Aquaculture research, tập 43, số 1, 2012

Aquaculture Research, 2012, 43, 1–13 doi:10.1111/j.1365-2109.2010.02785.x Effects of low dissolved oxygen on the digging behaviour and metabolism of the hard clam (Meretrix lusoria) An-Chin Lee,Yu-Ching Lee & Tzong-Shean Chin Department of Aquatic Biosciences, College of Life Science, National Chiayi University, Chiayi, Taiwan Correspondence: A-C Lee, Department of Aquatic Biosciences, College of Life Sciences, National Chiayi University, 300 University Road, Chiayi 600, Taiwan E-mail: aclee@mail.ncyu.edu.tw Abstract The dissolved oxygen (DO) concentration that induces the onset of anaerobic metabolism in hard clams was found to be 1.11mg O2 L À 1, at which time, the concentration of succinate in the body £uid was 4.4 mmol mL À When the DO concentration was o1.11mg O2 L À 1, the burial depth was signi¢cantly reduced, and succinate signi¢cantly accumulated in the body £uid After 24 h of anoxic exposure, succinate had accumulated in the gills and equal amounts of succinate and alanine had accumulated in the foot This indicates that carbohydrates in the gills and amino acids in the foot contribute to anaerobic energy production The accumulation rates of succinate and propionate in the body £uid were the highest compared with those in other tissues, while no accumulation of alanine in the body £uid was found The recovery rates of succinate in the body £uid and alanine in the foot were the highest compared with those in other tissues The results of this study suggest that the DO concentration in the bottom water of clam ponds should be maintained at ! 1.11mg O2 L À 1, and the anoxia-tolerant ability of hard clams can be assessed by the contents of carbohydrates Keywords: hard clam, critical concentration, DO, digging behaviour, anoxia Introduction Hard clams (Meretrix lusoria) are widely found throughout Asia, in Japan, Korea, China,Taiwan and © 2011 Blackwell Publishing Ltd elsewhere They naturally reside in the estuary of the Tanshui River in northern Taiwan and along the west coast of Taiwan Most hard clams sold commercially are cultured in ponds in Taiwan, as local estuaries are polluted by industrial pollutants during the early rainy season (Jeng & Wang 1976) However, massive die-o¡s of hard clams have occurred in ponds in times of high water temperatures (Tseng 1976) High stocking densities (1^2  106 seeds À 1) and supplementation of powdered ¢sh meal and fermented organic matter in ponds can result in a reducing layer in the sediments and, possibly, in low dissolved oxygen (DO) concentrations in the bottom waters of ponds (Hon 1988) Low DO may decrease the burial depth, alter the metabolism and ultimately cause massive die-o¡s of hard clams Clarifying the cause of these massive die-o¡s of hard clams is very important for the clam culture industry as they are an important economic bivalve, and their production ranked ¢rst among the malacofauna in Taiwan in 2008 (Fisheries Agency 2008/2010) Hard clams are commonly found either partially or completely buried in bottom sediments of their marine habitat, suggesting that they have developed the metabolic capacity to survive periods of hypoxia and anoxia When anaerobic conditions become severe, physiological and behavioural changes occur that are associated with high clam mortality The in£uence of environmental oxygen on adaptive biochemical and physiological survival strategies is well documented for a variety of facultative anaerobic mollusks, and data indicate that critical oxygen levels that trigger hypoxic and anoxic Digging behavior and metabolism of hard clams A-C Lee et al energy-producing pathways di¡er among species (Cheng, Chang & Chen 2004; Long, Brylawski & Seitz 2008) The current research concerns the relationship between environmental oxygen availability, and the burial behaviour and metabolism of hard clams DO concentrations considerably a¡ect the survival and behaviour of aquatic organisms (Long et al 2008) The DO concentration that induces the onset of anaerobic metabolism is de¢ned as the critical concentration, which varies with di¡erent bivalves (de Zwaan, Cortesi, van der Thillart, Brooks, Storey, Roos, van Lieshout, Cattani & Vitali1992; Le Moullac, Que¤au, Le Souchu, Pouvreau, Moal, Le Coz & Samain 2007) When hard clams experience hypoxia, they usually emerge from the bottom sediment to stay at a higher position in search of oxygen (Lee, Lin, Lin, Lee & Chen 2007) After emerging from the bottom, clams usually die within a short time if extra oxygen is not found Once environmental hypoxia occurs, the ability to obtain chemical energy through anaerobic metabolism a¡ects their survival The strategy adopted by marine mollusks exposed to hypoxia/anoxia requires a decrease in ATP turnover that involves coordinated down-regulation of ATP consumption and production (i.e through depression of the metabolic rate) In mollusks, metabolic rates decline by a factor of 10^20 or more (Storey & Storey 1990; Brooks & Storey 1997) The remaining lowATP demand is covered by the chemical energy produced through anaerobic metabolism Anaerobic metabolism has been widely studied in many kinds of invertebrates and vertebrates (de Zwaan 1983; Storey & Storey 1990; de Zwaan & Eertman 1996; Larade & Storey 2002) Under hypoxia/ anoxia, invertebrates can obtain energy by anaerobic metabolism that occurs under functional and environmental anoxia (Livingstone 1991; P˛rtner 2002) The lactate and opine pathways play important roles in functional anoxia, while the aspartate^succinate and glucose^succinate pathways are utilized during environmental anoxic survival (Livingstone 1991; Lee, Lee, Lee & Lee 2008; Lee & Lee 2010) Anoxic-tolerant mollusks exposed to hypoxia meet their energy demands mostly by relying on glycolysis However, alternative substrate-level phosphorylation with the accumulation of succinate or propionate occurs when the concentration of DO is lower than a critical concentration The initial responses to anoxia are coupled to the fermentation of glycogen and aspartate, causing the accumulation of succinate and alanine In the aspartate^succinate pathway, amino groups from aspartate are transferred to pyruvate to Aquaculture Research, 2012, 43, 1–13 form alanine, and their carbon skeleton, oxaloacetate, is then transformed to succinate in hypoxia When aspartate pools are depleted, succinate is mostly formed from glycogen, which involves the £ow of carbon through the phosphoenolpyruvate (PEP) branch point of glycolysis This is accomplished via inhibition of pyruvate kinase due to the accumulation of alanine and protons This favours the carboxylation of PEP as mediated by the PEP carboxykinase reaction to produce oxaloacetate that feeds the reaction of succinate synthesis (Bacchiocchi & Principato 2000; Larade & Storey 2002) Under prolonged anoxia, succinate is converted to propionate by a pathway inside the mitochondria that produces an extra mole of ATP per unit of succinate (Schulz, Kluytmans & Zandee 1982; Isani, Cattani, Zurzolo, Pagnucco & Cortesi 1995) A hallmark of anoxic adaptation is an evolved capacity for the rapid reversible entry into and return from metabolically depressed steady states This involves speci¢c control of key regulatory enzymes to reorganize metabolism and allow entry and arousal from anoxia (Larade & Storey 2002, 2009) The critical concentration of DO is an important parameter for managing clam culture However, information on the relationship between the DO concentration of seawater and the burial behaviour of hard clams is scant In addition, the study of anaerobic energy and its sources in tissues of hard clams so far has been super¢cial, although some results were reported (Lee et al 2008) Therefore, the aims of this study were to examine the e¡ects of the DO concentration on the burial behaviour of hard clams, to determine the critical DO concentration inducing the onset of anaerobic metabolism, and ¢nally to examine anaerobic energy sources in tissues of hard clams Material and methods Animals Hard clams (14.6 Æ 1.1g) were purchased from clam farms in the Taishi area (Yunlin County, southwestern Taiwan) Arti¢cial seawater (ASW) (20 g L À 1) was prepared by dissolving 1:50 (w/v) synthetic sea s salts (Meersaltz , Heinsberg, Germany) in tap water that was strongly aerated for at least weeks before use (Taiwan Water Corporation 2003) No chlorine was detected in the aerated water Three hundred clams were acclimated at room temperature (23^ 28 1C) in two 2000 L tanks, each of which contained 1300 L of 20 g L À ASW with a 10 cm depth of sand © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Aquaculture Research, 2012, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al on the bottom Clams that did not burrow into the sand were excluded Clams were fed the microalgae Isochrysis a¡ galbana, Chaetoceros muelleri and Tetraselmis chui at the respective ¢nal concentrations of  104,  104 and  104 cells mL À twice daily for10 days After days, 400 L of water from the tank was replaced with clean 20 g L À ASW No food was provided day before the experiments Design for water sampling The design for water sampling is presented in Fig The DO concentration of the sample was determined using the Winkler method (azide modi¢cation) (APHA, AWWA & WEF 1995) The end (opening) of a tube was placed on the top of the sediment in an aquarium.Water sampled from the top of the sediment was termed ‘bottom water’ in this study In order to avoid oxygen molecules in the tube and DO bottle with this design, nitrogen was used to £ush the tube and DO bottle when switch I was turned on and switch II was turned o¡ After nitrogen £ushing, a hand-operated vacuum pump was used to aspirate water through the tube when switch II was turned on and switch I was turned o¡ The DO bottle was used to collect sample water to determine the DO concentration Determination of the burial depth and succinate concentrations in the body £uid of hard clams Experiment I: Aquarium with an open-water surface Twelve aquaria [60 (L)  30 (W)  35 cm (H)], each of which contained 40 L of 20 g L À ASW with a cm depth of sand on the bottom, were used in this study Thirty hard clams were placed in each aquarium Mild aeration was applied to the aquarium overnight at 25 1C The experiment began on day 0, and thereafter, no aeration was applied The assessment of digging indices of the hard clams and the determination of DO concentrations in the bottom water were carried out every other day After assessing the digging indices and determining the DO concentration, 30 hard clams from one aquarium were sampled to determine the concentration of succinate in the body £uid This experiment was performed for 20 days Body £uid (extracellular £uid) from ¢ve clams was collected as a replicate Therefore, there were six replicates for each aquarium The digging index of the clams was scored as follows (Lee et al 2007): 1, completely dug into the sand; 0.9, completely dug into the sand but the top of the shell was exposed; 0.33 and 0.67, only 1/3 and 2/3, respectively, of