NOT TO BE CITED WHTHOUT PRIOR REFERENCE TO THE AUTHORS. Developmental stage. Control. High currents. Long photophase Transport in the weaning tanks

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1 Fast fish Assessment of stress level and welfare in sea bass Papandroulakis N., Pavlidis M., Lika K., Fanouraki E., Papadakis G., Asderis M., Anastasiadis P., Stefanakis S., Vardanis G. and P. Divanach. Institute of Aquaculture, Hellenic Center for Marine Research, Greece. Biology Dept, University of Crete, Greece Aim of work Investigate and evaluate in European seabass fish behavior husbandry stressors Develop non-invasive welfare indicators under different rearing systems management conditions and routine operations Workshop September, Krakow Methodology Experimental design Larval rearing systems Husbandry Changes in light regime (extended photophase with no food) Changes in water currents (high currents) Transportation Handling confinement air exposure On growing Net-pen cages Husbandry Ordinary conditions Feeding Extra-ordinary human actions Developmental stage Control High currents Long photophase Transport in the weaning tanks Transport in the sea cages Hatching mouth opening Flexion Post-flexion Metamorphosis Juveniles Adults o Collection and analysis of behavioral data (friendly for the producer) that could characterize stressing conditions in different developmental stages Orientation and velocity Distribution in depth Sea cages DATA: VIDEO SONAR Larval rearing Hatchery stages Season Density of eggs Origin Water quality Temperature Water renewal rate Photoperiod Winter - Spring - egg l -.A. - Nireus S.A. Tank filling with filtered sea water Renewal from deep drill Initially o C, gradually 7. ± o C Initially % d -, gradual increase to % on 3-3 dph Ambient Larvae were fed on Enriched rotifers ( ind ml - ), Enriched Artemia Instar (.-. ind ml - ) Artificial feed Daily addition of phytoplankton ( ± 3x 3 cells ml - ) until dph

2 Season Larval Rearing Density of eggs Origin Water quality Temperature Water renewal rate Photoperiod Winter - Spring egg l -.A. - Nireus S.A. Tank filling with filtered sea water Renewal from biological filter Initially o C, gradually 7. ± o C Initially % h -, gradual increase to %, 3 dph L:D Larvae were fed on Enriched rotifers ( ind ml - ), Enriched Artemia Instar (.-. ind ml - ) Artificial feed Daily addition of phytoplankton ( ± 3x 3 cells ml - ) until dph TL (mm) Growth performance (days) TL (mm) 3 3 (days) (days) (days) TL (mm) SURVIVAL % 7 TL (mm) Monitoring with video recordings Development of software (scales,9dph) Data analysis Video observation Image analysis Estimation of velocity (value and direction) Vertical distribution 3 3 FishTrace analysis consists of the following steps:. Importing the frameset.. Adjusting the Brightness & Contrast. 3. Adjusting the threshold.. Selecting the outline of the fish at the initial frame.. Calculating the surface of the fish at the initial frame.. Starting the analysis by providing the maximum and minimum surface of the fish at the initial frame. 7. Producing the output files in.csv format for velocity and relevant vertical distribution and an image displaying the direction of the fish movement. Screenshots from steps of the process Screenshot is the output image displaying the direction of the movement. Two additional.csv files are produced with records of the direction, the velocity and the vertical distribution of fish within the frameset.

