx 7.34 0.95 yx

Survival (%)

87.9 0.05 88.4 0.07

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Upon opening of the shipping bags, temperature, salinity, DO saturation, and pH were recorded, and water samples for nal ammonia chemistry analysis were collected. When water quality sampling was complete, volumetric subsamples of shipping water containing larvae were collected to assess survival. Total ammonia nitrogen (TAN) concentration of individual water samples was obtained via the salicylate method (Hach Company, Loveland, Colorado). Water quality and survival data were statistically analyzed with Stata release 10 (Stata Corp., College Station, Texas). Comparisons were made between treatments at similar periods as well as between time periods within treatments. All data were rst analyzed for normality (ShapiroWilk W ) and similarity of variance (F -test). Distributional analysis revealed multiple instances in which parametric assumptions were violated; thus, further analysis of means utilized Wilcoxon rank-sum tests to simplify statistical analysis (Zar 2009). Signicance level was set to 0.05 for all statistical testing. A variable describing the increase in TAN was calculated to standardize for differing initial TAN concentrations. Un-ionized ammonia (NH3 ) concentration and percent un-ionized ammonia (%NH3 ) were computed from TAN concentrations utilizing the equations described by Bell et al. (2007, 2008). The DO saturation (%) was converted to DO concentration (mg/L) via the equations reported by Benson and Krause (1984). All other percentage data were arcsine transformed before further analysis (Zar 2009).

RESULTS Survival was high (>80%) and statistically similar (P > 0.05) between control and probiotic treatments (Table 1). No statistical differences were found for initial temperature, salinity, DO concentration, or pH between treatments (P > 0.05; Table 1). Final water chemistry values within the closed shipping bags differed between the control and the probiotic treatment. Although the control (P = 0.0369) and probiotic treatment (P = 0.0463) experienced signicant decreases in pH by the end of the 24-h experimental period, nal pH was significantly higher in the control than in the probiotic treatment (P = 0.0495; Table 1). Final temperature was signicantly higher in control shipping bags (P = 0.0495) than in probiotic treatment bags; however, within both the probiotic treatment (P = 0.0495) and the control (P = 0.0495), ini-

tial and nal mean temperatures were signicantly different (Table 1). A signicant difference (P = 0.0495) was detected between treatments for nal DO concentration, with mean DO concentration being much higher in the probiotic treatment bags than in the control bags (Table 1). As would be expected, control nal mean DO concentration was signicantly lower than the initial concentration (P = 0.0495). Interestingly and converse to the trend observed in control bags, the nal mean DO concentration in the probiotic treatment was signicantly greater (P = 0.0495) than the initial DO concentration (Table 1). Treatment and control shipping bags experienced nonsignicant decreases in salinity between the initial and nal samples. Although not statistically different, the mean nal salinity in control bags was lower than the mean in probiotic treatment bags. No signicant difference was detected in initial mean TAN between the control and probiotic treatment shipping bags, although the mean initial NH3 concentration was signicantly higher for the control than for the probiotic treatment (P = 0.0463; Table 2). To eliminate potential bias due to differing initial TAN concentrations, the nal TAN was standardized by computing the increase in TAN between initial and nal samples. The increase in TAN for the control was signicantly greater (P = 0.0431) than that for the probiotic treatment (Table 2). Similarly, nal TAN and nal NH3 concentrations were both signicantly higher in control shipping bags (P = 0.0463) than in probiotic treatment bags (P = 0.0495; Table 2). Furthermore, mean TAN concentrations did not signicantly differ (P > 0.05) between initial and nal conditions for the probiotic treatment; however, TAN concentrations signicantly increased (P = 0.0339) in the control bags. Neither treatment exhibited a signicant difference between initial and nal NH3 concentrations (P > 0.05; Table 2). Final %NH3 was signicantly higher (P = 0.0495) for the control than for the probiotic treatment. Initial %NH3 was signicantly greater than nal %NH3 for the control (P = 0.0369), but the same trend was not observed for the probiotic treatment (P > 0.05; Table 2). DISCUSSION Upon termination of the trial after 24 h, no statistical difference was detected in larval survival between treatments, and overall survival was high for all replicates. Lack of substantial mortality despite degrading water quality conditions was

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TABLE 2. Mean ( SD) total ammonia nitrogen (TAN), increase in TAN at nal sample relative to initial sample, un-ionized ammonia (NH3 ), and percent NH3 (%NH3 ) in shipping bags during mock transport of yellown tuna larvae. Within a column, values sharing the same letter are signicantly different (p < 0.05).

