Aquaculture research, tập 41, số 1, 2010

Aquaculture Research, 2010, 42, 1^8 doi:10.1111/j.1365-2109.2010.02572.x REVIEW ARTICLE Effects of stocking density and algal concentration on the survival, growth and metamorphosis of Bobu Ivory shell, Babylonia formosae habei (Neogastropoda: Buccinidae) larvae Huaiping Zheng1,2, Caihuan Ke1, Zewen Sun2, Shiqiang Zhou1 & Fuxue Li1 State Key Laboratory of Marine Environmental Science, Department of Oceanography, Xiamen University, Xiamen, China Mariculture Research Center for Subtropical Shell¢sh & Algae, Shantou University, Shantou, China Correspondence: C Ke, State Key Laboratory of Marine Environmental Science, Department of Oceanography, Xiamen University, Xiamen 361005, China E-mail: chke@xmu.edu.cn Abstract Independent and combined e¡ects of stocking density and algal concentration on the survival, growth and metamorphosis of the Bobu Ivory shell Babylonia formosae habei larvae were assessed using a  factorial design with densities of 0.25, 0.5, 0.75, 1.00 and 1.50 larvae mL À and algal concentrations of 5, 10, 15, 20 and 25  104 cells mL À in the laboratory Larval growth, survival and metamorphosis were signi¢cantly a¡ected by both the independent e¡ects of stocking density and algal concentration and by their interaction The highest per cent survival (72.5%) and metamorphosis (49.5%), fastest growth (41.57 mm day À 1) and shortest time to initial metamorphosis (10 days) all occurred at the lowest stocking density and the highest algal concentration Both crowding and food limitation had independently negative impacts on the survival, growth and metamorphosis of larvae, and these negative impacts were further strengthened by the interaction of a higher stocking density and a lower algal concentration Moreover, the results suggest that stocking density and algal concentration obviously played di¡erent roles in determining larval survival and growth To maximize survival and growth, B formosae habei larvae should be reared at a lower stoking density of 0.25 larvae mL À and fed a higher algal concentration of 25  104 cells mL À in large-scale hatchery seed culture r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd Keywords: Babylonia formosae habei larvae, stocking density, Algal concentration, Survival, Growth, Metamorphosis Introduction Temperature, salinity, diet and rearing density are exogenous factors a¡ecting larval growth, settlement and metamorphosis (for a review, see Crisp 1974) Stocking density and algal concentration are two more important factors in£uencing the success of hatchery seed culture for planktotrophic larvae in marine molluscs, because they are easier to manipulate than other environmental factors in arti¢cial larval production systems Therefore, the e¡ects of stocking density or food concentration on larval survival, growth and metamorphosis have been well documented in marine molluscs (Fretter & Montgomery 1968; Pilkington & Fretter 1970; Perron & Turner 1977; Aldana-Aranda, Lucas, Brule, Salguero & Rendon 1989; Pechenik, Eyster, Widdows & Bayne 1990; Hansen 1991; His & Seaman 1992; Pechenik, Estrella & Hammer 1996; Avila, Grenier,Tamse & Kuzirian 1997; Preece, Shepherd, Clarke & Keesing 1997; Doroudi & Southgate 2000; Pechenik, Jarrett & Rooney 2002; Powell, Bochenek, John, Klinck & Hofmann 2002; Daume, Huchette, Ryan & Day 2003; Zhao, Qiu & Qian 2003; Zheng, Ke, Zhou & Li 2005; E¡ects of density and concentration on larvae H Zheng et al Aquaculture Research, 2010, 42, 1^8 Liu, Dong, Tang, Zhang & Xiang 2006; Yan, Zhang & Yang 2006; Mazo¤n-SuaŁstegui, Ru|¤ z-Ru|¤ z, ParresHaro & Saucedo 2008; Raghavan & Gopinathan 2008; Capo, Bardales, Gillette, Lara, Schmale & Serafy 2009; Rico-Villa & Robert 2009) In general, larvae, reared under a lower stocking density or higher food concentration conditions, have higher survival, faster growth and more metamorphosed individuals In contrast, larvae, reared crowing or lower food supply conditions show slower growth and development, less survival and metamorphic success, and a smaller size or a lower energy content at metamorphosis Most of these studies focused on the independent effect of stocking density or food concentration on larval survival, growth and metamorphosis; only a few studies involved their combined e¡ects (Doroudi & Southgate 2000; Powell et al 2002; Mazo¤n-SuaŁstegui et al 2008; Capo et al 2009) In large-scale hatchery seed culture practice, it is important to de¢ne a realistic strategy for maximizing larval growth and survival under optimal stocking density and diet concentration conditions Members of the gastropod genus Babylonia are found only in the Indo-Paci¢c region (Altena, Regteren & Gittenberger1981) Bobu Ivory shell B formosae habei only exists on the SE coast of China and generally inhabits the muddy/sandy subtidal zone at depths of 4^20 m (in summer) or 40^60 m (in winter) (Liu & Xiao 1998) The conch is a large marine gastropod (adult size 50^60 mm), whose meat is an important source of protein and is of considerable dietary and economic signi¢cance to the inhabitants of the coast of southern China In recent years, the Bobu Ivory shell has been widely cultured in the coast of southern China Larvae are generally reared at a density of 0.5 larvae mL À and fed an algal concentration of 10^20  104 cells mL À (Ke, Zheng, Zhu, Zhou & Li 2001; Zheng, Zhu, Ke, Zhou & Li 2001) However, the combined e¡ects of stocking density and algal concentration on larvae have been not studied Determination of their combined e¡ects may be an important step in developing more e⁄cient large-scale hatchery culture techniques for B formosae habei larvae culture Because the factorial design provides a greater precision for assessing the interactions between di¡erent factors and allows the interpolation of interactions at intermediate levels of the factors being tested (Doroudi & Southgate 2000), a  factorial design with densities of 0.25, 0.50, 0.75, 1.00 and 1.50 larvae mL À and algal concentrations of 5, 10, 15, 20 and 25  104 cells mL À was applied in the laboratory And the independent and combined e¡ects of larval density and algal concentration on the survival, growth and metamorphosis of B formosae habei larvae were investigated in the present study Materials and methods Acquisition of larvae Adults of B formosae habei were collected from the coast of Changle, Fujian Province (China), and maintained between 23 and 28 1C in two1m3 aquaria and fed with the razor clam Sinonovacula constricta (L.) Egg capsules containing fertilized eggs were deposited after several days, after which the parents were removed The resulting embryos were maintained for development by changing the seawater daily and by continuous aeration Most larvae escaped from their egg capsules after about week Larvae were isolated on a 200 mm sieve and transferred to 0.45 mm ¢ltered seawater All larvae tested in the experiment were released on the same day (day 0), but not necessarily by the same female Experimental design and larval rearing A  factorial design with stocking densities of 0.25, 0.50, 0.75, 1.00 and 1.50 larvae mL À and algal concentrations of 5, 10, 15, 20 and 25  104 cells mL À was used in the experiment Two replicate glass beakers were used for each treatment, and in all the experimental groups, larvae were initially reared in 400 mL of seawater As soon as the newly hatching veligers (0-day-larvae) were transferred to ¢ltered seawater, they were collected in pipettes and distributed into ¢fty glass beakers Each day, seawater was replaced with fresh 0.45 mm membrane-¢ltered seawater, food was replaced with a fresh diet and beakers were thoroughly cleaned with deionized water during water changes Larval density was maintained as in the original beakers from the beginning to the end of the experiment by adjusting the water volumes with daily water change Larvae were reared at 24^25 1C, 24^26% salinity and a constant photoperiod of 12 L:12 D using a 40 W £uorescent lamp, these being optimal for larval rearing (Ke et al 2001; Zheng et al 2001) Larvae were fed a unialgal diet of Dicrateria zhanjiangensis, this enrichment ensuring the rapid growth of B formosae habei with low mortality r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8 Aquaculture Research, 2010, 42, 1^8 E¡ects of density and concentration on larvae H Zheng et al through metamorphosis (Zheng et al 2001) Algal concentration was microscopically measured using a blood cell counter plate The algae was cultured in Walne’s medium and used when the stock cultures were in the exponential phase To avoid the introduction of algal medium into the larval containers, the stock cultures were centrifuged and the cells were suspended in 0.45 mm membrane-¢ltered seawater before feeding to the larvae (Lucas & Costlow 1979) cent metamorphosis were examined by ANOVA Because data on survival and metamorphosis were presented as percentages of the total amount, an arcsine-transformation was performed before analysis All statistical analyses were performed on a SAS System for windows (SAS 8.0, SAS Institute, Cary, NC, USA), and signi¢cance for all analyses was set to Po0.05 unless noted otherwise Measurement of larval growth and survival To determine larval growth, shell lengths (longest dimension) of 10 larvae from each replicate were randomly measured nondestructively every other day using a microscope equipped with an ocular micrometer (  64) Larvae were pipetted onto a microscope slide in a small volume of water and the water was quickly removed by a pipette to immobilize the larvae, and then shell length was measured After this, the larvae were immediately returned to their containers The average growth rate for each replicate was measured by regressing larval shell length over time for the ¢rst 10 days of larval life for each larva Larval survival was expressed by a percentage of numbers of live 10-old-day larvae to the initial numbers of larvae in each replicate De¢nition of larval metamorphosis Metamorphosis of B formosae habei larvae as described by Zheng et al (2005) using the de¢nition of Pechenik (1980) involves: (1) loss of the larval velum, (2) larval behaviour changing from free swimming to crawling, (3) shell changing from the larval £attenedelliptic pattern to the adult spiral pattern and (4) siphon being extended The time to initial metamorphosis was denoted as developmental duration from day larvae to the day the ¢rst juvenile occurred in each replicate Per cent metamorphosis was the ratio of the total numbers of metamorphosed larvae to the initial numbers of larvae in each replicate Statistic analyses The independent and combined e¡ects of larval density and algal concentration on per cent survival, growth rate, time to initial metamorphosis and per Results Larval survival Analyses of variance (Table 1) demonstrated that larval per cent survival was signi¢cantly a¡ected by the independent e¡ects of both stocking density and algal concentration and by their interaction, and estimation of variance components further indicated that the e¡ect of stocking density was more important than that of algal concentration Larvae survived less with increasing stocking density at the same algal concentration and survived more with increasing algal concentration at the same stocking density Of 10-old-day larvae, 72.5% survived at the lowest larval density and the highest algal concentration combination, which only survived 4.0% at the highest stocking density and the lowest algal concentration combination (Table 2) Larval growth The average growth rates of larvae at di¡erent combinations of stocking density and algal concentration are listed in Table Larvae grew more slowly as the stocking density increased regardless of the algal concentration and with decreasing algal concentration regardless of the stocking density The average growth rate was up to 41.57 mm day À when larvae were cultured at the lowest