Aquaculture research, tập 42, số 2, 2011

Aquaculture Research, 2011, 42, 161^168 doi:10.1111/j.1365-2109.2010.02545.x Low-density culture of red abalone juveniles, Haliotis rufescens Swainson 1822, recirculating aquaculture system and flow-through system Miroslava Vivanco-Aranda1, Cristian Jorge Gallardo-EscaŁrate2 & Miguel AŁ ngel del R|¤ o-Portilla1 Laboratorio de Gene¤tica, Departamento de Acuicultura, Centro de Investigacio¤n Cient|¤ ¢ca y de Ecuacio¤n Superior de Ensenada Ensenada, BC, Me¤xico Departamento de Oceanograf|¤ a, Centro de Biotecnolog|¤ a, Universidad de Concepcio¤n, Barrio Universitario s/n Casilla 160-C, Concepcio¤n, Chile Correspondence: M A del R|¤ o-Portilla, CICESE, Acuicultura, PO Box 434844, San Diego, CA 92143-4844, USA E-mail: mdelrio@ cicese.mx Abstract Commercial abalone culture is carried out using £ow-through systems with a high water volume exchange in Baja California, Mexico The objective of this work was to compare the growth rate and survival of red abalone cultured in two systems Flow through (daily water exchange rate of 800%) and recirculating systems consisted of a 250 L ¢breglass tank and constant aeration, but bio¢ltration in the recirculating system was provided with a 28 L (1ft3) bubble-washed bead ¢lter.Water variables were measured either daily (dissolved oxygen, temperature, pH and salinity) or three times a week (total ammonia nitrogen, nitrate-nitrogen, nitrite-nitrogen and alkalinity) Shell length was measured every weeks for 18 weeks Only the alkalinity and pH were signi¢cantly di¡erent due to the addition of sodium bicarbonate to the recirculating system Abalone growth rate was 26.1 Æ 15.96 mm day À in the recirculating systems and 22.21 Æ 18.69 mm day^1 in the £ow-through systems The ¢nal survival was 78.74% in the recirculating systems and 71.82% in the £ow-through systems Signi¢cant di¡erences in the ¢nal size and survival of the abalones were found between systems (Po0.05) Therefore, recirculating aquaculture systems is a feasible alternative for juvenile red abalone culture Keywords: red abalone, Haliotis rufescens, recirculating systems, growth, closed system Introduction Abalones, Haliotis, are marine gastropod molluscs, with a high commercial value There is a high inter- r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd est in abalone culture for commercial and stock enhancement In Baja California, abalone culture started in 1984 with the red abalone, Haliotis rufescens Swainson 1822 (Mazo¤n-SuaŁstegui, Mucinìo-D|¤ az & Bazu¤a-Sicre 1996), although blue, Haliotis fulgens Philippi 1845, and yellow, Haliotis corrugata Wood 1828, abalone culture are also of interest The £owthrough system is commonly used both commercially and for stock enhancement In this system, water is constantly introduced to provide good water quality to the organisms with an 800% daily water exchange This practice requires large amounts of water to be pumped and, thus, yields a high cost Recently, the abalone industry has implemented important technical changes to reduce costs and to improve the production procedures, and recirculating systems are considered to be a feasible option to reduce the pumping cost, but without reducing abalone growth Commercial recirculating systems have been used for nearly three decades (Masser, Rakocy & Losordo 1992) This type of system allows high environmental control and feasible cultivation conditions as well as a reduction of water and land (Masser et al 1992) Abalone requires a good water quality to obtain high growth rates (Basuyaux & Mathieu 1999) this involves the control and monitoring of di¡erent physical^chemical variables Among the most important are the temperature, dissolved oxygen, pH, salinity, concentrations of total ammonia nitrogen (TAN), nitrite-nitrogen (NO2-N), nitrate-nitrogen (NO3-N) and alkalinity In general, control of these variables will provide good water quality for abalone growth and development (Huchette, Koh & Day 2003) The 161 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al objective of this work was to evaluate and compare the growth rate and survival of red abalone cultured in a recirculating and in a £ow-through system Materials and methods Juvenile abalone Seven-month-old red abalone (batch 200D, produced by spawning 33 males and 73 females), were obtained from the commercial farm ‘Abulones Cultivados S.A de C.V.’ located in Ejido Ere¤ndira, Baja California, Me¤xico (31116 03300 N,116122 05300 W) Juvenile abalones were separated from substrata using CO2 and were transported on a wet sponge in plastic bags with oxygen at the Aquaculture Department (Departamento de Acuicultura) at the Centre for Scienti¢c Research and Higher Education in Ensenada (Centro de Investigacio¤n Cient|¤ ¢ca y de Educacio¤n Superior de Ensenda, CICESE) After arriving at our facilities, individual separation was not possible; thus, abalone distribution was based on the weight of several abalones, which were then transferred to culture systems In each tank 50 g of abalones were placed, which corresponds to a low stocking density of 0.070 kg m À 570 g cm À (2265 organisms m À 2) Culture systems One £ow-through system and one recirculating system were used in the growth trial Three individual £ow-through systems, each one with individual water supply plus three individual recirculating systems, were used Each one of the recirculating systems consisted of a cylindrical £at-bottom ¢breglass tank (250 L capacity) connected to a magdrive 500 pump (Danner Manufacturing, Islandia, NY, USA) and a 28.31L (1ft3) bubble wash bead ¢lter (Aquaculture System Technologies, New Orleans, LA, USA) as a bioclari¢er (Malone & Beecher 2000), with a backwash frequency of two to three times a week The bead ¢lters were acclimated for 60 days at 20 1C before the experiment as recommended by Malone and Beecher (2000) In the recirculating systems, the £ow rate was 30 L À The three individual £owthrough systems consisted of similar tanks (250 L capacity) mentioned above A £ow rate of 1.39 L À l was set in the three tanks for a daily water exchange rate of 800% The water used was pumped from the ocean and ¢ltered to 70 mm before reaching the tanks Constant aeration was provided throughout the experiment to both systems The experiment 162 Aquaculture Research, 2011, 42, 161^168 lasted months, beginning on October 2003 and concluding on 13 February 2004 Fifty grams of organisms were randomly allocated to each tank The initial mean shell length was 5.88 Æ 0.04 mm for abalones maintained in the recirculating system and 6.21 Æ 0.01mm in the £ow-through system All data are average Æ standard error Abalones were fed at a ratio of 70% of abalone weight per week with Macrocystis pyrifera according to the alimentation of the abalones in the commercial farms (N Garc|¤ a, pers comm.) Every week, macroalgae were removed and replaced with fresh ones Abalone and environmental variable measurements Every weeks, the shell length of 50 randomly selected animals per tank was measured using an electronic vernier calliper (precision to 0.01mm) model S225 (Fowler Company, Newton, MA, USA) Every day, temperature and dissolved oxygen were measured using an oxymeter model YSI 55 (YSI, Yellow Springs, OH, USA) On 24 November, in both systems, a heater (precision of Æ 0.55 1C) model Pro Heat II 150 W (Won Brother’s, Fredericksburg, VA, USA) was placed to maintain the temperature above 16 1C Salinity was measured using a temperature-compensated refractometer (conventional refractometer) and pH was measured using a Hanna Hi98127 meter (Hanna Instruments,Woonsocket, RI, USA) Alkalinity was measured two or three times a week with 1.6 N H2SO4 and a bromcresol green-methyl red indicator (Hach Company, Loveland, CO, USA) Because an alkalinity concentration between 80 and 200 mg L À of CaCO3 is optimal for bacterial survival in the bio¢lters (Loyless & Malone 1997), when alkalinity values declined below 100 mg L À of CaCO3, alkalinity was adjusted to 150 mg L À of CaCO3 with the addition of commercial sodium bicarbonate, Iris quality (Smart & Final, Tijuana, BCN, Mexico), to the recirculating systems according to the method described by Loyless and Malone (1997) The concentrations of TAN, NO2-N and NO3-N were determined two or three times per week using a saltwater master liquid test kit (Aquarium Pharmaceuticals, Chalfont, PA, USA); for coloration of each nutrient, the absorbance was read using a spectrophotometer Shimadzu UV-1201 (Shimadzu Scienti¢c Instruments, Columbia, MD, USA) Statistical analyses At the beginning of the experiment, abalone shell lengths were compared among tanks with an ANOVA r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 Aquaculture Research, 2011, 42, 161^168 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al after assumptions of normality and homogeneity of variances were met At the end of the experiment, the abalone shell lengths and the survival between the two culture systems were compared using a covariance analysis (ANCOVA) to determine the di¡erences among the slope of the two culture systems against time Di¡erences between treatment means were considered to be signi¢cant at Po0.05 Growth rates [(shell length at a given time À shell length at the time of the previous measurement)/shell length at the time of the previous measurement  100] were calculated for every period of measurement Survival was estimated by counting empty shells at each measurement time For the di¡erent physical^chemical variables (temperature, dissolved oxygen, pH, salinity,TAN, NO2-N, NO3-N and alkalinity), paired t-tests were carried out to determine whether there was a tendency for the data to have a higher value between culture systems using [recirculating datum] À [£owthrough datum] to calculate the t-value in the t-tests (paired comparisons, Sokal & Rohlf 1995) Statistical analyses were carried out using MINITAB for WINDOWS release 10.2 (Minitab, State Collage, PA, USA) Results The initial mean shell length (10 October) was 5.88 Æ 0.04 mm for abalones maintained in the recirculating system and 6.21 Æ 0.01mm in the £owthrough system (Fig 1) Signi¢cant di¡erences in the initial abalone size were found between systems (F(1,298) 12.25; Po0.01) The ¢nal mean shell length was 9.17 Æ 0.03 mm for organisms in the recirculating system and 9.01 Æ 0.02 mm for organisms in the £ow-through system (Fig 1) Marginally signi¢cant di¡erences were found between systems in the abalone growth rate (F(1,2996) 4.16; P 0.04) Therefore, throughout the experiment, abalones that were Figure Shell length (mm) of juvenile Haliotis rufescens in two culture systems Mean Æ SE Three £ow-through system (n 50150150 at each time point) Three recirculating systems (n 50150150 at each time point) Figure Growth rate of juvenile Haliotis rufescens in two culture systems Mean Æ SE Three £ow-through system (n 50150150 at each time point) Three recirculating systems (n 50150150 at each time point) Figure Survival (%) of juvenile red abalones Haliotis rufescens in two culture systems Mean Æ SE Three £ow-through system (n 50150150 at each time point) Three recirculating systems (n 50150150 at each time point) in the recirculating system grew more in comparison with the £ow-through system Abalone growth rates ranged from 6.43 to 58.64 (total mean 26.1) mm day À in the recirculating system and from 2.3 to 65.87 (total mean 22.21) mm day À in the £owthrough system (Fig 2) Juvenile survivals (at day126) were 78.74% for organisms in the recirculation system and 71.82% for organisms in the £ow-through system (Fig 3) No signi¢cant di¡erence was found in abalone survival (F(1,4) 3.78, P 0.12) Temperature, salinity and dissolved oxygen did not show a signi¢cant di¡erence between treatments (Table 1) The values of dissolved oxygen found in this work were below the saturation point (72.54% for the recirculating system and 71.62% for the £owthrough system), but never below mg L À Although in the £ow-through system, there was a tendency (paired samples test) to have a higher temperature (n 5128, t À 13.89, Po0.001), lower NO2-N concentration (n 45, t 2.53, P 0.015), r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 163 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al Aquaculture Research, 2011, 42, 161^168 Table Water quality parameters measured in two systems of culture of juvenile red abalones Haliotis rufescens Recirculating system Water parameters Mean Æ SE Temperature ( 1C) Dissolved oxygen (mg L À 1) Salinity (g L À 1) pH Alkalinity (mg L À 1) of CaCO3 TAN (mg L À 1) of CaCO3 NO2-N (mg L À 1) of CaCO3 NO3-N (mg L À 1) of CaCO3 16.07 7.08 35.35 8.13 139.00 0.11 0.04 1.08 Æ Æ Æ Æ Æ Æ Æ Æ 0.98 0.51 0.38 0.04 7.99 0.04 0.05 1.21 Flow-through system Min^Max Mean Æ SE 10.40–20.80 5.34–8.50 35.00–38.00 7.90–8.30 100.00–165.00 0.00–0.36 0.00–0.65 0.02–17.29 16.77 6.89 35.23 8.01 115.11 0.12 0.01 0.83 Æ Æ Æ Æ Æ Æ Æ Æ 0.88 0.55 0.29 