a clam was covered by sand; and 0, the clam was on top of the sand Therefore, the burial depth of hard clams could be indicated by a digging index: a smaller value of the digging index indicated a shallower burial depth Experiment II: Aquarium with 50% of the surface covered In order to limit the in£ux of oxygen molecules in the air into the water, 50% of the water surface in an aquarium was covered by a Styrofoam board Thirteen aquaria were used in this study Seven of them were used to study changes in the concentration of succinate in the body £uid The other six were used to study changes in the digging indices of hard clams and the DO concentration of bottom water in the tank The following conditions were similar to those Figure A design for water sampling © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al in experiment I In the group used to determine the concentration of succinate in the body £uid, 50% of the water surface of six aquaria was covered by a Styrofoam board after the hard clams from an aquarium were sampled to determine the concentration of succinate on day Thereafter, hard clams from an aquarium were sampled to determine the concentration of succinate every day In the group used to determine the digging indices of hard clams and the DO concentration of bottom water, 50% of the water surface of six aquaria was covered by a Styrofoam board after both parameters were determined on day Thereafter, these parameters were determined every day Normoxic and anoxic exposure of hard clams In total, 275 clams were placed in a tank with 275 L of 20 g L À aerated seawater overnight The DO was maintained at ! ppm Five clams were sampled as a replicate, and ¢ve replicates served as a normoxic group (0 h exposure) Twenty litres of 20 g L À ASW placed in a plastic bag with 50 glass jars ($ 130 mL) was bubbled with N2 for h After bubbling, 250 clams were placed in the plastic bag with N2 gas blowing over them Five clams were sealed in a glass jar inside the plastic bag Fifty glass jars were placed at 25 1C Five glass jars were, respectively, sampled at 8, 24, 48 and 64 h After 64 h of anoxic exposure, 20 glass jars with no dead clams were sampled as the materials for the recovery experiment The other jars were not used further in the experiment Recovery after 64 h of anoxic exposure Clams in 20 glass jars after 64 h of anoxic exposure were released into 100 L of ASW with aeration at 25 1C Twenty-¢ve clams were, respectively, sampled at 0,8, 24 and 48 h Five clams were pooled together as a replicate Their body £uid, digestive gland, gills, foot, adductor and mantle were sampled and, respectively, pooled There were ¢ve replicates for each sampling time Aquaculture Research, 2012, 43, 1–13 to the aquaria at 25 1C The digging indices of the hard clams were assessed at 0, 8, 24 and 48 h Collection of specimens, and extraction and determination of anaerobic end products in tissues Collection of specimens After the seawater in the mantle cavity had been drained out, the body £uid (extracellular £uid) was collected from shucked clams as quickly as possible After the body £uid of the clams had been sampled, the foot, digestive gland, adductor, gills and mantle of the ¢ve clams in the glass jar were, respectively, dissected and stored in liquid nitrogen until used Extraction of anaerobic end products in tissues The extraction of anaerobic end products was based on the methods described by Lee et al (2007, 2008) Frozen tissues were powdered in a steel mortar cooled with dry ice One gram of powdered tissue was homogenized in a homogenizer (PRO Scienti¢c, Oxford, CT, USA) with 4.5 mL of 6% cooled perchloric acid (PCA) on ice water The homogenate was centrifuged at 10 000 g for 20 at 1C The precipitate was extracted with a further mL of 6% cooled PCA After centrifugation, both supernatants were combined and neutralized with 2.08 mL of a 3.6 M KOH solution The contents were continuously mixed during neutralization to avoid the production of local alkalinity in the solution, and then the solution was kept on ice for 30 to allow the KClO4 to precipitate The precipitate was washed with mL of deionized water Both supernatants were combined and stored at À 20 1C for later analysis of anaerobic end products Nine millilitres of 6% cooled PCA was added to mL of body £uid The mixture was centrifuged at 10 000 g for 20 at 1C The supernatant was neutralized with 2.5 mL of 3.6 M KOH and placed on ice for 30 Subsequent steps were conducted similar to those described above Determination of anaerobic end products Digging indices during 48 h of recovery after 64 h of anoxic exposure Clams in 18 glass jars after 64 h of anoxic exposure were released and washed as soon as was possible with clean ASW Three aquaria as described above were used in this study Thirty hard clams were placed in each aquarium Mild aeration was applied Succinate and propionate in tissues were analysed by high-performance liquid chromatography (HPLC) using a Transgenomic ICSep ICE-ION-300 column (7.8  300 mm) using 0.001 N sulphuric acid as the mobile phase following the method described by Chiou, Lin and Shiau (1998) The £ow rate was 0.3 mL À The column temperature was set © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 2012, 43, 1–13 Analysis of data to 52 1C, and detection was monitored at 210 nm Succinate and propionate in the body £uid were determined by HPLC using a Mightysil RP-C18 GP column (4.6  250 mm, mm) using 10 mM H3PO4 as the mobile phase The £ow rate was 1mL À The concentration of alanine was determined using an enzymatic method (Grassl & Supp 1984) Among-treatment di¡erences in data from Table 1, and Figs 3, 4, and were determined using Duncan’s comparison test to determine where signi¢cant differences occurred The signi¢cance of all tests was accepted at Po0.05 The accumulation and recovery rates of anaerobic end products in tissues were calcu- Table Concentrations of succinate, alanine and propionate in tissues of hard clams after 0, 8, 24, 48 and 64 h of anoxic exposure Tissue Gills Digestive gland Foot Adductor Mantle Body fluidw Time of anoxic exposure (h) 24 48 64 64–0 24 48 64 64–0 24 48 64 64–0 24 48 64 64–0 24 48 64 64–0 24 48 64 64–0 Anaerobic end products (lmol g À wet weight) Succinate Ã2.34 Æ 0.33d b 8.73 10.2 15.3 Ã20.4 Ã18.0 ÃÃ10.2 14.7 19.6 23.6 ÃÃ29.9 ÃÃ19.7 0.94 3.11 8.08 12.0 Ã11.0 Ã10.1 Ã1.25 3.61 7.77 10.5 Ã11.6 Ã10.4 0.31 2.67 7.73 11.0 Ã10.9 ÃÃ10.6 0.70 11.5 32.9 48.3 ÃÃ50.3 ÃÃ49.6 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.43c 0.80c 0.57b 1.09ac 1.12b 0.21ea 0.53d 0.95c 1.55b 1.56ab 1.64b 0.06dc 0.15c 0.96b 0.81a 0.37ad 0.38c 0.07dc 0.08c 0.40b 0.59a 0.64ad 0.63c 0.12dd 0.33c 0.37b 0.65a 0.61ad 0.67c 0.17dcd 0.70c 0.56b 1.40a 1.73aa 1.70a Alanine Propionate ÃÃ3.36 Æ 0.19c b 3.58 3.94 5.13 7.57 4.21 Ã3.30 5.74 6.32 10.1 Ã10.2 Ã6.92 Ã8.18 14.0 14.2 16.4 ÃÃ18.3 Ã10.2 ÃÃ8.23 10.7 12.1 12.0 Ã12.5 4.23 Ã4.14 5.38 8.25 10.4 Ã9.22 Ã5.08 0.15 0.15 0.26 0.37 0.39 0.24 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.70c 0.29bc 0.55b 0.38ad 0.39c 0.51cb 0.82b 0.49b 0.39a 0.32ac 0.51b 0.83ca 0.45b 0.67b 0.65a 0.83aa 0.98a 0.32ba 0.78ab 1.49a 0.76a 0.94ab 1.19c 0.48cb 0.31c 0.24b 0.73a 0.35ab cd 0.21bc 0.02c 0.02 0.01 0.02 0.01e 0.02d 0.45 0.36 0.46 2.26 5.22 4.76 0.30 0.85 1.53 2.41 2.94 2.65 0.00 0.00 0.00 2.01 2.84 2.84 0.00 0.11 0.59 2.63 3.30 3.3 0.00 0.00 0.00 2.07 3.14 3.14 0.00 0.14 0.16 5.31 Ã8.73 Ã8.73 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 0.09ca 0.14c 0.19c 0.11b 0.94ab 0.92b 0.19ca 0.23bc 0.24b 0.37a 0.33ac 0.35c 0.00cb 0.00c 0.00c 0.21b 0.26ac 0.26bc 0.00db 0.07d 0.18c 0.15b 0.25abc 0.25bc 0.00cb 0.00c 0.00c 0.16b 0.54ac 0.54bc 0.00cb 0.06c 0.16c 0.28b 1.09aa 1.09a Accumulation is expressed as the di¡erence in the concentrations of anaerobic end products in tissues of hard clams between at and at 64 h of anoxic exposure Data are given as the mean Æ the standard error for ¢ve replicates (¢ve clams per replicate) Data of a speci¢c end product in a speci¢c tissue with di¡erent superscript letters signi¢cantly di¡er among di¡erent exposure times (Po0.05) Data of a speci¢c end product at or 64 h anoxic exposure with di¡erent subscript letters signi¢cantly di¡er among tissues (Po0.05) In a speci¢c tissue at a speci¢c exposure time, data of an end product with ‘ÃÃ’ are signi¢cantly greater than those with ‘Ã’, which are in turn signi¢cantly greater than those with no asterisks (Po0.05) wUnits of body £uid are mmol mL À © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 2012, 43, 1–13 decreased sharply from 6.31mg O2 L À on day of exposure to 3.22 mg O2 L À at day and 1.34 mg O2 L À at days of exposure at 25 1C (Fig 3) After days of exposure, the DO concentration of the bottom water had gradually decreased to 0.28 mg O2 L À Although no signi¢cant change in the digging indices of hard clams before days of exposure was found, the digging index (27.7) at days of exposure had dramatically decreased (22.2) at days of exposure This means that hard clams were obviously observed to have emerged from the sediment to some extent At the same time, the concentration of succinate in the body £uid of hard clams had greatly increased from 4.39 mmol mL À at days of exposure to 9.54 mmol mL À at days of exposure Therefore, the third day of exposure was when anaerobic metabolism began in hard clams The DO concentration of 1.11mg O2 L À on the third day of exposure can be de¢ned as the critical concentration of DO for hard clams During the experiment, only six dead clams were found lated by linear regressions using SigmaPlot software vers.10 from SPSS (Chicago, IL, USA) The simple correlation coe⁄cient of the straight line was determined Results Determination of burial depth and succinate level in the body £uid of hard clams The digging index of a hard clam that had completely dug into the sand was scored as1 Therefore, the maximum score for the digging index of an aquarium containing 30 clams was 30 Although the DO concentration in the bottom water dramatically decreased from 6.2 mg O2 L À on day of exposure to mg O2 L À at days of exposure, it was slightly higher at 4.33 mg O2 L À after 20 days of exposure at 25 1C (Fig 2) The depth of the water body in the aquaria was only 20 cm.Water £ow produced by the clams might have been su⁄cient to have created local circulation, which would have enhanced the di¡usion of oxygen molecules in the air into the water This level of DO does not cause stress that would affect the behaviour and metabolism of hard clams After 20 days of exposure, no signi¢cant changes in the digging indices or the concentration of succinate in the body £uid of hard clams were observed (Fig 2) No mortality was found during the experiment The DO concentration in the bottom water of the aquaria with 50% of the water surface covered Anaerobic end products under normoxia and anoxia The presence of anaerobic end products was examined in the body £uid and ¢ve tissues of M lusoria Under normoxia (day 0), the concentration of succinate was much higher in the digestive gland than in 20 30 (10) (9) (8) (7) (6) (5) (4) (3) (2) 25 Dissolved oxygen Digging indice Succinate 20 15 10 15 10 Succinate (µmol / ml) (11) Digging indices Dissolved oxygen (mg O2 / L) (12) 5 0 10 Exposure time (days) 15 20 Figure E¡ects of the dissolved oxygen (DO) concentration on the digging index and concentration of succinate in the body £uid of hard clams in aquaria (30 clams per aquarium) with an open surface at 25 1C Numbers in parentheses indicate the numbers of replicates for measurements of DO (n 2^12 aquaria) and the digging index (n 2^12 aquaria) There were six replicates in the measurement of succinate in the body £uid (¢ve clams per replicate) Data are given as the mean with the standard error © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 2012, 43, 1–13 a a a a Dissolved oxygen Digging indice Succinate 30 30 25 25 a b 20 c 15 b b d c c cd de d e 0 e ef de Exposure time (days) 20 15 10 10 5 0 Succinate (µmol / ml) a Digging indices Dissolved oxygen (mg O2 / L) f Figure E¡ects of the dissolved oxygen (DO) concentration on the digging index and concentration of succinate in the body £uid of hard clams in aquaria (30 clams per aquarium) in which 50% of the water surface was covered at 25 1C There were six replicates in the measurements of DO (an aquarium per replicate), the digging index (an aquarium per replicate) and succinate in the body £uid (¢ve clams per replicate) Data are given as the mean with the standard error Data with di¡erent letters signi¢cantly di¡er at di¡erent exposure times (Po0.05) The arrow indicates the position at the critical DO concentration the body £uid and other tissues (Table 1) However, the concentrations of alanine were signi¢cantly higher in the foot and adductor muscle than in the body £uid and other tissues After 64 h of anoxic exposure, increases in the concentrations of anaerobic end products were found in all tissues of the hard clam The greatest accumulations of succinate and propionate were found in the body £uid, while those of alanine were observed in the foot The concentration of alanine in the body £uid was minor compared with those in other tissues Among succinate, alanine and propionate, the accumulation of succinate was greater than those of alanine and propionate in all tissues, except the foot However, the accumulation of succinate in the foot was comparable to that of alanine Accumulation rate of anaerobic end products under anoxia Data from Table were used to calculate the accumulation rates of anaerobic end products in tissues Before 48 h of anoxic exposure, huge proportions of succinate and alanine had accumulated in the various tissues However, the accumulation of propionate in the tissues of hard clams before 24 h of anoxic exposure was minor Therefore, the anoxic © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 period of 0^48 h was used to calculate the accumulation rates of succinate and alanine, while 24^64 h was used for propionate All of the simple correlation coe⁄cients (g) in the calculation of accumulation rates of anaerobic end products in the tissues were ! 0.94, except those for alanine accumulations in the foot (g 50.82) and adductor (g 50.80) This was due to large amounts of alanine accumulating in the foot and adductor at h of anoxic exposure Among these six tissues, the accumulation rate of succinate in the body £uid was the highest (0.99 mmol h À mL À 1), while those in the other ¢ve tissues were comparable (0.19^0.27 mmol h À g À wet weight) (Table 2) More than half of the alanine had accumulated in the foot and adductor muscle at h of anoxic exposure (Table 1) Therefore, accumulation rates of alanine in the foot and adductor muscle were di⁄cult to compare with those in the other three tissues The order of the accumulation rates of alanine in tissues was the mantle (0.18 mmol h À g À wet weight) 4digestive gland (0.13 mmol h À g À wet weight) 4gills (0.04 mmol h À g À wet weight) The order of the accumulation rates of propionate in tissues was body £uid (0.22 mmol h À mL À 1) 4gills (0.12 mmol h À g À wet weight) 4mantle (0.08 mmol h À g À wet weight) 4foot (0.07 mmol h À g À wet weight) adductor muscle (0.07 mmol h À g À wet weight) 4digestive gland (0.04 mmol h À g À wet weight) Digging behavior and metabolism of hard clams A-C Lee et al Table Accumulation and recovery rates of anaerobic end products calculated from data in Table and Fig respectively Tissues Gills Anaerobic end product Succinate Alanine Propionate Digestive Succinate gland Alanine Propionate Foot muscle Succinate Alanine Propionate Adductor Succinate muscle Alanine Propionate Mantle Succinate Alanine Propionate Body fluidà Succinate Alanine Propionate Accumulation rate (lmol h À g À wet weight) c Recovery rate after h of normoxic exposure (lmol h À g À wet weight) 0.24 0.04 0.12 0.27 0.13 0.04 0.23 0.14 0.07 0.19 0.07 0.07 0.20 0.18 0.08 0.99 – 0.22 1.08 0.39 – 2.05 0.53 – 0.56 1.16 – 0.82 – – 0.76 0.35 – 2.84 – – 0.94 0.98 0.96 0.97 0.97 1.0 0.99 0.82 0.99 0.97 0.80 0.99 0.98 0.97 0.99 0.98 – 0.99 The accumulation of anaerobic end products was calculated by subtracting the mean value of ¢ve replicates at h from those at each sampling time The time period for calculating the accumulation rates of succinate and alanine was 0^48 h, and that for propionate was 24^64 h ÃUnits of body £uid are mmol h À mL À g is the simple correlation coe⁄cient for the calculation of the accumulation rate Succinate and alanine in tissues and digging indices of clams during the recovery period Rapid declines in the concentrations of succinate and alanine were observed in these tissues after h of recovery, except for alanine in the adductor (Fig 4) During aerobic recovery, the concentrations of metabolites returned to basal levels: succinate within h in the digestive gland and foot, and 24 h in the adductor, mantle and body £uid, while restoration in the gills required 48 h (Fig 4) In the digestive gland, foot and mantle, alanine concentrations had returned to basal levels within h, while 48 h was required in the gills The fastest recovery rate for succinate was in the body £uid (2.84 mmol h À mL À 1), followed by the digestive gland (2.05 mmol h À g À wet weight), with the slowest rate in the foot (0.56 mmol h À g À wet weight) (Table 2) However, the recovery rate of alanine in the foot was the highest compared with Aquaculture Research, 2012, 43, 1–13 those in other tissues No recovery rate of alanine was found in the adductor The pro¢le of digging indices of clams was similar to that of succinate concentrations in the body £uid After h of recovery, the concentration of succinate in the body £uid had decreased signi¢cantly (Fig 4f), and the digging indices of clams had increased signi¢cantly (Fig 5) Signi¢cant changes in the succinate concentration and digging indices were also observed at 8^24 h of recovery Discussion Tissue-speci¢c substrates of anaerobic metabolism The accumulation of succinate was accompanied by that of alanine in all tissues of the hard clam (M lusoria), except the gills, after 24 h of anoxic exposure A rapid increase in the concentration of succinate without alanine accumulation was found in the gills This indicates that carbohydrates contribute to anaerobic energy production in the gills However, equal amounts of succinate and alanine had accumulated in the foot of clams after 64 h of anoxic exposure, indicating that amino acids might contribute to anaerobic energy production in the foot, as the succinate formed was derived from the carbon skeletons of amino acids The digestive gland, adductor and mantle of hard clams seem likely to utilize both carbohydrates and amino acids as energy sources during anaerobic metabolism In general, aspartate pools represent the major cellular energy source mobilized in early anoxia (Zurburg & de Zwaan1981; Eberlee, Storey & Storey1983; Livingstone 1983), and other amino acids, such as alanine and glycine, acquire importance as energy substrates during subsequent recovery in normoxia (Eberlee et al 1983) However, Chiou et al (1998) found a 24% decrease in glutamate and a 60% increase in alanine as well as no signi¢cant change in aspartate in the edible meat of hard clams after days of aerial exposure at 20 1C Therefore, glutamate may play a certain role in the anaerobic metabolism of hard clams Unique anaerobic metabolism in gills The succinate that accumulated in the gills is not an intermediate of the glucose^succinate or aspartate^ succinate pathways Succinate might be derived from malate, a product of malic enzymes This speculation was supported by the observation that high activities of malic enzymes were found in the gills of the ribbed © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al Aquaculture Research, 2012, 43, 1–13 40 (a) (d) 30 Succinate 20 a 10 a Alanine b b c b Metabolites (µmol / g wet weight) 40 bc c c c (e) (b) 30 b a 20 a 10 b b b b b b a b b 40 b b a c (f) (c) c # a 30 a 20 b 10 b b b a b b b c c 0 24 48 24 Recovery after 64 h anoxic exposure (h) 48 Figure Concentrations of succinate and alanine in the gills (a), digestive gland (b), foot (c), adductor (d), mantle (e) and body £uid (f) of hard clams during 48 h of recovery after 64 h of anoxic exposure Each value represents the mean value from ¢ve determinations (¢ve clams per determination) with the standard error.Values for the same anaerobic end product with di¡erent letters di¡er signi¢cantly (Po0.05) #Units of body £uid are mmol mL À mussel Modiolus demissus, Mytilus edulis and Crassostrea virginica (Paynter, Karam, Ellis & Bishop 1985; Brodey & Bishop 1992) Optimum pH values for malate and pyruvate utilization by malic enzymes in the gills of M demissus were 8.5 and respectively (Brodey & Bishop 1992) Therefore, the direction of the reaction catalysed by malic enzymes in gill tissues of clams under anoxia would favour the production of malate, which is then reduced to succinate (Skorkowski 1988) The accumulation of succinate © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 was also solely observed in the gills of the common cockle Cardium edule after anoxic exposure (Meinardus & GÌde 1981) Relationships among the DO concentration, digging behaviours and anaerobic metabolism A low DO (o2 mg O2 L À 1) was found to reduce the burial depth of Macoma balthica (Long et al 2008) Aquaculture Research, 2012, 43, 139–148 Gibbons 1985; Billard, Cosson & Crim 1993; Cossson, Cosson, Andre & Billard 1995; Cosson, Groison, Suquet, Fauvel, Dreanno & Billard 2008) In ¢sh sperm, ATP stored before activation appears to be a factor limiting the duration of the motility period (Cosson 2010) A similar dependence also seems to occur with the content of TAN Adenosine triphosphate is also one of the factors a¡ecting the amplitude of £agella waves (Cosson 2010) and its level correlates positively with the spermatozoa motility and fertilizing ability (Bencic et al 1999a, b; Ingermann et al 2003; Zi˛etara, SzominŁska, Swierczynski et al 2004; Zilli et al 2004), although Billard, Linhart, Fierville and Cosson (1997) observed lower fertilization capacity of