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Temporal changes in the suitability of claywater as a greenwater substitute for rearing larval sablefish (Anoplopoma fimbria)
Aquaculture 470 (2017) 11–16
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Temporal changes in the suitability of claywater as a greenwater substitute for rearing larval sablefish (Anoplopoma fimbria) Jonathan S.F. Lee ⁎, Matthew A. Cook, Barry A. Berejikian, Frederick W. Goetz Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 7305 Beach Dr E, Port Orchard, WA 98366, USA
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Article history: Received 20 November 2015 Received in revised form 18 July 2016 Accepted 7 December 2016 Available online 10 December 2016 Keywords: Clay Algae Larvae Turbidity Feeding Wall-nosing
a b s t r a c t For some species, marine aquaculture facilities increase turbidity to improve larval feeding, growth, and survival, typically by mixing algae with seawater to make “greenwater.” Clay is a less expensive and inorganic potential algae substitute that has been shown to reduce bacterial levels relative to algae and promote equal or better growth and survival in some species. Sablefish (Anoplopoma fimbria) is a prime candidate species for aquaculture but the rearing of sablefish larvae has not yet been experimentally tested with clay. This study tested whether clay is a viable algae substitute for rearing sablefish, and whether the relative performance of clay versus algae varies as a function of time. In the first week of larval rearing, algae (Nannochloropsis) produced more than three times greater survival than clay (Kentucky Ball Clay OM4). However, switching from algae to clay at the beginning of the second week led to 1.5 times greater larval growth compared to tanks where algae was used in both weeks. The performance difference between clay and algae, despite equal turbidity, suggests that clay and algae have potentially harmful and beneficial effects in addition to turbidity effects, and that these effects change through time. However, because the first experiment was a replacement study, it was not possible to know whether algae produced better survival in week one because 1) clay, which might be harmful, was not present, 2) algae, which might be beneficial, was present, or 3) both. A further additive study was conducted to test the second possibility. The experiment found that adding algae to clay in the first week of larval rearing leads to greater growth and survival, suggesting that algae has beneficial effects beyond turbidity. We discuss some possible mechanisms for potential harmful and beneficial effects of algae and clay. Published by Elsevier B.V.
1. Introduction Turbid water is needed to successfully rear larvae of many marine species. When reared in non-turbid clear water, larvae of many marine species starve, possibly because they have trouble seeing live feed (Boehlert and Morgan, 1985). Clear water may also cause larval marine fish to swim unproductively against tank side walls (wall-nosing), which wastes energy and may cause deformities (Cobcroft et al., 2012). While high turbidity can be just as detrimental (Boehlert and Morgan, 1985; Carton, 2005; Utne-Palm, 2004), moderate turbidity can improve feeding and prevent wall-nosing (Boehlert and Morgan, 1985; Cobcroft et al., 2012). For marine fish larvae which typically rely heavily on visual cues (Blaxter, 1986; Utne-Palm, 2002), increasing turbidity in culture tanks may more closely mimic natural conditions Abbreviations: Low C, low concentration claywater; High C, high concentration claywater; Low C + G, low concentration claywater plus greenwater; G → G, greenwater during weeks 1 and 2; G → C, greenwater during week 1, then claywater during week 2; C → C, claywater during weeks 1 and 2. ⁎ Corresponding author. E-mail addresses: [email protected] (J.S.F. Lee), [email protected] (M.A. Cook), [email protected] (B.A. Berejikian), [email protected] (F.W. Goetz).
http://dx.doi.org/10.1016/j.aquaculture.2016.12.011 0044-8486/Published by Elsevier B.V.
(Utne-Palm, 2004) and promote normal behavior by enhancing visual contrast, by scattering light, or by reducing perceived predation risk for the larvae (Boehlert and Morgan, 1985; Utne-Palm, 2002). Most marine fish hatcheries achieve turbidity by adding algae to seawater. The resulting mixture is known as “greenwater” and can improve feeding, behavior, growth, and survival when compared to clear water (Cahu et al., 1998; Cobcroft et al., 2012; Palmer et al., 2007; Papandroulakis et al., 2002; Stuart and Drawbridge, 2011; van der Meeren et al., 2007). However, greenwater has drawbacks. Fish hatcheries can purchase dead algae or grow their own live algae, but purchasing algae is expensive and growing it can be unpredictable and labor intensive. Also, because algae is organic and decays, adding it to rearing tanks can promote bacterial growth and water fouling, particularly if dead algae is used (Attramadal et al., 2012). Nevertheless, greenwater continues to be widely used because there are few well-researched alternatives. Previous work has tested clay as an inexpensive algae substitute. Attramadal et al. (2012) showed that using clay instead of algae can promote larval Atlantic cod (Gadus morhua) survival and growth while reducing organic matter and bacterial abundance. Stuart et al. (2015) found that claywater with continuous feeding led to greater survival
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than claywater with batch feeding or greenwater with batch feeding in yellowtail amberjack (Seriola lalandi); the latter two treatments did not differ. Vibrio colonies were more abundant in the algal paste treatment than either claywater treatment. In other studies, Daugherty (2013) found a negative impact of claywater on yellowtail kingfish (Seriola l. lalandi) growth and survival, but no effect on cobia (Rachycentron canadum). Claywater had fewer Vibrio counts than greenwater, though Vibrio did not appear to affect growth or survival. Sablefish has been identified as a prime candidate species for aquaculture, with moist, firm flesh, and high prices off fishing vessels and in the retail market. In the wild, eggs are spawned and develop in deep waters (N200 m), but larvae are found at the surface (Kendall and Matarese, 1987). A significant cost associated with rearing larval sablefish is the price of algae for greenwater, which is used by the nascent North American sablefish aquaculture industry. Continued development of a domestic sablefish industry would be more feasible if rearing methods were refined to reduce costs and increase production quality and quantity. Currently, there are no published studies on the use of clay in sablefish aquaculture. This study asked whether clay is a viable alternative to algae, and whether the performance of clay differs as a function of time. Here, first-feeding sablefish larvae were reared for two weeks in three treatments: 1) greenwater for two weeks, 2) claywater for two weeks, or 3) greenwater for the first week and claywater for the second week. Comparisons were made for larval body weights among treatments at the end of the first and second weeks, and survival at the end of the second week. Follow-up studies then tested the effects of clay concentrations and algae-clay mixtures on behavior, growth, and survival. 2. Materials and methods Larvae originated from crosses of broodstock captured off the Washington coast. For spawning, egg fertilization, and hatching details see Cook et al. (2015). Experiments were carried out in 37 L tanks (40 cm tall, 37 cm diameter) with flat, white bottoms and black walls. A 3.3cm diameter center standpipe maintained water at a height of 36.5 cm. Plastic tubing (0.635 cm internal diameter) delivered filtered, ambient clear seawater to each tank at a rate of 270 mL/min. A 61-cm tall, 40-cm diameter cylinder made out of Reflectix insulation (Markleville, IN, USA) rested on top of each tank and was topped by a white plastic disk that sealed the tank from external light. A lighting fixture was attached to the underside of the white plastic disk. The lighting fixture contained an LED bulb (Satco S8813, Brentwood, NY, USA) above 14 layers of 1.5-mm stainless window mesh that reduced light intensity to 12 lx at the water surface. Light was on 24 h per day. Larvae were fed three times per day at a density of 20 rotifers per mL of tank water. Concentrated greenwater was made by mixing Nannochloropsis Instant Algae (Reed Mariculture, Campbell, CA, USA, 68 billion cells per mL) and green dye (“Green Shade Color,” Esco Foods, San Francisco, CA, USA; see below for ratios). To make concentrated claywater, Kentucky Ball Clay OM4 (Kentucky-Tennessee Clay Company, Roswell, GA, USA) was mixed with water for 2 min (1 min on “low” followed by 1 min on “high”) in a commercial blender (Waring MX1000XTX 3.5 HP). These concentrated solutions were kept suspended by aerating them in buckets. Peristaltic pumps (Anko Products, Bradenton, FL, USA) were activated by cycle timers (Cap Controllers, Perris, CA, USA) to deliver the concentrated solutions via manifolds to the experimental tanks. 2.1. Experiment 1—can claywater substitute for greenwater in weeks 1 and 2? First-feeding larvae were stocked into 37 L experimental tanks. Four broodstock crosses were equally represented among tanks (75 larvae per cross per tank; 300 total larvae in each tank). First-feeding larvae were reared under three different conditions: 1) greenwater for two weeks (n = 6 tanks, G → G), 2) claywater for two weeks (n = 6