density of 0.25 larvae mL À and fed the highest algal concentration of 25  104 cells mL À 1, whereas larvae only grew 17.06 mm daily when they were cultured at the highest density of 1.5 larvae mL À and fed the lowest algal concentration of  104 cells mL À Analyses of variance (Table 1) showed that the larval growth rate was not only a¡ected by the independent e¡ects of stocking density and algal concentration but also by their interaction, and the e¡ect of algal concentration was more signi¢cant than that of stocking density r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8 E¡ects of density and concentration on larvae H Zheng et al Aquaculture Research, 2010, 42, 1^8 Table Analyses of variance for traits in larval survival, growth rate, time to initial metamorphosis and per cent metamorphosis of Babylonia formosae habei larvae Traits Source df MS Per cent survival a b aÂb Error a b aÂb Error a b aÂb Error a b aÂb Error 4 16 25 1160.765856 292.964204 10.575150 1.439511 143.984948 336.739468 4.874038 0.329176 34.7026495 104.9052536 5.3123383 2.2554348 535.795754 224.698804 12.519950 0.280943 Growth rate Time to initial metamorphosis Per cent metamorphosis 4 14 23 4 16 25 Variance components (% of total) F 806.36ÃÃà 203.52ÃÃà 7.35ÃÃà 437.41ÃÃà 1022.98ÃÃà 14.81ÃÃà 15.39ÃÃà 46.51ÃÃà 2.36à 1907.14ÃÃà 799.80ÃÃà 44.56ÃÃà 115.01907 28.23891 4.56782 1.43951 13.91109 33.18654 2.27243 0.32918 2.98031 9.39207 2.97286 2.25543 52.32758 21.21789 6.11950 0.28094 (77.06%) (18.92%) (3.06%) (0.96%) (27.99%) (66.77%) (4.57%) (0.66%) (16.93%) (53.36%) (16.89%) (12.81%) (65.45%) (26.54%) (7.65%) (0.35%) a and b represents the stocking density (larvae mL À 1) and the algal concentration (cells mL À 1) respectively ÃPo0.05; ÃÃÃPo0.001 df, degrees of freedom; MS, mean squares Table Per cent survival (%) of Babylonia formosae habei larvae at di¡erent stocking density and algal concentration combinations Stocking density (larvae mL À 1) 0.25 0.50 0.75 1.00 1.50 Algal concentration ( 104 cells mL À 1) 10 15 20 25 35.0 32.0 14.0 8.5 4.0 50.0 38.0 20.0 16.5 7.75 65.0 44.0 26.0 20.0 12.0 70.0 48.5 32.0 26.0 14.0 72.5 52.0 38.0 30.0 20.0 Table Average growth rate (mm day À 1) of Babylonia formosae habei larvae at di¡erent stocking density and algal concentration combinations Stocking density (larvae mL À 1) 0.25 0.50 0.75 1.00 1.50 Algal concentration ( 104 cells mL À 1) Percentage metamorphosis 10 15 20 25 23.85 21.84 21.26 18.52 17.06 28.39 27.27 24.30 22.74 21.00 34.40 31.48 27.77 25.17 24.82 37.08 35.99 31.25 28.24 26.54 41.57 38.18 34.38 34.52 27.98 Per cent metamorphosis listed in Table decreased with increasing stocking density regardless of the algal concentration and increased with increasing algal concentration regardless of the stocking density No larvae survived to metamorphosis when they were cultured at the highest density of 1.5 larvae mL À and fed the lowest algal concentration combination of  104 cells mL À 1, but 49.5% larvae successfully metamorphosed to juveniles when they were cultured at the lowest density of 0.25 larvae mL À and fed the highest algal concen- Time to initial metamorphosis Table presents the time to initial metamorphosis for larvae cultured at di¡erent stocking density and algal concentration combinations Time to initial metamorphosis increased with increasing stocking density at the same algal concentration and reduced with increasing algal concentration at the same stocking density The time to initial metamorphosis was only 10 days when larvae were cultured at a density of 0.25 larvae mL À and fed an algal concentration of 25  104 cells mL À 1, whereas the time to initial metamorphosis was 20^26 days for larvae cultured at the lowest algal concentration of  104 cells mL À Stocking density and algal concentration exerted signi¢cantly independent and combined e¡ects on the time to initial metamorphosis (Table 1) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8 Aquaculture Research, 2010, 42, 1^8 E¡ects of density and concentration on larvae H Zheng et al Table Time to initial metamorphosis (day) of Babylonia formosae habei larvae at di¡erent stocking density and algal concentration combinations 10%, 9% and 5% respectively (Aldana-Aranda et al 1989) Of the ¢ve densities tested, 500 larvae L À yielded the least growth, and the most growth was obtained with 100 larvae L À In the abalone Haliotis rubra, survival at 64 days followed a negative power function of density at settlement (Daume et al 2003) Larvae of the nudibranch mollusk Hermissenda crassicornis grew 4.30 mm day À at the minimum larval density (1larvae mL À 1) and 1.60 mm day at the highest larval density (15 larvae À 1), and stocking density exhibited a clear negative e¡ect on larval growth (Avila et al 1997) The survival of Crassostrea gigas larvae through metamorphosis declined drastically when larval densities were increased above 1larvae mL À 1, and high density reduced the survival of larvae with low growth e⁄ciency (Powell et al 2002) Manila clam Ruditapes philippinarum larvae survived more and grew faster at larvae mL À than those at 15 and 20 larvae mL À (Yan et al 2006) For the California sea hare Aplysia californica larvae, the growth rates declined from approximately 16 mm day À at the lowest stocking densities to about mm d À at the highest, time to competency increased by about two-fold and survival rates declined from about 85% to 5% or less at a density ranging from 0.5 to larvae mL À (Capo et al 2009) All results exhibited an adverse relationship between larval survival/growth and stocking density Therefore, to maximize survival and growth, B formosae habei larvae should be cultured at a lower density in a large-scale hatchery culture Basch (1996) pointed out three possible e¡ects of larval density including: (1) larvae grazing particles to levels where detection or feeding e⁄ciency is reduced, (2) physical interactions between larvae or (3) accumulation of soluble wastes, reducing the feeding times or rates When the stocking density is lower, the density-dependent behaviour in relation to competition for resources such as space and food is slight (McShane1991; Preece et al.1997), and so larvae show higher survival and faster growth When the stocking density is higher, crowding conditions have a signi¢cant impact on larval survival and growth through chemical interactions and mechanical/physical interference such as collisions between swimming larvae (Sprung 1984; Avila et al 1997) and becoming entangled in mucus strings (Hansen1991) Moreover, it is di⁄cult for larvae at a high density to accumulate energy due to competition for food, social stress, and growth inhibitors, even when food is abundant (Crump 1981) Therefore, larvae have lower survival and slower growth when the stocking density is higher Stocking density (larvae mL À 1) 0.25 0.50 0.75 1.00 1.50 Algal concentration ( 104 cells mL À 1) 10 15 20 25 20.0 23.25 26.0 – – 16.0 20.25 22.0 23.0 22.25 14.75 16.75 17.5 19.0 18.75 12.5 15.0 16.75 16.25 18.5 10.5 13.0 15.25 15.75 16.75 ^, no larvae survived to metamorphosis Table Per cent metamorphosis (%) of Babylonia formosae habei larvae at di¡erent stocking density and algal concentration combinations Stocking density (larvae mL À 1) 0.25 0.50 0.75 1.00 1.50 Algal concentration ( 104 cells mL À 1) 10 15 20 25 15.0 3.5 0.25 0.0 0.0 35.5 10.0 4.5 3.0 1.75 39.5 13.75 11.75 5.9 2.8 41.0 19.25 17.75 10.3 3.1 49.5 32.5 27.75 14.8 11.3 tration of 25  104 cells mL À Per cent metamorphosis of larvae was a¡ected not only by the independent e¡ects of stocking density and algal concentration but also by their signi¢cantly combined e¡ect (Table 1) Discussion The independent e¡ects of stocking density on larval survival, growth and metamorphosis Larval culture density is an important factor in£uencing the success of hatchery seed culture of molluscs Although larger larval density may increase the yield of hatchery-produced spat, it may result in reduced growth and survival In the present study, stocking density had striking e¡ects on the survival, growth and metamorphosis of B formosae habei larvae Under the same algal concentration conditions, a high stocking density exerted negative e¡ects on larval survival, growth and metamorphosis Such negative e¡ects of crowding or density stress on larvae have been reported in other molluscs For milk conch Strombus costatus larvae, the survival rate for ¢ve larval densities tested (from100 to 500 larvae L À 1) was17%,18%, r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8 E¡ects of density and concentration on larvae H Zheng et al The independent e¡ects of algal concentration on larval survival, growth and metamorphosis Algal concentration is another important factor in£uencing the success of hatchery seed culture of molluscs Particularly, planktotrophic larvae survive less and grow slower when food is limited or scarce Such negative e¡ects of low algal concentration on larval survival, growth and metamorphosis were also observed in the present study Dicrateria zhanjiangensis has been used as a good unialgal diet to feed B formosae habei larvae, and supported rapid growth and high survival at a high concentration of 20  104 cells mL À (Zheng et al 2001, 2005) Furthermore, no detrimental e¡ect of this alga has been reported on mollusk larvae Therefore, the negative e¡ects of low algal concentration on larvae can be attributed to a de¢ciency in energy gain from food The negative e¡ects of food limitation on larvae have been documented in other molluscs (Fretter & Montgomery 1968; Pilkington & Fretter 1970; AldanaAranda et al 1989; Pechenik et al 1990, 1996; His & Seaman 1992; Strathmann, Fenaux, Sewell & Strathmann 1993; Avila et al 1997; Rico-Villa & Robert 2009) It is known that planktotrophic larvae feed on phytoplankton (and possibly other organic material in suspension) and are dependent on a net energy gain from such food for successful growth and development From the point of view of the individual’s physiological energetics, the energy and nutrients gained by the animal (in this case the larva) from the environment are distributed among the various metabolic requirements of maintenance, movement and growth (Bayne 1983) The energy and nutrients gained by the larvae from food are not able to meet the various normal metabolic requirements when food is scarce, and so it is inevitable that larvae survive less and grow slower Therefore, to maximize survival and growth, B formosae habei larvae should be fed a higher algal concentration in a large-scale hatchery culture Interaction of stocking density and algal concentration on larvae In general, the e¡ects of stocking density or algal concentration on the growth and survival of planktotrophic larvae have been studied for each factor separately, and reports on their combined e¡ects have been very scarce Doroudi and Southgate (2000) found that the interaction of algal ration and larval Aquaculture Research, 2010, 42, 1^8 density did not a¡ect the growth or the survival of 7or 20-day-old black-lip pearl oyster Pinctada margaritiferia larvae However, Powell et al (2002) found that a number of di¡erent combinations of culture conditions including stocking density, algal concentration, feeding frequency and food quality generated very complex interactions for the growth and development of Paci¢c oyster C gigas larvae In the present study, the combined e¡ects of stocking density and algal concentration on the growth and survival of B formosae habei larvae were very common Larvae survived more, grew faster and metamorphosed earlier at a lower stocking density and a higher algal concentration combination, whereas larvae survived less, grew slower and metamorphosed later at a higher stocking density and a lower algal concentration combination According to the present results, larvae should be cultured at a density of 0.25 larvae mL À and fed an algal concentration of 25  104 cells