0.03 4.76 0.05 0.01 0.33 Min^Max 12.90–20.90 5.23–8.28 34.00–37.00 7.80–8.20 95.00–140.00 0.01–0.49 0.00–0.05 0.01–4.61 Temperature, dissolved oxygen, salinity and pH (n 5128) Alkalinity, TAN, nitrite-nitrogen and nitrates-nitrogen (n 45) lower dissolved oxygen concentration (n 5128, t 513.51, Po0.001) and higher salinity (n 5128, t 2.45, P 0.016) Alkalinity and pH were signi¢cantly di¡erent between systems (Table 1) due to the addition of sodium bicarbonate in the recirculating system, where alkalinity and pH were higher (n 45, t 511.2, Po0.001; n 5128, t 25.8, Po0.001 respectively) There were no di¡erences between systems with TAN (n 45, t À 0.45, P 0.66) and NO3-N (n 45, t 0.94, P 0.35) between systems (Table 1) Discussion Abalone growth is in£uenced by environmental conditions, water quality (Leitman 1992; Hoshikawa, Sakai & Kijima 1998; Harris, Maguire, Edwards & Johns 1999), diet type (Viana, Cervantes-Trujano & SolanaSansores 1994; Capinpin Jr & Corre 1996; Viana, Cervantes-Trujano & Solana-Sansores 1996; Haaker, Parker, Barsky & Chun 1998; Lopez, Tyler & Viana 1998; Bautista-Teruel & Millamena 1999; Capinpin Jr, Toledo, Encena & Doi 1999), culture density (Day & Fleming 1992; Mgaya & Mercer 1995; Mgaya, Gosling, Mercer & Donlon 1995; Clarke & Creese 1998; ValdesUrriolagoitia 2000) and abalone size at the beginning of the experiment (Corazani & Illanes 1998; Trevelyan, Mendoza & Buckley 1998; Steinarsson & Imsland 2003) Abalone size is a principal factor a¡ecting the feeding rates of gastropods Generally, feeding rates per biomass unit are higher in smaller and faster growing juveniles than in larger abalone (Marsden & Williams, 1996) H rufescens fed with arti¢cial diets and di¡erent types of macroalgae produce similar growth rates as macroalgae (Table 2) It can be observed that red abalone growth rate/initial shell length (GR/ISL) 164 obtained in this study were similar to other GR/ISL values Furthermore,Table shows a comparison between studies carried out with arti¢cial diets against macroalgae with other abalone species The highest growth rates in most of these works were achieved by feeding abalones with an arti¢cial diet This may be explained by the fact that both the arti¢cial diets had higher protein and fat contents and produced the best growth rates in terms of the total weight and shell length (Capinpin & Corre1996), which suggests that diet type has a direct e¡ect on abalone growth rate In contrast to this study, for other abalone species of similar sizes growth rates of the100 mm day À growth rate of similar abalone size were obtained with abalone juveniles of Haliotis tuberculata (Linnaeus, 1758) fed with an arti¢cial diet (Lopez et al 1998) and with Japanese abalone Haliotis discus hannai Ino,1953 (Hoshikawa et al 1998) fed with diatoms The highest growth rates have been reported in juveniles between 10 and 40 mm shell length in other abalone species: Haliotis asinina Linnaeus, 1758 (Capinpin & Corre, 1996; Fermin 2002), black abalone Haliotis cracherodii Leach, 1814 (Leighton & Boolootian 1963), paua abalone Haliotis iris Gmelin, 1791 (Clarke & Creese 1998), H fulgens (Leighton, Byhower, Kelly, Hooker & Morse 1981), greenlip abalone Haliotis laevigata Donovan, 1808 (Gilroy & Edwards 1998) and H tuberculata (Lopez et al.1998), all fed with an arti¢cial diet An other aspect that a¡ects the growth rate is the culture density For abalone cultured in £ow-through systems, growth is inversely related to density (Mgaya & Mercer1995; Capinpin et al.1999;Valde¤s-Urriolagoitia 2000) Culture density has an inverse effect on abalone survival and may a¡ect abalone growth directly through competition for food and space However, in this study, density was not a critical factor for the growth rate because a low r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 Aquaculture Research, 2011, 42, 161^168 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al Table Data from di¡erent studies on the growth juvenile red abalones Haliotis rufescens Initial shell length (mm) Growth rate (mm month^1) GR GR/ISL Diet Source 10.00 34.51 33.70 34.88 35.94 8.26 8.26 4.00 4.00 4.00 21.00 0.47 1.23 1.67 0.70 1.34 1.03 1.79 1.13 1.00 0.58 2.97 0.047 0.036 0.049 0.020 0.037 0.125 0.217 0.283 0.250 0.145 0.141 Macroalgae Lessonia trabeculata Macadamia integifolia Utricularia rigida Artificial diet Macrocystis Microcladia Artificial diet Leitman (1992) Corazani and Illanes (1998) Steinarsson and Imsland (2003) 25.00 33.00 66.00 81.00 98.00 3.15 3.27 2.67 2.13 1.83 0.126 0.099 0.040 0.026 0.018 Caminaria digitataand Palmaria palmata (9:1) 5.88 6.21 0.78 0.67 0.133 0.108 Macrocystis pyrifera Trevelyan et al (1998) Valde´s-Urriolagoitia (2000) Present study Recirculating system Flow through system Initial shell length (ISL) is the average length of the abalone’s shell in the beginning of the experiment and growth rate (GR) is the monthly growth of the abalones shell (evaluated in millimetres) Table Data of di¡erent relating experimental studies for the growth juvenile of diverse species Species Haliotis fulgens Haliotis tuberculata Haliotis asinina Haliotis asinina Haliotis rubra Haliotis rufescens Initial shell length (mm) Growth rate (mm month^1) 10.00 25.00 42.00 15.30 15.20 19.60 23.80 16.80 15.80 15.20 15.80 15.90 19.00 33.91 34.23 5.88 6.21 GR/ISL Diet Source 2.71 1.74 0.71 1.75 1.07 1.61 1.67 1.81 4.07 6.67 7.33 7.43 4.20 1.30 0.90 0.271 0.070 0.017 0.114 0.070 0.082 0.070 0.108 0.258 0.439 0.464 0.467 0.221 0.038 Egregia laevigata and Macrocystis pyrifera Leighton et al (1981) Palmaria palmata Mgaya and Mercer (1995) Gracilaria bailinae Artificial diet Artificial diet Artificial diet Gracilaria heteroclada Artificial diet Bautista-Teruel and Millamena (1999) 0.78 0.67 0.133 0.108 Macrocystis pyrifera Capinpin et al (1999) Huchette et al (2003) Present study Recirculating system Flow through system Initial shell length (ISL) is the average length of the abalone’s shell in the beginning of the experiment and growth rate (GR) is the monthly growth of the abalones shell (evaluated in millimetres) stocking density was used for both culture systems to eliminate potential complications due to a high stocking rate The low stocking density used in this study can explain the highest survival rate obtained compared with the survival rates reported in other studies (Nie, Ji & Yan 1996, Park, Rho & Song 1995) Also, the di¡erences may be due to the size of the organisms and the duration of the experiment Therefore, it is possible to obtain high growth rates of H rufescens even on feeding with macroalgae if the physical^chemical variables are within the ranges of good water quality and low stocking density r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 165 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al Mortality not only depends on the culture density but also on inadequate handling (stress) of food transfer (Searcy-Bernal, Salas-Garza & Flores-Aguilar 1992) The mortality rate is also associated with the organism’s size and stage of development (Mazo¤n-SuaŁstegui et al.1996).Water quality variables like salinity, temperature, dissolved oxygen, pH and nitrogen wastes are also important factors governing the growth of abalones (Harris, Maguire, Edwards & Hindrum 1998) The results obtained in the present work for all physical^chemical variables are within the ranges of good water quality Special attention should be paid to the pH and alkalinity variables, because they were the only two quality parameters that di¡ered considerably between the £ow-through and the recirculating system due to the addition of sodium bicarbonate to the recirculating system However, the pH range observed in the recirculating system was similar to those shown in other studies with di¡erent abalone species (Nie et al.1996; Harris, Maguire, Edwards & Hindrum 1997; Harris et al 1998; Basuyaux & Mathieu 1999; Bautista-Teruel & Millamena 1999) The pH interval obtained in this study is within the pH values that promoted the activities of nitrifying bacteria and prevents ammonia toxicity (Loyless & Malone 1997, Malone & Beecher 2000) To our knowledge, there are no studies on the in£uence of alkalinity on abalone metabolism; however, the alkalinity values in the recirculating system (100^165 mg L À CaCO3) were within the ranges recommended for bacterial survival in bio¢lters (Masser et al 1992; Loyless & Malone 1997; Malone & Beecher 2000) Further studies on the e¡ect of alkalinity on abalone growth could be carried out; however, it is considered that low alkalinity and pH values may have a higher e¡ect on abalone growth than alkalinity values between 100 and 165 L À CaCO3, because these values were close to the alkalinity values found in the £ow-through system and in nature In the case of the waste nitrogen compounds, the TAN, NO2-N and NO3-N concentrations presented in this study are below the toxic levels reported for abalone (Harris et al 1997; Harris et al 1998) Basuyaux and Mathieu (1999) found that H laevigata mortality and growth were a¡ected by a concentration of 1mg TAN L À 1; they also found that a concentration of 1^5 mg NO2-N L À does not in£uence abalone growth, and in contrast, a concentration of mg NO2-N L À stimulates the growth in H tuberculata On the other hand, they report that toxic levels are from 8.5 to 15.4 NO2-N L À For the NO3-N, 166 Aquaculture Research, 2011, 42, 161^168 Table Cost analysis of the two systems of culture of juvenile red abalones Haliotis rufescens Cost (US$) Recirculating system Equipment cost Bubble wash bead filter Fibreglass tank PVC pipes Magdrive 500 pump Electric energy Pumping cost (per day) Pumping total cost (127 days) Total Difference between systems 571.67 154.30 30.86 51.02 0.09 11.43 819.28 Flow-through system 154.30 30.86 1.76 223.52 408.68 410.60 Costs calculated by tank in the experiment time The cost of water pumped from the ocean and the equipment associated was not considered Basuyaux and Mathieu (1999) reported that H tuberculata supports (without a¡ecting growth) a concentration range of 100^250 mg of NO3-N L À The low concentration of the waste nitrogen compounds (TAN, NO2-N and NO3-N) presented in this study was probably due to the low density of abalones used and had no e¡ect on abalone growth In the £ow-through system, nearly 15^30% of the production costs are associated with the maintenance of a high rate of exchange One way to decrease the cost of production associated with constantly pumping water through the abalone grow-out tanks can be with the use of recirculating systems (Badillo, Segovia & Searcy-Bernal 2007) For a recirculating system to be considered a closed system, it should have o10% of water exchange per day of the total volume of the system (Masser et al 1992; Loyless & Malone 1997) In our experiment, the water exchange was 4.8% of the total volume per day and the only water replaced was that lost by evaporation and bio¢lter back£ushes; thus, it can be considered as a closed system Table shows a cost analysis of the two culture systems used in this study In the recirculating system, the high initial costs are due to the equipment purchased (bead ¢lter) However, with the saving of the rate of water exchange in the recirculating systems, it is possible to recover the initial investment in a short period of time In this case, the investment recovery time is close to months An economic analysis to the commercial level is necessary and will be important for the choice of the optimum stocking density to maximize the produc- r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 Aquaculture Research, 2011, 42, 161^168 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al tion of H rufescens in recirculation culture systems With the present work, it is possible to state that recirculating systems are a feasible alternative for the culture of juvenile red abalone at a low density Acknowledgments The CONACYT supported the M.Sc studies of M.V.-A with a scholarship This study was partially ¢nanced by means of the CONACYT project ‘Genetic markers of abalone, Haliotis spp.’ (33018 B) and by CICESE project number 655 The authors are particularly grateful to the commercial farm ‘Abulones Cultivados S.A de C.V.’ for providing the abalone used in this research The authors also thank Marisela Aguilar-JuaŁrez for the support provided and Oscar B Del R|¤ o Zaragoza for the technical assistance References Badillo L., Segovia M & Searcy-Bernal R (2007) E¡ect of two stocking densities on the growth and mortality of the pink abalone Haliotis Corrugata in recirculating and £ow-through systems Journal of Shell¢sh Research 26, 801^807 Basuyaux O & Mathieu M (1999) Inorganic nitrogen and its e¡ect on growth of the abalone Haliotis tuberculata Linnaeus and the sea urchin Paracentrotus lividus Lamarck Aquaculture 174, 95^107 Bautista-Teruel M.N & Millamena O.M (1999) Diet development and evaluation for juvenile abalone, Haliotis asinina: protein/energy levels Aquaculture 178, 117^126 Capinpin E.C Jr & Corre K.G (1996) Growth rate of the Philippine abalone, Haliotis asinina fed an arti¢cial diet and macroalgae Aquaculture 144, 81^89 Capinpin E.C Jr, Toledo J.D., Encena V.C II & Doi M (1999) Density dependent growth of the tropical abalone Haliotis asinina in cage culture Aquaculture 171, 227^235 Clarke C.B & Creese R.G (1998) On-growing cultured abalone (Haliotis iris) in northern New Zealand Journal of Shell¢sh Research 17, 607^613 Corazani D & Illanes J.E (1998) Growth of juvenile abalone, Haliotis discus hannai Ino 1953 and Haliotis rufescens Swainson 1822, fed with di¡erent diets Journal of Shell¢sh Research 17, 663^666 Day R.W & Fleming A.E (1992) The determinants and measurement of abalone growth In: Abalone of theWorld (Biology, Fisheries and Culture) Proceedings of the 1st International Symposium on Abalone (ed by S.A Shepherd, M.J Tegner & S.A GuzmaŁn del Pro¤o), pp.141^168 Fishing News Books, Oxford, UK Fermin A.C (2002) E¡ects of alternative starvation and refeeding cycles on food consumption and compensatory growth of abalone, Haliotis asinina (Linnaeus) Aquaculture Research 33,197^202 Gilroy A & Edwards S.J (1998) Optimum temperature for growth of Australian abalone: preferred temperature and critical thermal maximum for blacklip abalone, Haliotis rubra (Leach), and greenlip abalone, Haliotis laevigata (Leach) Aquaculture Research 29, 481^485 Haaker P.L., Parker D.O., Barsky K.C & Chun C.S.Y (1998) Growth of red abalone, Haliotis rufescens (Swainson), at Johnsons Lee, Santa Rosa Island, California Journal of Shell¢sh Research 17,747^753 Harris J.O., Maguire G.B., Edwards S.J & Hindrum S.M (1997) E¡ect of nitrite on growth and oxygen consumption for juvenile greenlip abalone, Haliotis laevigata Donovan Journal of Shell¢sh Research 160, 395^401 Harris J.O., Maguire G.B., Edwards S.J & Hindrum S.M (1998) E¡ect of ammonia on the growth rate and oxygen consumption of juvenile greenlip abalone, Haliotis laevigata Donovan Aquaculture 160, 259^272 Harris J.O., Maguire G.B., Edwards S.J & Johns D.R (1999) Low dissolved oxygen reduces growth rate and oxygen consumption rate of juvenile greenlip abalone, Haliotis laevigata Donovan Aquaculture 174, 265^278 Hoshikawa H., Sakai Y & Kijima A (1998) Growth characteristics of the hybrid between pinto abalone, Hatiotis kamtsckatkana Jonas, and ezo abalone, H discus hannai Ino, under high and low temperature Journal of Shell¢sh Research 17, 673^677 Huchette S.M.H., Koh C.S & Day R.W (2003) Growth of juvenile blacklip abalone (Haliotis rubra) in aquaculture tanks: e¡ects of density and ammonia Aquaculture 219, 457^470 Leighton D.L & Boolootian R.A (1963) Diet and growth in the black abalone Haliotis cracherodii Ecology 44, 227^238 Leighton D.L., Byhower M.J., Kelly J.C., Hooker G.N & Morse D.E (1981) Acceleration of development and growth in young green abalone (Haliotis fulgens) using warmed ef£uent seawater Journal of the World Mariculture Society 12, 170^180 Leitman A (1992) The e¡ects of gas supersaturation on the behaviour, growth and mortality of red abalone, Haliotis rufescens (Swainson) In: Abalone of the World (Biology, Fisheries and Culture) Proceedings of the 1st International Symposium on Abalone (ed by S.A Shepherd, M.J Tegner & S.A GuzmaŁn del Pro¤o), pp 75^85 Fishing News Books, Oxford, UK Lopez L.M.,Tyler P.A & Viana M.T (1998) The e¡ect of temperature and arti¢cial diets on growth rates of juvenile Haliotis tuberculata (Linnaeus, 1758) Journal of Shell¢sh Research 17, 657^662 Loyless J.C & Malone R.F (1997) A sodium bicarbonate dosing methodology for pH management in freshwater-recirculating aquaculture systems American Fisheries Society 59,198^205 Malone R.F & Beecher L.E (2000) Use of £oating bead ¢lters to recondition recirculating waters in warm water aqua- r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 167 Recirculating and £ow through red abalone culture M.Vivanco-Aranda et al culture production systems Aquacultural Engineering 22, 57^73 Marsden I.D & Williams P.M.J (1996) Factors a¡ecting the grazing rate of the New Zealand abalone Haliotis iris Martyn Journal of Shell¢sh Research 15, 401^406 Masser M.P., Rakocy J & Losordo T.M (1992) Recirculating aquaculture tank production systems Management of recirculating systems Louisiana State University Agricultural Center 452, 1–11 Mazo¤n-SuaŁstegui J.M., Mucinìo-D|¤ az M & Bazu¤a-Sicre L.A (1996) Cultivo de abulo¤n Haliotis spp In: Estudio del Potencial Pesquero y Acu|¤ cola de Baja California Sur, Vol II (ed by M Casas-Va´ldez & G Ponce-Dı´ az), pp 475– 511 SEMARNAP, Gobierno del Estado de Baja California Sur, FAO, Instituto Nacional de la Pesca, UABCS, CIBNOR, CICIMAR, CETMAR Baja California Sur, Me´xico Mgaya Y.D., Gosling E.M., Mercer J.P & Donlon J (1995) Genetic variation at three polymorphic loci in wild and hatchery stocks of the abalone Halitios tuberculata Linnaeus Aquaculture 136,71^80 Mgaya Y.D & Mercer J.P (1995) The e¡ects of size grading and stocking density on growth performance of juvenile abalone, Haliotis tuberculta Linnaeus Aquaculture 136, 297^312 Nie Z.Q., Ji M.F & Yan J.P (1996) Preliminary studies on increased survival and accelerated growth of overwintering juvenile abalone, Haliotis discus hannai Ino Aquaculture 140, 177^186 Park M.E., Rho S & Song C.B (1995) Density e¡ect on the growth of juvenile abalones (Haliotis discus hannai) reared in the closed recirculating water system Bulletin of 168 Aquaculture Research, 2011, 42, 161^168 Marine Research Institute, Cheju National University 19, 93^102 Searcy-Bernal R., Salas-Garza A.E & Flores-Aguilar R.A (1992) Research in Me¤xico on the critical stage of abalone (Haliotis spp.) seed production In: Abalone of the World (Biology, Fisheries and Culture) Proceedings of the 1st International Symposium on Abalone (ed by S.A Shepherd, M.J Tegner & S.A GuzmaŁn del Pro¤o), pp 547^560 Fishing News Books, Oxford, UK Sokal R.R & Rohlf F.J (1995) Biometry The Principles and Practice of Statistics in Biological Research State University of New York at Stony Brook W H Freeman and Company, NewYork, NY, USA, 887pp Steinarsson A & Imsland A.K (2003) Size dependent variation in optimum growth temperature of red abalone (Haliotis rufescens) Aquaculture 224, 353^362 Trevelyan G.A., Mendoza J.L & Buckley B (1998) Increasing the yield of red abalone with the alga, Microcladia coulteri Journal of Shell¢sh Research 17, 631^633 Valde¤s-Urriolagoitia A.A (2000) Efecto de tres densidades de cultivo en la sobrevivencia y crecimiento de juveniles de abulo¤n rojo Haliotis rufescens en un laboratorio comercial Tesis de Licenciatura, UABC Ensenada, Me¤xico, 52pp Viana M.T., Cervantes-Trujano M & Solana-Sansores R (1994) Attraction and palatability activities in juvenile abalone (Haliotis fulgens): nine ingredients used in arti¢cial diets Aquaculture 127,19^28 Viana M.T., Cervantes-Trujillo M & Solana-Sansores R (1996) The use of silage made from ¢sh and abalone viscera as an ingredient in abalone feed Aquaculture 140, 87^98 r 2010 CICESE Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 161^168 Aquaculture Research, 2011, 42, 169^176 doi:10.1111/j.1365-2109.2010.02551.x Assay performance during validation of freezing channel catfish Ictalurus punctatus (Rafinesque) infected with a Gram-negative bacterium Julie Bebak1, Craig Shoemaker1, Covadonga Arias2 & Phillip Klesius1 USDA ARS AAHRU, Auburn, AL, USA Department of Fisheries and Allied Aquaculture, Auburn University, Auburn, AL, USA Correspondence: J Bebak, USDA ARS AAHRU, 990 Wire Rd., Auburn, AL 36832, USA E-mail: julie.bebak@ars.usda.gov Abstract Recovery of bacteria from infected ¢sh during population sampling can be a¡ected by factors including the type of assay, method of specimen preservation and concentration of bacteria present Consequently, before use in ¢eld sampling, methods should be validated The three objectives of this study were, ¢rst, to determine whether a channel cat¢sh Ictalurus punctatus (Ra¢nesque) ¢ngerling classi¢ed as positive for Gram-negative Edwardsiella ictaluri infection according to bacterial culture before freezing was also classi¢ed as positive after freezing, second, to determine how direct culture from the kidney (DIRECT), culture of homogenate (HOMOG) and standard PCR (PCR) agree with bacterial culture in terms of classifying ¢sh as positive or negative and third, to estimate diagnostic sensitivity (dSe) and diagnostic speci¢city (dSp) for DIRECT, HOMOG and PCR In fresh and frozen ¢sh, as bacterial concentration decreased, the ability of each assay to detect positive ¢sh also decreased, especially when there were o104 colony-forming units per gram (CFU g À 1) tissue HOMOG proved to be the most reliable at correctly classifying cat¢sh, whether they were subclinically or clinically infected PCR assay was the least reliable Overall, values for this study population for dSe were 0.66, 0.92 and 0.43, and for dSp were 0.86, 0.91 and 0.95, for DIRECT, HOMOG and PCR respectively Keywords: validation, freezing, Edwardsiella, cat¢sh, PCR, bacterial culture Introduction Studies conducted on ¢eld populations of ¢sh may require sampling large numbers of individuals to achieve su⁄cient statistical power For example, r 2010 Blackwell Munksgaard No claim to original US government works studies that estimate prevalence, incidence or other epidemiological measures generally require sampling hundreds, or even thousands, of ¢sh Assuming 100% diagnostic sensitivity (dSe) and speci¢city (dSp), for a prevalence estimate in a population of 100 000 animals, an error of 5%, and con¢dence of 95%, the sample size for simple random sampling will be 138 animals if the expected prevalence is 10% and 384 animals if the expected prevalence is 50% (Thrus¢eld 1995) If the animals sampled are warmwater pond-cultured ¢sh species, then sampling will most likely occur in an environment with daytime temperatures that range from an average of about 27 1C to 40 1C, processing ¢sh will proceed slowly and the distance from laboratory facilities would preclude transporting large numbers of dead ¢sh for laboratory analysis while still maintaining sample integrity The transport of live ¢sh back to the laboratory may be considered, but would likely be constrained by the need to keep groups of ¢sh separated and the lack of adequate culture facilities at the destination One possible solution to this problem would be to place dead ¢sh on dry ice (i.e at least À 78.2 1C), and then transport them to the laboratory for storage at À 80 1C and processing at a later date Freezing can result in the inactivation of bacteria and a loss of infectious units (Sheridan 1997; Archer 2004) Suomalainen, Reunanen, Ijas, Valtonen and Tiirola (2006) froze enriched Flavobacterium columnare from rainbow trout skin mucus at À 20 1C for an unspeci¢ed length of time and then thawed it at 23 1C and found that bacterial DNA could not be detected with PCR after freezing Electron microscopy revealed that frozen then thawed bacterial cells had disintegrated They hypothesized that bacterial Dnases, lyases and proteases present in F columnare were responsible for the destruction of 169 Tea tree oil anaesthesia of common carp G J Hajek Aquaculture Research, 2011, 42, 296^300 Table Duration of induction of general anaesthesia and recovery (mean Æ SD) of common carp (n 512) exposed to ¢ve concentrations of tea tree oil Concentration (mL L À 1) 0.2 0.3 0.4 0.5 0.6 Inductionà time (s) Recoveryw time (s) 686ABC Æ 263 355 Æ 168 227D Æ 79 361 Æ 133 181Aa Æ 55 324 Æ 72 145B Æ 44 335 Æ 82 107CDa Æ 25 306 Æ 120 Mean values with the same capital letter superscript, within a line, are signi¢cantly di¡erent at P 0.01; lowercase letter superscript indicates a signi¢cant di¡erence at P 0.05 ÃInduction time is the time necessary to reach a state of general anaesthesia at which the ¢sh is fully handleable ^ no motion or reaction to being taken out of the water wRecovery time is the time required for the ¢sh to regain the ability to swim properly, measured from the transfer to the recovery tank Table Mean (Æ SD) times of the onset of disturbances in ventilation, appearing during the exposition, measured from the start of immersion Exposition (min) 15 Ventilation disturbances (s) Regaining of continuous ventilation (s) First caudal fin movement (s) Recovery (s) Mortality (%)à 432 141AB 234ABa 405ABa 30 Æ Æ Æ Æ 74 37 97 90 388 536A 683A 908A 45 Æ Æ Æ Æ 57 276 329 359 425 689B 1590B 1789B 41.6 60 Æ Æ Æ Æ 94 381 1200 1231 408 552 1620a 1868a 83.3 75 Æ Æ Æ Æ 75 357 764 752 385 Æ 110 – – – 100 Mean (Æ SD) times of the onsets of recovery process symptoms, measured from the transfer to the recovery tank, plus mortality, all of which were a result of prolonged exposure of common carp (n 512) to tea tree oil at a concentration of 0.5 mL L À Mean values with the same capital letter