tanks, C → C), and 3) greenwater for one week followed by claywater for one week (n = 6 tanks, G → C). Greenwater was maintained at 0.021 mL Nannochloropsis instant algae and 0.005 mL of green dye (“green shade color,” Esco Foods, San Francisco, CA, USA) per L of tank water. Claywater was maintained at 12 mg of Kentucky ball clay OM4 per L of tank water. These concentrations produced equal Secchi-disk turbidities between treatments (Carolina turbidity tube, Carolina Biological Supply Company, Burlington, NC, USA). In the first week, twelve tanks received greenwater and six tanks received claywater (18 tanks total). On day 8, six of the tanks that had previously received greenwater were switched to claywater. The six tanks that were switched to claywater were selected so that the average body weight at switch-over was equal between the G → G and G → C treatments. 2.1.1. Sampling Fifteen larvae were weighed together for each tank on day 8. Larvae were removed from each tank, overdosed in MS-222, weighed wet, and then weighed again after drying overnight in a 110 °C oven. That weight was divided by 15 to arrive at “average larval dry weight.” On day 15, tanks were drained, survivors were counted, and all survivors were bulk-weighed after drying overnight for each tank (“total dry biomass”). Quantification of survivors required stressful larval handling and was therefore only done at the end of the experiment. 2.1.2. Statistics Separate analyses were conducted to isolate effects during weeks one and two. To isolate the effect of claywater versus greenwater during week one, C → C and G → C treatments were compared with Wilcoxon tests in terms of survival after week two and total dry biomass after week two. Since these two treatments both used claywater during week two, any difference could be attributed to the use of claywater versus greenwater in week one. At the end of week one, six tanks had contained claywater and 12 tanks had contained greenwater. We conducted a Wilcoxon test to compare average larval dry weights between the six claywater and 12 greenwater tanks. To isolate the effect of claywater versus greenwater during week two, G → G and G → C treatments were compared with Wilcoxon tests in terms of growth during week two, survival after week two, and total dry biomass after week two. Growth during week two was calculated for each tank as the difference in average larval dry weights between the end of weeks 1 and 2. Since these two treatments both used greenwater in week one, any difference could be attributed to the use of claywater versus greenwater in week two. 2.2. Experiment 2—what is the best clay concentration during week two? Three different clay concentrations were compared in an eight-day rearing trial using one-week-old larvae. The larvae had initially been stocked at first-feeding from three broodstock crosses into a 2000 L holding tank in greenwater. After eight days, 250 larvae were transferred into each of 18 tanks (37 L) for the experiment. The 18 tanks were evenly divided among low, medium, and high clay concentration treatments: 3, 9, and 15 mg clay per L tank water, respectively (n = 6 tanks per treatment). A nephelometer (HI 93703, Hanna Instruments, Woonsocket, RI, USA) measured turbidity to be 2.4, 11.1, and 13.9 NTU in the low, medium, and high concentration treatments, respectively. 2.2.1. Sampling For each tank on day four, an observer counted the number of larvae visible from the surface that were touching tank walls (wall-nosing) and the number that were not touching tank walls. Counts were made with a hand tally counter and took about ten seconds per tank. On the eighth day, the tanks were drained and larvae counted. All surviving larvae were weighed with the same methods used in Experiment 1.
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2.2.2. Statistics The percentage of larvae that were wall-nosing was compared among treatments with a Kruskal-Wallis test and followed by pairwise Bonferroni-corrected Wilcoxon tests. Survival, total dry biomass, and average larval dry weight were compared among treatments with Kruskal-Wallis tests.
2.3.1. Sampling For each tank on day four, an observer quantified the number of larvae visible from the surface that were touching tank walls (wall-nosing) and the number that were not touching tank walls. On the eighth day, the tanks were drained and larvae enumerated. All surviving larvae were weighed with the same methods used in Experiment 1.
2.3. Experiment 3—does greenwater have additional benefits beyond turbidity during week one?
2.3.2. Statistics The percentage of larvae that were wall-nosing was compared among treatments with a Kruskal-Wallis test. Survival, total dry biomass, and average larval weight were compared among treatments with Kruskal-Wallis tests and followed by pairwise Bonferroni-corrected Wilcoxon tests.
Two-hundred first-feeding larvae from one broodstock cross and 90 first-feeding larvae from a second broodstock cross were stocked into each 37 L experimental tank (290 total larvae in each tank). The larvae were reared under three treatment conditions for one week: 1) low concentration of claywater (“Low C,” n = 6 tanks, 10.5 mg clay per L tank water), 2) high concentration of claywater (“High C,” n = 6 tanks, 13.5 mg clay per L tank water), 3) low concentration of claywater plus greenwater (“Low C + G,” n = 6 tanks, 10.5 mg clay and 0.0235 mL algae per L tank water). The High C and Low C + G treatments had equal turbidity as measured by the nephelometer. Turbidity levels were chosen based on results from Experiment 2, which suggested no differences in growth or survival between the 9 and 15 mg clay per L clay concentrations, despite behavioral differences.
3. Results 3.1. Experiment 1 Tanks with greenwater during week one (G → C) had higher survival and higher total dry biomass at the end of the experiment than tanks with claywater during week one (C → C, Fig. 1a–b, survival: Z = 2.01, P b 0.05; total dry biomass: Z = 2.00, P b 0.05). The G → C and G → G treatments did not differ from the C → C treatment in average larval dry weight at the end of week one (Fig. 1c, Z= − 1.37, P N 0.05).
Fig. 1. Experiment 1, week-one effect (a–c) and week-two effect (d–f) of clay versus algae on mean (+SE) survival, total dry biomass, and larval dry weight over two weeks. Tanks received either clay or algae during week one, then clay during week 2. Experiment began with first-feed larvae. Symbols represent individual data points (tanks).
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Survival and total dry biomass did not differ between G → C and G → G treatments (Fig. 1d–e; survival: Z = − 0.72, P N 0.05; total dry biomass: Z = − 0.88, P N 0.05). Tanks with claywater during week two (G → C) had higher growth during week two than tanks with greenwater during week two (G → G, Fig. 1f; Z = −2.32, P b 0.05). 3.2. Experiment 2 Higher clay concentrations were associated with less wall-nosing (Fig. 2; H = 14.76, P b 0.001). All treatments differed from each other (Bonferroni-corrected post-hoc comparisons: 20–60 Z = − 2.81, P b 0.05; 20–100 Z = 2.81, P b 0.05; 60–100 Z = 2.64, P b 0.05). There were no differences in survival, total dry biomass, or average larval dry weight among treatments (Fig. 3a–b; survival: H = 4.16, P N 0.05; total dry biomass: H = 3.89, P N 0.05; average larval dry weight: H = 0.11, P N 0.05). 3.3. Experiment 3 Wall-nosing did not differ among Low C, High C, and Low C + G treatments (H = 2.21, P N 0.05). Survival, total dry biomass, and average larval dry weight were greater in the “Low C + G” treatment than in the “Low C” and “High C” treatments (P b 0.05, Fig. 4a–c, Table 1). 4. Discussion In the current study, experiments showed that the suitability of greenwater versus claywater can vary as a function of time, within a single species. In the first week of the two-week experiment, greenwater led to greater survival than claywater. However, switching to claywater at the beginning of the second week led to greater growth than when larvae were kept on greenwater for both weeks. These data suggest that claywater is not only a cheaper alternative to greenwater but can also produce larger fish, provided that it does not replace greenwater too early in rearing. Daugherty (2013) found that the effects of greenwater versus claywater on growth and survival varied between cobia (Rachycentron canadum) and yellowtail kingfish (Seriola lalandi). Most published studies report that claywater can produce equal or better growth and survival than greenwater (Attramadal et al., 2012; Bjornsdottir, 2010; Stuart et al., 2015), although it is unknown whether studies finding poorer claywater performance are less likely to be published.