stripped milt than intratesticular spermatozoa of Silurus glanis despite their higher ATP stores Some authors have detected a direct relationship between the sperm ATP levels and the swimming speed (Lahnsteiner, Berger, Weismann & Patzner 1998; Ingermann et al 2003; Burness et al 2005) but such a relationship has not been found in other analyses (Burness et al 2004) In salmonids (¢sh with external fertilization), ATP is mainly accumulated before the phase of motility, because in this phase, the yield of oxidative phosphorylation is very low (Christen et al 1987; Lahnsteiner et al 1999), and therefore, the value of ATP and AEC concentrations could be the basis for sperm quality determination In salmonids, low levels of energy resources are su⁄cient to sustain motility for 30^60 s at the initial velocity of 100^150 mm s À (Christen et al 1987; Saudrais et al 1998; Lahnsteiner et al 1999) or 220 mm s À (Cosson, Cosson & Billard1991) In Siberian sturgeon and turbot, the oxidative phosphorylation is more e¡ective than in salmonids as after the rapid decrease in ATP, its level remains constant in the later swimming phase (Billard, Cosson, Fierville, Brun, Rouault & Williot 1999; Dreanno, Cosson, Suquet, Seguin, Dorange & Billard 1999) In our study, we obtained a large intermale ATP content variation and no relation between the ATP content and the percentage of motile cells, which is consistent with the results of other authors (Lahnsteiner, Berger,Weismann & Patzner1996; Billard et al.1999; Burness et al 2005) No signi¢cant correlations between per cent of motile spermatozoa and ATP but a positive correlation with the ADP content observed by us suggest that spermatozoa have limited capacity to maintain a stable level of ATP and little ATP hydrolysis before analysis Moreover, the amount of hydrolysed ATP could di¡er among males because of di¡erent rates of metabolism resulting from di¡erent: sperm Energetic parameters salmon spermatozoa K Dziewulska et al density, seminal plasma composition, contamination, height of milt layer during transport and others During storage, ADP concentrations in African cat¢sh change only slightly, while ATP depletion is associated with AMP increase (Zie˛ tara,  SzominŁska, Rurangwa, Ollevier, Swierczynski & Skorkowski 2004; Zie˛ tara, SzominŁska, Swierczynski et al 2004) Therefore, the correlation between per cent of motile spermatozoa could be more expressed with ADP than ATP In our study in the group of ¢sh where the per cent of motile spermatozoa was close to 0% and the mean values of AEC and ATP were signi¢cantly lower, the concentrations of hypoxanthine were signi¢cantly higher in comparison with the ¢sh with the high AEC value The contents of xanthine and inosine in the sperm cells were very low, and similarly, in all studied ¢sh, uric acid was not detected (data not presented) It seems that in salmonids, hypoxanthine is the ¢nal product of adenine nucleotide catabolism similarly as in African cat¢sh spermatozoa (Zie˛ tara, SzominŁska, Rurangwa et al 2004), but in the salmon, the concentration of hypoxanthine was much lower than in African cat¢sh (Zie˛ tara, SzominŁska, Rurangwa et al 2004) Until now, there are few data available on energetic metabolism in ¢sh spermatozoa Limited information is available on substrates and metabolite levels of energy resources (Terner & Korsh 1963; Billard & Cosson 1990; Lahnsteiner, Patzner & Weismann 1992, 1993; Lahnsteiner et al 1999) and the enzymes involved in energetic nucleotide metabolism in ¢sh sperm (Lahnsteiner et al 1996, 1998; Weismann, Weismann & Patzner 1997; Rurangwa, Biegniewska, Slominska, Skorkowski & Ollevier 2002; Mansour, Lahnsteiner & Berger 2003; Gronczewska, Zie˜tara, Biegniewska & Skorkowski 2003; Grzyb, Rychzowski, Biegniewska & Skorkowski 2003; Grzyb & Skorkowski 2006; Zie˛ tara, Biegniewska, Rurangwa, Swierczynski, Ollevier & Skorkowski 2009) Among various topics, the role of phosphocreatine participation in the energetic balance (Christen et al 1987; Robitaille, Mumford & Brown 1987; Saudrais et al 1998; Dreanno, Cosson, Suquet, Seguin et al 1999; Dreanno, Seguin, Cosson, Suquet & Billard 2000; Ingermann 2008) and the regulation of axonemal activity by the ratio between ATP and ADP should be studied (Yoshimura, Nakano & Shingyoji 2007; Cosson et al 2008) This knowledge will help to determine sperm quality, adjust conditions to allow longer storage of milt and obtain better fertilization results © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 139–148 145 Energetic parameters salmon spermatozoa K Dziewulska et al References Aas G.H., Refsite T & Gjerde B (1991) Evaluation of milt quality of Atlantic salmon Aquaculture 95, 125^132 Alavi A.M.H & Cosson J (2005) Sperm motility and fertilizing ability in the Persian sturgeon Acipenser persicus Aquaculture Research 36, 841^850 Atkinson D.E (1968) The energy charge of the adenylate pool as a regulatory parameters Interaction with feedback modi¢ers Biochemistry 7, 4030^4034 Benau D & Terner Ch (1980) Initiation, prolongation and reactivation of the motility of salmonid spermatozoa Gamete Research 3, 247^257 Bencic D.C., Krisfalusi J.G., Cloud J.G & Ingermann R.L (1999a) ATP level of chinook salmon (Oncorhynhus tschawytcha) sperm following in vitro exposure to the various oxygen tensions Fish Physiology and Biochemistry 20, 389^397 Bencic D.C., Krisfalusi J.G., Cloud J.G & Ingermann R.L (1999b) Maintenance of steelhead trout (Oncorhynchus mykiss) sperm at di¡erent in vitro oxygen tensions alters ATP levels and cell functional characteristics Fish Physiology and Biochemistry 21, 193^200 Billard R & Cosson M.-P (1990) The energetics of ¢sh sperm motility In: Controls of Sperm Motility: Biological and Clinical Aspects (ed by C Gagnon), pp.153^174 CRC Press, Boca Raton, FL, USA Billard R & Cosson M.P (1992) Some problems related to the assessment of sperm motility in freshwater ¢sh Journal of Experimental Zoology 261,122^131 Billard R., Cosson J & Crim L.W (1993) Motility and survival of halibut sperm during short term storage Aquatic Living Resources 6, 67^75 Billard R., Linhart O., Fierville F & Cosson J (1997) Motility of European cat¢sh Silurus glanis spermatozoa in testes and in milt Polish Archives of Hydrobiology 44, 115^122 Billard R., Cosson J., Fierville F., Brun R., Rouault T & Williot P (1999) Motility analysis of the Siberian sturgeon Acipenser baerii spermatozoa Journal of Applied Ichthyology 15, 199^203 Burness G., Casselman S.J., Schulte-Hostedde A.I., Moyes Ch.D & Montgomerie R (2004) Sperm swimming speed and energetics vary with sperm competition risk in bluegill (Lepomis macrochirus) Behavioral Ecology and Sociobiology 56, 65^70 Burness G., Moyes Ch.D & Montgomerie R (2005) Motility, ATP levels and metabolic enzyme activity of sperm from bluegill (Lepomis macrochirus) Comparative Biochemistry and Physiology Part A 140, 11^17 Christen R., Gatti J.-L & Billard R (1987) Trout sperm motility The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the £agellar movement European Journal of Biochemistry 160, 667^671 Cosson J (2010) Frenetic activation of ¢sh spermatozoa £agella entails short-term motility, portending their 146 Aquaculture Research, 2012, 43, 139–148 precocious decadence Journal of Fish Biology 76, 240^279 Cosson J., Groison A.-L., Suquet M., Fauvel C., Dreanno C & Billard R (2008) Studying sperm motility in marine ¢sh: an overview on the state of the art Journal of Applied Ichthyology 24, 460^486 Cosson M.-P., Billard R., Gatti J.-L & Christen R (1985) Rapid and quantitative assessment of trout spermatozoa motility using stroboscopy Aquaculture 46,71^75 Cosson M.-P., Cosson J & Billard R (1991) Synchronous triggering of trout sperm is followed by an invariable set sequence parameters whatever the incubation medium Cell Motility and the Cytoskeleton 31, 159^176 Cossson M.-P., Cosson J., Andre F & Billard R (1995) The cAMP/ATP relationship in the activation of trout sperm motility: their interaction in membrane-deprived models and in live spermatozoa Cell Motility and the Cytoskeleton 31, 159^176 Dreanno C., Cosson J., Suquet M., Cibert C., Fauvel C., Dorange G & Billard R (1999) E¡ects of osmolality, morphology perturbations and intracellular nucleotide content during the movement of sea bass (Dicentrarchus labrax) spermatozoa Journal of Reproduction and Fertility 116, 113^125 Dreanno C., Cosson J., Suquet M., Seguin F., Dorange G & Billard R (1999) Nucleotide content, oxidative phosphorylation, morphology, and fertilizing capacity of turbot (Psetta maxima) spermatozoa during the motility period Molecular Reproduction and Development 53, 230^243 Dreanno C., Seguin F., Cosson J., Suquet M & Billard R (2000) H1-NMR and 31P-NMR analysis of energy metabolism of quiescent and motile turbot (Psetta maxima) spermatozoa Journal of Experimental Zoology 286, 513^522 Gatti J.-L., King S.M., Moss A.G & Witman G.B (1989) Outer arm dynein from trout spermatozoa The Journal of Biological Chemistry 264,11450^11457 Gibbons B.H., Baccetti B & Gibbons I.R (1985) Motility of the 910 £agellum of Anguilla sperm Cell Motility 5,333^350 Gronczewska J., Zi˛etara M.S., Biegniewska A & Skorkowski E.F (2003) Enzyme activities in ¢sh spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengus Comparative Biochemistry and Physiology 134B, 399^406 Grzyb K & Skorkowski E.F (2006) Puri¢cation and some properties of two creatine kinase isoforms from herring (Clupea harengus) spermatozoa Comparative Biochemistry and Physiology 144B,152^158 Grzyb K., Rychzowski M., Biegniewska A & Skorkowski E.F (2003) Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributylin Comparative Biochemistry and Physiology 134C, 207^213 Ingermann R.L (2008) Energy metabolism and respiration in ¢sh spermatozoa In: Fish Spermatology (ed by S.M.H © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 139–148 Aquaculture Research, 2012, 43, 139–148 Alavi, J.J Cosson, K Coward & G Ra¢ee), pp 241^266 Alpha Science, Oxford, UK Ingermann R.L., Robinson M.L & Cloud J.G (2003) Respiration of steelhead trout sperm: sensitivity to pH and carbon dioxide Journal of Fish Biology 62,13^23 Kazakov R.V (1979) Produtsirovanie spermy karlikovymi samtsami atlanticheskogo lososya i kharakteristika ee rybovodnogo kachestva Sbornik Nauchnykh Trudov GosNIORKH 139, 40^47 Lahnsteiner F., Patzner R.A & Weismann T (1992) Monosaccharides as energy resources during motility of spermatozoa in Leuciuscus cephalus (Cypinidae, Teleostei) Fish Physiology and Biochemistry 10, 283^289 Lahnsteiner F., Patzner R.A & Weismann T (1993) Energy resources of spermatozoa of the rainbow trout Oncorhynchus mykiss (Pisces,Teleostei) Reproduction, Nutrition, Development 33, 349^360 Lahnsteiner F., Berger B.,Weismann T & Patzner R.A (1996) Motility of spermatozoa of Alburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism Fish Physiology and Biochemistry 15, 167^179 Lahnsteiner F., Weismann T & Patzner R.A (1997) Aging processes of rainbow trout semen during storage The Progressive Fish Culturist 59, 272^279 Lahnsteiner F., Berger B.,Weismann T & Patzner R.A (1998) Determination of semen quality of the rainbow trout Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoal metabolism Aquaculture 163, 163^181 Lahnsteiner F., Berger B & Weismann T (1999) Sperm metabolism of the teleost ¢shes Chalcalburnus chalicoides and Oncorhynchus mykiss and its relation to motility and viability Journal of Experimental Zoology 284, 454^465 Mansour N., Lahnsteiner F & Berger B (2003) Metabolism of intratesticular spermatozoa of a tropical teleost ¢sh (Clarias gariepinus) Comparative Biochemistry and Physiology Part B 135, 285^296 Morisawa M & Okuno M (1982) Cyclic AMP induces maturation of trout sperm axoneme to initiate motility Nature 298,703^704 Morisawa M., Suzuki K & Morisawa S (1983) E¡ects of potassium and 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J., Jeulin C., Fierville F & Billard R (1998) Initiation of carp spermatozoa motility and early ATP reduction after milt contamination by urine Aquaculture 160, 317^328 Robitaille P.