Period Initial Final

Treatment Control Probiotic Control Probiotic

TAN (mg/L) 0.3 0.1 z 0.1 0.1 0.7 0.1 zy 0.2 0.0 y

Increase in TAN (mg/L)

NH3 (mg/L) 0.009 0.003 z 0.002 0.004 z 0.007 0.0007 y 0.001 0.0003 y

%NH3 3.1 0.1 z 1.0 1.8a 1.0 0.1 zy 0.7 0.1 y

0.4 0.1 z 0.1 0.1 z

a This value is affected by the lack of detection of TAN in two of the three probiotic replicates; these zero values reduced the mean %NH3 . For the one replicate that initially contained NH3 , the initial %NH3 was 3.1%, the same as the mean for the control.

surprising, especially for the control shipping bags. Consideration of interactions between the various water chemistry factors explains this intriguing outcome. A signicant difference in nal DO concentration was observed between treatments. Similarly, Gomes et al. (2008) reported a higher DO concentration after transport of adult freshwater shes treated with Bacillus spp. probiotics. In the present study, the difference in DO concentration may have been associated with stress reduction for probiotic-treated larvae, which could have reduced their oxygen consumption. The increases in DO concentration in probiotic treatments between the initial and nal measurements could have resulted from human error. However, the substantial DO reduction in control bags and the marginal DO increases in probiotic treatment bags were consistent across all replicates within each treatment, suggesting that these were actual trends. Gomes et al. (2008, 2009) noted reduced stress (as measured by decreases in cortisol levels and reduced ion ux) with incorporation of Bacillus spp. probiotics during transport. In our study, nal mean salinity was lower than initial salinity in treatment and control shipping bags, with nal salinity for the probiotic treatment remaining more similar to initial values; this is an intriguing result given the closed-system nature of the trial. Stress response and the ability to maintain hypo-osmoregulation may have been improved in probiotic-treated replicates, although these factors were not investigated in the present study. In the extensively studied zebrash Danio rerio, the hypothalamicpituitaryinterrenal (HPI) axis and related cortisol production are developed at hatching (Alsop and Vijayan 2009) and are reactive to stressors such as seawater exposure at 1 d posthatch (Alderman and Bernier 2009) and handling stress at 2 d posthatch (Alsop and Vijayan 2008). Similarly, cortisol production and an active HPI axis have been demonstrated in Asian seabass Lates calcarifer at 4 h posthatch (SampathKumar et al. 1995), and exposure to water-soluble crude oil fractions increased cortisol expression in yolk sac larvae of turbot Scophthalmus maximus at 2 d posthatch (Stephens et al. 1997). Cortisol plays important roles in osmoregulation via control of Na+,K+-ATPase activity, ionocyte size and density, and drinking rate (Varsamos et al. 2005) and is necessary for hypo-osmoregulation by larval summer ounder Paralichthys

dentatus in seawater (Veillette et al. 2007). Thus, previously suggested stress remediation and improved osmoregulatory capabilities at this early developmental stage are plausible. The observation of signicantly different nal temperatures between treatments seems implausible because the shipping boxes were stored together in a temperature-controlled room. Possible explanations include human error during measurement or location of the shipping boxes in relation to air conditioning vents. Signicant differences between initial and nal mean temperatures were anticipated as ambient water temperature (about 27 C) lowered to match storage room air temperature (24 C). Reductions in temperature are commonly found in real shipping situations, where ice packs are utilized to cool shipping water temperature to the lower end of the transported organisms viable temperature range. A signicant difference in nal pH was detected between treatments, with the trend for lower pH occurring in probiotictreated shipment bags (Table 1). This difference probably resulted from the direct uptake of ammonia by probiotic bacteria, resulting in differing H+ concentrations. In control shipping bags, increased TAN concentrations could have led to the increased uptake of free H+ by NH3 , thus yielding the observed higher pH. Bacillus spp. have long been recognized to utilize multiple nitrogen sources, including both NH3 and NH4 +, for catabolism of proteins (Stadtman and Ginsburg 1974; Donohue and Bernlohr 1981). Without buffer utilization, signicant differences between initial and nal pH were anticipated. This is readily explained by the metabolic production of CO2 and subsequent carbonic acid (H2 CO3 ) formation from reaction with water molecules (Millero 2002). Similar reductions in pH are typical of closed transport systems (Estudillo and Duray 2003; Benetti et al. 2007; Colburn et al. 2008). Final pH values (minimum observed pH = 7.23) were well above 24-h LC50 (lethal concentration resulting in 50% mortality of test organisms in 24 h) values published for other larval shes (porgies [Sparidae]: pH = 5.06, soles [Soleidae]: pH = 4.51, cods [Gadidae]: pH = 4.99; Brownell 1980). The reduced pH potentially increased physiological stress; for example, carbonic anhydrase activity and related exchange of HCO3 as the waste product of respiration are reduced at lower pH (Brauner 2008).