Fig. 2. Experiment 2, mean (+SE) number of larvae wall-nosing versus swimming normally in open water in low, medium, and high concentration claywater. Black bars represent larvae swimming in open water. Gray bars represent wall-nosing larvae.
Fig. 3. Mean (+ SE) survival (a) and total dry biomass (b) in low, medium, and high concentration claywater after one week. Experiment began with one-week-old larvae. Symbols represent individual data points (tanks).
In the first week of Experiment 1, greenwater promoted greater survival and greater total dry biomass than claywater under conditions of equal turbidity. The higher survival during week one in Experiment 1 may have been caused by the absence of harmful properties of clay, beneficial properties of greenwater, or both. Clay exposure in week one could have damaged young developing larval gills or guts at their earliest and most sensitive stages (Hess et al., 2015; Muncy et al., 1979), and algae might provide probiotics that seed larval guts with beneficial bacteria (Verschuere et al., 2000). The additive experiment (Experiment 3) tested whether greenwater has benefits beyond turbidity effects during week 1. The “Low C” treatment (10.5 mg clay per L tank water) had the same amount of clay as the “Low C + G” treatment, but the latter had better growth and survival, showing that algae leads to better growth and survival even when clay concentration is kept constant. One potentially confounding factor is that turbidity was greater in the “Low C + G” treatment than the “Low C” treatment, but the “High C” treatment (13.5 mg clay per L tank water) controlled for this. Turbidity in “High C” was equal to “Low C + G,” yet growth and survival were equal between “High C” and “Low C” treatments, indicating that the greater performance of “Low C + G” was due to the presence of algae and not to higher turbidity. These results do not preclude the possibility that clay or algae may each have some harmful effects during the first week of larval rearing; they just suggest that, controlling for turbidity effects, there are benefits of algae over clay beyond those attributable to turbidity. Beyond showing a beneficial effect of algae, the additive experiment shows that a clay and algae mixture could potentially be applied during
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Fig. 4. Mean (+SE) survival (a), total dry biomass (b), and larval dry weight (c) in lowconcentration clay (Low C), high-concentration clay (High C), and low-concentration clay plus algae (Low C + G) after one week. Experiment began with first-feed larvae. Symbols represent individual data points (tanks).
the first week after first feeding. Dose-response experiments for algae in “Low C + G” mixtures could identify the minimum concentration of algae that can be used to maintain algal benefits while minimizing algal expense and organic input.
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Algae could benefit larval growth and survival in week 1 through a variety of potentially compatible mechanisms. There might be a probiotic effect early in larval rearing where larval guts are seeded with beneficial bacteria (Verschuere et al., 2000). Algae could also stimulate gut enzymes (Cahu et al., 1998) or feeding in young larvae. Algae can release chemical compounds that have been shown to stimulate feeding behavior in juvenile and adult fish and birds (Charlson et al., 1987; DeBose et al., 2008; Nakajima et al., 1990; Nevitt et al., 1995). These algal compounds might also stimulate feeding in larval fish, which could be particularly important as young larvae learn to eat and capture prey. Larvae might also be able to directly consume algae (van der Meeren et al., 2007), though ingestion of large amounts of algae would be more likely if we had used larger algae that could have been captured by filter-feeding larval gill rakers (van der Meeren, 1991). These hypotheses require further experimentation. Previous experiments have shown that claywater tanks have less bacterial growth than greenwater tanks (Attramadal et al., 2012; Bjornsdottir, 2010; Daugherty, 2013; Stuart et al., 2015). This bacterial mechanism may be consistent with our observation that claywater did not show a benefit until the second week of larval rearing. In the first week of rearing, perhaps bacterial levels were low even in greenwater treatments because tanks and larvae were relatively clean when larvae were first introduced, and bacteria concentrations in tanks and on larvae may not have had enough time to increase to a detrimental level. During the second week, perhaps bacterial concentrations increased to the point where bacteria-reducing properties of clay led to greater larval growth in the claywater treatment. Future sablefish experiments that couple a time-series of microbial sampling with performance measures would be informative. Understanding the beneficial effects, harmful effects, and temporal dynamics of these effects of clay and algae may also help us understand and predict when claywater or greenwater will produce higher growth or survival. For example, species may differ in their ability to tolerate higher loads of harmful bacteria or in the degree to which algae stimulates feeding. If there is a species that is naturally more tolerant of higher bacterial levels, then a claywater-induced reduction in bacterial loads may not be a benefit, and greenwater might produce higher growth or survival. Or if there is a species that is a naturally voracious feeder, then any feeding stimulants potentially released by algae may not be important, and claywater might then produce higher growth or survival. The type of clay that is used could affect results, since different clay types can differ in antibacterial properties (Morrison et al., 2014; Williams et al., 2011), and can differ in particle size and thus have different effects on larval vision or larval gills and guts. This study used Kentucky OM4 because this type of clay has been commonly used in previous studies and in commercial aquaculture, but future studies on other clay types would be informative. Understanding these mechanisms might also raise awareness of potential methodological influences on results. For example, methodological details such as stocking density, the frequency of tank cleaning, the duration of the experiment, rearing temperature, and larval age might determine whether claywater or greenwater is found to produce greater growth or survival in a particular experiment or rearing situation (e.g. see Daugherty, 2013). In this study with sablefish, a one-week experiment stocked with first-feed larvae would conclude that greenwater produces greater
Table 1 Effect of low-concentration clay (Low C), low-concentration clay plus algae (Low C + G), and high-concentration clay (High C) on survival, total dry biomass, and average dry weight after one week. Experiment began with first-feed larvae. Response
Survival Total biomass Average weight
Overall effect
Bonferroni-corrected pairwise comparisons
All treatments
Low C + G vs Low C
Low C + G vs High C
Low C vs High C
H = 9.06, P b 0.05 H = 10.88, P b 0.01 H = 9.95, P b 0.01
Z = 2.49, P b 0.05 Z = 2.65, P b 0.05 Z = −2.65, P b 0.05
Z = −2.49, P b 0.05 Z = −2.81, P b 0.05 Z = −2.46, P b 0.05
Z = 0.24, P N 0.05 Z = 0.24, P N 0.05 Z = 0.10, P N 0.05