mL À in order to maximize the survival and growth in B formosae habei hatchery culture practice Di¡erence between the e¡ects of stocking density and algal concentration on larval survival and growth A more interesting and important ¢nding from the present study is that stocking density and algal concentration obviously exert di¡erent impacts on larval survival and growth, that is, stocking density played a more important role than algal concentration in determining larval survival, whereas algal concentration played a more important role than stocking density in determining larval growth This result has been not reported in those previous studies From an ecological point of view, it is reasonable to draw such conclusions because larval density and algal concentration represent two di¡erent types of factors ^ spatial and nutritional Competition for space in a crowded living space with an increase in physical collision or interference may be a major and direct factor resulting in the lower survival of larvae, whereas an increase in physical interference in a crowded living space may be a causal or an indirect factor resulting in slow larval growth by reducing feeding e⁄ciency (Rasheed & Bull 1992) This can be supported by the fact that percentage survival was very low under higher density conditions even in the presence of abundant food resources The energy and nutrients gained by larvae may be reallocated to maintain survival ¢rst rather than growth when r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8 Aquaculture Research, 2010, 42, 1^8 E¡ects of density and concentration on larvae H Zheng et al food is scarce (Pechenik et al 1996) Therefore, competition for nutrition during food limitation may be the principal factor resulting in slow larval growth This can be supported by larvae surviving longer, combined with very slow growth, at a low food concentration in the present study or when fully deprived of food (Zheng et al 2005) In conclusion, both stocking density and algal concentration independently had signi¢cant impacts on the survival and growth of B formosae habei larvae; their combined e¡ects were also signi¢cant Larvae survived more, grew faster and metamorphosed earlier at a lower stocking density and a higher algal concentration combination, whereas larvae survived less, grew slower and metamorphosed later at a higher stocking density and a lower algal concentration combination In a large-scale hatchery seed culture of B formosae habei, larvae should be reared at a lower stocking density of 0.25 larvae mL À and fed a higher algal concentration of 25  104 cells mL À in order to maximize larval survival and growth Wilbur), pp 299^343 Academic Press, New York, NY, USA Capo T.R., Bardales A.T., Gillette P.R., Lara M.R., Schmale M.C & Serafy J.E (2009) Larval growth, development, and survival of laboratory-reared Aplysia californica: effects of diet and veliger density Comparative Biochemistry and Physiology Part C 149, 215^223 Crisp D.J (1974) Factors in£uencing the settlement of marine invertebrate larvae In: Chemoreception in Marine Organisms (ed by P.T Grant & A.M Mackie), pp 177^263 Academic Press, NewYork, NY, USA Crump M.L (1981) Energy accumulation and amphibian metamorphosis Oecologia 49,167^169 Daume S., Huchette S., Ryan S & Day R.W (2003) Nursery culture of Haliotis rubra: the e¡ect of cultured algae and larval density on settlement and juvenile production Aquaculture 236, 221^239 Doroudi M.S & Southgate P.C (2000) The in£uence of algal ration and larval density on growth and survival of blacklip pearl oyster Pinctada margaritifera (L.) larvae Aquaculture Research 31, 621^626 Fretter V & Montgomery M.C (1968) The treatment of food by some prosobranch veligers Journal of the Marine Biological Association of the United Kingdom 48, 499^520 Hansen B (1991) Feeding behaviour in larvae of the opisthobranch Philine aperta II Food size spectra and particle selectivity in relation to larval behaviour and morphology of the velar structures Marine Biology 111, 263^270 His E & Seaman M.N.L (1992) E¡ects of temporary starvation on the survival, and on subsequent feeding and growth, of oyster (Crassostrea gigas) larvae Marine Biology 114, 277^279 Ke C.H., Zheng H.P, Zhu J.X., Zhou S.Q & Li F.X (2001) E¡ects of temperature on survival, growth and metamorphosis of larvae Babylonia formosae habei (Gastropoda: Buccinidae) Transactions of the Chinese Society of Malacology 9, 70–76 (in Chinese with English abstract) Liu B.Z., Dong B.,Tang B.J., Zhang T & Xiang J.H (2006) Effect of stocking density on growth, settlement and survival of clam larvae, Meretrix meretrix Aquaculture 258, 344^349 Liu D.J & Xiao S (1998) Preliminary study on ecology of Babylonia formosae Journal of Fisheries Science of China 5, 93^96 (in Chinese) Lucas J.S & Costlow J.D (1979) E¡ects of various temperature cycles on the larval development of the gastropod mollusca Crepidula fornicata Marine Biology 51, 111^117 Mazo¤n-SuaŁstegui J.M., Ru|¤ z-Ru|¤ z K.M., Parres-Haro A & Saucedo P.E (2008) Combined e¡ects of diet and stocking density on growth and biochemical composition of spat of the Cortez oyster Crassostrea corteziensis at the hatchery Aquaculture 284, 98^105 McShane P.E (1991) Density-dependent mortality of recruits of the abalone Haliotis rubra (Mollusca: Gastropoda) Marine Biology 110, 385^389 Acknowledgments We thank Dr Haihui Ye and Donghui Guo for their kind help in this experiment.We also thank Ms Kirsty A Mattinson (Cantab MA) and Professor I J Hodgkiss for helping to revise the manuscript This work was supported in part by the Earmarked Fund for Modern Agro-industry Technology Research System (No nycytx-47) and Research Project of Technical Exploitation of Fujian Province (No.: 98 À Z À 8) References Aldana-Aranda D., Lucas A., Brule T., Salguero E & Rendon F (1989) E¡ects of temperature, algal food, feeding rate and density on the larval growth of the milk conch (Strombus costatus) in Mexico Aquaculture 76, 361^371 Altena C.O., Regteren V & Gittenberger E (1981) The genus Babylonia (Prosobranchia, Buccinidae) Zoologische Verhandelingen 188,1^57 Avila C., Grenier S.,Tamse C.T & Kuzirian A.M (1997) Biological factors a¡ecting larval growth in the nudibranch mollusc Hermissenda crassicornis 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Abundance of food a¡ects relative size of larval and postlarval structures of a molluscan veliger Reference: Biological Bulletin 185, 232^239 Yan X.W., Zhang G.F & Yang F (2006) E¡ects of diet, stocking density, and environmental factors on growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum larvae Aquaculture 253, 350^358 Zhao B., Qiu J.W & Qian P.Y (2003) E¡ects of food availability on larval development in the slipper limpet Crepidula onyx (Sowerby) Journal of Experimental Marine Biology and Ecology 294, 219^233 Zheng H.P, Zhu J.X., Ke C.H., Zhou S.Q & Li F.X (2001) E¡ects of three microalgae on survival, growth and metamorphosis of larvae Babylonia formosae habei (Gastropoda: Buccinidae) Transactions of the Chinese Society of Malacology 9, 77–84 (in Chinese with English abstract) Zheng H.P., Ke C.H., Zhou S.Q & Li F.X (2005) E¡ects of starvation on larval growth, survival and metamorphosis of Ivory shell Babylonia formosae habei Altena et al., 1981 (Neogastropoda: Buccinidae) Aquaculture 243, 357^366 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1^8 Aquaculture Research, 2010, 42, 9^13 doi:10.1111/j.1365-2109.2010.02482.x Growth and survival of juvenile lined seahorse, Hippocampus erectus (Perry), at different stocking densities Dong Zhang1,2,Yinghui Zhang2, Junda Lin2 & Qiang Lin2,3 East China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Shanghai, China Vero Beach Marine Laboratory, Florida Institute of Technology,Vero Beach, FL, USA Key Laboratory of Tropical Marine Environmental Dynamics, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China Correspondence: D Zhang, East China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Shanghai 200090, China E-mail: zd_¢t@hotmail.com Abstract The lined seahorse, Hippocampus erectus (Perry), is an important species in both medicinal and aquarium trades The aim of this study was to evaluate the effects of stocking density (1, and individuals L À 1) on the growth performance and survival of the early-stage juvenile H erectus The height (HT), wet weight, weight gain (WG) and speci¢c growth rate (SGR) were a¡ected signi¢cantly by the stocking density during the 40-day study The HT,WG and SGR of the seahorse at and juveniles L À were signi¢cantly higher than that at juveniles L À The survival of juveniles at the three stocking densities was not signi¢cantly di¡erent at day 25 (90.3 Æ 4.5%, 86.7 Æ 4.2% and 86.2 Æ 3.8% for 1, and juveniles L À respectively), but was signi¢cantly di¡erent at day 40 (87.8 Æ 3.9%, 69.6 Æ 4.2% and 52.9 Æ 2.8% for 1, and juveniles L À respectively) For the early-stage juvenile H erectus, we recommend a stocking density of juveniles L À 1, but the density should be reduced to1^2 juveniles L À to avoid reduced and variable growth and high mortality after 25 days Keywords: Hippocampus erectus (Perry), density, growth, survival, seahorse Introduction Although there have been attempts at commercial culturing of seahorses for over 50 years (Zou 1958), r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd signi¢cant breakthroughs in culture techniques have only occurred in the last 10 years This has in part been the result of increasing culture e¡orts since all 33 recognized seahorse species were listed on Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2004) due to overexploitation of the wild populations to meet the growing demand in Chinese medicine and ornamental market (Vincent 1996; Lourie, Vincent & Hall1999) More than10 seahorse species have been reared successfully in captivity, such as Hippocampus abdominalis (Woods 2000a, b; 2005), Hippocampus comes (Job, Buu & Vincent 2006), Hippocampus erectus (Correa, Chung & Manrique 1989; Scarratt 1995; Lin, Lin & Zhang 2008), Hippocampus kuda (Job, Do & Hall 2002; Lin, Lu & Gao 2006; Lin, Gao, Sheng, Chen, Zhang & Lu 2007), Hippocampus reidi (Olivotto, Avella, Sampaolesi, Piccinetti, Ruiz & Carnevali 2008), Hippocampus subelongatus (Payne & Rippingale 2000) and Hippocampus trimaculatus (Sheng, Lin, Chen, Gao, Shen & Lu 2006) Research e¡orts have focused on the e¡ects of food type and feed regimes and various environmental factors, including temperature, salinity, light intensity and photoperiods, on the growth and survival of juvenile seahorses to establish appropriate rearing protocols (e.g Payne & Rippingale 2000; Woods 2000b, 2005; Lin et al 2006, 2008; Olivotto et al 2008) However, the e¡ect of stocking density on the growth and survival of seahorses in the early juvenile stages has received much less attention, although it is recognized Nutritional status of R decussatus juveniles S Fathallah et al Grizzle R.E., Bricelj V.M & Shumway S.E (2001) Physiological ecology of Mercenaria mercenaria In: Biology of the Hard Clam (ed by J.N Kraeuter & M Castagna), pp 305^ 382 Elsevier Science, Amsterdam, the Netherlands Hudson R.G & Isom B.G (1984) Rearing of juveniles of the freshwater mussel (Unionidae) in a laboratory setting Nautilus 98,129^135 Huettel M & Rusch A (2000) Transport and degradation of phytoplankton in permeable sediment Limnology and Oceanography 45, 534^549 Johnson M.W., Rooker J.R., Gatlin D.M & Holt G.J (2002) Effects of variable ration levels on direct and indirect measures of growth in juvenile red drum (Sciaenops ocellatus) Journal of Experimental Marine Biology and Ecology 274, 141^157 Jˇrss K., Bittorf T & Vokler T (1986) In£uence of salinity and food deprivation on growth, RNA/DNA ratio and certain enzyme activities in rainbow trout (Salmo gardneri Richardson) Comparative Biochemistry and Physiology 83B, 425^433 Jˇrss K., Bittorf T.,Vokler T & Wacke R (1987) E¡ects of temperature, food deprivation and salinity on growth, RNA/ DNA ratio and certain enzyme activities in rainbow trout (Salmo gairdneri Richardson) Comparative Biochemistry and Physiology 87B, 241^253 Koueta N., Castro B.G & Boucaud-Camou E (2000) Biochemical indices for instantaneous growth estimation in young cephalopod Sepia o⁄cinalis L Journal of Marine Science 57, 1^7 Lodeiros C.J.M., Fernandez R.L., Bonmati A., Himmelman J.H & Chung k.S (1996) Relation of RNA/DNA ratios to growth for the scallop Euvola (Pecten) ziczac in suspended culture Marine Biology 126, 245^251 Lopez G.R & Holopainen I.J (1987) Interstitial suspensionfeeding by Pisidium spp (Pisidiidae: Bivalvia): a new guild in the lentic benthos? American Malacological Bulletin 5, 21^30 Medhioub M.N (1993) La conchyliculture en Tunisie Projet Tunis/92/002 Direction ge¤ne¤rale de la PeŒche et de l’Aquaculture PNUD/FAO, 83pp Medhioub M.N & LimayemY (1995) E¡et d’un substrat sur la survie et la croissance de la palourde Ruditapes decussatus en phase de pre¤grossissement Bulletin de l’institut National des Scienti¢que Technologique Oce¤ anographiques PeŒche SalammboŒ 22, 33^38 Paterson K.J & Nell J.A (1997) E¡ect of di¡erent growing techniques and substrate types on the growth and survival of the clams Tapes dursatus (Lamarck) and Katelysia rhytiphora (Lamy) Aquaculture Research 28,707^715 146 Aquaculture Research, 2010, 42, 139–146 Puigcerver M (1996) Analysis on individual growth of juvenile carpet shell clams,Tapes decussatus (L.), in three di¡erent rearing conditions employed in Ebro’s Delta, Spain Aquaculture Research 27, 399^404 Reid R.G.B., Mcmahon R.E, Foighil D.O & Finnigan R (1992) Anterior inhalant currents and pedal feeding in bivalves Veliger 35, 93^104 Robinson S.M & Ware D (1988) Ontogenetic development of growth rates in larval Paci¢c herring, Clupea harengus pallasi, measured with RNA/DNA ratios in the Strait of Georgia, British Columbia Canadian Journal of Fish Aquatic Science 45, 1422^1429 Saiz E., Calbet A., Fara A & Berdalet E (1998) RNA content of copepods as a tool for determining adult growth rates in the ¢eld Limnology and Oceanography 43, 465^470 TanakaY., Satoh K.,Yamada H.,TakebeT., Nikaido H & Shiozawa S (2008) Assessment of the nutritional status of ¢eld-caught larval Paci¢c blue¢n tuna by RNA/DNA ratio based on a starvation experiment of hatchery-reared ¢sh Journal of Experimental Marine Biology and Ecology 354, 56^64 Urban E.R & Langdon C.J (1984) Reduction in costs of diets for the American oyster (Crassosstrea virginica) (Gmelin), by the use of non-algal supplements Aquac 38, 277^291 Vrede T., Persson J & Aronsen G (2002) The in£uence of food quality (P:C ratio) on RNA/DNA ratio and somatic growth rate of Daphnia Limnology and Oceanography 47, 487^494 Wagner M., Durbin E & Buckley L (1998) RNA/DNA ratios as indicators of nutritional condition in the copepod Calanus ¢nmarchichus Marine Ecology Progress Series 162, 173^181 Wang S.Y & Stickle W.B (1986) Changes in nucleic acid concentration with starvation in the blue crab Callinectes sapidus Rathbun Journal of Crustacean Biology 6, 49^56 Weiss M.B., Curran P.B., Peterson B.J & Gobler C.J (2007) The in£uence of plankton composition and water quality on hard clam (Mercenaria mercenaria L.) populations across Long Island’s south shore lagoon estuaries (New York, USA) Journal of Experimental Marine Biology and Ecology 345,12^25 Wright D.A & Hetzel E.W (1985) Use of RNA/DNA ratios as an indicator of nutritional stress in the American oyster, Crassostrea virginica Marine Ecology Progress Series 25, 199^206 Yeager M., Cherry D.S & Neves R.J (1994) Feeding and burrowing behaviours of juvenile rainbow mussels, Villosa iris (Bivalvia:Unionidae) Journal of the North American Benthological Society 13, 217^222 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 139^146 Aquaculture Research, 2010, 42, 147^152 doi:10.1111/j.1365-2109.2010.02532.x SHORT COMMUNICATION First report of nodavirus outbreak in cultured juvenile shi drum, Umbrina cirrosa L., in Greece Pantelis Katharios1 & Costas S Tsigenopoulos2 Hellenic Centre for Marine Research (HCMR) Crete, Institute of Aquaculture,Thalassocosmos, Heraklion, Crete, Greece Hellenic Centre for Marine Research (HCMR) Crete, Institute of Marine Biology and Genetics,Thalassocosmos, Heraklion, Crete, Greece Correspondence: P Katharios, Hellenic Centre for Marine Research (HCMR) Crete, Institute of Aquaculture, Thalassocosmos, PO Box 2214, Gournes Pediados, 715 00 Heraklion, Crete, Greece E-mail: katharios@her.hcmr.gr Shi drum, Umbrina cirrosa L is considered an ideal candidate for the diversi¢cation of aquaculture, especially in the Mediterranean region where sea bass and sea bream over-production has led to signi¢cant economic suppression of the industry Shi drum can be easily adapted to rearing conditions, has good growth rates and its £esh is highly appreciated by consumers (Barbaro, Francescon, Bertotto, Bozzato, Di Maria, Patarnello, Furlan & Colombo 2002) Certain issues should be addressed for the successful introduction of a new species to aquaculture and for its ¢nal domestication, among which nutritional requirements, reproduction and health/welfare are the most critical Concerning the latest, U cirrosa has been reported to be susceptible to ¢sh nodavirus (Bovo, Nishizawa, Maltese, Borghesan, Mutinelli, Montesi & De Mas 1999), one of the most pathogenic viruses known causing severe mortalities in a broad range of cultured marine ¢sh species Viruses of the family Betanodaviridae are the causative agents of viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (OIE 2006) The disease has worldwide distribution and the list of the susceptible ¢sh containing more than 30 species is constantly growing (OIE 2006) The disease is characterized by neurological abnormalities (erratic swimming, spiral movements with belly-up) and a distinct vacuolization of the nerve tissue (brain, retina) (OIE 2006) Mortality associated with the disease is severe, reaching 100% depending on age, with younger ¢sh being more susceptible (OIE 2006) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd In this paper, we describe the clinical signs of juvenile shi drums withVNN, including the course of the disease, the associated histopathology, detection of the virus with RT-PCR and con¢rmation by sequence analysis Although the disease has already been reported in shi drum (Comps,Trindade & Delsert 1996; Bovo et al 1999; DallaValle, Negrisolo, Patarnello, Zanella, Maltese, Bovo & Colombo 2001), this is the ¢rst description of the disease in this species containing information about rearing conditions and mortalities su¡ered, and the ¢rst report of nodavirus infection in cultured shi drum in Greece The study was carried out at the facilities of the Institute of Aquaculture, Hellenic Centre for Marine Research, Crete, Greece Eggs were obtained in June 2005 from a 3-year-old broodstock (F1 generation from wild-caught stock from Cyprus), after hormonal induction of spawning using a single GnRHa injection (10 mg kg À 1) (Mylonas, Kyriakou, Sigelaki, Georgiou, Stephanou & Divanach 2004) Larval rearing was performed using the mesocosm technology as described previously (Zaiss, Papadakis, Maingot, Divanach & Mylonas 2006) Following weaning and when ¢sh had reached 0.3 g mean weight at 46 days post hatching (dph), they were transferred to the ongrowing facilities of the Institute One hundred twenty-¢ve thousand ¢sh were distributed in ¢ve 10 m3 tanks Water (salinity: 35 g L À 1) was supplied directly from the sea and from a shallow well at a 50:50 ratio At the time when the ¢rst mortalities were observed (72 dph), water temperature was 22.5 1C, water renewal was 100% per hour and ¢sh 147 Nodavirus in shi drum P Katharios & C S Tsigenopoulos density was 6.5 kg m À (average ¢sh weight 2.6 g) The behaviour of the ¢sh was monitored daily Forty ¢sh of 5.8 Æ 1.8 g mean weight were sampled at the beginning of October (110 dph) when clinical signs of the disease were evident Both sick and apparently healthy ¢sh were sampled Twenty ¢sh were sampled for histological analysis and the rest for RT-PCR Excised brains and eyes from ¢sh exhibiting clinical signs as well as from apparently healthy ¢sh were ¢xed in 4% formaldehyde:1% glutaraldehyde (McDowell & Trump 1976), dehydrated in a 70^95% ethanol series and embedded in glycol methacrylate resin (Technovit 7100, Heraeus Kulzer, Wehrheim, Germany) Serial sections (3^5 mm thick) were obtained using a microtome (Biocut 2035, Reichert Jung, Wetzlar, Germany) with disposable blades After drying, slides were stained with methylene blue/azure II/basic fuchsin (Bennett, Wyrick, Lee & McNeil 1976) and examined by light microscopy For PCR analysis, brain tissues were submerged in liquid nitrogen upon dissection and stored at À 80 1C before RNA extraction Total RNAwas extracted using NucleoSpin RNA II (Macherey-Nagel GmbH, Dˇren, Germany) and the RNA samples were kept at À 80 1C until use One-step RT-PCR was carried out using the RobusT I RT-PCR kit (Finnzymes, Espoo, Finland) with all components mixed in one tube and reverse transcription automatically followed by PCR without additional steps The reactions were set up according to the supplier’s recommendation and mL of the RNA solution was used in a 50 mL reaction, which also contained 10 pmol of each primer and the concentration of MgCl2 was adjusted to 1.5 mM Reverse transcription was carried out for 30 at 48 1C, followed by at 94 1C to inactivate reverse transcriptase; 35 cycles of 95 1C for 30 s, 57 1C for 30 s and 72 1C for 45 s followed by a ¢nal extension step of 10 Following the supplier’s guidelines, positive and negative (without AMV reverse transcriptase) control reactions were prepared using upstream and downstream control primers and RNA supplied with the kit Two pairs of primers were tested: primers VNNV1 (5 -ACACTG GAGTTTGAAATTCA-3 0) and VNNV2 (5 -GTCTTGT TGAAGTTGTCCCA-3 0) were used to amplify a fragment covering 604 bp in the variable region of the coat protein gene [red-spotted grouper NNV-RGNNV type (Dalla Valle et al 2001)] and primers F2 (5 CGTGTCAGTCATGTGTCGCT-3 0) and R3 (5 -CGAGTC AACACGGGTGAAGA-3 0) to amplify a fragment of 426 bp [striped jack NNV-SJNNV type (Nishizawa, Mori, Nakai, Furusawa & Muroga 1994; Oh, Jung, Kim, Rajendran, Kim, Choi, Kim & Kim 2002)] 148 Aquaculture Research, 2010, 42, 147^152 The PCR products were run on a 1.0% agarose gel Tris-acetate^EDTA  and were stained with ethidium bromide to visualize amplicons Bands of expected size were cut and puri¢ed from the gel and cloned following the manufacturer’s guidelines with the pCR II TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) Sequencing was performed using a 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) and in both directions using theT7 and SP6 primers and the Big Dye Terminator v3.0 Cycle Sequencing Kit (Applied Biosystems) For phylogenetic analysis, nucleotide sequences of representative betanodavirus strains (TNV, TPNNV, SJNNV, BFNNV and RGNNV) were used (see also Skliris, Krondiris, Sideris, Shinn, Starkey & Richards 2001; Johansen, Sommerset, TÖrud , Korsnes, Hjortaas, Nilsen, Nerland & Dannevig 2004; Cutrin, Dopazo, Thiery, Leao, Olveira, Barja & Bandin 2007; To¡olo, Negrisolo, Maltese, Bovo, Belvedere, Colombo & Valle 2007) Nodavirus sequences were aligned using MEGA 4.0 (Tamura, Dudley, Nei & Kumar 2007) and the neighbourjoining analysis was performed using the Kimura2 parameter model of nucleotide substitution Statistical support for internal nodes was based on 10 000 bootstrap replicates The a¡ected ¢sh presented clinical signs typical of nodavirus infection, including spiral movement on the surface of the water, erratic and uncoordinated swimming, lethargy at later stages of the disease and swimming with the belly up usually on the surface of the water, darkening of the skin and swim bladder