-M.L., Mumford K.G & Brown G.G (1987) 31P nuclear magnetic resonance study of trout spermatozoa at rest, after motility, and during short-term storage Biochemistry and Cell Biology 65, 474^485 Rurangwa E., Biegniewska A., Slominska E., Skorkowski E.F & Ollevier F (2002) E¡ect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility Comparative Biochemistry and Physiology Part C 131, 335^344 Saudrais C., Fierville F., Loir M., Le Rumeur E., Cibert C & Cosson J (1998) The use of phosphocreatine plus ADP as energy source for motility of membrane-derived trout spermatozoa Cell Motility and the Cytoskeleton 41, 91^ 106 Smolenski R.T., Lachno D.R., Ledingham S.J & Yacoub M.H (1990) Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation Journal of Chromatography 527, 414^420 Suquet M., Dreanno C., Dorange G., Normant Y., Quemener L., Gaignon J.L & Billard R (1998) The ageing phenomenon of turbot spermatozoa: e¡ects on morphology, motility and concentration, intracellular ATP content, fertilization, and storage capacities Journal of Fish Biology 52, 31^41 Terner Ch & Korsh G (1963) The oxidative metabolism of pyruvate, acetate and glucose in isolated ¢sh spermatozoa Journal of Cellular and Comparative Physiology 62, 243^249 Vladic› T.V & JÌrvi T (2001) Sperm quality in alternative reproductive tactics of Atlantic salmon: the importance of loaded ra¥e mechanism Proceedings of the Royal Society London B 264, 219^226 Vladic› T.V., Afzelius B.A & Bronnikov G.E (2002) Sperm quality as re£ected through morphology in salmon alternative life histories Biology of Reproduction 66, 98^105 Yoshimura A., Nakano I & Shingyoji C (2007) Inhibition by ATP and activation by ADP in the regulation of £agellar movement in sea urchin Cell Motility and the Cytoskeleton 64,777^793 Zi˛etara M.S., SzominŁska E., Rurangwa E., Ollevier F.,  Swierczyn Ł ski J & Skorkowski E.F (2004) In vitro adenine nucleotide catabolism in African cat¢sh spermatozoa © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 139–148 147 Energetic parameters salmon spermatozoa K Dziewulska et al Comparative Biochemistry and Physiology Part B 138, 385^389 Zi˛etara M.S., SzominŁska E., Swierczynski J., Rurangwa E., Ollevier F & Skorkowski E.F (2004) ATP content and adenine nucleotide catabolism in African cat¢sh spermatozoa stored in various energetic substrates Fish Physiology and Biochemistry 30, 119^127 Zie˛ tara M.S., Biegniewska A., Rurangwa E., Swierczynski J., Ollevier F & Skorkowski E.F (2009) Bioenergetic of ¢sh 148 Aquaculture Research, 2012, 43, 139–148 spermatozoa during semen storage Fish Physiology and Biochemistry 35, 607^614 Zilli L., Schiavone R., Zonno V., Storelli C & Vilella S (2004) Adenosine triphosphate concentration and D-glucuronidase activity as indicators of sea bass semen quality Biology of Reproduction 70, 1679^1684 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 139–148 Aquaculture Research, 2012, 43, 149–153 doi:10.1111/j.1365-2109.2011.02795.x SHORT COMMUNICATION Evaluation of geosmin and 2-methylisoborneol off-flavour in smoked rainbow trout fillets using instrumental and sensory analyses Paul V Zimba1, Kevin K Schrader2, Grethe Hyldig3, Bjarne W Strobel4 & Niels O G JÖrgensen5 Center for Coastal Studies,Texas A&M University-Corpus Christi, Corpus Christi,TX, USA USDA-ARS, Natural Products Utilization Research Unit,Thad Cochran Research Center, University, MS, USA Division of Seafood Research, National Food Institute,Technical University of Denmark, Lyngby, Denmark Department of Basic Sciences and Environment, University of Copenhagen, Copenhagen, Denmark Department of Agriculture and Ecology, University of Copenhagen, Copenhagen, Denmark Correspondence: N O G JÖrgensen, Department of Agriculture and Ecology, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Copenhagen, Denmark E-mail: nogj@life.ku.dk Taste and odour compounds (TOCs) produced by certain species of cyanobacteria (Smith, Boyer & Zimba 2008) and actinomycete bacteria (Guttman & van Rijn 2008) can accumulate in the £esh of ¢sh and subsequently have signi¢cant negative impacts on the aquaculture industry including the following: (1) economic losses to the producers from delayed harvest due to an unpalatable and unmarketable product; and (2) consumer dissatisfaction, which hampers the growth of the industry Common TOC problems are believed to decrease the sale of channel cat¢sh Ictalurus punctatus (Ra¢nesque) by 30% in the United States of America (Engle, Pounds & van der Ploeg 1995) In Europe, tainting of ¢sh by TOCs has been observed in several countries, e.g in the French rainbow trout aquaculture industry (Selli, Prost & Serot 2009), and, in the United Kingdom, up to 20% of the trout producers have experienced seasonal problems (Robertson, Hammond, Jauncey, Beveridge & Lawton 2006) Smith et al (2008) cited studies identifying o¡-£avour occurrence in Nile tilapia, shrimp, Atlantic salmon, rainbow trout, cat¢sh species, American lobster, largemouth bass and white sturgeon The latter two occurrences involved water recirculation systems Water quality and availability impact site location of aquaculture facilities In many countries, includ- © 2011 Blackwell Publishing Ltd ing Denmark, restrictions in water consumption for aquaculture have been implemented The goal is to reduce the consumption of natural water resources in traditional £ow-through aquaculture and increase construction of recirculating production systems Unfortunately, recirculation of the water increases the risk of accumulation of TOCs, such as geosmin and 2-methylisoborneol (MIB), because these TOCs are slowly degraded by microorganisms in aerobic bio¢lters (Guttman & van Rijn 2009) Production of rainbow trout Oncorhynchus mykiss (Walbaum) in Denmark requires a 9-month growing season to achieve a fresh-weight of 300^350 g ¢sh Fat content of the ¢sh is typically about 5% Most of the rainbow trout produced are salted, smoked over beech chips, vacuum-packed in polyethylene ¢lm ¢lled with 30% CO2 and 70% N2, and sold in the European market In Denmark, recent data on geosmin and MIB concentrations in the water of 41000 m3 concrete production basins con¢rm 5^10-fold higher TOC levels than in £ow-through systems (N O G JÖrgensen & B.W Strobel, unpubl obs.) Smoked ¢llets of trout produced in recirculating aquaculture systems (RAS) have occasionally led to consumer complaints about o¡-£avours, but the levels and composition of TOCs in the £esh of the ¢sh cultured in these systems have not been determined In addition, there has not 149 Off-flavour in smoked rainbow trout fillets PV Zimba et al Aquaculture Research, 2012, 43, 149–153 been an evaluation of salting and smoking the ¢llets on £avour quality due to ‘masking’of the o¡-£avours The purpose of this current study was to quantitatively assess if smoked trout ¢llets from the recirculated systems retained volatile aromatics contributing to the o¡-£avours and, if so, the potential bene¢ts of smoking and salting on improving £avour quality by masking any residual TOCs For this study, rainbow trout were bred in a 1200 m3 RAS supplied with groundwater, and bio¢lter techniques were used for water treatment (cleaning), with approximately 50% of the water volume replenished daily The commercial system and exact location in Denmark is not identi¢ed by request of the owner Standard Danish commercial methods for feeding, grow-out, harvesting and processing were used to produce the ¢nal product of smoked trout ¢llets The smoked ¢llets were kept at À 20 1C for about months before analyses of o¡-£avours and the common TOC compounds geosmin and MIB Before conducting instrumental analysis, frozen ¢llets (sampling set of 12) were partially thawed For each ¢llet, one 20 g portion was cut from the anterior end of the ¢llet and used to obtain distillate with microwave distillation using the procedures of Lloyd and Grimm (1999) Distillates were analysed using headspace SPME/GC/MS as previously described by Lloyd, Lea, Zimba and Grimm (1998) and as modi¢ed by Schrader, Nanayakkara, Tucker, Rimando, Ganzera and Schaneberg (2003) For sensory analysis, the sensory panel consisted of six assessors that had been selected, tested and speci¢cally trained in descriptive analysis (ISO 11035) of rainbow trout (Hyldig 2009a) The vocabulary for the sensory pro¢ling was developed during four sessions using samples of smoked rainbow trout with known content of MIB and geosmin, i.e the compounds were not added The ¢rst session was qualitative, i.e., to develop a list of attributes describing odour, £avour and texture of rainbow trout The following three sessions were quantitative, i.e the assessors were trained to evaluate the descriptors on a linear scale Each attribute was evaluated using a 15cm unstructured linear scale with two anchor points that were ‘little’ and ‘much’ of attribute intensity The anchor points were placed 1.5 and 13.5 cm from on the scale (Meilgaard, Civille & Carr 2007) The sensory attributes analysed were the following: (1) odour (e.g smoke, wood tar, sweet, sourish, fusty/mouldy and muddy); and (2) £avour (e.g smoke, sweet, salty, sourish, oily, earthy, mouldy/fusty and muddy); (3) texture (juicy and ¢brousness); and (4) light/dark col- our for appearance (Hyldig 2009b) The evaluations were performed in separated booths under normal daylight and at ambient temperature The assessors used water and £at bread to clean the palate between samples Each sample was minced and mixed before subsampling into individual porcelain bowls covered with porcelain lids The ¢llet size was insu⁄cient for replicate analyses by each assessor A sample without any sensory detectable MIB and geosmin was used as reference The instrumental analyses demonstrated that all 12 smoked trout samples contained both MIB and geosmin The levels of MIB were at least one order of magnitude greater than geosmin levels in each respective ¢llet; MIB was the dominant odour component (Fig 1) The MIB concentrations ranged from 4.8 to 19.7 mg kg À while geosmin ranged from 0.27 to 0.59 mg kg À Observations by the sensory