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survival than claywater, whereas a one-week experiment stocked with one-week-old larvae might conclude that claywater produces better growth with no survival differences. Further experiments are needed to determine whether claywater leads to greater growth or survival than greenwater from weeks three to five. After week five, turbid water is no longer needed for normal behavior (MC, personal observations). Clay provides a significant cost savings over algae. Currently, Kentucky OM4 clay retails for $1.89 per kg, whereas Nannochloropsis algae retails for $61.70 per L. Thus, 1 L of greenwater costs 57 times more than 1 L of claywater. Discounts for bulk purchases are likely available for both clay and algae. Clay is typically available for pickup at local pottery supply stores whereas algae must be cultured or shipped frozen from producers. In Experiment 1, 1 L of claywater required 12 × 10−6 kg of clay while 1 L of greenwater required 21 × 10−6 L of algal paste. A typical 5000 L tank used in aquaculture would turn over about six times per day, and use greenwater for 35 days at an algal cost of $1361 per tank. If the tanks were instead switched to claywater on day eight, the expense per tank would be $272.30 for algae in the first seven days plus $19.05 for clay for the remaining 28 days. The experiments in this study suggest that in addition to the significantly cheaper cost, clay can also produce larger and potentially more fish if used at the proper rearing stage. Future studies that define the mechanisms and temporal dynamics of beneficial and harmful effects of clay and algae may enable the creation of “hybrid” seawater additives that combine the benefits of clay and algae while minimizing harmful effects. For example, if clay produces better growth than algae because it does not promote the growth of harmful bacteria, but greenwater releases chemicals that stimulate feeding, then adding algal extracts to clay may allow hatcheries to rear larvae with the benefits of algae (turbidity, feeding stimulants) but without the costs (increased bacterial growth due to high organic input), and achieve higher growth and survival than with either clay or algae alone. Acknowledgements We thank Megan Sorby for discussion and for suggesting that clay might be harmful early in larval rearing. Mike Rust and Ken Webb provided valuable suggestions on beneficial mechanisms of algae and harmful effects of clay. Linda Rhodes and Rachel Poretsky provided expertise on potential bacterial mechanisms. Lyle Britt advised on larval fish vision and tank setup. Jeff Atkins, Rob Endicott, Bill Fairgrieve, Doug Immerman, Andy Jasonwicz, Cort Jensen, Ken Massee, Sean Oden, Jose Reyes-Tomassini, and Tom Wade provided valuable help and advice with live feeds, maintenance, and general husbandry. Funding was provided by the National Oceanic and Atmospheric Administration Fisheries Office of Aquaculture, and by a grant from Washington Sea Grant, University of Washington, pursuant to National Oceanic and Atmospheric Administration Award No. NA14OAR4170078. The views expressed herein are those of the authors and do not necessarily reflect the views of the National Oceanic and Atmospheric Administration or any of its sub-agencies. References Attramadal, K.J.K., Tondel, B., Salvesen, I., Oie, G., Vadstein, O., Olsen, Y., 2012. Ceramic clay reduces the load of organic matter and bacteria in marine fish larval culture tanks. Aquac. Eng. 49, 23–34.
Bjornsdottir, R., 2010. The Bacterial Community During Early Production Stages of Intensively Reared Halibut (Hippoglossus hippoglossus L.). School of Health Sciences, Faculty of Medicine. University of Iceland, Reykjavik, Iceland. Blaxter, J.H.S., 1986. Development of sense organs and behavior of teleost larvae with special reference to feeding and predator avoidance. Trans. Am. Fish. Soc. 115, 98–114. Boehlert, G.W., Morgan, J.B., 1985. Turbidity enhances feeding abilities of larval Pacific herring, Clupea harengus Pallasi. Hydrobiologia 123, 161–170. Cahu, C.L., Infante, J.L.Z., Peres, A., Quazuguel, P., Le Gall, M.M., 1998. Algal addition in sea bass (Dicentrarchus labrax) larvae rearing: effect on digestive enzymes. Aquaculture 161, 479–489. Carton, A.G., 2005. The impact of light intensity and algal-induced turbidity on first-feeding Seriola lalandi larvae. Aquac. Res. 36, 1588–1594. Charlson, R.J., Lovelock, J.E., Andreae, M.O., Warren, S.G., 1987. Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate. Nature 326, 655–661. Cobcroft, J.M., Shu-Chien, A.C., Kuah, M.K., Jaya-Ram, A., Battaglene, S.C., 2012. The effects of tank colour, live food enrichment and greenwater on the early onset of jaw malformation in striped trumpeter larvae. Aquaculture 356, 61–72. Cook, M.A., Massee, K.C., Wade, T.H., Oden, S.M., Jensen, C., Jasonowicz, A., Immerman, D.A., Goetz, F.W., 2015. Culture of sablefish (Anoplopoma fimbria) larvae in four experimental tank designs. Aquac. Eng. 69, 43–49. Daugherty, Z.N., 2013. Effects of Algae Paste Substitutes on the Larval Rearing Performance and Microbial Communities in the Culture of Cobia (Rachycentron canadum) and Yellowtail Kingfish (Seriola lalandi). University of Miami, Dissertation. DeBose, J.L., Lema, S.C., Nevitt, G.A., 2008. Dimethylsulfoniopropionate as a foraging cue for reef fishes. Science 319, 1356. Hess, S., Wenger, A.S., Ainsworth, T.D., Rummer, J.L., 2015. Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: impacts on gill structure and microbiome. Sci. Rep. 5. Kendall, A.W., Matarese, A.C., 1987. Biology of eggs, larvae, and epipelagic juveniles of sablefish, Anoplopoma fimbria, in relation to their potential use in management. Mar. Fish. Rev. 49, 1–13. Morrison, K.D., Underwood, J.C., Metge, D.W., Eberl, D.D., Williams, L.B., 2014. Mineralogical variables that control the antibacterial effectiveness of a natural clay deposit. Environ. Geochem. Health 36, 613–631. Muncy, R.J., Atchison, G.J., Bulkley, R.V., Menzel, B.W., Perry, L.G., Summerfelt, R.G., 1979. Effects of Suspended Solids and Sediment on Reproduction and Early Life of Warmwater Fishes: A Review. Corvallis Environmental Research Laboratory, Corvallis, OR, p. 101. Nakajima, K., Uchida, A., Ishida, Y., 1990. Effect of a feeding attractant, dimethyl-betapropiothetin, on growth of marine fish. Nippon Suisan Gakkaishi 56, 1151–1154. Nevitt, G.A., Veit, R.R., Kareiva, P., 1995. Dimethyl sulfide as a foraging cue for Antarctic Procellariiform seabirds. Nature 376, 680–682. Palmer, P.J., Burke, M.J., Palmer, C.J., Burke, J.B., 2007. Developments in controlled greenwater larval culture technologies for estuarine fishes in Queensland, Australia and elsewhere. Aquaculture 272, 1–21. Papandroulakis, N., Divanach, P., Kentouri, M., 2002. Enhanced biological performance of intensive sea bream (Sparus aurata) larviculture in the presence of phytoplankton with long photophase. Aquaculture 204, 45–63. Stuart, K.R., Drawbridge, M., 2011. The effect of light intensity and green water on survival and growth of cultured larval California yellowtail (Seriola lalandi). Aquaculture 321, 152–156. Stuart, K., Rotman, F., Drawbridge, M., 2015. Methods of microbial control in marine fish larval rearing: clay-based turbidity and passive larval transfer. Aquac. Res. 2015, 1–11. Utne-Palm, A.C., 2002. Visual feeding of fish in a turbid environment: physical and behavioural aspects. Mar. Freshw. Behav. Physiol. 35, 111–128. Utne-Palm, A.C., 2004. Effects of larvae ontogeny, turbidity, and turbulence on prey attack rate and swimming activity of Atlantic herring larvae. J. Exp. Mar. Biol. Ecol. 310, 147–161. van der Meeren, T., 1991. Algae as first food for cod larvae, Gadus morhua L.: filter feeding or ingestion by accident? J. Fish Biol. 39, 225–237. van der Meeren, T., Mangor-Jensen, A., Pickova, J., 2007. The effect of green water and light intensity on survival, growth and lipid composition in Atlantic cod (Gadus morhua) during intensive larval rearing. Aquaculture 265, 206–217. Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64, 655–671. Williams, L.B., Metge, D.W., Eberl, D.D., Harvey, R.W., Turner, A.G., Prapaipong, P., PoretPeterson, A.T., 2011. What makes a natural clay antibacterial? Environ. Sci. Technol. 45, 3768–3773.