superscript, within a line, are signi¢cantly di¡erent at P 0.01; the lowercase letter superscript indicates a signi¢cant di¡erence at P 0.05 ÃThe number of ¢sh that did not recover passively returned to a normal position Transfer to the recovery tank resulted in a return to consciousness The recovery process consisted of the following phases: Motionlessness Motionless carp breathing continuously could be found in a normal body position at the bottom of the tank or drifting under the surface of the water First movement After148 Æ 95 s the ¢sh started to move or even swim Recovery After 335 Æ 82 s ¢sh swam normally The lowest concentration of TTO causing general anaesthesia in less than was 0.5 mL L À The induction lasted and 25 s, and recovery and 35 s (Table 1).With all the solutions, the recovery time was dose-independent and lasted up to All the ¢sh recovered from anaesthesia No mortalities or negative symptoms were recorded within the next 10 days Experiment Prolonged exposure resulted in motionlessness Only ventilatory movements with a gradually decreasing 298 amplitude were observed (3^4 min) After min, intervals in regular ventilation started to appear (Table 2) Next, alternate periods of regular and irregular breathing were noted At about 8^10 of exposure, most of the ¢sh breathed irregularly or underwent temporary apnoea, passively maintaining normal body position or with lateral inclination However, at up to 30 min, some ¢sh had continuous ventilation periods Two specimens were still breathing weakly even after a 60-min exposure A reversed order of events was observed during the recovery process First, weak, irregular ventilatory movements appeared Then the ¢sh regained a continuous breathing pattern with increasing amplitude and frequency Caudal ¢n movement preceded the regaining of the normal body position and swimming Often, the carp started to move forward while still lying laterally Finally, the ¢sh swam e⁄ciently Exposures of 15 and 30 allowed all the ¢sh to recover The recovery time after a 15-min exposure was about min, being signi¢cantly shorter than after longer-lasting immersions None of the ¢sh survived 75 of intoxication No delayed mortalities of the recovered ¢sh were observed within a 10-day observation period r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 296^300 Aquaculture Research, 2011, 42, 296^300 Discussion To the author’s knowledge, the general anaesthetic potency of tea tree oil in ¢sh has not been evaluated before Its local anaesthetic properties have, on occasions, been mentioned in general TTO descriptions but it is hard to ¢nd any detailed documentation The inhalation of TTO did not a¡ect the behaviour of mice (Lim, Seo, Lee, Pyo & Lee 2005) However, there are studies describing the neuronal-depressant activity of other essential oils with a composition similar to that of TTO (Leal-Cardoso, Moreira, da Cruz, de Morais, Lahlou & Coelho-de-Souza 2004) or terpene ingredients alone (Brodin & Roed 1984; Peana, D’Aquila, Chessa, Moretti, Serra & Pippia 2003; De Sousa, Quintans-Junior & Almeida 2007) Gas chromatography/mass spectrometry identi¢ed terpinen-4-ol, a-terpineol and 1.8-cineole as the water-soluble components of TTO (Hart, Brand, Carson, Riley, Prager & Finlay-Jones 2000), and it is probably thanks to these chemicals that TTO has the effect described in the present study Some of their speci¢c physiological properties have already been determined The local anaesthetic activity of terpineol (the authors not specify the drug; similar to terpineol, Sigma o¡ers a mixture of isomers with the presence of a-terpineol, $ 65%) in vivo in the rabbit conjunctival re£ex test and in vitro in a rat phrenic nervehemidiaphragm technique was demonstrated by Ghelardini, Galeotti and Mazzanti (2001) Similar in vitro results were obtained by Moreira, Cruz, Lopes, Albuquerque and Leal-Cardoso (2001) While determining the mechanism with whichTTO reduced a histamine-induced skin in£ammation, it was found that a solution of all the water-soluble components of TTO causes a signi¢cant reduction in response to sensory nerve stimulation (Hart et al 2000) Studied separately, terpinen-4-ol had no e¡ect on the sensory nerves but it modulated vasodilation and plasma extravasation The 1.8-cineole acted directly on sensory nerves and a-terpineol displayed both anti-oedema and local anaesthetic properties In another study, feeding rats and mice with 1.8-cineole resulted in decreased chemical nociception (Santos & Rao 2000) Finally, treating mice with terpinen-4-ol, the main component of TTO, caused a decrease in the spontaneous motor activity It also produced a dosedependent increase in the sleep duration and had an anticonvulsant e¡ect, indicating a depressant impact on the central nervous system (de Sousa, No¤brega, de Morais & de Almeida 2009) It can therefore be concluded that the dose-dependent changes in the behaviour of the ¢sh exposed to Tea tree oil anaesthesia of common carp G J Hajek TTO, as described in the present study, result from the anaesthetic properties of this agent Further work should clarify the pharmacologic mechanisms of this action Among several classi¢cations of ¢sh anaesthesia stages, the one most similar to the presently described observations is a more general scheme of Schoettger and Julin (1967, from Mattson & Riple 1989) Also in that study, it was observed that at the stage of a partial loss of equilibrium, the ¢sh displayed ‘uncoordinated movement, followed by active, erratic swimming’ The anaesthesia level at which the exposure to TTO was ¢nished (Experiment I in the present paper) should be considered to be light anaesthesia (not surgical), which would correspond with the transition from stage to stage of Schoettger and Julin’s scheme (1967) The criterion assessing handleability applied in the present study was a reaction to handling and exposition to the air Such stimuli have a multifactor action on mechanoreceptors in the ¢sh skin and mechano- and chemo-receptors in the gill arches Because the loss of equilibrium was not evident even in the deeply a¡ected ¢sh (Experiment II), the responsiveness to handling appeared to be a more suitable indicator of anaesthesia progress The sensitivity to surgical stimuli was not tested The practical requirements of a safe and e⁄cient ¢sh anaesthetic, established by Gilderhus and Marking (1987), applied by several research teams (Weyl, Kaiser & Hecht 1996; Hseu, Yeh, Chu & Ting 1998; Park, Hur, Im, Seol, Lee & Park 2008), are easily accomplished using TTO Its potency, estimated by comparison of data in the literature, can be considered to be similar to that of 2-phenoxyethanol (Mohamed 1999;Vel|¤ s› ek & SvobodovaŁ 2004; Lambooij, Pilarczyk, Bialowas, Reimert, Andre¤ & van de Vis 2009), while it seems to be about 10 times weaker than clove oil (Vel|¤ s› ek, SvobodovaŁ, Piac›kovaŁ, Groch & NepejchalovaŁ 2005; Hajek, Kzyszejko & Dziaman 2006) Such a level of anaesthetic potency should be taken into account when TTO is used to treat ¢sh TTO costs about 30% more than clove oil and it is much less e⁄cient, and so it is unlikely that it will gain much popularity as a ¢sh anaesthetic But is it all that TTO can o¡er to aquaculture? Melaleuca oil is well known for its several medical advantages To the author’s knowledge, only its antifungal property has been tested in ¢sh TTO was e¡ective in ceasing the activity of Aphanomyces invadans zoospore (Campbell et al 2001) and e⁄ciently controlled Saprolegnia fungus on rainbow trout eggs (Marking et al 1994) Therefore, r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 296^300 299 Tea tree oil anaesthesia of common carp G J Hajek the anaesthetic properties described in this study and its other features described in the literature, make tea tree oil worthy of interest and further investigation References Brodin P & Roed A (1984) E¡ects of eugenol on rat phrenicnerve diaphragm preparations Archives of Oral Biology 29, 611–615 Campbell R.E., Lilley J.H., Panyawachira V & Kanchanakhan S (2001) In vitro screening of novel treatments for Aphanomyces invadans Aquaculture Research 32, 223–233 Carson C.F & Riley T.V (2001) Safety, e⁄cacy and provenance of tea tree (Melaleuca alternifolia) oil Contact Dermatitis 45, 65^67 Carson C.F., Riley T.V & Cookson B.D (1998) E⁄cacy and safety of tea tree oil as a topical antimicrobial agent Journal of Hospital Infection 40, 175^178 De Sousa D.P., Quintans-Junior L.J & Almeida R.N (2007) Evolution of the anticonvulsant activity of a-terpineol Pharmaceutical Biology 45, 69–70 De Sousa D.P., No¤brega F.F., de Morais L.C & de Almeida R.N (2009) Evaluation of the anticonvulsant activity of terpinen-4-ol Zeitschrift fˇr Naturforschung C 64, 1^5 Ghelardini C., Galeotti N & Mazzanti G (2001) Local anaesthetic activity of monoterpenes and phenylpropanes of essential oils Planta Medica 67, 564^566 Gilderhus P.A & Marking L.L (1987) Comparative e⁄cacy of 16 anaesthetic chemicals on rainbow trout North American Journal of Fisheries Management 7, 288^292 Greay S.J., Ireland D.J., Kissick H.T., Levy A., Beilharz M.W., Riley T.V & Carson C.F (2010) Induction of necrosis and cell cycle arrest in murine cancer cell lines by Melaleuca alternifolia (tea tree) oil and terpinen-4-ol Cancer Chemotherapy and Pharmacology 65, 877^888 Hajek G.J., Kzyszejko B & Dziaman R (2006) The anaesthetic e¡ect of clove oil on common carp, Cyprinus carpio L Acta Ichthyologica et Piscatoria 36, 93^97 Hart P.H., Brand C., Carson C.F., RileyT.V., Prager R.H & FinlayJones J.J (2000) Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses in£ammatory mediator production by activated human monocytes In£ammation Research 49, 619^626 Hseu J.R.,Yeh S.L., Chu Y.T & Ting Y.Y (1998) Comparison of e⁄cacy of ¢ve anesthetics in goldlined sea bream, Sparus sarba Acta ZoologicaTaiwanica 9, 35^41 International Organization for Standardization (2004) ISO 4730:2004, Oil of Melaleuca, Terpinen- 4-ol Type (Tea Tree Oil) ISO, Geneva, Switzerland Kristo¡ersen S.S., Atkin P.A & Shen¢eld G.M (1996) Uptake of alternative medicine Lancet 347, 569–573 300 Aquaculture Research, 2011, 42, 296^300 Lambooij B., Pilarczyk M., Bialowas H., Reimert H.G.M., Andre¤ G & van de Vis H (2009) Anaesthetic properties of propiscin (Etomidaat) and 2-phenoxyethanol in the common carp (Cyprinus carpio L.), neural and behavioural measures Aquaculture Research 40, 1328–1333 Leal-Cardoso J.H., Moreira M.R., da Cruz G.M., de Morais S.M., Lahlou M.S & Coelho-de-Souza A.N (2004) E¡ects of essential oil of Alpinia zerumbet on the compound action potential of the rat sciatic nerve Phytomedicine 11, 549^553 LimW.C., Seo J.M., Lee C.I., Pyo H.B & Lee B.C (2005) Stimulative and sedative e¡ects of essential oils upon inhalation in mice Archives of Pharmacal Research 28,770^774 Marking L.L., Rach J.J & Schreier T.M (1994) Evaluation of antifungal agents for ¢sh culture The Progressive Fish Culturist 56, 225^231 Mattson N.S & Riple T.H (1989) Metomidate, a better anesthetic for cod (Gadus morhua) in comparison with benzocaine, MS-222, chlorobutanol and phenoxyethanol Aquaculture 83, 89^94 Mohamed S.J (1999) Comparative e⁄cacy of four anesthetics on common carp Cyprinus carpio L Acta Ichthyologica et Piscatoria 29, 91^97 Moreira M.R., Cruz G.M.P., Lopes M.S., Albuquerque A.A.C & Leal-Cardoso J.H (2001) E¡ects of terpineol on the compound action potential of the rat sciatic nerve Brazilian Journal of Medical and Biological Research 34, 1337^ 1340 Park M.O., HurW.J., Im S.Y., Seol D.W., Lee J & Park I.S (2008) Anesthetic e⁄cacy and physiological responses to clove oil-anesthetized kelp grouper Epinephelus bruneus Aquaculture Research 39, 877^884 Peana A.T., D’Aquila P.S., Chessa M.L., Moretti M.D.L., Serra G & Pippia P (2003) (-)-Linalool produces antinociception in two experimental models of pain European Journal of Pharmacology 460, 37^41 Santos F.A & RaoV.S (2000) Anti-in£ammatory and antinociceptive e¡ects of1.8-cineole a terpenoid oxide present in many plant essential oils Phytotherapy Research 14, 240^ 244 Schoettger R.A & Julin A.M (1967) E⁄cacy of MS-222 as an anesthetic on four salmonids U.S Fish andWildlife Service Investigation in Fish Control 13, 1^5 Vel|¤ s› ek J & SvobodovaŁ Z (2004) Anaesthesia of common carp (Cyprinus carpio L.) with 2-phenoxyethanol: acute toxicity and e¡ects on biochemical blood pro¢le Acta Veterinaria Brno 73, 247^252 Vel|¤ s› ek J., SvobodovaŁ Z., Piac›kovaŁ V., Groch L & NepejchalovaŁ L (2005) E¡ects of clove oil anaesthesia on common carp (Cyprinus carpio L.).Veterinarni Medicina 50, 269^275 Weyl O., Kaiser H & Hecht T (1996) On the e⁄cacy and mode of action of 2-phenoxyethanol as an anaesthetic for gold¢sh, Carassius auratus, at di¡erent temperatures and concentrations Aquaculture Research 27, 757–764 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 296^300 Aquaculture Research, 2011, 42, 301^307 doi:10.1111/j.1365-2109.2010.02638.x Effects