panel con¢rmed the presence of TOC in the smoked ¢llets and mainly attributed to the o¡-odour and o¡-£avour from both MIB (mouldy/fusty) and geosmin (muddy) (Fig 2) The intensity of the o¡-£avour from MIB and geosmin was signi¢cantly higher than the other odours in most samples, but especially noteworthy was the intense fusty/mouldy odour originating from MIB in sample ‘Fish 8’ Interestingly, Fish and both had a high content of MIB (statistically identical) (Fig.1), yet Fish had a higher score for mouldy/fusty £avour and odour (Fig 2) Possibly, the saltier £avour and smoked (wood tar) odour of Fish masked the mouldy/fusty attributes Sensory pro¢les illustrating the tested odour, £avour and texture attributes are shown for a low, medium and high score ¢sh with respect to £avour in Fig The sensory plots indicate that even though the intensity of smoke and wood tar was relatively high, the odours and £avours from MIB and geosmin were easily detectable and identi¢able In previous studies with channel cat¢sh, MIB levels were found to in some cases to vary greatly between the two ¢llets from the same ¢sh (K K Schrader, unpubl obs.) In the current study, di¡erent ¢llets from the same ¢sh were used for sensory analysis and instrumental analysis, and this reason may account for some of the di¡erences (e.g Fish 4) between the intensity of MIB-related o¡-£avour (Fig 2) compared with the MIB levels in the same ¢sh (Fig 1) The levels of MIB in the sampled trout ¢llets were typically 20-fold above geosmin levels This di¡erence was also re£ected in the sensory pro¢les that were dominated by the mouldy/fusty £avour and 150 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 149–153 Aquaculture Research, 2012, 43, 149–153 Off-flavour in smoked rainbow trout fillets PV Zimba et al Figure Geosmin and 2-methylisoborneol (MIB) levels in rainbow trout ¢sh ¢llets from a commercial Danish production facility Mean concentrations Æ SD shown (n 3) Figure Intensity scores from human sensory evaluation of o¡-odour (O) and o¡-£avour (F) from 2-methylisoborneol (MIB) (fusty/mouldy) and from MIB and geosmin (muddy) Asterisks (Ã) indicate ¢sh for which sensory pro¢les are shown in Fig odour that are characteristic of MIB Geosmin was previously reported to be the major TOC in rainbow trout (Robertson et al 2006), and our study is the ¢rst to report on the high levels of MIB in cultured rainbow trout Unfortunately, the levels of geosmin and MIB could not be measured analytically in the basin © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43 , 149–153 151 Off-flavour in smoked rainbow trout fillets PV Zimba et al Aquaculture Research, 2012, 43, 149–153 Figure Sensory pro¢les for Fish 3,7, and a reference ¢sh Intensity of the sensory attributes ranges from (no score; centre of plot) to 12 (maximum score; perimeter of plot) The letters ‘O’, ‘F’and ‘T’ refer to odour, £avour and texture respectively water, but the higher MIB levels in the ¢sh £esh indicates that MIB was likely at a substantially higher concentration than geosmin Geosmin and MIB are produced by certain microorganisms that are abundant in aquaculture systems, such as cyanobacteria and actinomycetes (Zaitlin & Watson 2006; Smith et al 2008; Schrader & Summerfelt 2010) Recently, Guttman and van Rijn (2008) observed a large release of MIB from aerobic bio¢lter material in tilapia ponds (up to 175 ng L À 1) and concluded that the compound was produced by Streptomyces bacteria In Danish rainbow trout basins with water recirculation, levels of geosmin and MIB in water of 30^40 and 5^15 ng L À 1, respectively, have typically been observed (N O G JÖrgensen & B W Strobel, unpubl obs.) Variable thresholds for human sensory detection of MIB and geosmin are reported for ¢sh For channel cat¢sh, Grimm, Lloyd and Zimba (2004) reported a threshold of 0.1^0.2 mg kg À for MIB and 0.25^ 0.5 mg kg À for geosmin while Robertson, Jauncey, Beveridge and Lawton (2005) suggested a threshold of 0.7 mg kg À for MIB and 0.9 mg kg À for geosmin in rainbow trout Thus, depending upon the applied threshold level from these two studies, the level of MIB in our sampled trout was 10^25 times or 80^ 200 times above the detection threshold while geos- 152 levels in the ¢sh appeared to be just below the threshold It is well established that o¡-£avours are assimilated into ¢sh tissue, with removal being a slow process [reviewed in Smith et al (2008) and Tucker (2000)] Natural depuration e¡orts can be slow and in£uenced by temperature, fat content of ¢sh, as well as the amount of o¡-£avour compound accumulated (Dionigi, Johnsen & Vinyard 2000) Therefore, postharvest approaches in mitigating geosmin and MIBrelated o¡-£avour can be useful when the taint is still present Bett, Ingram, Grimm, Vinyard, Boyette and Dionigi (2000) demonstrated that masking of MIB and geosmin o¡-£avour in cat¢sh with spices such as lemon-pepper and Cajun mixes is e¡ective In this study, we anticipated that processed smoking of trout ¢llets would result in lowered sensory perception of o¡-£avour by potential removal of volatile aromatics during the smoking process and the accumulation of additional intense odorous compounds from the beech smoke into the ¢llets Beech wood smoke largely consists of lignin degradation products, primarily hemicelluloses and cellulose, with minor contribution by furans from lignin breakdown but no terpenes (Omrani, Masson, Pizzi & Mansouri 2008) As demonstrated in our study, the high detected intensities of odours and £avours such as © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 149–153 Aquaculture Research, 2012, 43, 149–153 Off-flavour in smoked rainbow trout fillets PV Zimba et al smoke and wood tar from the smoking process did not hamper sensory detection of the strong fusty/ mouldy and muddy odours and £avours Therefore, the smoking process was not e¡ective in reducing or masking the respective odours and £avours of geosmin and MIB at the levels detected in the trout £esh As more recirculation production systems are established, management to prevent o¡-£avour occurrence is essential to maintain customer acceptance of product quality Development of alternative processing lines (e.g spice addition) may provide alternatives in situations when o¡-£avour ¢sh are inadvertently found Hyldig G (2009b) Sensory descriptors In: Handbook of Seafood and Seafood Products Analysis, ed by L.M.L Nollet & F Toldra), pp 481^498 Taylor & Francis, NewYork, NY, USA Lloyd S.W & Grimm C.C (1999) Analysis of 2-methylisoborneol and geosmin in cat¢sh by microwave distillation-solid-phase microextraction Journal of Agricultural Food and Chemistry 47,164^169 Lloyd S.W., Lea J.M., Zimba P.V & Grimm C.C (1998) Rapid analysis of geosmin and 2-methylisoborneol in water using solid phase micro extraction procedures.Water Research 32, 2140^2146 Meilgaard M.M., Civille G.V & Carr T (2007) Sensory Evaluation Techniques, 4th edn Taylor & Francis, New York, NY, USA Omrani P., Masson E., Pizzi A & Mansouri H.R (2008) Emission of gases and degradation volatiles from polymeric wood constituents in friction welding of wood dowels Polymer Degradation and Stability 93,794^799 Robertson R.F., Jauncey K., Beveridge M.C.M & Lawton L.A (2005) Depuration rates and the sensory threshold concentration of geosmin responsible for earthy-musty taint in rainbow trout, Onchorhynchus mykiss Aquaculture 245, 89^99 Robertson R.F., Hammond A., Jauncey K., Beveridge M.C.M & Lawton L.A (2006) An investigation into the occurrence of geosmin responsible for earthy-musty taints in UK farmed rainbow trout, Onchorhynchus mykiss Aquaculture 259,153^163 Schrader K.K & Summerfelt S.T (2010) Distribution of o¡£avor compounds and isolation of geosmin-producing bacteria in a series of water recirculating systems for rainbow trout culture North AmericanJournal of Aquaculture 72, 1^9 Schrader K.K., Nanayakkara N.P.D., Tucker C.S., Rimando A.M., Ganzera M & Schaneberg B.T (2003) Novel derivatives of 9,10-anthraquinone are selective algicides against the musty-odor cyanobacterium Oscillatoria perornata Applied and Environmetnal Microbiology 69, 5319^5327 Selli S., Prost C & Serot T (2009) Odour-active and o¡-odour components in rainbow trout (Oncorhynchus mykiss) extracts obtained by microwave assisted distillation-solvent extraction Food Chemistry 114, 317^322 Smith J.L., Boyer G.L & Zimba P.V (2008) A review of cyanobacterial odorous and bioactive metabolites: impacts and management alternatives in aquaculture Aquaculture 280, 5^20 Tucker C.S (2000) O¡-£avor problems in aquaculture Reviews in Fisheries Science 8,1^44 Zaitlin B & Watson S.B (2006) Actinomycetes in relation to taste and odour in drinking water: myths, tenets and truths.Water Research 40,1741^1753 Acknowledgments We thank Agustson-Hevico for providing the smoked ¢sh ¢llets Veterinarian Niels H Henriksen, Danish Aquaculture Association, kindly provided information on RAS in Denmark The technical assistance of Dewayne Harries and Phaedra Page is greatly appreciated The study was supported by The Danish Food IndustryAgency, grant No 3310-06-00121, to NOGJ References Bett K.L., Ingram D.A., Grimm C.C., Vinyard B.T., Boyette K.D.C & Dionigi C.P (2000) Alteration of the sensory perception of the muddy/earthy odorant 2-methylisoborneol in channel cat¢sh Ictalurus punctatus ¢llet tissues by addition of seasonings Journal of Sensory Studies 15, 459^472 Dionigi C.P., Johnsen P.B & Vinyard B.T (2000) The recovery of £avor quality by channel cat¢sh North American Journal of Aquaculture 62,189^194 Engle C.R., Pounds G.L & van der Ploeg M (1995) The cost of o¡-£avour Jorunal of the World Aquaculture Society 26, 297^306 Grimm C.C., Lloyd S.W & Zimba P.V (2004) Instrumental versus sensory detection of o¡-£avors in farm-raised channel cat¢sh Aquaculture 236, 309^319 Guttman L & van Rijn J (2008) Identi¢cation of conditions underlying production of geosmin and 2-methylisoborneol in a recirculating system Aquaculture 279, 85^91 Guttman L & van Rijn J (2009) 2-Methylisoborneol and geosmin uptake by organic sludge derived from a recirculating aquaculture system.Water Research 43, 474^480 Hyldig G (2009a) Sensory aspects of heat treated seafood In: Handbook of Seafood and Seafood Products Analysis, ed by L.M.L Nollet & F Toldra), pp 499^514 Taylor & Francis, NewYork, NY, USA Keywords: geosmin, 2-methylisoborneol, o¡-£avour, rainbow trout © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43 , 149–153 153 Aquaculture Research, 2012, 43, 154–159 doi:10.1111/j.1365-2109.2011.02805.x SHORT COMMUNICATION Seasonal variations in the intestinal microbiota of farmed Atlantic salmon (Salmo salar L.) Maria Befring Hovda1, Ramon Fontanillas2, Charles McGurk2, Alex Obach2 & Jan Thomas Rosnes1 No¢ma NorconservAS, Stavanger, Norway Skretting Aquaculture Research Centre, Stavanger, Norway Correspondence: M B Hovda, No¢ma NorconservAS, MÔltidets Hus, Richard Johnsensgt 4, N-4021 Stavanger, Norway E-mail: maria.hovda@no¢ma.no Seasonal variations in seawater conditions, including temperature and bacterial £ora populations could be