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Temporal changes in the suitability of claywater as a greenwater substitute for rearing larval sablefish (Anoplopoma fimbria) Jonathan S.F. Lee ⁎, Matthew A. Cook, Barry A. Berejikian, Frederick W. Goetz Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 7305 Beach Dr E, Port Orchard, WA 98366, USA
a r t i c l e
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Article history: Received 20 November 2015 Received in revised form 18 July 2016 Accepted 7 December 2016 Available online 10 December 2016 Keywords: Clay Algae Larvae Turbidity Feeding Wall-nosing
a b s t r a c t For some species, marine aquaculture facilities increase turbidity to improve larval feeding, growth, and survival, typically by mixing algae with seawater to make “greenwater.” Clay is a less expensive and inorganic potential algae substitute that has been shown to reduce bacterial levels relative to algae and promote equal or better growth and survival in some species. Sablefish (Anoplopoma fimbria) is a prime candidate species for aquaculture but the rearing of sablefish larvae has not yet been experimentally tested with clay. This study tested whether clay is a viable algae substitute for rearing sablefish, and whether the relative performance of clay versus algae varies as a function of time. In the first week of larval rearing, algae (Nannochloropsis) produced more than three times greater survival than clay (Kentucky Ball Clay OM4). However, switching from algae to clay at the beginning of the second week led to 1.5 times greater larval growth compared to tanks where algae was used in both weeks. The performance difference between clay and algae, despite equal turbidity, suggests that clay and algae have potentially harmful and beneficial effects in addition to turbidity effects, and that these effects change through time. However, because the first experiment was a replacement study, it was not possible to know whether algae produced better survival in week one because 1) clay, which might be harmful, was not present, 2) algae, which might be beneficial, was present, or 3) both. A further additive study was conducted to test the second possibility. The experiment found that adding algae to clay in the first week of larval rearing leads to greater growth and survival, suggesting that algae has beneficial effects beyond turbidity. We discuss some possible mechanisms for potential harmful and beneficial effects of algae and clay. Published by Elsevier B.V.
1. Introduction Turbid water is needed to successfully rear larvae of many marine species. When reared in non-turbid clear water, larvae of many marine species starve, possibly because they have trouble seeing live feed (Boehlert and Morgan, 1985). Clear water may also cause larval marine fish to swim unproductively against tank side walls (wall-nosing), which wastes energy and may cause deformities (Cobcroft et al., 2012). While high turbidity can be just as detrimental (Boehlert and Morgan, 1985; Carton, 2005; Utne-Palm, 2004), moderate turbidity can improve feeding and prevent wall-nosing (Boehlert and Morgan, 1985; Cobcroft et al., 2012). For marine fish larvae which typically rely heavily on visual cues (Blaxter, 1986; Utne-Palm, 2002), increasing turbidity in culture tanks may more closely mimic natural conditions Abbreviations: Low C, low concentration claywater; High C, high concentration claywater; Low C + G, low concentration claywater plus greenwater; G → G, greenwater during weeks 1 and 2; G → C, greenwater during week 1, then claywater during week 2; C → C, claywater during weeks 1 and 2. ⁎ Corresponding author. E-mail addresses: [email protected] (J.S.F. Lee), [email protected] (M.A. Cook), [email protected] (B.A. Berejikian), [email protected] (F.W. Goetz).
http://dx.doi.org/10.1016/j.aquaculture.2016.12.011 0044-8486/Published by Elsevier B.V.
(Utne-Palm, 2004) and promote normal behavior by enhancing visual contrast, by scattering light, or by reducing perceived predation risk for the larvae (Boehlert and Morgan, 1985; Utne-Palm, 2002). Most marine fish hatcheries achieve turbidity by adding algae to seawater. The resulting mixture is known as “greenwater” and can improve feeding, behavior, growth, and survival when compared to clear water (Cahu et al., 1998; Cobcroft et al., 2012; Palmer et al., 2007; Papandroulakis et al., 2002; Stuart and Drawbridge, 2011; van der Meeren et al., 2007). However, greenwater has drawbacks. Fish hatcheries can purchase dead algae or grow their own live algae, but purchasing algae is expensive and growing it can be unpredictable and labor intensive. Also, because algae is organic and decays, adding it to rearing tanks can promote bacterial growth and water fouling, particularly if dead algae is used (Attramadal et al., 2012). Nevertheless, greenwater continues to be widely used because there are few well-researched alternatives. Previous work has tested clay as an inexpensive algae substitute. Attramadal et al. (2012) showed that using clay instead of algae can promote larval Atlantic cod (Gadus morhua) survival and growth while reducing organic matter and bacterial abundance. Stuart et al. (2015) found that claywater with continuous feeding led to greater survival
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than claywater with batch feeding or greenwater with batch feeding in yellowtail amberjack (Seriola lalandi); the latter two treatments did not differ. Vibrio colonies were more abundant in the algal paste treatment than either claywater treatment. In other studies, Daugherty (2013) found a negative impact of claywater on yellowtail kingfish (Seriola l. lalandi) growth and survival, but no effect on cobia (Rachycentron canadum). Claywater had fewer Vibrio counts than greenwater, though Vibrio did not appear to affect growth or survival. Sablefish has been identified as a prime candidate species for aquaculture, with moist, firm flesh, and high prices off fishing vessels and in the retail market. In the wild, eggs are spawned and develop in deep waters (N200 m), but larvae are found at the surface (Kendall and Matarese, 1987). A significant cost associated with rearing larval sablefish is the price of algae for greenwater, which is used by the nascent North American sablefish aquaculture industry. Continued development of a domestic sablefish industry would be more feasible if rearing methods were refined to reduce costs and increase production quality and quantity. Currently, there are no published studies on the use of clay in sablefish aquaculture. This study asked whether clay is a viable alternative to algae, and whether the performance of clay differs as a function of time. Here, first-feeding sablefish larvae were reared for two weeks in three treatments: 1) greenwater for two weeks, 2) claywater for two weeks, or 3) greenwater for the first week and claywater for the second week. Comparisons were made for larval body weights among treatments at the end of the first and second weeks, and survival at the end of the second week. Follow-up studies then tested the effects of clay concentrations and algae-clay mixtures on behavior, growth, and survival. 2. Materials and methods Larvae originated from crosses of broodstock captured off the Washington coast. For spawning, egg fertilization, and hatching details see Cook et al. (2015). Experiments were carried out in 37 L tanks (40 cm tall, 37 cm diameter) with flat, white bottoms and black walls. A 3.3cm diameter center standpipe maintained water at a height of 36.5 cm. Plastic tubing (0.635 cm internal diameter) delivered filtered, ambient clear seawater to each tank at a rate of 270 mL/min. A 61-cm tall, 40-cm diameter cylinder made out of Reflectix insulation (Markleville, IN, USA) rested on top of each tank and was topped by a white plastic disk that sealed the tank from external light. A lighting fixture was attached to the underside of the white plastic disk. The lighting fixture contained an LED bulb (Satco S8813, Brentwood, NY, USA) above 14 layers of 1.5-mm stainless window mesh that reduced light intensity to 12 lx at the water surface. Light was on 24 h per day. Larvae were fed three times per day at a density of 20 rotifers per mL of tank water. Concentrated greenwater was made by mixing Nannochloropsis Instant Algae (Reed Mariculture, Campbell, CA, USA, 68 billion cells per mL) and green dye (“Green Shade Color,” Esco Foods, San Francisco, CA, USA; see below for ratios). To make concentrated claywater, Kentucky Ball Clay OM4 (Kentucky-Tennessee Clay Company, Roswell, GA, USA) was mixed with water for 2 min (1 min on “low” followed by 1 min on “high”) in a commercial blender (Waring MX1000XTX 3.5 HP). These concentrated solutions were kept suspended by aerating them in buckets. Peristaltic pumps (Anko Products, Bradenton, FL, USA) were activated by cycle timers (Cap Controllers, Perris, CA, USA) to deliver the concentrated solutions via manifolds to the experimental tanks. 2.1. Experiment 1—can claywater substitute for greenwater in weeks 1 and 2? First-feeding larvae were stocked into 37 L experimental tanks. Four broodstock crosses were equally represented among tanks (75 larvae per cross per tank; 300 total larvae in each tank). First-feeding larvae were reared under three different conditions: 1) greenwater for two weeks (n = 6 tanks, G → G), 2) claywater for two weeks (n = 6