of temperature and salinity on oxygen consumption of tawny puffer Takifugu flavidus juvenile Yonghai Shi1,2, Genyu Zhang1, Jianzhong Liu1 & Weiling Zang2 Shanghai Fisheries Research Institute, Shanghai, China College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China Correspondence: Y Shi, Shanghai Fisheries Research Institute, Shanghai 200433, China E-mail: yonghais@163.com Abstract The respiratory rates of Tawny pu¡er Takifugu £avidus juvenile were measured at four temperatures (20, 23, 26 and 29 1C) and seven salinities (5, 10, 15, 20, 25,30 and 35 g L À 1) The results showed that both temperature and salinity signi¢cantly a¡ected the oxygen consumption of tawny pu¡er juvenile The oxygen consumption rate (OCR) increased signi¢cantly with an increase in the temperature from 20 to 29 1C Over the entire experimental temperature range (20^29 1C), the Q10 value was 1.59, and the lowest Q10 value was found between 23 and 26 1C The optimal temperature for the juvenile lies between 23 1C and 26 1C The OCR at 25 g L À was the highest among all salinity treatments The OCRs show a parabolic relationship with salinity (5^35 g L À 1) From the quadratic relationship, the highest OCR was predicted to occur at 23.56 g L À The optimal salinity range for the juvenile is from 23 to 25 g L À The results of this study are useful towards facilitating an increase in the production of the species juvenile culture Keywords: tawny pu¡er, Takifugu £avidus, temperature, salinity, oxygen consumption, juvenile Introduction Pu¡er ¢sh are widely distributed, with approximately 100 species in the world Some species are important ¢sheries’ resources and appear to be promising for aquaculture (Yang & Chen 2005) Tawny pu¡er Takifugu £avidus is mainly distributed in the inshore waters of the East China Sea, Yellow Sea and Bohai Bay (Yang, Zhang & Kuang 1991) Classi¢ed as a coastal temperate bottom ¢sh, it is not known for undertaking long-distance migration, but only seasonal r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd migration (Zhang, Shi, Zhu, Liu & Zang 2010) Tawny pu¡er is a species found in China considered to have potential for aquaculture because of its commercial importance and high market value (Shi, Zhang, Zhu, Liu & Zang 2010) However, because of environmental degradation, over¢shing and other factors, the danger of tawny pu¡ers being under threat has been increasingly highlighted (Shi, Zhang, Zhu,Yan, Liu & Zhu 2009) This is despite the arti¢cial breeding being conducted to continue meeting consumer demand (Shi et al 2009) Relatively little is known about the environmental requirements to support the juvenile stages of this species In aquaculture, successful production depends to a large extent on the survival rate of the juvenile stages Therefore, it is essential to determine the optimal culture conditions for each stage This optimum can be determined using two approaches: one is directly from the analysis of mortality and growth rates.We have determined the optimal temperature and salinity range (23^29 1C, 15^ 25 g L À respectively) for larvae culture of tawny pu¡er (Shi et al 2010; Zhang et al 2010) Another is indirectly by more analysis and evaluation of the intensity of respiratory metabolism This is a good indicator of the general physiological activity of organisms (Yagi, Ceccaldi & Gaudy 1990), because it takes into account the energy consumption and utilization (Meade, Doeller, Kraus & Watts 2002) The intensity of respiratory metabolism under di¡erent conditions of salinity and temperature is also useful for estimating the requirements of water exchange as a function of the density of organisms (SpanopoulosHernaŁndez, Mart|¤ nez-Palacios, Vanegas-Pe¤rez, Rosas & Ross 2005) in the culture of tawny pu¡er juvenile Metabolic rate is the most fundamental biological rate as it represents the rate of energy uptake, transformation and allocation (Brown, James, Allen, 301 Temperature and salinity e¡ect on tawny pu¡er juvenile Y Shi et al Savage & West 2004) Oxygen consumption is a widely studied indicator of metabolic rate (LyytikÌinen & Jobling 1998), and measurements of oxygen consumption rate (OCR) are often used to examine energy utilization and stress in aquatic animals and to determine the environmental conditions that result in maximal utilization of input energy for weight gain in an organism (Meade et al 2002) Many biotic and abiotic factors can a¡ect the oxygen consumption in aquatic organisms (Brett 1987), including activity (Dalla Via 1987), body mass (SpanopoulosHernaŁndez et al 2005; Daoud, Chabot, Audet & Lambert 2007), diet (Dall & Smith 1986), environmental temperature, salinity, oxygen concentration (Claireaux & Lagarde're 1999), circadian rhythms (Tian, Dong, Wang & Wu 2004; Chang, Jeong, Min, Neill & Fontaine 2005) and stocking density (Dalla Via,Villani, Gasteiger & NiederstÌtter 1998) The most important abiotic factors are temperature and salinity (Brett 1987), especially in the estuarine and coastal ecosystem for aquaculture; in such systems, water temperature and salinity vary diurnally and seasonally Temperature directly a¡ects the rate of all biological processes and salinity places an osmoregulatory demand on organisms (Spanopoulos-HernaŁndez et al 2005) To date, there have been many studies on the e¡ects of temperature and salinity on the oxygen consumption in teleosts, for example, sea bass Dicentrarchus labrax (Claireaux & Lagarde're 1999), the leopard shark Triakis semifasciata (Miklos, Katzmana & Cech 2003), the Japanese £ounder Paralichthys olivaceus (Kim, Yoon, Kim, Gil & Lee 2005) and the largemouth bass Micropterus salmoides (D|¤ az, Re, GonzaŁlez, SaŁnchez, Leyva & Valenzuela 2007) However, there is no literature report on the e¡ects of those abiotic factors on the oxygen consumption of pu¡er ¢sh The aim of the present study is to investigate the e¡ects of temperature and salinity on the oxygen consumption of tawny pu¡er juveniles The optimal temperature and salinity range for juvenile culture of the species are further determined by analysis and evaluation of the intensity of respiratory metabolism The result of this study is deemed useful in facilitating an increase in the production of this species through juvenile culture Materials and methods Juvenile collection and acclimation Tawny pu¡er juveniles were obtained by arti¢cial breeding (Shi et al 2009) The juveniles were kept in 302 Aquaculture Research, 2011, 42, 301–307 20 m3 concrete tanks with a salinity of 13.5 g L À and a temperature of 22.0^24.0 1C About 1650 juveniles (23 days after hatching) were harvested randomly from the concrete tanks and 150 juveniles were allocated to each of 200 L circular tanks containing brackish water (13.5 g L À 1; 23.0 1C) Four temperatures (20, 23, 26 and 29 1C) and seven salinities (5,10,15, 20, 25, 30 and 35 g L À 1) were selected Original water was either diluted with freshwater to reach salinities below 13.5 g L À or hypersaline seawater (Zhoushan, Zhejiang, China) to obtain salinities above 13.5 g L À Salinity was maintained by adding freshwater if necessary Temperature and salinity gradients were adjusted gradually at a rate of1 1C h À and g L À h À respectively (Fielder, Bardsley, Allan & Pankhurst 2005) When the ¢nal temperature and salinity was reached, it was maintained for 7^10 days to ensure full adaptation During acclimation, the juveniles were fed with Artemia sp Nauplii twice daily at 09:00 and 15:00 hours (Shi et al 2009) Dead juveniles, faeces and other debris were siphoned out every day Fifty per cent of the water in each tank was replaced with a fresh medium daily Each tank was provided with gentle aeration under natural light (600^800 lx) and a light^dark cycle of 14:00^10:00 hours To avoid interference with post-prandial metabolism of food and faeces excretion, acclimated juveniles were kept unfed for 24 h before experimentation To minimize the possible in£uence on oxygen consumption caused by body weight, narrow weight ranges (mean Æ SD fresh weight, 0.28 Æ 0.04 g) were used During the acclimation period, dissolved oxygen was 5.0^ 6.5 mg L À 1, pH was 8.0^8.5, salinity was 13.5 g L À (in the temperature acclimation experiment) and temperature was 22.0^23.0 1C (in the salinity acclimation experiment) Experiment design and physiological measurements Closed-chamber respiration methods were used to determine OCR (Zheng, Jin, Li, Bai & Dong 2008) The chamber was a 1.5 L transparent plastic cylindrical bottle Four temperatures at 13.5 g L À salinity and seven salinities at 22.5^22.6 1C were tested in triplicate Each temperature or salinity treatment held four respiration chambers; three respiration chambers held acclimated juveniles (each chamber inclusive of ¢ve juveniles) Another respiration chamber with no juvenile was used simultaneously in each r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 301^307 Aquaculture Research, 2011, 42, 301^307 Temperature and salinity e¡ect on tawny pu¡er juvenile Y Shi et al treatment as a control so that corrections could be made Each temperature treatment was conducted in di¡erent water baths equipped with thermoregulators and immersion heaters, and all salinity treatments were placed in a water bath To minimize the e¡ects of stress response on the oxygen consumption of juveniles, specimens were acclimated in the chambers until their gill covers £apped equably (approximately 30 min) All measurements were performed under natural light (600^800 lx) at the same time of the day (08:00 hours) Oxygen uptake was measured using a polarographic oxygen electrode (YSI-58 with stirrer) To minimize the possible in£uence on oxygen consumption caused by oxygen saturation, each measurement lasted for 1^3 h until oxygen saturation reached about 60% (Meade et al 2002; Zheng et al 2008) To ensure oxygen saturation before taking physiological measurements, seawater was fully aerated for at least 1h in a 100 L tank before ¢lling the experimental chambers To ensure about 60% of oxygen saturation at the end of measurements, all measurement times were estimated according to di¡erent temperatures and salinities through previous experiments Specimens were ¢nally measured for their fresh weight (the nearest 0.0001g) (Saucedo et al 2004): Q10 ¼  10=ðT2 ÀT1 Þ R2 R1 where R2 and R1 are the OCRs at temperaturesT2 and T1 respectively The e¡ects of salinity and temperature on the OCR were analysed using a one-way analysis of variance, followed by Duncan’s multiple-range tests The signi¢cance level was set at Po0.05 Statistical analyses were conducted using the SPSS 13.0 software Results E¡ect of temperature on the oxygen consumption Temperature strongly a¡ected the oxygen consumption of tawny pu¡er juvenile The OCR increased signi¢cantly (Po0.05) with an increase in temperature from 20 to 29 1C The linear equation between the OCR and temperature was established (r2 50.9973) (Fig 1) It can be seen that between 23 and 26 1C, the Q10 value was the lowest (1.48) and between 20 and 23 1C, the Q10 value was the highest (1.75) (Table 1) Over the entire experimental temperature range (20^29 1C), the Q10 value was 1.59 Daily water measurement Statistical analyses Data were used to determine the OCR, based on the following equation (Saucedo, Ocampo, Monteforte & Bervera 2004; Zheng et al 2008): R¼ ðCt0 À Ct1 ÞV ðt1 À t0 ÞW where R is the OCR and expressed as the fresh weight-speci¢c rate (mg O2 g À h À 1), t0 and t1 are the start and the ¢nish times (h) of the measurement period, Ct is the oxygen concentration in water (mg O2 L À 1) at time t,W is the body weight of the juveniles (g fresh weight) andV is the volume of the respiratory chamber (L) Using the calculated values of OCR, the Q10 value in oxygen consumption was calculated as follows E¡ect of salinity on the oxygen consumption Salinity also signi¢cantly a¡ected the oxygen consumption of tawny pu¡er juvenile The OCR at d 0.9 Oxygen Consumption (mg O2 g–1 h–1) Salinity, temperature, pH and dissolved oxygen were measured daily using theYSI Model:30-10 FT salinity meter (0.1g L À 1;Yellow Spring Instrument Company, Yellow Springs, OH, USA),YSI Model: 30-10 FT (0.1 1C), YSI Model: No pH 100 (0.1 pH unit) and YSI Model: 58 dissolved oxygen meter (0.1mg L À 1) respectively c 0.8 b 0.7 0.6 a y = 0.032x – 0.0891 R2 = 0.9979 0.5 0.4 17 20 23 26 Temperature (°C) 29 32 Figure Regression analysis de¢ned the relationship between temperature and the oxygen consumption rate (mean Æ SD, n 3) of tawny pu¡er juveniles at 13.5 g L À salinity Di¡erent letters above each bar indicate signi¢cant di¡erences among treatments (Po0.05) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 301^307 303 Temperature and salinity e¡ect on tawny pu¡er juvenile Y Shi et al Table Mean values of Q10 coe⁄cients in tawny pu¡er juveniles at di¡erent temperatures Temperature range ( 1C) Q10 coefficients 20–23 20–26 20–29 23–26 23–29 26–29 1.75 1.61 1.59 1.48 