3 Velocity b. Direction : am 9: am : am Normal: _dark lights-on feeding feeding Stress: _ lights-on air-high currents feeding During feeding d3fed Ordinary conditions d3f a. Value velocity (BL/sec),,,,3, 7 9 Direction ( o ) DL-M 7-D-L-M BS-D-A-M S3-D-A-M BF-D-F-M F3D-F-M AF3-D-F-M d d d3 7 9, "" Velocity (BL/sec) DL-M 7-D-L-M BS-D-A-M S3-D-A-M BF-D-F-M F3D-F-M AF3-D-F-M "",,,,3,, d3fed Direction ( o ) DL-I 7-D-L-I BS-D-A-I S3-D-A-I BF-D-F-I F3D-F-I AF3-D-F-I d d d3 7 d3f 9 "" DL-I 7-D-L-I BS-D-A-I S3-D-A-I BF-D-F-I F3D-F-I AF3-D-F- "" d d d % % % % % % % % % % % % % % % % % % % % % % % % Vertical distribution a % % a % % a % % a % % % % % % % % % % % % % % % % % % % a % f % fed % fed % % % % % % % % % % % % % % % % % % % % fed % % f % % f % % f % % % % % % % % % % % % % % % % % % f Control f fed % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % a a a3 a % % % % % % % % % % % % % % % % % % fed fed f % % % % % % % % % % % % % % % % % % % % % a f fed3 fed % % % % % % % % % % % % % % % % % % % % % % % % % f7 f % % % % % % % % % % % % fed f f7 f % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % %

4 f Control % % f % fed Conclusions % % % % % % % % % % % % % % % Fish respond differently according to the rearing system fed fed f % % % % % % % % % % % % fish respond more intense i.e. more natural, in the different conditions % % % % % f % % f % Velocity and distribution can be used as behavioral indices % % % % % % % % % % Use of classical and noninvasive physiological stress indicators for the assessment of husbandry stressors Whole body Cortisol concentration in larvae under different rearing systems Ontogenesis of cortisol response Assessment of stressors in early ontogeny F (ng g - ) F (ng g - ) hatch mouth opening flexion post fl/fins all fins melanophores scales control stressed hatch mouth opening flexion post fl/fins all fins melanophores scales control stressed Is the use of non-invasive methods for stress evaluation applicable? It is feasible to measure cortisol levels released into the water by the larvae in both rearing systems Transportation stress in larvae and juveniles A. Larval transportation It is not always possible the interpretation the observed differences in water cortisol levels under different stressors due to difficulties in estimation : Larval cortisol (ng g - ) Before During of the actual biomass of the releasing rate Cortisol release rate into the water.±. ng g - h - In closed systems reliable estimates

5 B. Juvenile transport simulation Conclusions Cortisol release rate (ng g- h-) Temperature = ±. o C Dissolved O =.7 ± 3 mg l -,,,,,,,,,, Weight 3 ± 7. g (mean ± SD) a kg m-3 a kg m-3 a b bc b c c b b b b (h) The European sea bass show high cortisol concentrations during early ontogeny Juveniles and adult fish show high unstressed plasma cortisol concentrations The measurement of released cortisol levels into the water is a reliable non-invasive method for the evaluation of husbandry stressors in juvenile and adult fish On-growing stages During ordinary conditions (calm) feeding extra-ordinary human actions, (stress) Results Observing feeding behavior 3 3 3,,,,,, Temperature ( o C), / / / / / / / / / / mes-7 mes7- int-7 int7- Nmes Nint T-7 T7- TN7-

6 Calm Analysis of echograms // //7 7//7 //7 // 9/9/ Weighted mean position every s Conditions: calmmes calmint fedmes fedint strmes strint :: :: ::3 :: :3: 3:: 3:: :9:3 :3: 3 3aprMes 3aprint feedmes StressMes feedint StressInt Mean position Feeding // //7 7//7 //7 // 9/9/ S-m S-i N-m N-i Stress // //7 7//7 //7 // 9/9/ SF-m SF-i SS-m NS-m SS-i NS-i calmmes vs calmint fedmes vs fedint strmes vs strint comment SEP OCT NOV low E currents 9JAN7 strong currents APR7 7APR7 strong west currents MAY7 strong west currents MAY7 hot JUN7 strong East currents JUN7 strong East currents JUL7 AUG7 strong East currents 7OCT7 DEC7 low E currents 3JAN strong west currents 3ARP strong East currents Absolute difference in mean transposition Comparison between groups,,,,,, OCT7N OCT7N stress only -sunset OCT7N OCT7N sunrise JANN JANN APRN,, :: :: ::3 :: :3: 3:: 3:: :9:3 :3: Mes Int feedmes StressMes feedint StressInt Mean transposition,, Feeding // //7 7//7 //7 // 9/9/,, // //7 7//7 //7 // 9/9/ SF-m Calm SF-i,, S-m N-m S-i N-i Stress // //7 7//7 //7 // 9/9/ SS-m SS-i calmmes vs calmint fedmes vs fedint strmes vs strint SEP OCT NOV 9JAN7 APR7 7APR7 MAY7 MAY7 JUN7 JUN7 JUL7 AUG7 7OCT7 DEC7 3JAN 3APR OCT7N OCT7N OCT7N OCT7N JANN JANN APRN