tanks, C → C), and 3) greenwater for one week followed by claywater for one week (n = 6 tanks, G → C). Greenwater was maintained at 0.021 mL Nannochloropsis instant algae and 0.005 mL of green dye (“green shade color,” Esco Foods, San Francisco, CA, USA) per L of tank water. Claywater was maintained at 12 mg of Kentucky ball clay OM4 per L of tank water. These concentrations produced equal Secchi-disk turbidities between treatments (Carolina turbidity tube, Carolina Biological Supply Company, Burlington, NC, USA). In the first week, twelve tanks received greenwater and six tanks received claywater (18 tanks total). On day 8, six of the tanks that had previously received greenwater were switched to claywater. The six tanks that were switched to claywater were selected so that the average body weight at switch-over was equal between the G → G and G → C treatments. 2.1.1. Sampling Fifteen larvae were weighed together for each tank on day 8. Larvae were removed from each tank, overdosed in MS-222, weighed wet, and then weighed again after drying overnight in a 110 °C oven. That weight was divided by 15 to arrive at “average larval dry weight.” On day 15, tanks were drained, survivors were counted, and all survivors were bulk-weighed after drying overnight for each tank (“total dry biomass”). Quantification of survivors required stressful larval handling and was therefore only done at the end of the experiment. 2.1.2. Statistics Separate analyses were conducted to isolate effects during weeks one and two. To isolate the effect of claywater versus greenwater during week one, C → C and G → C treatments were compared with Wilcoxon tests in terms of survival after week two and total dry biomass after week two. Since these two treatments both used claywater during week two, any difference could be attributed to the use of claywater versus greenwater in week one. At the end of week one, six tanks had contained claywater and 12 tanks had contained greenwater. We conducted a Wilcoxon test to compare average larval dry weights between the six claywater and 12 greenwater tanks. To isolate the effect of claywater versus greenwater during week two, G → G and G → C treatments were compared with Wilcoxon tests in terms of growth during week two, survival after week two, and total dry biomass after week two. Growth during week two was calculated for each tank as the difference in average larval dry weights between the end of weeks 1 and 2. Since these two treatments both used greenwater in week one, any difference could be attributed to the use of claywater versus greenwater in week two. 2.2. Experiment 2—what is the best clay concentration during week two? Three different clay concentrations were compared in an eight-day rearing trial using one-week-old larvae. The larvae had initially been stocked at first-feeding from three broodstock crosses into a 2000 L holding tank in greenwater. After eight days, 250 larvae were transferred into each of 18 tanks (37 L) for the experiment. The 18 tanks were evenly divided among low, medium, and high clay concentration treatments: 3, 9, and 15 mg clay per L tank water, respectively (n = 6 tanks per treatment). A nephelometer (HI 93703, Hanna Instruments, Woonsocket, RI, USA) measured turbidity to be 2.4, 11.1, and 13.9 NTU in the low, medium, and high concentration treatments, respectively. 2.2.1. Sampling For each tank on day four, an observer counted the number of larvae visible from the surface that were touching tank walls (wall-nosing) and the number that were not touching tank walls. Counts were made with a hand tally counter and took about ten seconds per tank. On the eighth day, the tanks were drained and larvae counted. All surviving larvae were weighed with the same methods used in Experiment 1.
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2.2.2. Statistics The percentage of larvae that were wall-nosing was compared among treatments with a Kruskal-Wallis test and followed by pairwise Bonferroni-corrected Wilcoxon tests. Survival, total dry biomass, and average larval dry weight were compared among treatments with Kruskal-Wallis tests.
2.3.1. Sampling For each tank on day four, an observer quantified the number of larvae visible from the surface that were touching tank walls (wall-nosing) and the number that were not touching tank walls. On the eighth day, the tanks were drained and larvae enumerated. All surviving larvae were weighed with the same methods used in Experiment 1.
2.3. Experiment 3—does greenwater have additional benefits beyond turbidity during week one?
2.3.2. Statistics The percentage of larvae that were wall-nosing was compared among treatments with a Kruskal-Wallis test. Survival, total dry biomass, and average larval weight were compared among treatments with Kruskal-Wallis tests and followed by pairwise Bonferroni-corrected Wilcoxon tests.
Two-hundred first-feeding larvae from one broodstock cross and 90 first-feeding larvae from a second broodstock cross were stocked into each 37 L experimental tank (290 total larvae in each tank). The larvae were reared under three treatment conditions for one week: 1) low concentration of claywater (“Low C,” n = 6 tanks, 10.5 mg clay per L tank water), 2) high concentration of claywater (“High C,” n = 6 tanks, 13.5 mg clay per L tank water), 3) low concentration of claywater plus greenwater (“Low C + G,” n = 6 tanks, 10.5 mg clay and 0.0235 mL algae per L tank water). The High C and Low C + G treatments had equal turbidity as measured by the nephelometer. Turbidity levels were chosen based on results from Experiment 2, which suggested no differences in growth or survival between the 9 and 15 mg clay per L clay concentrations, despite behavioral differences.
3. Results 3.1. Experiment 1 Tanks with greenwater during week one (G → C) had higher survival and higher total dry biomass at the end of the experiment than tanks with claywater during week one (C → C, Fig. 1a–b, survival: Z = 2.01, P b 0.05; total dry biomass: Z = 2.00, P b 0.05). The G → C and G → G treatments did not differ from the C → C treatment in average larval dry weight at the end of week one (Fig. 1c, Z= − 1.37, P N 0.05).
Fig. 1. Experiment 1, week-one effect (a–c) and week-two effect (d–f) of clay versus algae on mean (+SE) survival, total dry biomass, and larval dry weight over two weeks. Tanks received either clay or algae during week one, then clay during week 2. Experiment began with first-feed larvae. Symbols represent individual data points (tanks).
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Survival and total dry biomass did not differ between G → C and G → G treatments (Fig. 1d–e; survival: Z = − 0.72, P N 0.05; total dry biomass: Z = − 0.88, P N 0.05). Tanks with claywater during week two (G → C) had higher growth during week two than tanks with greenwater during week two (G → G, Fig. 1f; Z = −2.32, P b 0.05). 3.2. Experiment 2 Higher clay concentrations were associated with less wall-nosing (Fig. 2; H = 14.76, P b 0.001). All treatments differed from each other (Bonferroni-corrected post-hoc comparisons: 20–60 Z = − 2.81, P b 0.05; 20–100 Z = 2.81, P b 0.05; 60–100 Z = 2.64, P b 0.05). There were no differences in survival, total dry biomass, or average larval dry weight among treatments (Fig. 3a–b; survival: H = 4.16, P N 0.05; total dry biomass: H = 3.89, P N 0.05; average larval dry weight: H = 0.11, P N 0.05). 3.3. Experiment 3 Wall-nosing did not differ among Low C, High C, and Low C + G treatments (H = 2.21, P N 0.05). Survival, total dry biomass, and average larval dry weight were greater in the “Low C + G” treatment than in the “Low C” and “High C” treatments (P b 0.05, Fig. 4a–c, Table 1). 4. Discussion In the current study, experiments showed that the suitability of greenwater versus claywater can vary as a function of time, within a single species. In the first week of the two-week experiment, greenwater led to greater survival than claywater. However, switching to claywater at the beginning of the second week led to greater growth than when larvae were kept on greenwater for both weeks. These data suggest that claywater is not only a cheaper alternative to greenwater but can also produce larger fish, provided that it does not replace greenwater too early in rearing. Daugherty (2013) found that the effects of greenwater versus claywater on growth and survival varied between cobia (Rachycentron canadum) and yellowtail kingfish (Seriola lalandi). Most published studies report that claywater can produce equal or better growth and survival than greenwater (Attramadal et al., 2012; Bjornsdottir, 2010; Stuart et al., 2015), although it is unknown whether studies finding poorer claywater performance are less likely to be published.