1.51 1.55 Oxygen Consumption (mg O2 g–1 h–1) 0.7 e 0.6 c d d b b 0.5 a y = –0.00036 x2 + 0.01696 x + 0.38310 0.4 R2 = 0.91620 0.3 10 15 20 25 Salinity (‰) 30 35 40 Figure Regression analysis de¢ned the relationship between salinity and oxygen consumption rate (mean Æ SD, n 3) of tawny pu¡er juveniles at 22.5^ 22.6 1C Di¡erent letters above each bar indicate signi¢cant di¡erences among treatments (Po0.05) 25 g L À was the highest (Po0.05) among all the salinity treatments The OCR increased (Po0.05) signi¢cantly with an increase in salinity from to 25 g L À 1, whereas it signi¢cantly decreased (Po0.05) with an increase in salinity from 25 to 35 g L À The relationship between salinity and the OCR was modelled by a quadratic equation (r2 50.9162) (Fig 2) From the equation, the highest OCR was predicted to occur at 23.56 g L À Discussion Several factors may reduce the ability to obtain accurate measurements of resting oxygen consumption, including the oxygen concentration, diet, circadian rhythms, activity and the stress of handling To minimize those e¡ects, in experiments, we made some measures as follows: the narrow dissolved oxygen concentration ranges in all the measurements were 304 Aquaculture Research, 2011, 42, 301–307 maintained at (4.0^6.5 mg O2 L À 1), the specimens were kept unfed only for 24 h before all the measurements, the measurements were obtained during the same time of the day and specimens were acclimated for approximately 30 in the chambers before all the measurements respectively Hence, all OCR measurements were assumed to be indicative of tawny pu¡er juvenile resting metabolism Within the suitable temperature range, a higher temperature can improve the metabolic rate of aquatic organisms, whereas, beyond that range, excessively high temperatures may not be bene¢cial and may even have negative e¡ects (Saucedo et al 2004) This pattern has been con¢rmed for some teleosts Claireaux and Lagarde're (1999) showed that sea bass (D labrax) metabolic rate increased sharply as temperature increased from 10 to 20 1C, whereas it declined slightly at a higher temperature (25 1C) For the marbled rock¢sh Sebastiscus marmoratus, Kita, Tsuchida and Setoguma (1996) found that OCR increased with the temperature from10 to 23 1C, but declined at 26 1C In the present study, the negative e¡ect was not observed in tawny pu¡er juvenile over the experimental temperature range (20^29 1C); the OCR continued to increase throughout the entire temperature range, and the relationship between the OCR and temperature clearly ¢t the linear model (r2 50.9979) It may be hypothesized that the experimental temperatures (20^29 1C) were in the suitable range for tawny pu¡er juvenile Similar results have been obtained in many teleosts, for example, the leopard shark T semifasciata (Miklos et al 2003), the carp Labeo rohita (Das, Pal, Chakraborty, Manush, Sahu & Mukherjee 2005), the largemouth bass M salmoides (Lace¤pe'de) (D|¤ az et al 2007) and the miiuy croaker Miichthys miiuy (Basilewsky) (Zheng et al 2008) The temperature coe⁄cient (Q10) represents the degree of sensitivity of an organism to temperature, and it is a measure of the metabolic capacity of aquatic organisms to make adjustments after a temperature change (Spanopoulos-HernaŁndez et al 2005) Within the suitable temperature range, the lowest Q10 value occurs at optimal temperature, and the Q10 values increase on either side of the optimum (Yukihira, Lucas & Klumpp 2000; Zheng et al 2008); beyond that range, the Q10 values may decrease at both ends of the temperature scale, i.e a lower Q10 value may also occur at the lower and the upper temperature threshold (Dalla Via, Villani, Gasteiger & NiederstÌtter 1998; Claireaux & Lagarde're 1999) In the present study, the experimental temperatures r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 301^307 Aquaculture Research, 2011, 42, 301^307 Temperature and salinity e¡ect on tawny pu¡er juvenile Y Shi et al (20^29 1C) were in the suitable range (the initial conclusions in this study), the lowest Q10 value (1.48) was recorded between 23 and 26 1C and the Q10 value was recorded for 1.59 over the entire range (20^29 1C) Based on the Q10 values, we can come to the conclusion that the optimal temperature range for tawny pu¡er juvenile was from 23 to 26 1C This optimal temperature is close to the lower limit of the optimal temperature range for the larvae of this species (23^ 29 1C), which was observed in our previous studies (Shi et al 2010) Salinity also a¡ects the metabolic rate of aquatic organisms Some authors found that the OCR of aquatic organisms should be lowest at the isosmotic point (Dalla Via 1987; Aristizabal-Abud 1992), especially in freshwater species (Parry & Potts 1965) However, other authors also indicated that energetic cost associated with ionic and osmotic regulation was minimal within the salinity range that was normal for the species and life stage (Morgan & Iwama 1991), and changes in the metabolic rate following a change in salinity expressed the sum of metabolic processes and simultaneous behaviour change rather than the energy expenditure of pure osmotic work (Styczynska-Jurewicz 1970) In euryhaline species, alterations in the metabolic rate with varying salinity may be very small (Claireaux & Lagarde're 1999), especially after a long period of acclimation for salinity In the present study, Tawny pu¡er is known as euryhaline species (Shi et al 2009; Zhang et al 2010), The OCRs show a parabolic relationship with salinity (5^35 g L À 1), The OCR signi¢cantly increased with an increase in salinity from to 25 g L À 1, whereas it decreased signi¢cantly with an increase in salinity from 25 to 35 g L À The highest OCR was predicted to occur at 23.56 g L À from the quadratic relationship The decrease on either side of the optimum is a fairly common phenomenon with marine organisms This metabolic reaction corresponds to type de¢ned by Kinne (1962) Based on this ¢nding, we can come to the conclusion that the optimal salinity range for tawny pu¡er juvenile was from 23 to 25 g L À This optimal salinity is close to the upper limit of the optimal salinity range for larvae of this species (15^25 g L À 1) (Zhang et al 2010) In fact, it is at these optimal salinities that the energy needs for osmotic regulation appear to be the lowest and that growth is the greatest (Yagi et al 1990) Similar results have been reported in the studies of by Yagi et al (1990), in which maximal respiration rates were recorded at optimal salinity (25^31g L À 1) In the present study, we only separately investigated the e¡ects of temperature and salinity on the oxygen consumption of tawny pu¡er juvenile, while the combined e¡ects of salinity and temperature on the oxygen consumption have been demonstrated in some other aquatic organisms (Yagi et al 1990; Spanopoulos-HernaŁndez et al 2005) Therefore, further studies are needed to clarify the combined e¡ect of salinity and temperature on the oxygen consumption of tawny pu¡er juvenile In addition, it must be stressed that the e¡ects of temperature and salinity on a given species should not be the same for all life stages; physiological variations are age-dependent, and hence, the metabolic responses can vary (Spanopoulos-HernaŁndez et al 2005) In summary, both temperature and salinity signi¢cantly a¡ected the oxygen consumption of tawny pu¡er juvenile The OCR increased with an increase in temperature from 20 to 29 1C The optimal temperature for the juvenile lies between 23 1C and 26 1C, based on the lowest Q10 value The OCRs show a parabolic relationship with salinity (5^35 g L À 1), decreasing on either side of the optimum The optimal salinity range for the juvenile is from 23 to 25 g L À The results of this study are useful in facilitating an increase in the production of the species juvenile culture Acknowledgments We would like to thank Zhiwen Zhang, Jiabo Xu and Xiaodong Zhu for their assistance during the experiments.We are also grateful to the two anonymous reviewers for their helpful suggestions and English corrections, which considerably improved the manuscript This study was supported by the Agriculture Commission and the Sciences and Technology Commission of Shanghai References Aristizabal-Abud E.O (1992) E¡ects of salinity and weight on routine metabolism in the juvenile croaker, Micropogonias furnieri (Desmarest 1823) Journal of Fish Biology 40, 471^472 Brett J (1987) Environmental factors a¡ecting growth In: Fish Physiology, Vol (ed by W.H Hoare, D.J Randall & S.R Brett), pp 252^259 Academic Press, NewYork, NY, USA Brown J.H., James F.G., Allen A.P., Savage V.M & West G.B (2004) Toward a metabolic theory of ecology Ecology 85, 1771^1789 Chang Y.J., Jeong M.H., Min B.H., Neill W.H & Fontaine L.P (2005) E¡ects of photoperiod, temperature, and ¢sh size on oxygen consumption in the black porgyAcanthopagrus r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 301^307 305 Temperature and salinity e¡ect on tawny pu¡er juvenile Y Shi et al schlegli Journal of Fisheries Science and Technology 8, 142^150 Claireaux G & Lagarde're J.P (1999) In£uence of temperature, oxygen and salinity on the metabolism of the European sea bass Journal of Sea Research 42, 157^168 Dall W & Smith D.M (1986) Oxygen consumption and ammonia excretion in fed and starved tiger prawns, Penaeus esculentus Haswell Aquaculture 55, 23^33 Dalla Via G.J (1987) E¡ects of salinity and temperature on oxygen consumption in a freshwater population of Palaemonetes antennarius (Crustacea, Decapoda) Comparative Biochemistry and Physiology 88A, 299^305 Dalla Via J., Villani P., Gasteiger E & NiederstÌtter H (1998) Oxygen consumption in sea bass ¢ngerling Dicentrarchus labrax exposed to acute salinity and temperature changes: metabolic basis for maximum stocking density estimations Aquaculture 169, 303^313 Daoud D., Chabot D., Audet C & Lambert Y (2007) Temperature induced variation in oxygen consumption of juvenile and adult stage of the northern shrimp Pandalus borealis Journal of Experimental Marine Biology and Ecology 347, 30^40 Das T., Pal A.K., Chakraborty S.K., Manush S.M., Sahu N.P & Mukherjee S.C (2005) Thermal tolerance, growth and oxygen consumption of Labeo rohita fry (Hamilton, 1822) acclimated to four temperatures Journal of Thermal Biology 30, 378^383 D|¤ az F., Re A.D., GonzaŁlez R.A., SaŁnchez L.N., Leyva G & Valenzuela F (2007) Temperature preference and oxygen consumption of the largemouth bass Micropterus salmoides (Lace¤pe'de) acclimated to di¡erent temperatures Aquaculture Research 38, 1387^1394 Fielder D.S., Bardsley W.J., Allan D.L & Pankhurst P.M (2005) The e¡ects of salinity and temperature on growth and survival of Australian snapper, Pagrus auratus larvae Aquaculture 250, 201^214 Kim W.S.,Yoon S., Kim J.M., Gil J.W & Lee T.W (2005) E¡ects of temperature changes on the endogenous rhythm of oxygen consumption in the Japanese £ounder Paralichthys olivaceus Fisheries Science 71, 471^478 Kinne O (1962) Physiology of estuarine organisms with special reference to salinity and temperature; generai aspects In: Estuaries (ed by G.H Lau¡), pp 525^540 American Association for the Advancement of Science, Washington, DC, USA Kita J.,Tsuchida S & Setoguma T (1996) Temperature preference and tolerance, and oxygen consumption of the marbled rock¢sh, Sebastiscus marmoratus Marine Biology 125, 467^471 LyytikÌinen L & Jobling M (1998) The e¡ect of temperature £uctuations on oxygen consumption and ammonia excretion of underyearling Lake Inari Arctic charr Journal of Fish Biology 52,1186^1198 Meade M.E., Doeller J.E., Kraus D.W & Watts S.A (2002) Effects of temperature and salinity on weight gain, oxygen 306 Aquaculture Research, 2011, 42, 301–307 consumption rate, and growth e⁄ciency in juvenile redclaw cray¢sh Cherax quadricarinatrcs Journal of theWorld Aquaculture Society 33, 188^198 Miklos P., Katzmana S.M & Cech J.J Jr (2003) E¡ect of temperature on oxygen consumption of the leopard shark,Triakis semifasciata Environmental Biology of Fishes 66, 15^18 Morgan J.D & Iwama G.K (1991) E¡ects of salinity on growth, metabolism, and ionic regulation in juvenile rainbow trout and steelhead trout (Oncorhynchus mykiss) and fall chinook salmon (Oncorhynchus tshawytscha) Canadian Journal of Fisheries and Aquatic Sciences 48, 2083^2094 Parry G & Potts W.T.W (1965) Sodium balance in the fresh water prawn, Palaemonetes antennarius Journal of Experimental Biology 42, 415^421 Saucedo P.E., Ocampo L., Monteforte M & Bervera H (2004) E¡ect of temperature on oxygen consumption and ammonia excretion in the Cala¢a mother-of-pearl oyster, Pinctada mazatlanica (Hanley, 1856) Aquaculture 229, 377^387 Shi Y.H., Zhang G.Y., Zhu Y.Z., Yan Y.L., Liu J.Z & Zhu J.M (2009) Study on arti¢cial breeding techniques of Takifugu £avidus in Estuary Area of Hangzhou Bay In: Modern Fisheries and Sustainable Development, Vol (ed by X.L Zhang & J Liu), pp 165^172 Ocean