over-in£ation The disease was ¢rst observed in one of the ¢ve on-growing tanks around 70 dph All the ¢sh of the speci¢c tank were a¡ected presenting some of the aforementioned signs Because of the extent of the infection (100% of the population was a¡ected), it was decided to discontinue the culture and kill all the ¢sh of this tank (N 23800, age: 85 dph) However, the clinical signs of the disease appeared in the other four populations weeks later (100 dph) The reason for this delay in the spread of the disease in the other tanks is unclear The mortality thereafter had a gradual increase reaching 36% (estimated for the four remaining populations) months after the ¢rst mortality was recorded (Fig 1) At this point, there was substantial evidence to suspect VNN and it was decided to terminate the culture by killing all the ¢sh in all the tanks in order to avoid horizontal transmission of the disease to other susceptible species hosted in the facility r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 147^152 Aquaculture Research, 2010, 42, 147^152 Nodavirus in shi drum P Katharios & C S Tsigenopoulos 100 110 120 130 140 Time (days post hatching) 150 Cumulative mortality (%) 40 35 30 25 20 15 10 90 Tank 40 35 30 25 20 15 10 40 35 30 25 20 15 10 150 Cumulative mortality (%) Cumulative mortality (%) Cumulative mortality (%) Tank 40 35 30 25 20 15 10 90 100 110 120 130 140 Time (days post hatching) Tank 90 100 120 140 110 130 Time (days post hatching) 150 Tank 90 100 110 130 120 140 Time (days post hatching) 150 Figure Cumulative mortality of the reared shi drum population withVNN It should be noted that this graph represents the mortality recorded in four of the ¢ve on-growing tanks since the ¢sh of the ¢rst tank were killed at day 84 due to the extent of infection Vacuolation of the retina of the eyes was the most consistent histopathological ¢nding in most of the ¢sh examined (Fig 2) Vacuoles of varying size (5^ 40 mm) and shapes were found mostly in the outer nuclear layer Vacuolation to a smaller degree was also observed in the inner nuclear layer a¡ecting mostly the bipolar and the amacrine cells In some cases, vacuoles were also observed in the internal plexiform layer It should be noted that retina of some apparently healthy ¢sh exhibited vacuoles, although much smaller in size and in number No lesions or VNN signs (Munday, Langdon, Hyatt & Humphrey 1992) were observed in the brain samples examined (e.g necrosis, vacuolation, macrophage aggregations, etc.) From the two PCR assays with RGNNVand SJNNV primers, only the ¢rst one yielded an amplicon of the expected size; this was the ¢rst indication of the type of NNV detected in shi drum and refers to both sick and apparently healthy ¢sh Consequently, after cloning and sequencing 16 clones, three di¡erent 605-bplong isolates were identi¢ed: isolate 01 was found in 14 clones and the two others (02 and 03) di¡ered from the major one by only one transition Sequences were submitted to GenBank under accession numbers FJ480948^FJ480950 When ‘blasted’ in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), all three isolates (U cirrosa_NNV_01, U cirrosa_NNV_02, U cirrosa_NNV_03) were grouped with RGNNV-type iso- Figure Resin-embedded and polychrome-stained section of the retina of VNN-a¡ected shi drum juvenile Note the vacuolation of the retina in both external (arrowheads) and internal nuclear layer (arrows) lates For the sake of simplicity, we chose only the RGNNV-type isolates from ¢sh found in European waters to study and show the phylogenetic relationship of 31 isolates All three isolates are clearly grouped with RGNNV isolates and are di¡erent from those reported previously: 95.3% identity with isolate Uc-1 (501bp, AJ277811) of DallaValle et al (2001) and 94.9% with isolate It/24/Sdr (682 bp, AF175517) of Skliris et al (2001) Lastly, two sub-lineages could be identi¢ed in the RGNNV clade; this is concordant with previous results (Thiery, Cozien, De Boisseson, r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 147^152 149 Nodavirus in shi drum P Katharios & C S Tsigenopoulos Kerbart-Boscher & Nevarez 2004; Cutrin et al 2007) Present sequences, however, not appear to cluster with either of the two sub-lineages Based on the clinical signs of the a¡ected ¢sh (erratic and spiral swimming, darkening of the skin, swim bladder over-in£ation), the course of the disease, histopathological lesions, RT-PCR and sequence analysis, we conclude that the aetiological factor of the disease is a virus of the Betanodaviridae family Nodavirus infections have been reported in shi drum from the Mediterranean region (Comps et al 1996; Bovo et al 1999; Dalla Valle et al 2001), but this is the ¢rst detailed description of the disease in this species VNN was ¢rst reported in Greece a¡ecting juvenile and adult sea bass reared in £oating cages in 1995 (Le Breton, Grisez, Sweetman & Ollevier 1997) Since then, the disease became the most serious threat to European sea bass aquaculture The Greek hatcheries taking into consideration this threat have applied preventive measures against the disease including screening of broodstock with ELISA and PCR, and water disinfection These measures have proven successful because the incidences of nodavirus infection in sea bass have signi¢cantly decreased (personal communication with hatchery managers) However, the need for diversi¢cation of aquaculture production has led to the introduction of new ¢sh species that can be susceptible to VNN, a possibility that may have been overlooked by the producers Furthermore, restrictions in water resources and rearing sites made aquaculturists look to freshwater as an alternative water source for sea bass rearing Therefore, since the ¢rst report of VNN in Greece in 1995, other species have been reported to be a¡ected by nodavirus including gilthead sea bream, Sparus aurata (Bitchava, Xylouri, Fragkiadaki, Athanassopoulou, Papanastassopoulou & Sabatakou 2007), dusky grouper, Epinephelus marginatus (Katharios, Papadakis, Prapas, Dermon, Ampatzis & Divanach 2005), European sea bass grown in freshwater (Athanassopoulou, Billinis, Psychas & Karipoglou 2003) and sturgeon, Acipenser sp (Athanassopoulou, Billinis & Prapas 2004) In this study, the onset of the disease (¢sh age 70 dph) was observed about a month after transport of the ¢sh from mesocosm tanks of the hatchery to the on-growing unit of the same facility The ¢sh at that time were at the early juvenile stage, which is a critical period characterized by high losses in ¢sh affected by the disease especially in susceptible species (Munday, Kwang & Moody 2002) Nodaviruses can 150 Aquaculture Research, 2010, 42, 147^152 spread both horizontally between ¢sh and vertically from broodstock to o¡spring (Munday et al 2002) In our case, the water of the hatchery is used untreated for both broodstock and juvenile ¢sh; thus, the possibility of virus transport from the sea water cannot be excluded The spawners have been obtained from wild stock originated from Cyprus, and they were raised in our facilities without presenting any signs of the disease in the past Following this outbreak, blood samples were taken from all spawners and serums were sent to Aquatic Diagnostics (Stirling, Scotland) where they were analysed by ELISA for the presence of anti-Nodavirus antibodies The results were negative for all ¢sh The mode of infection is therefore more likely to be horizontal It is not clear, however, why there was a delay in the onset of the disease between tanks It is interesting that in terms of histopathology, the lesions were con¢ned to the retina of the eye; no pathological alterations were observed in the brain In the ¢rst published record of the disease in shi drum (Comps et al 1996), the authors reported that the ¢sh presented signs of the disease, the virus was detected using in situ hybridization in tissue sections of mesencephalon and medulla oblongata; however, there was no vacuolation or necrosis in the brain tissue As stressed in the review of Munday et al (2002), it is not clear if the authors examined the eyes of shi drum for possible histopathological alterations Comps et al (1996) suggested that there might be a di¡erent pattern of host’s tissue alteration depending on species Later, Grotmol, Bergh and Totland (1999) studying the pathomorphological and immunohistochemical characteristics of the disease in Atlantic halibut suggested that the spread of the virus could be either from the eye or the vascular bed to the brain The opposite route (from brain to the retina of the eye) was suggested in nodavirus-infected spotted wol⁄sh, Anarichas minor (Johansen, Amundsen, Dannevig & Sommer 2003) The absence of lesions in the brain is in agreement with the earlier report of the disease in shi drum (Comps et al 1996) Immunohistochemical analysis would probably shed light on the route of infection regarding this study; however, this was not possible due to inadequate ¢xation and tissue processing In the present study, we used RT-PCR and sequencing to identify the type of betanodavirus isolates in U cirrosa.We used both a more ‘universal’ primer pair for nodaviruses (VNNV, Dalla Valle et al 2001) and a speci¢c primer pair for SJNNV (Nishizawa et al.1994) as recent studies suggest that nodavirus isolates r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 147^152 Aquaculture Research, 2010, 42, 147^152 Nodavirus in shi drum P Katharios & C S Tsigenopoulos Acknowledgments We thank S Stefanakis and M.Vassilakis for technical support in the rearing of the ¢sh, M Papadaki for histology and S Kollias for RT-PCR References Figure Unrooted neighbour-joining phylogenetic tree resulting from the analysis of 612 bp of nervous necrosis virus partial gene for coat protein coming from 31 ¢sh nodaviruses (new isolates: U cirrosa_NNV_01, U cirrosa_NNV_02, U cirrosa_NNV_03) Numbers on nodes are bootstrap support values after 10 000 replicates and the length of branches is proportional to the number of nucleotide substitutions (see scale bar) identi¢ed so far in Mediterranean waters are either RGNNV- or SJNNV-type betanodaviruses (Thiery et al 2004; Cutrin et al 2007) The use of SJNNV-speci¢c primers failed to amplify a PCR product, whereas the VNNV pair of primers resulted in a PCR product, which after sequencing proved to be an RGNNV-type isolate Following a phylogenetic analysis, the topology obtained (Fig 3) is consistent with the abovementioned result and U cirrosa new isolates are clustered with others of the RGNNV-type strains Furthermore, our analysis showed that these isolates are genetically di¡erent from those already obtained in U cirrosa (95.1% mean identity) and form a branch that does not group with any of the two sub-lineages also identi¢ed in RGNNV-type isolates One possible explanation for this could come from what we observe in Fig.3; it appears that the clustering of the different nodavirus haplotypes is not species speci¢c but most probably related to di¡erent strain outbreaks in space (geographic region) and time Athanassopoulou F., Billinis C., Psychas V & Karipoglou K (2003) Viral encephalopathy and retinopathy of Dicentrarchus labrax (L.) farmed in fresh water in Greece Journal of Fish Diseases 26, 361^365 Athanassopoulou F., Billinis C & Prapas T (2004) Important disease conditions of newly cultured species in intensive freshwater farms in Greece: ¢rst incidence of nodavirus infection in Acipenser sp Diseases of Aquatic Organisms 60, 247^252 Barbaro A., Francescon A., Bertotto D., Bozzato G., Di Maria I., Patarnello P., Furlan F & Colombo L (2002) More e¡ective induction of spawning with long-acting GnRH agonist in the shi drum, Umbrina cirrosa L (Sciaenidae, Teleostei), a valuable candidate for Mediterranean mariculture Journal of Applied Ichthyology 18,192^199 Bennett H.S., Wyrick A.D., Lee S.W & McNeil J.H (1976) Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains Stain Technology 51,71^97 Bitchava