Fig. 2. Experiment 2, mean (+SE) number of larvae wall-nosing versus swimming normally in open water in low, medium, and high concentration claywater. Black bars represent larvae swimming in open water. Gray bars represent wall-nosing larvae.
Fig. 3. Mean (+ SE) survival (a) and total dry biomass (b) in low, medium, and high concentration claywater after one week. Experiment began with one-week-old larvae. Symbols represent individual data points (tanks).
In the first week of Experiment 1, greenwater promoted greater survival and greater total dry biomass than claywater under conditions of equal turbidity. The higher survival during week one in Experiment 1 may have been caused by the absence of harmful properties of clay, beneficial properties of greenwater, or both. Clay exposure in week one could have damaged young developing larval gills or guts at their earliest and most sensitive stages (Hess et al., 2015; Muncy et al., 1979), and algae might provide probiotics that seed larval guts with beneficial bacteria (Verschuere et al., 2000). The additive experiment (Experiment 3) tested whether greenwater has benefits beyond turbidity effects during week 1. The “Low C” treatment (10.5 mg clay per L tank water) had the same amount of clay as the “Low C + G” treatment, but the latter had better growth and survival, showing that algae leads to better growth and survival even when clay concentration is kept constant. One potentially confounding factor is that turbidity was greater in the “Low C + G” treatment than the “Low C” treatment, but the “High C” treatment (13.5 mg clay per L tank water) controlled for this. Turbidity in “High C” was equal to “Low C + G,” yet growth and survival were equal between “High C” and “Low C” treatments, indicating that the greater performance of “Low C + G” was due to the presence of algae and not to higher turbidity. These results do not preclude the possibility that clay or algae may each have some harmful effects during the first week of larval rearing; they just suggest that, controlling for turbidity effects, there are benefits of algae over clay beyond those attributable to turbidity. Beyond showing a beneficial effect of algae, the additive experiment shows that a clay and algae mixture could potentially be applied during
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Fig. 4. Mean (+SE) survival (a), total dry biomass (b), and larval dry weight (c) in lowconcentration clay (Low C), high-concentration clay (High C), and low-concentration clay plus algae (Low C + G) after one week. Experiment began with first-feed larvae. Symbols represent individual data points (tanks).
the first week after first feeding. Dose-response experiments for algae in “Low C + G” mixtures could identify the minimum concentration of algae that can be used to maintain algal benefits while minimizing algal expense and organic input.
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Algae could benefit larval growth and survival in week 1 through a variety of potentially compatible mechanisms. There might be a probiotic effect early in larval rearing where larval guts are seeded with beneficial bacteria (Verschuere et al., 2000). Algae could also stimulate gut enzymes (Cahu et al., 1998) or feeding in young larvae. Algae can release chemical compounds that have been shown to stimulate feeding behavior in juvenile and adult fish and birds (Charlson et al., 1987; DeBose et al., 2008; Nakajima et al., 1990; Nevitt et al., 1995). These algal compounds might also stimulate feeding in larval fish, which could be particularly important as young larvae learn to eat and capture prey. Larvae might also be able to directly consume algae (van der Meeren et al., 2007), though ingestion of large amounts of algae would be more likely if we had used larger algae that could have been captured by filter-feeding larval gill rakers (van der Meeren, 1991). These hypotheses require further experimentation. Previous experiments have shown that claywater tanks have less bacterial growth than greenwater tanks (Attramadal et al., 2012; Bjornsdottir, 2010; Daugherty, 2013; Stuart et al., 2015). This bacterial mechanism may be consistent with our observation that claywater did not show a benefit until the second week of larval rearing. In the first week of rearing, perhaps bacterial levels were low even in greenwater treatments because tanks and larvae were relatively clean when larvae were first introduced, and bacteria concentrations in tanks and on larvae may not have had enough time to increase to a detrimental level. During the second week, perhaps bacterial concentrations increased to the point where bacteria-reducing properties of clay led to greater larval growth in the claywater treatment. Future sablefish experiments that couple a time-series of microbial sampling with performance measures would be informative. Understanding the beneficial effects, harmful effects, and temporal dynamics of these effects of clay and algae may also help us understand and predict when claywater or greenwater will produce higher growth or survival. For example, species may differ in their ability to tolerate higher loads of harmful bacteria or in the degree to which algae stimulates feeding. If there is a species that is naturally more tolerant of higher bacterial levels, then a claywater-induced reduction in bacterial loads may not be a benefit, and greenwater might produce higher growth or survival. Or if there is a species that is a naturally voracious feeder, then any feeding stimulants potentially released by algae may not be important, and claywater might then produce higher growth or survival. The type of clay that is used could affect results, since different clay types can differ in antibacterial properties (Morrison et al., 2014; Williams et al., 2011), and can differ in particle size and thus have different effects on larval vision or larval gills and guts. This study used Kentucky OM4 because this type of clay has been commonly used in previous studies and in commercial aquaculture, but future studies on other clay types would be informative. Understanding these mechanisms might also raise awareness of potential methodological influences on results. For example, methodological details such as stocking density, the frequency of tank cleaning, the duration of the experiment, rearing temperature, and larval age might determine whether claywater or greenwater is found to produce greater growth or survival in a particular experiment or rearing situation (e.g. see Daugherty, 2013). In this study with sablefish, a one-week experiment stocked with first-feed larvae would conclude that greenwater produces greater
Table 1 Effect of low-concentration clay (Low C), low-concentration clay plus algae (Low C + G), and high-concentration clay (High C) on survival, total dry biomass, and average dry weight after one week. Experiment began with first-feed larvae. Response
Survival Total biomass Average weight
Overall effect
Bonferroni-corrected pairwise comparisons
All treatments
Low C + G vs Low C
Low C + G vs High C
Low C vs High C
H = 9.06, P b 0.05 H = 10.88, P b 0.01 H = 9.95, P b 0.01
Z = 2.49, P b 0.05 Z = 2.65, P b 0.05 Z = −2.65, P b 0.05
Z = −2.49, P b 0.05 Z = −2.81, P b 0.05 Z = −2.46, P b 0.05
Z = 0.24, P N 0.05 Z = 0.24, P N 0.05 Z = 0.10, P N 0.05
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survival than claywater, whereas a one-week experiment stocked with one-week-old larvae might conclude that claywater produces better growth with no survival differences. Further experiments are needed to determine whether claywater leads to greater growth or survival than greenwater from weeks three to five. After week five, turbid water is no longer needed for normal behavior (MC, personal observations). Clay provides a significant cost savings over algae. Currently, Kentucky OM4 clay retails for $1.89 per kg, whereas Nannochloropsis algae retails for $61.70 per L. Thus, 1 L of greenwater costs 57 times more than 1 L of claywater. Discounts for bulk purchases are likely available for both clay and algae. Clay is typically available for pickup at local pottery supply stores whereas algae must be cultured or shipped frozen from producers. In Experiment 1, 1 L of claywater required 12 × 10−6 kg of clay while 1 L of greenwater required 21 × 10−6 L of algal paste. A typical 5000 L tank used in aquaculture would turn over about six times per day, and use greenwater for 35 days at an algal cost of $1361 per tank. If the tanks were instead switched to claywater on day eight, the expense per tank would be $272.30 for algae in the first seven days plus $19.05 for clay for the remaining 28 days. The experiments in this study suggest that in addition to the significantly cheaper cost, clay can also produce larger and potentially more fish if used at the proper rearing stage. Future studies that define the mechanisms and temporal dynamics of beneficial and harmful effects of clay and algae may enable the creation of “hybrid” seawater additives that combine the benefits of clay and algae while minimizing harmful effects. For example, if clay produces better growth than algae because it does not promote the growth of harmful bacteria, but greenwater releases chemicals that stimulate feeding, then adding algal extracts to clay may allow hatcheries to rear larvae with the benefits of algae (turbidity, feeding stimulants) but without the costs (increased bacterial growth due to high organic input), and achieve higher growth and survival than with either clay or algae alone. Acknowledgements We thank Megan Sorby for discussion and for suggesting that clay might be harmful early in larval rearing. Mike Rust and Ken Webb provided valuable suggestions on beneficial mechanisms of algae and harmful effects of clay. Linda Rhodes and Rachel Poretsky provided expertise on potential bacterial mechanisms. Lyle Britt advised on larval fish vision and tank setup. Jeff Atkins, Rob Endicott, Bill Fairgrieve, Doug Immerman, Andy Jasonwicz, Cort Jensen, Ken Massee, Sean Oden, Jose Reyes-Tomassini, and Tom Wade provided valuable help and advice with live feeds, maintenance, and general husbandry. Funding was provided by the National Oceanic and Atmospheric Administration Fisheries Office of Aquaculture, and by a grant from Washington Sea Grant, University of Washington, pursuant to National Oceanic and Atmospheric Administration Award No. NA14OAR4170078. The views expressed herein are those of the authors and do not necessarily reflect the views of the National Oceanic and Atmospheric Administration or any of its sub-agencies. References Attramadal, K.J.K., Tondel, B., Salvesen, I., Oie, G., Vadstein, O., Olsen, Y., 2012. Ceramic clay reduces the load of organic matter and bacteria in marine fish larval culture tanks. Aquac. Eng. 49, 23–34.