expected to in£uence the intestinal microbiota of ¢sh In order to improve the understanding of the in£uence of environment and diet on the bacterial composition, further studies of farmed Atlantic salmon (Salmo salar L.) intestinal microbiota are required It has been reported that through manipulation of dietary composition, the microbiota can potentially be changed and controlled (RingÖ, Sperstad, Myklebust, Refstie & Krogdahl 2006; Bakke-McKellep, Penn, Salas, Refstie, Sperstad, Landsverk, RingÖ & Krogdahl 2007; RingÖ, Sperstad, Kraugerud & Krogdahl 2008) As diet, also geographic location may in£uence the intestinal microbiota (Holben,Williams, Gilbert, Saarinen, Sarkilahti & Apajalahti 2002), and seasonal variations could exert a similar e¡ect Recently, the bacterial £ora of Atlantic salmon have been widely studied (e.g Holben et al 2002; Bakke-McKellep et al 2007; Hovda, Lunestad, Fontanillas & Rosnes 2007; Liu, Zhou, Yao, Shi, He, HÖlvold & RingÖ 2008; RingÖ et al 2008) The intestinal microbiota of ¢sh has been shown to be more variable than ¢rst suggested, particularly in data generated from studies since the introduction of molecular-based methods The use of molecular biological methods decreases possible biases commensurate with approaches reliant on bacterial cultivation The use of such methods are based on analyses of the bacterial DNA using polymerase chain reaction (PCR) and 16S rDNA sequencing, and further bacterial identi¢cation This approach is well established in the study of microbial 154 ecology, and several authors have used such methods in the study of ¢sh intestine (Verner-Je¡reys, Shields, Bricknell & Birkbeck 2003; Huber, Spanggaard, Appel, Rossen, Nielsen & Gram 2004; Martin, Ross, Quetin & Murray 2006; Pond, Stone & Alderman 2006; Hovda, Lunestad, Fontanillas et al 2007; Kim, Brunt & Austin 2007; RingÖ et al 2008; Bjornsdottir, Johannsdottir, Coe, Smaradottir, Agustsson, Sigurgisladottir & Gudmundsdottir 2009; Merri¢eld, Burnard, Bradley, Davies & Baker 2009) Denaturing gradient gel electrophoresis (DGGE) enables studies of complex bacterial communities, and additional sequencing makes identi¢cation of the species present in the sample possible The method provides information about the predominant bacterial £ora in the sample, regardless of their ability to grow on agar Furthermore, the methodology can be used to study population dynamics and for time studies, such as studying seasonal variations in the gut microbiota Denaturing gradient gel electrophoresis compares samples based on base di¡erences in the GC content of the DNA sequence of di¡erent bacteria By analysing the same 16S rDNA region, bacterial variations can be detected There are, to our knowledge, only three studies from the last decade examining the gut microbiota during an annual cycle, all using ¢sh from ponds, in Saudi Arabia, Japan and France (Al-Harbi & Naim Uddin 2004; Hagi, Tanaka, Iwamura & Hoshino 2004; Naviner, Giraud, Le Bris, Armand, Mangion & Ganiere 2006).Whereas, some older papers also discuss the bacterial £ora of gut, using bacterial cultivation (Liston1956; Okuzumi & Awano1983) This is the ¢rst © 2011 Blackwell Publishing Ltd Aquaculture Research, 2012, 43, 154–159 Seasonal variations in the intestinal microbiota of Salmo salar L MB Hovda et al study investigating the microbial diversity in the gut as a function of seawater seasonal variations The purpose of this work was to describe seasonal variations in the predominant intestinal microbiota of farmed Atlantic salmon This experiment was conducted using Atlantic salmon (S salar L.) raised in sea cages at the Skretting Research Station in Lerang (Norway) from July 2006 until June 2007 The ¢sh were fed to satiation with a mm commercial diet (Skretting AS, Stavanger, Norway), based on ¢sh meal, ¢sh oil and extracted soybean meal, in addition to wheat, rapeseed oil and vitamin premix During the year, ¢ve ¢sh from each cage were collected in August, October, November, February, March, April, May and June, and killed using high doses of anaesthesia (tricaine methanesulphonate, Finquel MS 222, Argent Chemical Laboratories, Remond,WA, USA) The gut was divided into mid-gut (intermediate portion of the digestive system including the pyloric caeca and proximal intestine) and hind-gut (distal portion of the digestive system that corresponds to the distal intestine) The gut contents of ¢ve ¢sh, from each cage, were squeezed out, and the gut was rinsed three times using $ mL of peptone water, before the contents were pooled, corresponding to the method of RingÖ et al (2006) to collect both adherent and non-adherent bacteria The pooling of the samples was performed to minimize the individual variations in the ¢sh (Spanggaard, Huber, Nielsen, Nielsen, Appel & Gram 2000; Hovda, Lunestad, Fontanillas et al 2007) The seawater temperature and salinity were measured daily During the sampling period from August 2006 to June 2007, the seawater temperature varied between 18.8 1C (August) and 5.5 1C (March), whereas the salinity was between 30% (October) and 26.13% (May) Identi¢cation of the total bacterial microbiota was performed by analyses of the genomic bacterial DNA using PCR and DGGE Bacterial DNA was puri¢ed from the intestine samples, using QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) The PCR reaction was performed using universal primers for the 16S rDNA variable V3 region The PCR and DGGE were performed as described by Hovda, Lunestad, Fontanillas et al (2007), using the forward primer BA338f (5 -ACT CCT ACG GGA GGC AGC AG) including a 40-base GC clamp at the -end, and the reverse primer UN518r (5 -ATT ACC GCG GCT GCT GG) Denaturing gradient gel electrophoresis was performed with a 30^55% gradient After DGGE, the DNA fragments to be nucleotide sequenced were excised, re-run and con¢rmed on DGGE, before PCR ampli¢cation using the forward primer BA338f without the GC clamp Sequencing was performed at the University of Bergen Sequencing Facility (Bergen, Norway) Searches in BLAST from GenBank were used to ¢nd the closest known relatives to the partial 16S rDNA sequences (121^160 bp), and deposited in GenBank (HQ420280-94) To determine seasonal variations, during a 1-year cycle, in the predominant intestinal microbiota of farmed Atlantic salmon the methodologies of DGGE and further sequencing were used Thus, the predominant bacterial £ora present was detected from analyses of the 16S rDNA V3 region Samples were taken from both the mid- and hind-gut There were no observed di¡erences in the bacterial pro¢les between the mid- and hind-gut, these results were in accordance with Hovda, Lunestad, Fontanillas et al (2007) Using PCR-temporal temperature gradient gel electrophoresis Navarrete, Espejo and Romero (2009) observed similar microbiota in the stomach, pyloric caeca and intestine of juvenile Atlantic salmon During the sample collection period of a year, the predominant bacteria were analysed on the gel, visualized as pro¢les representing the di¡erent months (Fig 1) From the August and October samples, 29 bands were excised from the gel for sequencing and identi¢cation Table summarizes the sequencing result, using the BLAST database, of the 15 visible bands (Fig 1) from the total 29 bands To ensure that identical migration pattern of bands from di¡erent pro¢les represented the same bacterium, and not other bacteria with the same migration, some bands were excised in duplicate from di¡erent pro¢les Bands 1^5, and 10 were excised from both the August and October pro¢les All the sequenced bands possessed high similarities to previously detected bacteria (497%) The predominant intestinal bacterial £ora identi¢ed consisted of Lactobacillus fermentum, Lactococcus lactis, Vibrio sp.,Vibrio ichthyoenteri,Weissella cibaria, Photobacterium phosphoreum and Bacillus sp Among the samples, two bands represented sequences not assigned as bacteria: chloroplast and an unidenti¢ed eukaryotic 18S DNA sequence These two bands may represent traces of plants or other eukaryotes eaten by the ¢sh, and has also been observed previously using the universal primers for the V3 region (Jensen, ÒvreÔs, Bergh & Torsvik 2004; Hovda, Lunestad, Fontanillas et al 2007; Hovda, Lunestad, Sivertsvik & Rosnes 2007; Hovda, Sivertsvik, Lunestad, Lorentzen & Rosnes 2007; Navarrete et al 2009) © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 154–159 155 Seasonal variations in the intestinal microbiota of Salmo salar L MB Hovda et al Aquaculture Research, 2012, 43, 154–159 The sequenced bands were compared with the bands in Fig 1, and the bacterial diversity during the year was identi¢ed (Table 2) Table shows the comparison of the year cycle DGGE pro¢les, annotating Figure Denaturing gradient gel electrophoresis pro¢les of the bacterial diversity of Atlantic salmon intestine The lanes represent samplings during a 1-year cycle from August to June The bands indicated by numbers were sequenced and described in Table the sequenced bands to the pro¢les Some bands appeared in pro¢les other than the August and October sampling; these where, however, not excised from the gel Hence, some additional and unsequenced bacteria may be included as part of the gut microbiota The year cycle DGGE analyses of the gut £ora from Atlantic salmon revealed that some bacteria were present in the gut during the entire period, whereas other bacteria varied throughout the year The lactic acid bacteria (LAB) bacteria L fermentum, L lactis and Weissella sp were, in addition to chloroplast, found at all the sampling points during the year The highest numbers of bands were present in August and October with, nine bands representing six bacteria from the former month and eight bands representing ¢ve bacterial species from the latter The bacteria Vibrio spp., P phosphoreum, Bacillus sp and uncultivable spirochete were only found at some sampling points Kim et al (2007) suggested that DGGE underestimates the bacterial diversity, compared with 16S rDNA clone libraries, explaining the relatively few bacteria detected in the present study On the other hand, the carnivorous diet of salmon has also been suggested to explain the low numbers of species observed (Navarrete et al 2009) Lactic acid bacteria are known to be bene¢cial for the ¢sh and they might produce bacteriocins against pathogens; furthermore, they can serve as probiotics with a proposed e¡ect as biological control agents The ¢ndings of L fermentum, L lactis and W cibaria as the most predominant bacteria in the samples are of special interest, as these bacteria are known to have a bene¢cial e¡ect in nutritional processes Table 16S rDNA V3-sequence similarities to closest relatives using DNA from the respective bands in the denaturing gradient gel electrophoresis (DGGE) gel shown in Fig Band no Closest relative in GenBank database (accession number) Identity (%) Accession number GenBank 10 11 12 13 14 15 Weissella cibaria (AB494716.1) Lactobacillus fermentum (AM117175.1) L fermentum (EF460496.1) L fermentum (AM411110.1) Lactococcus lactis (AM411114.1) Vibrio ichthyoenteri (AB274764.1) Vibrio sp (EF033421.1) Vibrio sp (AS159568.1) Unidentified 18S rDNA-DGGE band Photobacterium phosphoreum (AY780009.1) P phosphoreum (AY435164.1) P phosphoreum (AY780009.1) Bacillus sp (DQ649444.1) Photobacterium sp (DQ317672.1) Angiosperm environmental, chloroplast (DQ889979.1) 99 98 99 98 99 99 99 100 100 99 98 100 99 97 100 HQ420280 HQ420281 HQ420282 HQ420283 HQ420284 HQ420285 HQ420286 HQ420287 HQ420288 HQ420289 HQ420290 HQ420291 HQ420292 HQ420293 HQ420294 156 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 154–159 Aquaculture Research, 2012, 43, 154–159 Seasonal variations in the intestinal microbiota of Salmo salar L MB Hovda et al Table Seawater temperature and sequencing results where the sequenced bands are compared with the denaturing gradient gel electrophoresis (DGGE) gel pro¢les (Fig.1) Bacterium month À August October November December February March May June Seawater temperature ( 1C) Lactobacillus fermentum Lactococcus lactis Vibrio ichthyoenteri Vibrio sp Weissella sp./ Weissella cibaria Photobacterium phosphoreum Bacillus sp Unculturable spirochete gene 18S rRNA sequence Angiosperm environmental, chloroplast No of bands No of bacteria No of lactic acid bacteria (LAB) Percentage LAB 18.8 X X X X 14.7 X X 11.8 X X 9.1 X X X X 7.5 X X X X 5.5 X X 9.9 X X 14.2 X X X X X X X X X X 50 X X X X X X X X X 75 X 3 100 X X X 43 X 3 100 X 60 X 60 X X X 60 Bands were collected for sequencing from the August to October pro¢les, and the result compared in the gel BalcaŁzar, Vendrell, de Blas, Ruiz-Zarzuela, Muzquiz and Girones (2008) studied, under in vitro conditions, the ability of L fermentum and L lactis to inhibit ¢sh adhesion of several ¢sh pathogens Their ¢ndings indicate that these bacteria suppress the growth of the tested pathogens Lactococcus lactis has been found to have an antimicrobial e¡ect in turbot (Villamil,Tafalla, Figueras & Novoa 2002) This and other papers suggest that these bacteria may serve as probiotic candidates (Gomez & Balcazar 2008) As the bacterial pro¢le showed consistent bacteria throughout the sampling period, a comparison of the intensity of the bands could indicate the relative amount of the bacteria present during the year Because the handling and methodology for DNA extraction and PCR are equal for all samples, and the DNA volumes applied on the gel were similar (10 mL), the band intensity of the di¡erent pro¢les might re£ect the relative or semi-quantitative amount of the bacteria (Ferris & Ward1997) Drawbacks of this assumption include variability of PCR-ampli¢cation and hence some bacteria may not be ampli¢ed e⁄ciently Navarrete, Magne, Mardones, Riveros, Opazo, Suau, Pochart and Romero (2010) showed that di¡erent primers ampli¢ed the gut microbiota with di¡erent speci¢city, resulting in di¡erent band intensity on the gel Others have, however, used the assumption that the band intensity correspond to bacterial abundance in the sample (Calvo-Bado, Pettitt, Parsons, Petch, Morgan & Whipps 2003; Magne, Abely, Boyer, Morville, Pochart & Suau 2006) The band intensities were highest for bands of W cibaria, L fermentum and L lactis, during the entire sampling period, except the February and June sampling, re£ecting these bacteria as the most consistent bacteria In the February sample, the most prominent bands represented Vibrio spp Vibrio ichthyoenteri has been reported as a ¢sh pathogen, responsible for bacterial enteritis in Japanese £at¢sh and mass mortalities in Asian hatcheries (Muroga, Yasunobu, Okada & Masumura 1990; Muroga 2001; Gauger, Smolowitz, Uhlinger, Casey & Gomez-Chiarri 2006) Photobacterium phosphoreum is a bacterium known to spoil fresh ¢sh stored in air and modi¢ed atmosphere, and it has been suggested to contaminate the ¢sh ¢llet from the gut content This bacterium has also been found previously in the gut of salmon (Holben et al 2002; Hovda, Lunestad, Fontanillas et al 2007) Several publications have studied the microbiota composition of the ¢sh intestine, but many of these used cultivation before 16S DNA sequencing (RingÖ et al 2006; Bakke-McKellep et al 2007; RingÖ et al 2008) These results may be biased by the drawbacks of cultivation Our experiment uses direct DNA extraction, without prior cultivation and pure culture analyses The experiment in this paper did not identify seasonal variations in the gut microbiota In August, we observed the highest temperature (18.8 1C) and the highest number of bacteria in the gut Although the water temperature varied during the year (Table 2), © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 154–159 157 Seasonal variations in the intestinal microbiota of Salmo salar L MB Hovda et al Aquaculture Research, 2012, 43, 154–159 there were no direct correlation observed between the water temperature and the bacterial diversity The bacteria obtained may be suggested to be part of the adherent microbiota of the gut, as little e¡ect of water temperature variations was observed This is in contrast to Hagi et al (2004) and Al-Harbi and Naim Uddin (2004) who reported that the bacterial load varied seasonally in tilapia, using cultivation, phenotypic analyses and biochemical tests of pure cultures to determine the species present However, the carnivorous features of the salmonid digestive tract di¡er signi¢cantly to those of herbivorous tilapiines Hagi et al (2004) focused on investigation of seasonal changes in the LAB composition in ¢sh intestines from three carp species and channel cat¢sh They studied the 16S rDNA of bacterial isolates in addition to random ampli¢ed polymorphic DNA analysis of L lactis and Lactococcus ra⁄nolactis strains taken during the annual cycle, and observed varying bacterial count of LAB, as a response to the water temperature, and strain variations within the species As no e¡ect of the seasonal variation and seawater was observed in this study, the identi¢ed microbiota may be dominated by the adherent bacterial £ora of the farmed Atlantic salmon The main ¢nding from the annual sampling is that the intestinal microbiota of the Atlantic salmon in this experiment consisted of LAB throughout the year cycle, whereas other bacterial species likeVibrio spp were only detected in certain sample points Furthermore, no major di¡erences were observed in the gut microbiota population in response to the seasonal variations of the seawater Acknowledgment The authors are thankful to Òystein Olsen, former bachelor student at the University of Stavanger, for the excellent work at the laboratory during the time at No¢ma Norconserv Thanks to Skretting Aquaculture Research Centre (Stavanger, Norway) for providing the salmon References Al-Harbi A.H & Naim Uddin M (2004) Seasonal variation in the intestinal bacterial £ora of hybrid tilapia (Oreochromis niloticus  Oreochromis aureus) cultured in earthen ponds in Saudi Arabia Aquaculture 229, 37^44 Bakke-McKellep A.M., Penn M.H., Salas P.M., Refstie S., Sperstad S., Landsverk T., RingÖ E & Krogdahl — (2007) 158 E¡ects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.) 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Improving cat¢sh spawning success National Warmwwater Aquaculture Center News 4, 2 Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS, USA [ ] 18 Published [2011] This article is a US Government Work and is in the public domain in the USA, Aquaculture Research, 43, 14–18 Aquaculture Research, 2012, 43, 19–25 doi:10.1111/j.1365-2109.2011.02796.x Heritability... www.fao.org/¢/statist/FISOFT/FISHPLUS (accessed February 2009) © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 19–25 Aquaculture Research, 2012, 43, 19–25 Gall G.A.E & Bakar Y (2002) Application of mixed-model techniques to ¢sh breed improvement: analysis of breeding-value selection to increase 98-day body weight in tilapia Aquaculture 212, 93^113 Gilmour A.R., Gogel B.J., Cullis B.R., Welham S.J & Thompson... for trial 1, 94 Æ 5% for trial 2 and 84 Æ 6% for trial 3 (Table 2) Discussion The tanks used in the present study were adequate for the ongrowing of O maya juveniles The growth rate and survival observed revealed that this species © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 26–31 Ongrowing of Octopus maya in large outdoor tanks P Domingues et al Aquaculture Research, 2012, 43, 26–31... (mm), claw length (mm) and body weight (g) of Macrobrachium rosenbergii at 2, 5 and 6 months after reaching the post-larva stage by sex and rearing condition Aquaculture Research, 2012, 43, 19–25 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 19–25 Heritability of growth-related traits varied considerably with ages At 2 months after reaching the PL stage, h2 of CL (0.35 Æ 0.15) and BW (0.26... were also reported for other crustaceans, [e.g h2 for growthrelated traits was higher for the immature male than © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 19–25 Heritability for growth traits in GFP N Kitcharoen et al Aquaculture Research, 2012, 43, 19–25 Table 3 Phenotypic correlation (rP, below diagonal) for carapace length (CL), body length (BL), total length (TL), claw length (ClL)... mussel (Mytilus galloprovincialis) farming on oxygen consumption and nutrient recycling in a eutrophic coastal lagoon Hydrobiologia 550,183^198 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Aquaculture Research, 2012, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al Pan L.Y (2009) Puri¢cation and application of succinyl thiokinase Master thesis, Graduate Institute of... 1.11mg O2 L À 1 Anaerobic metabolism is initiated at a DO of o1.11mg O2 L À 1 Therefore, the concentration of DO in the bottom water of clam © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 1–13 Aquaculture Research, 2012, 43, 1–13 Digging behavior and metabolism of hard clams A-C Lee et al ponds should be maintained above this critical concentration Increasing the air^water exchange rate can... Society 30, 98^106 Association of O⁄cial Analytical Chemists (AOAC) (1990) O⁄cial Methods of Analysis, 15th edn AOAC,Washington, DC, USA © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 26–31 Aquaculture Research, 2012, 43, 26–31 Ongrowing of Octopus maya in large outdoor tanks P Domingues et al Boucher-Rodoni R & Mangold K (1985) Ammonia excretion during feeding and starvation in Octopus... AB, Bromma, Swe- 34 Figure 1 Electron micrograph depicting the gross view of cells in the hepatopancreas of control Penaeus monodon  5000 © 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 32–43 Aquaculture Research, 2012, 43, 32–43 Ultrastructural changes caused byAFB1 in P monodon R Gopinath et al Figure 2 The ultrastructural view of hepatopancreas of control shrimp showing intact cell membrane... quadratic equation is y 5 À 0.0171x210.8056x10.4455; r2 599.3% [ ] 16 Published [2011] This article is a US Government Work and is in the public domain in the USA, Aquaculture Research, 43, 14–18 Aquaculture Research, 2012, 43, 14–18 0%; therefore, the approximate margin of safety is o3-fold Current approved treatments for fungus control on cat¢sh eggs are expensive and have potential human health