K., Xylouri E., Fragkiadaki E., Athanassopoulou F., Papanastassopoulou M & Sabatakou O (2007) First incidence of clinical signs of nodavirus infection in sea bream, Sparus auratus L Israeli Journal of Aquaculture ^ Bamidgeh 59, 3^9 Bovo G., Nishizawa T., Maltese C., Borghesan F., Mutinelli F., Montesi F & De Mas S (1999) Viral encephalopathy and retinopathy of farmed marine ¢sh species in Italy Virus Research 63, 143^146 Comps M., Trindade M & Delsert C (1996) Investigation of ¢sh encephalitis viruses (FEV) expression in marine ¢shes using DIG-labelled probes Aquaculture 143, 113^ 121 Cutrin J.M., Dopazo C.P.,Thiery R., Leao P., Olveira J.G., Barja J.L & Bandin I (2007) Emergence of pathogenic betanodaviruses belonging to the SJNNV genogroup in farmed ¢sh species from the Iberian Peninsula Journal of Fish Diseases 30, 225^232 Dalla Valle L., Negrisolo E., Patarnello P., Zanella L., Maltese C., Bovo G & Colombo L (2001) Sequence comparison and phylogenetic analysis of ¢sh nodaviruses based on the coat protein gene Archives ofVirology 146,1125^1137 Grotmol S., Bergh O & Totland G.K (1999) Transmission of viral encephalopathy and retinopathy (VER) to yolk-sac larvae of the Atlantic halibut Hippoglossus hippoglossus: occurrence of nodavirus in various organs and a possible route of infection Diseases of Aquatic Organisms 36, 95^ 106 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 147^152 151 Nodavirus in shi drum P Katharios & C S Tsigenopoulos Johansen R., Amundsen M., Dannevig B.H & Sommer A.I (2003) Acute and persistent experimental nodavirus infection in spotted wol⁄sh Anarhichas minor Diseases of Aquatic Organisms 57, 35^41 Johansen R., Sommerset I., TÖrud B., Korsnes K., Hjortaas M.J., Nilsen F., Nerland A.H & Dannevig B.H (2004) Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.) Journal of Fish Diseases 27, 591^601 Katharios P., Papadakis I.E., Prapas A., Dermon C.R., Ampatzis K & Divanach P (2005) Mortality control of viral encephalopathy and retinopathy in 01 grouper Epinephelus marginatus after prolonged bath in dense Chlorella minutissima culture Bulletin of the European Association of Fish Pathologists 25, 28^31 Le Breton A., Grisez L., Sweetman J & Ollevier F (1997) Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax (L.) Journal of Fish Diseases 20, 145^151 McDowell E.M & Trump B.F (1976) Histologic ¢xatives suitable for diagnostic light and electron microscopy Archives of Pathology and Laboratory Medicine 18, 405^414 Munday B.L., Langdon J.S., Hyatt A & Humphrey J.D (1992) Mass mortality associated with a viral-induced vacuolating encephalopathy and retinopathy of larval and juvenile barramundi, Lates calcarifer Bloch Aquaculture 103, 197^ 211 Munday B.L., Kwang J & Moody N (2002) Betanodavirus infections of teleost ¢sh: a review Journal of Fish Diseases 25,127^142 Mylonas C.C., Kyriakou G., Sigelaki I., Georgiou G., Stephanou D & Divanach P (2004) Reproductive biology of the shi drum (Umbrina cirrosa) in captivity and induction of spawning using GnRHa Israeli Journal of Aquaculture ^ Bamidgeh 56,75^92 Nishizawa T., Mori I.K., Nakai T., Furusawa I & Muroga K (1994) Polymerase chain reaction (PCR) ampli¢cation of 152 Aquaculture Research, 2010, 42, 147^152 RNA of striped jack nervous necrosis virus (SJNNV) Diseases of Aquatic Organisms 18,103^107 Oh M.J., Jung S.J., Kim S.R., Rajendran K.V., KimY.J., Choi T.J., Kim H.R & Kim J.D (2002) A ¢sh nodavirus associated with mass mortality in hatchery-reared red drum, Sciaenops ocellatus Aquaculture 211, 1^7 OIE (2006) Viral encephalopathy and retinopathy In: Manual of DiagnosticTests forAquatic Animals (ed by T Nakai), pp.131^141 OIE, Paris Skliris G.P., Krondiris J.V., Sideris D.C., Shinn A.P., Starkey W.G & Richards R.H (2001) Phylogenetic and antigenic characterization of new ¢sh nodavirus isolates from Europe and Asia.Virus Research 75, 59^67 Tamura K., Dudley J., Nei M & Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0 Molecular Biology and Evolution 24, 1596^ 1599 Thiery R., Cozien J., De Boisseson C., Kerbart-Boscher S & Nevarez L (2004) Genomic classi¢cation of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-¢sh species speci¢city Journal of GeneralVirology 85, 3079^3087 To¡olo V., Negrisolo E., Maltese C., Bovo G., Belvedere P., Colombo L & Valle L.D (2007) Phylogeny of betanodaviruses and molecular evolution of their RNA polymerase and coat proteins Molecular Phylogenetics and Evolution 43, 298^308 Zaiss M.M., Papadakis I.E., Maingot E., Divanach P & Mylonas C.C (2006) Ontogeny of the digestive tract in shi drum (Umbrina cirrosa L.) reared using the mesocosm larval rearing system Aquaculture 260, 357^368 Keywords: Nodavirus,VER, disease, histology, RT-PCR r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 147^152 Aquaculture Research, 2010, 42, 153^156 doi:10.1111/j.1365-2109.2010.02598.x SHORT COMMUNICATION Sperm motility of Rhamdia quelen studied using computer-assisted analysis by open-source software Eduardo AntoŒnio Sanches1, Robie Allan Bombardelli2, Ronan Maciel Marcos2, Giovano Neumann2, Ce¤sar Pereira Rebechi de Toledo2 & Elizabeth Romagosa3 Aquaculture Centre, State University of Saìo Paulo, Jaboticabal, Saìo Paulo, Brazil State University of West ParanaŁ,Toledo, ParanaŁ, Brazil Fishery Institute ^ APTA, SAA, Saìo Paulo, Saìo Paulo, Brazil Correspondence: E Romagosa, Av Francisco Matarazzo, 455, AŁgua Branca, Saìo Paulo, SP 05001-000, Brazil E-mail: eromagosa@pesca sp.gov.br Fish sperm quality is often assessed using subjective methods, among which progressive sperm motility and sperm vigour are the ones most frequently observed However, these measurements have raised doubts regarding their validation because the values obtained vary considerably, and basically depend on the experience of a single observer (Rurangwa, Kime, Ollevier & Nash 2004) Lately, using speci¢c software, ¢sh sperm analyses have been conducted with computer programs such as the computer-assisted sperm analysis (CASA) (Ravinder, Nasaruddin, Majumdar & Shivaji 1997; Wilson-Leedy & Ingermann 2007) The importance of CASA has been demonstrated in studies on optimization of gametes (Rurangwa et al 2004), conservation of semen (Ravinder et al 1997; Rurangwa, Volckaert, Huyskens, Kime & Ollevier 2001) and as indicators of water pollution (Kime, Van Look, Mcallister, Huyskens, Rurangwa & Ollevier 2001) Di⁄culties in the assessment of sperm motility using the CASA have been observed, such as the short duration of the motility of the spermatozoa after activation, the high cost of system implementation and the process of standardization for the di¡erent species of teleost ¢sh (Wilson-Leedy & Ingermann 2007) Thus, the present study was carried out with the objective of standardizing the method of CASA in jundiaŁ (Rhamdia quelen) using open-source software The experiment was carried out at the Laboratory of Reproduction Technology of Aquacultured Animals (LATRAAC) in the State University of West Para- r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd naŁ (UNIOESTE), located at the Centre of Research in Environmental Aquaculture (CPAA), in Toledo, ParanaŁ, Brazil (24146 048.3100S; 53143 025.7700 W) Fourteen jundiaŁ (R quelen) males, with a mean total weight of 324.00 Æ 73.85 g, which released semen under slight abdominal pressure, were used Individually, the males were hormonally induced with crude carp pituitary extract (CCPE), with a single dose of 2.5 mg CCPE kg À (Bombardelli, M˛rschbÌcher, Campagnolo, Sanches & Syperreck 2006), for increased the sperm dilution The semen was collected after 240 h-degree (sum of water temperature in relation to time) after hormonal induction 4.9 Æ 1.4 mL of semen was collected from each male for the measurement of sperm concentration and sperm motility by one trained observer in an optical microscope 40  ; this motility evaluation was characterized using a subjective method The same semen samples were used to capture video and for further evaluation in software CASA, characterized as an objective method For the determination of sperm concentration, the sperm cell count was performed in a Neubauer hematimetric chamber (Wirtz & Steinmann 2006), using the dilution 1:1000 of semen:bu¡ered formol saline [30% NaCl1.8%; 20% phosphate bu¡er (71% of Na2HPO4 4.33% and 29% of KH2PO4 4.45%); 12.5% formaldehyde 37%; and 37.5% distilled water] In order to obtain a suitable seminal dilution for the capture of the videos, the semen was previously diluted in inactivating solution LATRAAC-N6 (patent 153 Sperm motility of jundiaŁ E A Sanches et al pending) at a proportion of1:500.The semen previously diluted in LATRAAC-N6 was diluted in an activating solution (distilled water) at a proportion of 1:3 Afterwards, 10 mL of the mixture was placed in a mirrored Neubauer chamber (100 mm deep), which was transferred to the light microscope to capture the videos A Basler 602fc camera attached to a trinocular Nikon microscope was used The videos were captured using the software AMCAP (Basler Vision Technologies, Ahrensburg, Germany) at a rate of 60 frames s À The videos were captured in format Ã.avi, edited in the software VIRTUALDUB-1.9.0 (virtualdub.org) and exported as a sequence of images in format Ã.jpg The images corresponding to 1s of video were opened, edited in the software IMAGEJ (National Institutes of Health, USA, http:// rsb.info.nih.gov/ij/) and compiled using the application CASA (University of California and Howard Hughes Medical Institute, USA) Video processing was performed based on the description of the necessary components for the uti- Aquaculture Research, 2010, 42, 153^156 lization of the application CASA, an open-source software available at http://wilson-leedy.com/CASA/ instructions.html The con¢guration of the dialogue box of the application CASA was performed based on the images of the tracks covered by the spermatozoa that had been generated by the application (Fig 1a) For the validation of those images, they were compared with the images generated by the software IMAGEJ in Image/ Stacks/Z Project/Projection Type: max intensity (Fig 1b) Moreover, di¡erent con¢gurations were tested with the purpose of increasing the capacity of the application to identify the spermatozoa moving slowly or immotile spermatozoa Spermatozoa considered to be moving slowly or that were immotile exhibited a grey track, while the others exhibited a black track Therefore, the con¢gurations were tested so as to produce similar graphs (Fig 1a and b), and with no grey tracks (Fig 1b) Analyses of sperm motility using the subjective method Figure Image of the spermatozoa tracks generated by the application CASA (a), and generated by the software IMAGEJ (b) Dialogue box generated by the application CASA inside the software IMAGEJ with the con¢gurations used in the analyses of the sperm motility of jundiaŁ (Rhamdia quelen) (c) 154 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 153^156 Aquaculture Research, 2010, 42, 153^156 Sperm motility of jundiaŁ E A Sanches et al were only used for the standardization of the con¢gurations of the application CASA within 15 s of sperm activation The con¢gurations that exhibited similar motility and spermatozoa track rates were adopted as the standard for the analyses (Fig 1c) Analyses of motility were performed 15, 25 and 35 s after the start of sperm activation, with1s of video at each instant For the semen originating from each male analyses were performed in triplicate, evaluating motility (MOT), the curvilinear velocity (VCL ^ the velocity between each frame evaluated), the average path velocity (VAP ^ the velocity between each frame using an average path at a rate of 1/6 of the frame rate used), the straight line velocity (VSL ^ the velocity between the ¢rst and the last frame analysed), linearity (LIN ^ obtained by the division of VSL byVAP), wobble (WOB ^ obtained by the division of VAP byVCL), progression (PROG ^ the average distance