Press, Beijing, China Shi Y.H., Zhang G.Y., Zhu Y.Z., Liu J.Z & Zang W.L (2010) Effects of temperature on fertilized eggs and larvae of tawny pu¡er Takifugu £avidus Aquaculture Research (in press), doi:10.1111/j.1365-2109.2009.02460.x Spanopoulos-HernaŁndez M., Mart|¤ nez-Palacios C.A., Vanegas-Pe¤rez R.C., Rosas C & Ross L.G (2005) The combined e¡ects of salinity and temperature on the oxygen consumption of juvenile shrimps Litopenaeus stylirostris (Stimpson,1874) Aquaculture 244, 341^348 Styczynska-Jurewicz E (1970) Bioenergetics of osmoregulation in aquatic animals Polskie Archiwum Hydrobiologii 17, 295–302 Tian X.L., Dong S.L.,Wang F & Wu L.X (2004) The e¡ects of temperature changes on the oxygen consumption of juvenile Chinese shrimp Fenneropenaeus chinensis Osbeck Journal of Experimental Marine Biology and Ecology 310, 59^72 Yagi H., Ceccaldi H.J & Gaudy R (1990) Combined in£uence of temperature and salinity on oxygen consumption of the pink shrimp, Palaemon serratus (Pennant) (Crustacea, Decapoda, Palaemonidae) Aquaculture 86,77^92 Yang Z & Chen Y.F (2005) E¡ect of temperature on incubation period and hatching success of obscure pu¡erTakifugu obscurus (Abe) eggs Aquaculture 246, 173^179 Yang Z.F., Zhang H.Q & KuangY.H (1991) Studies on biology of Takifugu £avidus in Bohai Bay Marine Sciences Bulletin 10, 44^47 (in Chinese with English abstract) Yukihira H., Lucas J.S & Klumpp D.W (2000) Comparative e¡ects of temperature on suspension feeding and energy r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 301^307 Aquaculture Research, 2011, 42, 301^307 Temperature and salinity e¡ect on tawny pu¡er juvenile Y Shi et al budgets of the pearl oysters, Pinctada margaritifera and P maxima Marine Ecology Progress Series 195, 179^188 Zhang G.Y., Shi Y.H., Zhu Y.Z., Liu J.Z & Zang W.L (2010) E¡ects of salinity on embryos and larvae of tawny pu¡er Takifugu £avidus Aquaculture 302,71^75 Zheng Z.M., Jin C.H., Li M.Y., Bai P.F & Dong S.L (2008) E¡ects of temperature and salinity on oxygen consumption and ammonia excretion of juvenile miiuy croaker, Miichthys miiuy (Basilewsky) Aquaculture International 16, 581^589 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 301^307 307 Aquaculture Research, 2011, 42, 308^312 doi:10.1111/j.1365-2109.2010.02623.x SHORT COMMUNICATION Mud crab, Scylla tranquebarica (Decapoda: Portunidae), a new host for the white spot syndrome virus Ayyaru Gopalakrishnan1, Mayalagu Rajkumar1,2, Jun Sun2, Min Wang3 & Kesavanandha Senthil Kumar4 Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai,Tamil Nadu, India Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China College of Marine Life Sciences, Ocean University of China, Qingdao, China Society of Aquaculture Professionals,Vettuvankeni, Chennai,Tamil Nadu, India Correspondence: Prof Dr Jun Sun, Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7th Nanhai Road, Qingdao 266071, China E-mail: phytoplankton@163.com In recent years, viral diseases have become important limiting factors for shrimp production throughout the world (Flegel 1997) The white spot syndrome virus (WSSV) is one of the most prevalent virus, which infects and causes disease that are responsible for the mass mortality of cultured shrimp in India and other countries In India alone, the net loss has been estimated at several million dollars per year due to this pathogen (Anonymous1996).TheWSSV has awide range of hosts, including 40 species of crustaceans (Cai, Huang,Wang, Song, Sun,Yu, Zhang & Yang1995; Chang, Chen & Wang 1998; Lightner, Hasson, White & Redman 1998; Wang, Lo, Chang & Kou1998) The occurrence of WSSV in wild crabs Calappa lophos (Herbst, 1785), Portunus sanguinolentus (Herbst, 1785), Portunus pelagicus (Linnaeus, 1758), Charybdis sp., Helice tridens (De Haan, 1835) and Scylla serrata (Forsskal, 1755) has been documented clearly (Chang et al 1998; Kanchanaphum,Wongteerasupaya, Sitidilokratana, Boonsaeng, Panyim,Tassanakajon,Withyachumnarnkul & Flegel 1998; Chen, Lo, Chiu, Chang & Kou 2000; Corbel, Zuprizal, Shi, Huang, Sumartono, Arcier & Bonami 2001) Natural WSSV infection has also been found in both captured and cultured specimens of mud crab, S serrata, in Taiwan (Lo, Ho, Peng, Chen, Hsu, Chiu, Chang, Liu, Su, Wang & Kou 1996) and in Thailand (Flegel 1997; Kanchanaphum et al 1998) Chang et al (1998) and Wang et al (1998) 308 foundWSSV infection but without signi¢cant loss or viral disease in avariety of marine crabs and spiny lobsters Scylla tranquebarica (Fabricius,1798) is one of the candidate species that has widely been used and cultured as mono- and polyculture along with both ¢n- and shell¢sh It is also being reared in cages and pens in southeast Asia S tranquebarica is larger than S serrata It grows comparatively faster then S serrata Its availability in the wild is very common in backwater and mangrove environments Its entry into shrimp ponds is possible, because some farmers culture this species in cultured ponds neighbouring/adjacent to shrimp ponds (extensive/modi¢ed extensive) The aim of this study was to document WSSV infection in a new candidate species of mud crab (S tranquebarica) and compare experimental infection of mud crabs by WSSV isolates to infection in the penaeid shrimp Penaeus monodon We have con¢rmedWSSV infection using polymerase chain reaction (PCR) of moribund crabs and provide light and electron microscopical evidence that the mud crab is a new host for this virus (light microscope: MAGNUS LIVE USB 2.0 VIEWER) Samples of S tranquebarica infected with WSSV were collected from a crab-cultured farm at Cuddalore in south India The cultured ponds had been stocked with crabs collected from the wild, weighing between 325 and 450 g at a rate of 1/2 m2 in a 0.8 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd Aquaculture Research, 2011, 42, 308^312 area The crabs were fed with trash ¢sh at the rate of 10% of biomass at four intervals of 06:00,12:00,18:00 and 00:00 hours per feed The trash ¢sh, particularly the ¢n¢sh, are predominantly sardines and other juvenile ¢n¢sh caught in the trawl The lethargic (sluggish) and moribund crabs were tested for WSSV using the commercial nested PCR method (Applied Biosystems Inc., Foster City, CA, USA) to con¢rm the WSSV and samples were collected for morphological examination as described below White spots were spotted on the inner surface of the crab shell (Fig 1a) Some spots were found only in moribund and dead crabs, and not in active crabs, even though they were infected with WSSV (PCR con¢rmed) Although crabs started dying after the 90th day of culture, crabs were collected for morphological studies on the 110th day of culture For histopathological studies, the crab gills and hepatopancreas were ¢xed in Davidson’s ¢xative and processed for routine para⁄n sections that were stained with haematoxylin and eosin staining (Lightner 1996) White spot syndrome virus-infected crab tissues taken for electron microscopic studies were ¢xed in 3% glutaraldehyde (pH 7.2), washed in phosphate bu¡er (pH 7.2), post-¢xed in1% osmium tetroxide and washed in bu¡er, dehydrated through an ascending series of graded alcohol from 50% to 100% cleared in propylene oxide The tissue was further in¢ltrated by propylene oxide and embedded in epoxy resin The embedded mould was kept in an incubator (Technico Laboratories Product, Chennai, India) at 60 1C for 48 h, and it was cooled down Semi-thin sections of mm were cut using an ultra microtome (Leica ultracut UCT, Leica Ltd., Germany) with a glass knife and stained with 1% toludine blue Ultrathin sections were taken on a copper grid and stained with uranyl acetate and Reynold’s solution The sections were examined using a transmission electron microscope (TEM) (Phillips model 201-C, Phillips Electron Instruments, Mahwah, NJ, USA) For scanning electron microscopy (SEM), the crab carapace and the gill were ¢xed immediately in 2.5% glutaraldehyde in 0.2 M phosphate bu¡er at a pH of 7.2 (JEOL JSM-5610LV SEM, JEOL Ltd., Tokyo, Japan) The samples were post-¢xed with 1% osmium tetroxide in the same bu¡er, dehydrated through a graded series of ethanol and critical point dried The samples were coated with gold and observed under a SEM Healthy P monodon (Fabricius, 1798) (5 g mean weight) were reared at the rate density of 10 animals per tub (50 L) (control and experiment both in triplicate) with su⁄cient aeration The arti¢cial feed had Mud crab is a new host for WSSV A Gopalakrishnan et al been was withheld for 24 h before the start of the experiment Once a day, the shrimps were fed crab muscle containing WSSVat a rate of 0.28 g g À of shrimp body weight For the control, beef liver was given (to avoid the WSSV completely) Water salinity and temperature were maintained constant for both control and experiment Mortality was observed daily and dead shrimps were removed from the tank every day A water-borne infection trial was performed using the ¢ltrate of crab muscle from the infected S tranquebarica Muscle from the infected crab was removed and homogenized in brackish water (20 ppt) at 1C at a ratio of 1:9 After being centrifuged at 8518 g for min, the supernatant £uid was ¢ltered through a 0.45 mm membrane and diluted 5000 times with brackish water, for use as water-borne inoculation (Chou, Huang,Wang, Chiang & Lo1995) The shrimps were immersed in these diluted ¢ltrates for h Controls were exposed similarly to sterilized brackish water After inoculation, shrimps were kept in plastic tubs (50 L) with aeration The white spots are smaller (3 mm) and denser in the crab (Fig 1a) than in the shrimp P monodon (Fig 1b) Scanning electron microscopy showed that in the spontaneously infected crabs, the white spots were clear and looked amoeboidal (di¡erent in shape and size) in nature Apart from the white spots, bacteria could also be seen (Fig.1c) The SEM study on gill lamellae revealed muddy debris in the WSSV-infected crab gills (Fig.1d) Because of virus infection, the fouling of microbes and muddy debris would result The ultrathin sections of the gill tissue from spontaneously viral-diseased moribund crab showed nonoccluded enveloped viral particles with rod-shaped morphology (Fig.1e) The rod-shaped virions were aggregated in the nuclei of infected gill cells (Fig 1f) Mature virions were localized in the periphery of the nuclei and, less densely, in the cytoplasm (Fig 1e) Virions of di¡erent morphologies were evident in the nuclei The aggregations of viral particles were evidenced in the nucleus when compared with cytoplasm The virions are di¡erent in their morphologies, they are rod shaped and also ovoidal in shape, and empty viral capsids are also found in the nucleus It is densely arranged in the nucleus than in the cytoplasm Further, it could be seen that sometimes the gill lamellae were fouled with bacteria (Fig.1g) Empty capsids were present in the cytoplasm of the cell and were scattered randomly; partial capsids could also be seen in the cytoplasm (Fig 1f) The histological sections of hepatopancreas and gills showed some pathological changes (Fig 1h) In the r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 308^312 309 Mud crab is a new host for WSSV A Gopalakrishnan et al Aquaculture Research, 2011, 42, 308^312 Figure White spot syndrome virus (WSSV) on Scylla tranquebarica [(a) light photo micrograph of S tranquebarica of inner side carapace surface with white spots]; (b) Penaeus monodon carapace with white spots; (c) scanning electron microscopic of the white spots and bacteria in the inner carapace of S tranquebarica; (d) scanning electron microscopic of muddy debris deposition in the gill of S tranquebarica; (e) electron dense viral particles in the nucleus of the gill cell of S tranquebarica; (f) transmission electron micrograph of viral particles in the cytoplasm; (g) transmission electron micrograph of bacterial fouling in the WSSV-infected moribund crab gill lamellae; (h) light micrograph of hepatopancreas of S tranquebarica showing the inclusion bodies; (i) light micrograph of gill lamellae of S tranquebarica; (j) scanning electron micrograph of gill lamellae thickening bacterial fouling in the WSSV-infected moribund crab; (k) cumulative mortality of Penaeus monodon exposed to the WSSV through water and ingestion routes hepatopancreas, degenerated cells with hypertrophy were observed Basophilic inclusions can be seen in the crab hepatopancreas, and it is a characteristic of WSSV infection Darkly stained inclusions bodies could also be seen in the sections Gill cells were also characterized by hypertrophied nuclei and eosinophilic to basophilic inclusions (Fig 1i) The gillchoked (clogging) crab lamellae were characterized by epithelial thickening and intra-cellular necrosis 310 (Fig 1j) The normal epithelial thickness was mm, due to the fouling thickness that was mm due to infection WSSV infection occurred primarily through water and ingestion Both water and ingestion routes are the