7 Calm 3 Comparisons between trials of Same season Same Mean Weight Mean position Feeding S-m N-m S-i N-i Stress 3 3 SF-m SF-i SS-m NS-m SS-i NS-i JAN JAN JAN JAN JAN JAN NMJAN - NMAPR NIJAN NIAPR SMJAN7 SMAPR7 - SMJAN - SMAPR SIJAN7 SIAPR7 SIJAN SIAPR JAN JAN JAN JAN JAN JAN F F 7F F 7F F F F 7F F 7F F NMJANF NMAPRF - NIJANF NIAPRF SMJAN7F - SMAPR7F SMJANF SMAPRF SIJAN7F SIAPR7F SIJANF SIAPRF JAN JAN JAN JAN JAN JAN S S 7S S 7S S S S 7S S 7S S NMJANS NMAPRS - NIJANS NIAPRS - POS SMAPR7 SIAPR7 SMJAN SIJAN SMAPR7 SIAPR7 SMJAN SIJAN SMAPR7F SIAPR7F SMJANF SIJANF SMAPR7F SIAPR7F SMJANF - SIJANF SMAPR7S SIAPR7S SMJAN7S SIJANS SMAPR7S - SIAPR7S SMJANS SIJANS SMJAN7S SMAPR7S - - SMJANS - SMAPRS - SIJAN7S SIAPR7S SIJANS SIAPRS Calm, Mean transposition, JAN JAN JAN JAN JAN JAN NMJAN - NMAPR - NIJAN - NIAPR SMJAN7 - SMAPR7 - - SMJAN - SMAPR - - SIJAN7 - SIAPR7 SIJAN SIAPR 3 3 S-m S-i N-m N-i J JAN JAN JAN JAN JAN AN F F 7F F 7F F F F 7F F 7F F NMJANF NMAPRF Feeding Stress NIJANF NIAPRF SMJAN7F SMAPR7F - - -,, SMJANF - - SMAPRF - - SIJAN7F - SIAPR7F - SIJANF SIAPRF,, J JAN JAN JAN JAN JAN AN APR APR APR APR PR APR S S 7S S 7S S S S 7S S 7S S NMJANS NMAPRS - NIJANS NIAPRS SMJAN7S SMAPR7S SMJANS - - SMAPRS - - SIJAN7S - SIAPR7S SIJANS SIAPRS SF-m SF-i SS-m NS-m SS-i NS-i

8 DIFF SMAPR7 SIAPR7 SMJAN SIJAN Conclusions SMAPR SIAPR7 - - SMJAN SIJAN SIAPR7F SMJANF SIJANF SMAPR7F SMAPR7F SIAPR7F - - SMJANF - SIJANF SIAPR7S SMJAN7S SIJANS SMAPR7S SMAPR7S SIAPR7S - - SMJANS - Fish respond to different actions by changing their position The position of the fish populations is different according to the various conditions tested A more conservative index is the transposition of the populations Results obtained are reproducible in time and location SIJANS Conclusions (cont) Populations from both rearing systems respond similar in the different conditions tested During feeding and stress, reactions are more intense There are differences between conditions with the mesocosm populations to react more intensively During stress mesocosm populations are moving deeper Position transposition can be used as an index of behavioral response in cages