of the sperm from its origin on the average path in all the frames analysed) and the number of spermatozoa monitored (WilsonLeedy & Ingermann 2007) The parameters of CASA were subjected to an analysis of variance to a level of signi¢cance of 5% When there was evidence of some e¡ect, the Tukey test was applied, to the same level of signi¢cance The motility rates evaluated for objective and subjective methods were subjected to the Pearson correlation analysis at a level of signi¢cance of 5% The semen of jundiaŁ R quelen presented 4.508 Æ 1.573  1010 spermatozoa mL À 1, and the ¢nal seminal dilution of1:1500 (semen:diluent1water) resulted in the capture of the videos exhibiting an average of 43 Æ 18 spermatozoa per ¢eld, which was suitable for the motility parameters (Wilson-Leedy & Ingermann 2007) The subjective motility rate within 15 s after activation was 59.17 Æ 21.01% of motile spermatozoa There was a positive correlation (Po0.05) between the motility rates for objective (MOBJ) and subjective (MSUBJ) methods (r 0.6927; P 0.0000004; MOBJ 38.898910.4475MSUBJ) The time elapsed after activation did not in£uence (P40.05) sperm linearity However, it in£uenced (Po0.05) the other sperm parameters negatively (Table 1) According to Amann and Katz (2004), the ¢rst studies on the CASA were described in the mid1980s, with sperm analyses in mammals In ¢sh, those studies started only in the mid-1990s (Rurangwa et al 2004); therefore, there is a need to standardize and optimize the speci¢c protocols for each ¢sh species (Wilson-Leedy & Ingermann 2007), because several factors need to be considered, such as the sperm concentration, chamber depth and diluents used (Kime et al 2001; Rurangwa et al 2004) Rapid, exact and objective analyses are fundamental for the determination of the quality of ¢sh sperm during the short period of time during which the spermatozoa remain viable Until now, assessments on the parameters of sperm motility of South American tropical cat¢sh species using CASA have not been performed Thus, the open-source software CASA may be an important tool in future research related to ¢sh reproduction focused on quality and aspects of sperm activation The spermatozoa of jundiaŁ (R quelen) lose their viability according to the time elapsed after activa- Table Sperm parameters of the jundiaŁ (Rhamdia quelen) obtained in 1s of image,15, 25 and 35 s after sperm activation Time after activation (s) Parameters 15 MOT (%) VCL (mm s À 1) VAP (mm s À 1) VSL (mm s À 1) LIN (%) WOB (%) PROG (mm) 65.37 77.47 58.67 46.51 78.07 76.65 1202.80 25 Æ Æ Æ Æ Æ Æ Æ 13.57a 14.97a 9.01a 6.58a 13.52 9.36a 163.89a 41.10 68.36 45.96 35.75 78.15 68.62 964.63 Pà 35 Æ Æ Æ Æ Æ Æ Æ 17.55b 16.90b 9.47b 7.35b 7.83 11.31b 234.57b 25.59 66.40 42.43 34.42 81.85 63.36 885.16 Æ Æ Æ Æ Æ Æ Æ 13.34c 16.08b 10.46b 7.91b 7.88 12.70b 342.69b 0.0000 0.0048 0.0000 0.0000 0.1667 0.0000 0.0000 Di¡erent letters on the same line indicate signi¢cant di¡erence by the Tukey test to a level of 5% of signi¢cance ÃProbability obtained in the analysis of variance Means values and standard deviations MOT, motility rate; VCL, curvilinear velocity; VAP, average path velocity; VSL, straight line velocity; LIN, linearity; WOB, wobble; PROG, progression r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 153^156 155 Sperm motility of jundiaŁ E A Sanches et al tion The application CASA may be used as a rapid, exact and objective method in sperm analyses of tropical ¢sh The con¢guration established for the dialogue box of the application CASA (Fig 1c) may be used for the sperm analysis of the jundiaŁ References Amann R.P & Katz D.F (2004) Re£ections on CASA after 25 years Journal of Andrology 25, 317^325 Bombardelli R.A., M˛rschbÌcher E.F., Campagnolo R., Sanches E.A & Syperreck M.A (2006) Dose inseminante para fertilizac°aìo arti¢cial de ovo¤citos de jundiaŁ Rhamdia quelen (Quoy & Gaimardm,1824) Revista Brasileira de Zootecnia 35, 1251^1257 Kime D.E., Van Look K.J.W., Mcallister B.G., Huyskens G., Rurangwa E & Ollevier F (2001) Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in ¢sh Comparative Biochemistry and Physiology Part C 130, 425^433 Ravinder K., Nasaruddin K., Majumdar K.C & Shivaji S (1997) Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp fol- 156 Aquaculture Research, 2010, 42, 153^156 lowing short-term storage of semen Journal of Fish Biology 50,1309^1328 Rurangwa E., Kime D.E., Ollevier F & Nash J.P (2004) The measurement of sperm motility and factors a¡ecting sperm quality in cultured ¢sh Aquaculture 234, 1^28 Rurangwa E.,Volckaert F.A.M., Huyskens G., Kime D.E & Ollevier F (2001) Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African cat¢sh (Clarias gariepinus) Theriogenology 55,751^769 Wilson-Leedy J.G & Ingermann R.L (2007) Development of a novel CASA system based on open source software for characterization of zebra¢sh sperm motility parameters Theriogenology 67, 661^672 Wirtz S & Steinmann P (2006) Sperm characteristics in perch Perca £uviatilis L Journal of Fish Biology 68, 1896^ 1902 Keywords: CASA, ¢sh, reproduction, semen, sperm velocity, spermatozoa r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 153^156 Aquaculture Research, 2010, 42, 157^160 doi:10.1111/j.1365-2109.2010.02599.x SHORT COMMUNICATION Daily feeding patterns and self-selection of dietary oil in Nile tilapia Rodrigo Fortes da Silva1, Francisco J Mart|¤ nez1, Morris Villarroel2 & Francisco J SaŁnchez-VaŁzquez1 Department of Physiology, Faculty of Biology, University of Murcia, Murcia, Spain Department of Animal Production, ETSI Agronomy, Polytechnic University of Madrid, Madrid, Spain Correspondence: R Fortes da Silva, Department of Physiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain E-mail: fortesrs@yahoo.com.br The e⁄ciency of food utilization is high in farmed ¢sh, because feed quality and quantity and the timing of its distribution are optimized to meet the metabolic requirements Fish feed rhythmically, consuming meals at speci¢c times during the day or night (Madrid, Boujard & SaŁnchez-VaŁzquez 2001), although their circadian rhythm is more £exible than higher vertebrates, with interspeci¢c and even intraspeci¢c di¡erences (Oishi 1991) The ingestion of the ideal mix and quantity of nutrients to achieve the nutritional need is termed as the intake target (Simpson & Raubenheimer 2001) Macronutrient selection in ¢sh has been investigated in laboratory studies (Vivas, Rubio, SaŁnchez-VaŁzquez, Mena, Garc|¤ a Garc|¤ a & Madrid 2006), leading one to expect that ¢sh are able to match their intake target by adjusting the intake of di¡erent food types (Rubio, SaŁnchez-VaŁzquez & Madrid 2005) The aim of the present work was to investigate the feeding rhythms of Nile tilapia provided with di¡erent sources of dietary lipid by means of self-feeders s A population of 90 male o¡spring (GMT ) with an average weight of 142 Æ 68 g (mean Æ SD) was used for this experiment, which was carried out in the ¢sh laboratory at the University of Murcia, Spain Fish were divided randomly into six tanks in a closed system, 15 ¢sh per tank, and the total biomass was recorded every fortnight until the end of the experiment The water temperature was maintained at 30 Æ 1C and the photoperiod was 12L:12D (lights on at 07:00 hours) Three self-feeders per tank (Eheim 3581, Deizisau, Germany) that delivered three di¡erent diets, supplying approximately three pellets each r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd (about 0.27 g), were connected to a computer that continuously recorded the activation of the demand-sensor, which were submerged 1.5 cm under the water surface The three diets contained 35.0% crude protein and 9.4 MJ DE kg À (Table 1), but di¡erent sources of oil (12.8% linseed,12.1% soy and 12.8% ¢sh oil) In phase 1, ¢sh were given free access to the three self-feeders (days 0^20), and then in phase (day 21), feeds were switched over among feeders The feed remaining in each feeder was weighed and the feed container was re¢lled daily The presence of uneaten food pellets was visually checked daily and the real feed intake was calculated Diet moisture was determined by drying samples for 24 h at 110 1C to a constant weight, crude protein was calculated using the Kjeldahl method (N  6.25%), crude lipids by diethyl ether extraction, ash by heating at 450 1C for 24 h and nitrogen-free extract as the remainder of crude protein, crude fat and ash Feed consumption rates were expressed as gram per 100 g BWday À and the relative oil selection was expressed as the percentage of diet, considering the total of the diets to 100% The data were analysed using chronobiology software (TEMPS, v.1,179 by Dr D|¤ ez Noguera, Barcelona University) and SPSS by one-way ANOVA (Po0.05) using Tukey’s test The results showed two peaks of feeding activity around lights on (07:00 hours) and lights o¡ (19:00 hours) (Fig 1) On average, feeding activity during the light phase accounted for 46% of the total daily food demands The same crepuscular feeding pattern was observed for the three diets with soy, linseed or ¢sh oil 157 Table Proximate composition and fatty acid composition of the experimental diets Ingredients (%) SD Soybean meal-51 (hamlet) 30.1 Pea-52 30.0 Wheat bran 22.8 Linseed oilà – Soybean oilà 12.0 Fish oilà 0.7 L-lysine HCL-178 – 0.1 DL-methionine Celite 2.0 Premix vit 0.1 Premix mineral 0.1 Calcium phosphate 1.1 Calculated chemical composition (%) Dry matter 92.3 Ash 7.9 Crude protein 35.1 % of total fatty acids 14:0 0.5 Æ 0.1 16:0 14.6 Æ 0.3 16:1n7 0.1 Æ 0.1 18:0 6.5 Æ 0.1 18:1n9 26.6 Æ 0.4 18:1n7 2.1 Æ 0.1 18:2n6 0.2 Æ 0.3 18:3n3 3.4 Æ 0.1 20:1n9 0.3 Æ 0.1 20:4n6 – 20:5n3 – 22:6n3 0.4 Æ 0.0 SFA 22.2 Æ 0.2 MUFA 29.1 Æ 0.2 PUFA 47.8 Æ 0.5 TUFA 76.9 Æ 0.2 PUFA/SFA 2.1 Æ 0.2 LD FD 30.1 30.0 22.1 12.7 – – – 0.1 2.0 0.1 0.1 1.1 30.1 30.0 22.1 – – 12.7 92.3 7.9 35.1 92.3 7.9 35.1 0.3 9.1 0.3 4.2 22.3 0.8 28.6 30.3 0.8 0.6 13.6 23.4 59.5 82.9 4.4 0.1 2.0 0.1 0.1 1.1 Æ Æ Æ Æ Æ Æ Æ Æ Æ – – Æ Æ Æ Æ Æ Æ 0.2 4.8 Æ 0.2 0.1 8.8 Æ 0.1 0.0 0.3 Æ 0.0 0.1 5.0 Æ 0.1 0.5 18.8 Æ 0.1 0.1 0.8 Æ 0.0 0.0 9.4 Æ 0.1 0.3 1.6 Æ 0.1 0.1 11.3 Æ 0.3 2.7 Æ 0.2 17.5 Æ 0.2 0.1 5.4 Æ 0.3 0.3 21.6 Æ 0.2 0.0 24.0 Æ 0.0 0.3 36.6 Æ 0.3 0.3 60.6 Æ 0.1 0.1 1.7 Æ 0.0 ÃOils added Results are given as mean Æ SD (n 3) The fatty acid compositions of the diets were analysed as described by Fontagne¤, Corraze and Bergot (2000) SD, soy oil diet; LD, linseed oil diet; FD, ¢sh oil diet; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; n-3, omega-3; n-6, omega-6 Although feeding during the light phase has been suggested for optimal growth, feed e⁄ciency and survival of tilapia ¢ngerlings (Biswas & Takeuchi 2002; El-Sayed & Kawanna 2004), a recent paper reported diurnal as well as nocturnal and arrhythmic activity patterns in tilapia under light/dark conditions (Vera, Cairns, SaŁnchez-VaŁzquez & Migaud 2009) The dual phasing (diurnal/nocturnal) behaviour of ¢sh was ¢rst reported in European sea bass (Dicentrarchus labrax) held under the same controlled environmental conditions (SaŁnchez-VaŁzquez, Mart|¤ nez, Zamora & 158 Number of demands Feeding behaviour and self-selection in tilapia R Fortes da Silva et al Aquaculture Research, 2010, 42, 157^160 25 20 Linseed oil 15 Fish oil Soy oil 10 5 11 13 15 17 19 21 23 Time (h) Figure Average daily waveform feeding activities for all groups tilapia kept under laboratory conditions (12L:12D) The black bars at the top of each graph indicate the length of the dark phase.Values represent the mean Æ SEM of 90 animals during the 46 days of the experimental period Madrid 1994), but it seems to be a common feature among many ¢sh species When tilapias were presented with the three experimental diets, they took days to establish a clear pattern of preference for linseed lipid diets and ¢sh oil diets On day 20, after switching over the feeds, tilapia needed days to re-establish their previous preferences (Fig 2) The average intake of