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Differences in initial TAN concentrations between probiotic treatment and control shipping bags were most likely due to the action of bacterial probiotics upon the samples because TAN analysis of these water samples was not conducted until completion of the shipment packing. Despite a lack of signicant difference for initial TAN concentration, a signicant difference in initial NH3 concentration was detected. The lack of consistent signicant differences across both variables suggests a marginal actual difference in initial ammonia chemistry between the probiotic treatment and control shipping bags. Signicant differences in the nal TAN concentration and the increase in TAN were observed with the inclusion of Bacillus spp. probiotics (Table 2); similarly, nal NH3 concentrations were signicantly different between the probiotic treatment and the control. These reductions concur with other reports of Bacillus spp. probiotic inclusion in open-container transport situations (Gomes et al. 2008, 2009) and recirculating aquaculture systems (Chen and Chen 2001; Lalloo et al. 2007). The equilibrium TAN is partially dependent upon pH and temperature, and NH3 increases relative to NH4 + with increases in both inuential factors (Johansson and Wedborg 1980; Bell et al. 2007, 2008). Within both probiotic treatment and control bags, nal NH3 concentrations did not signicantly differ from initial concentrations. This is less surprising for the probiotic treatment, in which TAN concentrations did not signicantly change between initial and nal samples. This observation must be considered in conjunction with the signicant difference between initial and nal %NH3 in the control bags (Table 2). A similar trend would most likely have been observed for the probiotic treatment; for the one replicate in which initial TAN was observed, the %NH3 was 3.1%, which was the same as the mean for the control. For the probiotic treatment and the control, decreases in pH and temperature acted benecially by reducing %NH3 and thus the NH3 concentration, despite the increased nal TAN concentration. Despite high nal TAN concentrations in the control (Table 2), signicant differences between estimated survival rates were not detected (Table 1). Information regarding environmental TAN and NH3 tolerance in early life stages of yellown tuna is not available. Wedemeyer (1997) suggested that NH3 levels remain at or below 0.02 mg/L (1.17 M) during intensive sh culture. Computed nal NH3 concentrations (control range = 0.0010.007 mg/L [0.060.4 M]) were well below the chronic exposure values suggested by Wedemeyer (1997). Furthermore, exposure to NH3 concentrations of 0.55 mg/L (32.3 M) and higher caused 50% mortality in larval red drum Sciaenops ocellatus within 2448 h of exposure (Holt and Arnold 1983). Given that the NH3 values from the study by Holt and Arnold (1983) were orders of magnitude higher than those calculated here, it is not surprising that survival was not immediately inuenced in the present study. Physiological stress most likely resulted from the high TAN concentrations

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and would be manifested as modication of amino acid metabolism, enzyme induction, impairment of ion exchange, and utilization of more energetically demanding ureolytic nitrogenous excretion (Mommsen and Walsh 1992; Randall and Tsui 2002; Terjesen 2008). Although immediate survival was not negatively impacted, posttrial survival and growth were not monitored, and conclusions regarding observed survival must therefore be made with caution. In coldwater marine species, ammonia excretion peaks in the period after hatching (Rnnestad and Fyhn 1993) due to release of ammonia accumulated during the embryonic stage (Terjesen 2008). Although decreasing pH results in lower NH3 concentrations, lower pH also decreases ammonia toxicity tolerance, and in seawater NH4 + remains toxic due to the enhanced permeability of shes to this ion (Erickson 1985; Eddy 2005). These conclusions have been conrmed to apply to larval marine nsh (Miller et al. 1990). Our trial suggests a few of the potential benets of probiotic incorporation during the simulated transport of yellown tuna yolk sac larvae; these benets include reduced TAN concentrations, decreased oxygen utilization, and potentially decreased stress, as suggested by reduction in mean salinity uctuation. However, the pH shifts related to probiotic utilization, which resulted in both potentially positive and negative consequences, should not be ignored. To eliminate pH shifts and reduce this source of stress, further study is needed to determine proper buffer concentrations for use during probiotic-treated shipments of yolk sac larvae. Investigation of bacterial proliferation and community succession during shipping and further study of bacterial colonization in early marine teleost larvae could detect additional benets from probiotic incorporation during transport of yolk sac larvae and other life stages. Current shipping protocols are successful without probiotic additions, but the marginal increases in cost could yield improvements that facilitate transport success and posttransport survival and growth. ACKNOWLEDGMENTS We appreciate the assistance and support of those with the IATTC and the staff of Achotines Laboratory in Panama. We also thank the participants of the 3rd University of Miami-IATTC Workshop on Physiology and Aquaculture of Pelagics, with Emphasis on Reproduction and Early Developmental Stage of Yellown Tuna, Thunnus albacares, conducted in 2005 for this assistance in the completion of this trial. This study was partially (20052006) supported by Florida Sea Grant College Program grant numbers RLRA40 and RLRA42. REFERENCES
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