Bjornsdottir, R., 2010. The Bacterial Community During Early Production Stages of Intensively Reared Halibut (Hippoglossus hippoglossus L.). School of Health Sciences, Faculty of Medicine. University of Iceland, Reykjavik, Iceland. Blaxter, J.H.S., 1986. Development of sense organs and behavior of teleost larvae with special reference to feeding and predator avoidance. Trans. Am. Fish. Soc. 115, 98–114. Boehlert, G.W., Morgan, J.B., 1985. Turbidity enhances feeding abilities of larval Pacific herring, Clupea harengus Pallasi. Hydrobiologia 123, 161–170. Cahu, C.L., Infante, J.L.Z., Peres, A., Quazuguel, P., Le Gall, M.M., 1998. Algal addition in sea bass (Dicentrarchus labrax) larvae rearing: effect on digestive enzymes. Aquaculture 161, 479–489. Carton, A.G., 2005. The impact of light intensity and algal-induced turbidity on first-feeding Seriola lalandi larvae. Aquac. Res. 36, 1588–1594. Charlson, R.J., Lovelock, J.E., Andreae, M.O., Warren, S.G., 1987. Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate. Nature 326, 655–661. Cobcroft, J.M., Shu-Chien, A.C., Kuah, M.K., Jaya-Ram, A., Battaglene, S.C., 2012. The effects of tank colour, live food enrichment and greenwater on the early onset of jaw malformation in striped trumpeter larvae. Aquaculture 356, 61–72. Cook, M.A., Massee, K.C., Wade, T.H., Oden, S.M., Jensen, C., Jasonowicz, A., Immerman, D.A., Goetz, F.W., 2015. Culture of sablefish (Anoplopoma fimbria) larvae in four experimental tank designs. Aquac. Eng. 69, 43–49. Daugherty, Z.N., 2013. Effects of Algae Paste Substitutes on the Larval Rearing Performance and Microbial Communities in the Culture of Cobia (Rachycentron canadum) and Yellowtail Kingfish (Seriola lalandi). University of Miami, Dissertation. DeBose, J.L., Lema, S.C., Nevitt, G.A., 2008. Dimethylsulfoniopropionate as a foraging cue for reef fishes. Science 319, 1356. Hess, S., Wenger, A.S., Ainsworth, T.D., Rummer, J.L., 2015. Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: impacts on gill structure and microbiome. Sci. Rep. 5. Kendall, A.W., Matarese, A.C., 1987. Biology of eggs, larvae, and epipelagic juveniles of sablefish, Anoplopoma fimbria, in relation to their potential use in management. Mar. Fish. Rev. 49, 1–13. Morrison, K.D., Underwood, J.C., Metge, D.W., Eberl, D.D., Williams, L.B., 2014. Mineralogical variables that control the antibacterial effectiveness of a natural clay deposit. Environ. Geochem. Health 36, 613–631. Muncy, R.J., Atchison, G.J., Bulkley, R.V., Menzel, B.W., Perry, L.G., Summerfelt, R.G., 1979. Effects of Suspended Solids and Sediment on Reproduction and Early Life of Warmwater Fishes: A Review. Corvallis Environmental Research Laboratory, Corvallis, OR, p. 101. Nakajima, K., Uchida, A., Ishida, Y., 1990. Effect of a feeding attractant, dimethyl-betapropiothetin, on growth of marine fish. Nippon Suisan Gakkaishi 56, 1151–1154. Nevitt, G.A., Veit, R.R., Kareiva, P., 1995. Dimethyl sulfide as a foraging cue for Antarctic Procellariiform seabirds. Nature 376, 680–682. Palmer, P.J., Burke, M.J., Palmer, C.J., Burke, J.B., 2007. Developments in controlled greenwater larval culture technologies for estuarine fishes in Queensland, Australia and elsewhere. Aquaculture 272, 1–21. Papandroulakis, N., Divanach, P., Kentouri, M., 2002. Enhanced biological performance of intensive sea bream (Sparus aurata) larviculture in the presence of phytoplankton with long photophase. Aquaculture 204, 45–63. Stuart, K.R., Drawbridge, M., 2011. The effect of light intensity and green water on survival and growth of cultured larval California yellowtail (Seriola lalandi). Aquaculture 321, 152–156. Stuart, K., Rotman, F., Drawbridge, M., 2015. Methods of microbial control in marine fish larval rearing: clay-based turbidity and passive larval transfer. Aquac. Res. 2015, 1–11. Utne-Palm, A.C., 2002. Visual feeding of fish in a turbid environment: physical and behavioural aspects. Mar. Freshw. Behav. Physiol. 35, 111–128. Utne-Palm, A.C., 2004. Effects of larvae ontogeny, turbidity, and turbulence on prey attack rate and swimming activity of Atlantic herring larvae. J. Exp. Mar. Biol. Ecol. 310, 147–161. van der Meeren, T., 1991. Algae as first food for cod larvae, Gadus morhua L.: filter feeding or ingestion by accident? J. Fish Biol. 39, 225–237. van der Meeren, T., Mangor-Jensen, A., Pickova, J., 2007. The effect of green water and light intensity on survival, growth and lipid composition in Atlantic cod (Gadus morhua) during intensive larval rearing. Aquaculture 265, 206–217. Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64, 655–671. Williams, L.B., Metge, D.W., Eberl, D.D., Harvey, R.W., Turner, A.G., Prapaipong, P., PoretPeterson, A.T., 2011. What makes a natural clay antibacterial? Environ. Sci. Technol. 45, 3768–3773.