linseed diet was signi¢cantly higher than that of the soy and the ¢sh oil diets (Po0.05) (Fig 3) The preferential intake of linseed and ¢sh oil diets could be due to their better palatability or most likely due to their better nutritional value Indeed, ¢sh make dietary selections based on their ‘nutritional wisdom’ to compose a balanced diet that best ¢ts to their nutritional demands (Simpson & Raubenheimer 2001) In general, marine ¢sh require n-3 HUFA, while freshwater ¢sh such as tilapia require n-6 fatty acids (Rodr|¤ guez, Pe¤rez, Diaz, Izquierdo, FernaŁndez-palacios & Lorenzo 1997) Although marine ¢sh oil is the main source of lipid used in the formulation of commercial aquafeeds, providing essential n-3 fatty acids, a few studies showed the possibility of replacing ¢sh oil by vegetal oil in ¢sh meal-based diets for tilapia without negative e¡ects on growth performance, feed e⁄ciency or body-organ indices (Ng 2006; Bahurmiz & Ng 2007) Moreover, linseed oil in tilapia feed increased the nutritional value of adult Nile tilapia according to Tonial, Stevanato, Matsushita, De Souza, Furuya and Vicentainer (2009) In addition, some ¢sh species have the ability to elongate and desaturate C18 fatty acids to higher molecular weight n-3 HUFA, while other species not have this ability (Ibeas, Cejas, Go¤mez, Jerez & Lorenzo 1996) The apparent r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 157^160 Aquaculture Research, 2010, 42, 157^160 Feeding behaviour and self-selection in tilapia R Fortes da Silva et al Phase Phase Diet selection % (in grams) 80 70 60 50 Linseed oil 40 Soybean oil 30 Fish oil 20 10 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Time (Days) Total Intake (g / 100 g BW day–1) Figure Selection of three diets with di¡erent sources of oil expressed in (%) considering the total of diets to be100%, and the corresponding standard errors (SEM) by Nile tilapia in the 46 days and two phases of the experimental period After the feed demand had stabilized in the day 20, feeds were switched between feeders to reduce the possible preference for the string sensor 2.5 c b 1.5 a 0.5 Fish oil Soy oil Linseed oil Figure E¡ect of diets with di¡erent sources of oil on tilapia intake Di¡erent letters indicate statistically di¡erent cumulative feed intakes (Po0.05, one-way ANOVA and Tukey’s test) digestibility of fatty acids decreases with increasing fatty acid chain length and increases with increasing unsaturation of the fatty acid (Ng, Sigholt & Bell 2004) Adding linseed oil in diet may have positive effects for membrane absorption and may probably affect food selection According to Geurden, Jutfelt, Olsen and Sundell (2009), rainbow trout (Oncorhynchus mykiss) pre-fed linseed oil might modify the membrane £uidity and the physicochemical properties of the apical membrane This may in turn facilitate passive fat acid di¡usion, or, in the case of carrier-mediated fat acid transport, increase the expression, access or function of the carrier proteins anchored in the apical membrane The hypothesis proposed for dietary selection is that the source of macronutrients could be detected by gastrointestinal receptors during digestion, as they are released inside the stomach and then pass into the intestine These receptors would trigger signals (neural activity and hormones) informing brain centres about the nutritional properties of food and modifying feeding behaviour in ¢sh (Rubio et al 2005; Almaida-pagan, Rubio, Mendiola, De Costa & Madrid 2006; Vivas et al 2006) In conclusion, our ¢ndings reveal that Nile tilapia can use self-feeders e⁄ciently to ¢nely adjust food intake according to the diet composition, the diet containing linseed oil being the most selected Besides, tilapia displayed two daily peaks of feeding associated with lights on/o¡, which should be taken into account to optimize the feeding schedules (e.g meal frequency and timing of food delivery) under intensive aquaculture conditions Acknowledgments This research was supported by the Spanish Ministry of Education and Science (MEC) by project (AGL2007-66507-CO2/ACU) to F.J.S.V References Almaida-Pagan P.F., Rubio V.C., Mendiola P., De Costa J & Madrid J.A (2006) Macronutrient selection through postingestive signals in sharpsnout seabream fed gelatine capsules and challenged with protein dilution Physiology and Behavior 88, 550^558 Bahurmiz O.M & Ng W.K (2007) E¡ects of dietary palm oil source on growth, tissue fatty acid composition and nutri- r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 157^160 159 Feeding behaviour and self-selection in tilapia R Fortes da Silva et al ent digestibility of red hybrid tilapia, Oreochromis sp., raised from stocking to marketable size Aquaculture 262, 382^392 Biswas A.K & Takeuchi T (2002) E¡ect of di¡erent photoperiod cycles on metabolic rate and energy loss of fed and unfed adult tilapia Oreochromis niloticus: part II Fisheries Science 68, 543^553 El-Sayed A.F.M & Kawanna M (2004) E¡ects of photoperiod on the performance of farmed Nile tilapia Oreochromis niloticus: I Growth, feed utilization e⁄ciency and survival of fry and ¢ngerlings Aquaculture 231, 393^402 Fontagne¤ S., Corraze G & Bergot P (2000) Response of common carp Cyprinus carpio larvae to di¡erent dietary levels and forms of supply of medium-chain fatty acids Aquatic Living Resources 13, 429^437 Geurden I., Jutfelt F., Olsen R.E & Sundell K.S (2009) A vegetable oil feeding history a¡ects digestibility and intestinal fatty acid uptake in juvenile rainbow trout Oncorhynchus mykiss Comparative Biochemistry and Physiology 152, 552^559 Ibeas C., Cejas J., Go¤mez T., Jerez S & Lorenzo A (1996) In£uence of dietary n-3 HUFA levels on juvenile gilthead sea bream growth and tissue fatty acid composition Aquaculture 142, 221^235 Madrid J.A., Boujard T & SaŁnchez-VaŁzquez F.J (2001) Feeding rhythms In: Food Intake in Fish (ed by D Houlihan, M Jobling & T Boujard), pp 189^215 Blackwell Science, Oxford, UK Ng W.K (2006) Palm oil: Malaysia’s gift to the global aquafeed industry In: Asian Aquafeeds: Current Developments in the Aquaculture Feed Industry, Vol 13 (ed by W.K Ng & C.K Ng), pp 40^54 Malaysian Fisheries Society Occasional Publication, Kuala Lumpur Ng W.K., Sigholt T & Bell J.G (2004) The in£uence of environmental temperature on the apparent nutrient and fatty acid digestibility in Atlantic salmon (Salmo salar L.) fed ¢nishing diets containing di¡erent blends of ¢sh oil, rapeseed oil and palm oil Aquaculture Research 35,1228^1237 160 Aquaculture Research, 2010, 42, 157^160 Oishi T (1991) Fishes In: Handbook of Chronobiology, Vol (ed byY Chiba & K Takahashi), pp.69^78 Tokyo Asakura Shoten,Tokyo Rodr|¤ guez C., Pe¤rez J.A., Diaz M., Izquierdo M.S., FernaŁndezPalacios H & Lorenzo A (1997) In£uence of the EPA/DHA ratio in rotifers on gilthead sea bream (Sparus aurata) larval development Aquaculture 150,77^89 Rubio V.C., SaŁnchez-VaŁ zquez F.J & Madrid J.A (2005) Fish macronutrient selection through post-ingestive signals: e¡ect of selective macronutrient deprivation Physiology and Behavior 84, 651^657 SaŁnchez-VaŁzquez F.J., Mart|¤ nez M., Zamora S & Madrid J.A (1994) Design and performance of an accurate demand feeder for the study of feeding behaviour in sea bass, Dicentrarchus labrax L Physiology and Behavior 56, 789^794 Simpson S.J & Raubenheimer D (2001) A framework for the study of macronutrient intake in ¢sh Aquaculture Research 32, 421^432 Tonial I.B., Stevanato F.B., Matsushita M., De Souza N.E., FuruyaW.M & Vicentainer J.V (2009) Optimization of £axseed oil feeding time length in adult Nile tilapia (Oreochromis niloticus) as a function of muscle omega-3 fatty acids composition Aquaculture Research 15, 564^568 Vera L.M., Cairns L., SaŁnchez-VaŁzquez F.J & Migaud H (2009) Circadian rhythms of locomotor activity in the Nile tilapia Oreochromis niloticus Chronobiology International 26, 666^681 Vivas M., Rubio V.C., SaŁnchez-VaŁ zquez F.J., Mena C., Garc|¤ a Garc|¤ a B & Madrid J.A (2006) Dietary self-selection in sharpsnout seabream (Diplodus puntazzo) fed paired macronutrient feeds and challenged with protein dilution Aquaculture 251, 430^437 Keywords: self-selection, feeding, behaviour, oil, tilapia, self-feeders r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 157^160 [...]... (Ra¢nesque) Aquaculture 178, 77^88 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 9^13 Aquaculture Research, 2010, 42, 9^13 Job S., Buu D & Vincent A (2006) Growth and survival of the tiger tail seahorse, Hippocampus comes Journal of World Aquaculture Society 37, 322^327 Job S.D., Do H.H & Hall H.J (2002) Culturing the oceanic seahorse Hippocampus kuda Aquaculture. .. co-cultured in lantern nets Aquaculture 256, 510^520 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21 21 Aquaculture Research, 2010, 42, 22^30 doi:10.1111/j.1365-2109 .2010. 02488.x Cryopreservation of sperm from natural and sex-reversed orange-spotted grouper (Epinephelus coioides) Taweesin Peatpisut & Amrit N Bart Aquaculture and Aquatic Resources... culture ponds in October reached 44.1mg g À 1, which was higher than that in the other months, and no signi¢cant di¡erences were observed among other months (P40.05) Particulate organic carbon £ux in the two ponds ranged from 377.0 to r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21 Aquaculture Research, 2010, 42, 14^21 Sedimentation in sea cucumber... hand counted in each incubation beaker 18 h after fertilization 100 0 100 0 100 0 Aquaculture Research, 2010, 42, 22–30 Cryopreservation of sperm from Epinephelus coioides T Peatpisut & A N Bart r 2010 Blackwell Munksgaard No claim to original US government works, Aquaculture Research, 42, 22^30 Aquaculture Research, 2010, 42, 22^30 Cryopreservation of sperm from Epinephelus coioides T Peatpisut &... total particulate matter; POC, particulate organic carbon; PON, particulate organic nitrogen; TP, total phosphorus; Chl a, chlorophyll a r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21 17 Aquaculture Research, 2010, 42, 14–21 Sedimentation in sea cucumber culture ponds Y Ren et al 1.2 POC contents in the sediment, mgg–1 10 PON TP 1.0 8 6 4 2 0.0... SD (n 510) 18 Discussion Previous studies have shown that OM deposition is a key process in the open sea (Takahashi 1986; r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21 POC, PON and TP contents, mgg–1 Aquaculture Research, 2010, 42, 14^21 10 Sedimentation in sea cucumber culture ponds Y Ren et al Sea cucumber Without sea cucuber 8 6 4 2 0 POC... Seasonal £uxes of pelagic diatoms in the Subarctic Paci¢c, 1982^1983 Deep-Sea Research Part A Oceanographic Research Papers 33, 1225^1251 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21 Aquaculture Research, 2010, 42, 14^21 Wassmann P (1993) Regulation of vertical export of particulate organic matter from the euphotic zone by planktonic heterotrophs... quality of sea cucumber culture ponds There were no signi¢cant di¡erences in the water quality (temperature, salinity, dissolved oxygen r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 14^21 15 Aquaculture Research, 2010, 42, 14–21 Sedimentation in sea cucumber culture ponds Y Ren et al 10 Chl a content, ugL–1 (DO), pH, Chl a and content of suspended... each tank were weighed The weight gain [WG 5100  (¢nal body weight À initial body weight)/initial body weight (g)], the speci¢c growth r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 9^13 Aquaculture Research, 2010, 42, 9^13 Growth of seahorse at di¡erent densities D Zhang et al rate [SGR 5100  (ln ¢nalWW À ln initialWW)/time] and the condition factor... of tagged fish 11.8 18.4 26.9 16.2 17.2 90 90 90 90 270 (6.2) (1.7) (4.6) (6.2) (6.5) Combined large, medium and small ¢sh are included r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 31^39 Aquaculture Research, 2010, 42, 31^39 Harbour, Australia) that were inserted posterior to the dorsal ¢n, a method that has been successfully used for turbot juveniles