prime routes for WSSV infection Infection through the oral ingestion route occurs when WSSV-infested crabmeat is used as feed Infection will be also through the water route when the infested crabmeat is dissolved in the culture medium r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 308^312 Aquaculture Research, 2011, 42, 308^312 (water) Following ingestion, the infected shrimps showed white spots inside the carapace The mortality of the shrimp reached100% by the 23rd day In the case of the water-borne infection trial, mortality has been recorded till the 23rd day, and after that, no mortality was recorded up to the end of the experiment Clear white spots were observed in all the dead shrimps In the case of controls, no mortality was observed up to the end of the experiment (Fig 1k) Unlike shrimp, crabs infected with WSSV in ponds su¡er chronic mortalities The WSSV is extremely virulent and has a wide range of hosts (Lo, Ho, Chen, Liu, Chiu,Yeh, Peng, Hsu, Liu, Chang, Su,Wang & Kou 1997) It is virulent to all the crustaceans, but comparatively, it is more severe on shrimps then on crabs in terms of outbreak (mortalities in cultured ponds) In crab farms, the mortality is chronic in nature, but in shrimp, mass mortalities are very common In most of the shrimp farms, nets are placed around the ponds to prevent the crab from entering and infecting the farms It is for any number of crabs, including freshwater crabs around the farms The crabs are considered as carriers of WSSV (Supamattaya, Holfmann, Booyaratpalin & Kanchanaphum 1998) Therefore, the shrimp farmers eradicate the crabs when preparing the ponds Crabs are eliminated during the pond-preparation stage itself by drying and ploughing the pond bottom thoroughly Besides, the ponds are disinfected with chlorine (sodium hypochloride 30 ppm) before stocking the crabs The cannibalistic behaviour of the crabs has also been evidenced during outbreaks Dead and mutilated crabs were also observed during outbreaks During the viral disease outbreaks, other entrants such as the shrimps P monodon, Panaeus semisulcatus (De Haan, 1844) and Metapenaeus spp were found, which were also infected, but no mortality has been observed in them Other crabs such as P pelagicus and Thalamita spp were also found dead in the pond during the outbreak Both ultrastructural and infectivity observations have shown that WSSV was the causative agent for the crab mortalities in the pond Extensive tissue damage occurs in the crabs’ gill tissue, which is su⁄cient to interfere with the normal physiological function In the present study, the viral particles were concentrated around the periphery of the nucleus of the gill cell Also, empty viral capsids were located in the central portion of the a¡ected cytoplasm and they were enveloped, as reported previously by Durand, Lightner, Redman and Bonami (1997) in P monodon The SEM study of the white spots in the carapace shows clear amoeboidal white Mud crab is a new host for WSSV A Gopalakrishnan et al spots and bacterial fouling in the inner shell, which is in agreement with results reported by authors Wang, Lee, Najiah, Shari¡ and Hassan (2000) Bacterial fouling was observed in the moribund crab gill tissue Both viral particles and bacterial fouling occurred in gill cells Apart from the primary infection (virus), the bacterial infection further aggravates the situation and stresses the organisms, while gill thickening due to haemolytic in¢ltration and chronic fouling by the bacteria further stress the organisms Supamattaya et al (1998) reported the protozoan fouling in the viral-infected shrimps The light microscopic study of hepatopancreas reveals the occurrence of basophilic hypertrophied nuclei, eosinophilic to basophilic inclusions and cellular necrosis, as reported by Wang, Lo, Leu, Chou, Yeh, Chou, Tung, Chang, Su and Kou (1995) In the WSSV infectivity investigation, 95.25% mortality has been observed on the 17th day through the oral ingestion route, but in the case of water-borne infection, it was 95% on the 23rd day Earlier, Chou et al (1995) recorded 100% infection in 14 days for the water-borne infection route, but only in days for the oral ingestion route In the current investigation, 100% mortality did not occur by either route of infection, and furthermore, the duration for the mortality to occur was longer than in earlier investigations This could have been due to lower virulence by this virus strain, or alternatively, the shrimp might have developed resistance against the virus, or both The present study was the ¢rst report describing that white spots could be seen even in crabs (moribund carapace) Further, the mud crab S tranquebarica was a host, not just a carrier Acknowledgments We would like to thank Prof T Balasubramanian, Director and Mr T M.Vasanthan, Special O⁄cer, Centre of Advanced Study in Marine Biology, Annamalai University for their valuable suggestions during the study We also would like to express our gratitude to Dr Ian Jenkinson for his valuable comments We thank the crab and shrimp farmers and the technicians who provided the specimens for the analysis The Christian Medical College, Vellore, Tamil Nadu, India for TEM work and SEM by Central Instrumentation Laboratory of Annamalai University are greatly acknowledged This work was also supported by Chinese Academy of Sciences Research Fellowship for International Young Researchers to M Rajkumar r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 308^312 311 Mud crab is a new host for WSSV A Gopalakrishnan et al hosted by J Sun, and Knowledge Innovation Project of The Chinese Academy of Sciences (KZCX2-YWQN-205 and KZCX2-YW-213-2) to J Sun References Anonymous (1996) Report of Marine Products Export Development Agency The Hindu, June 4, p 17 Cai S., Huang J., Wang C., Song X., Sun X., Yu J., Zhang Y & Yang C (1995) Epidemiological studies on the explosive epidemic disease of prawn in 1993^1994 Journal of Fisheries of China 19,112^117 Chang P.S., Chen H.C & WangY.C (1998) Detection of white spot syndrome associated baculovirus in experimentally infected wild shrimp, crab and lobsters by in situ hybridization Aquaculture 164, 233^242 Chen L.L., Lo C.F., Chiu Y.L., Chang C.F & Kou G.H (2000) Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata Diseases of Aquatic Organisms 40,157^161 Chou H.Y., Huang C.Y., Wang C.H., Chiang H.C & Lo C.F (1995) Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan Diseases of Aquatic Organisms 23, 165^173 Corbel V., Zuprizal Z., Shi C., Huang I., Sumartone C., Arcier J.M & Bonami J.R (2001) Experimental infection of European crustaceans with white spot syndrome virus (WSSV) Journal of Fish Diseases 24, 377^382 Durand S., Lightner D.V., Redman R.M & Bonami J.R (1997) Ultra structure and morphogenesis of white spot syndrome baculovirus (WSSV) Diseases of Aquatic Organisms 29, 205^211 Flegel T.W (1997) Special topic review: major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand World Journal of Microbiology and Biotechnology 13, 433^ 442 Kanchanaphum P., Wongteerasupaya C., Sitidilokratana N., Boonsaeng V., Panyim S., Tassanakajon A., Withyachumnarnkul B & Flegel T.W (1998) Experimental transmission of white spot syndrome virus (WSSV) from crabs to shrimp Penaeus monodon Diseases of Aquatic Organisms 34,1^7 312 Aquaculture Research, 2011, 42, 308^312 Lightner D.V (1996) A Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid Shrimp World Aquaculture Society, Baton Rouge, LA, USA Lightner D.V., Hasson K.W.,White B.L & Redman R.M (1998) Experimental infection of Western hemisphere penaeid shrimp with Asian white spot syndrome virus and Asian yellow head virus Journal of Aquatic Animal Health 10, 271^281 Lo C.F., Ho C.H., Peng S.E., Chen C.H., Hsu H.C., Chiu Y.L., Chang C.F., Liu K.F., Su M.S., Wang C.H & Kou G.H (1996) White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods Diseases of Aquatic Organisms 27, 215^225 Lo C.F., Ho C.H., Chen C.H., Liu K.F., ChiuY.L.,Yeh P.Y., Peng S.E., Hsu H.C., Liu H.C., Chang C.F., Su M.S.,Wang C.H & Kou G.H (1997) Detection and tissue tropism of white spot syndrome baculovirus (WSBV) in captured brooders of Penaeus monodon with a special emphasis on reproductive organs Diseases of Aquatic Organisms 30, 53^72 Supamattaya K., Holfmann R.W., Booyaratpalin S & Kanchanaphum R (1998) Experimental transmission of white spot syndrome virus (WSSV) from black tiger shrimp Penaeus monodon to the sand crab Portunus pelagicus, mud crab, Scylla serrata and krill Acetes sp Diseases of Aquatic Organisms 32,79^85 Wang C.H., Lo C.F., Leu J.H., Chou C.M., Yeh P.Y., Chou H.Y., Tung M.C., Chang C.F., Su M.S & Kou G.H (1995) Puri¢cation and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon Diseases of Aquatic Organisms 23, 239^242 WangY.C., Lo C.F., Chang P.S & Kou G.H (1998) Experimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan Aquaculture 164, 221^231 Wang Y.G., Lee K.L., Najiah M., Shari¡ M & Hassan M.D (2000) A new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS) caused by virus Diseases of Aquatic Organisms 41, 9^18 Keywords: mud crab, Scylla tranquebarica,WSSV r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 308^312 [...]... molecular detection of Flavobacterium columnare Applied and Environmental Microbiology 72, 1702–1704 Thrus¢eld M (1995) Veterinary Epidemiology, 2nd edn Blackwell Science Ltd, Oxford, UK, 483pp r 2010 Blackwell Munksgaard No claim to original US government works, Aquaculture Research, 42, 169^176 Aquaculture Research, 2011, 42, 188^195 doi:10.1111/j.1365-2109.2010.02579.x Sperm capacitation of the shrimp... spike contains abundant micro¢laments (c) Two prominent electron-dense triangular dots (arrows) at the base of the spike 190 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 188^195 Aquaculture Research, 2011, 42, 188^195 Sperm capacitation of L vannamei S Aungsuchawan et al Figure 2 Transmission electron microscopy of S- and T-sperm of Litopenaeus vannamei... of the cap region of S-sperm, especially those that were embedded in the spermatophoric matrix, r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 188^195 191 Sperm capacitation of L vannamei S Aungsuchawan et al Aquaculture Research, 2011, 42, 188–195 were observed (Fig 3a) The two lines were less prominent in T-sperm (Fig 3b), compared with those of the... when a bright red spot of the P-tyr signal appeared at the centre of FM (d, arrowhead) All pictures use the same scale bar 192 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 188^195 Aquaculture Research, 2011, 42, 188^195 without microtubules or centrioles, and unlike Macrobrachium rosenbergii sperm, which possessed a pair of centrioles in closely associated... glucose Biology of Reproduction 64, 1350^1357 Vacquire V.D (1998) Evolution of gamete recognition proteins Science 281, 1995^1998 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 188^195 Aquaculture Research, 2011, 42, 188^195 Vanichviriyakit R., Kruevaisayawan H., Weerachatyanukul W., Tawipreeda P., Withyachumnarnkul B., Pratoomchat B., Chavadej J & Sobhon... Mammalian fertilization The Physiology of Reproduction (ed by E Knobil & JD Neill), pp 189^317 Raven Press, NewYork, NY, USA r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 188^195 195 Aquaculture Research, 2011, 42, 196^209 doi:10.1111/j.1365-2109.2010.02606.x Evaluating the use of Lactobacillus acidophilus as a biocontrol agent against common pathogenic... gr.2 and 20.33 mm for S xylosus, while the inhibition zone for S agalactiae was 21.17 mm (Al-Dohail et al 2008) r 2010 Universiti Sains Malaysia Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 196^209 Aquaculture Research, 2011, 42, 196^209 L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Table 1 De¢nition of ¢sh groups and treatments used for the feeding... non-probiotic controls, whereas the non-probiotic treatment di¡ered signi¢cantly from both controls in their ESR values r 2010 Universiti Sains Malaysia Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 196^209 Aquaculture Research, 2011, 42, 196^209 L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Table 2 Haematological parameters of Clarias gariepinus at 7 days... any noticeable di¡erence in the Ig levels between the non-probiotic treatments and the non-probiotic control r 2010 Universiti Sains Malaysia Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 196^209 201 Aquaculture Research, 2011, 42, 196–209 L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al Table 3 Haematological parameters of clarias gariepinus at 21... mild effects observed in the infected ¢sh fed the probiotic diet during the same period (Fig 1; images 7Psx, r 2010 Universiti Sains Malaysia Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 196^209 Aquaculture Research, 2011, 42, 196^209 L acidophilus as a probiotic in African cat¢sh M A Al-Dohail et al (a) 7NPsx 7NPc 7NPah 7NPsa 7Pah 7Psa A B A 7Pc 7Psx A A A B (b)