First evidences of the effect of polyunsaturated fatty acids in fish neural activity and their implications in behaviour

First evidences of the effect of polyunsaturated fatty acids in fish neural activity and their implications in behaviour Tibiábin Benítez Santana Grupo de Investigación en Acuicultura Instituto Canario de Ciencias Marinas Programa de Doctorado: Acuicultura: Producción controlada de animales acuáticos Instituto Universitario de Sanidad Animal y Seguridad Alimentaria Universidad de las Palmas de Gran Canaria Being a thesis submitted for the degree of Doctor of Phylosophy in the University of Las Palmas de Gran Canaria, 2011. Directors: Prof. Marisol Izquierdo & Prof. Reiji Masuda A mi padre, Pepe AB. “As long as our brain is a mystery, the universe, the reflection of the structure of the brain will also be a mystery” Ramón y Cajal I I In n nd d de e ex x x Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .V Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .XI List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .XIII Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVII 1. Introduction 1.1 Significance of fish lipid nutrition in larviculture . . . . . . . . . . . . .1 1.2 Essential fatty acids and their role in neural function . . . . . . . . .3 1.3 Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..10 1.4 Mauthner cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 2. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 3. Material and Methods 3.1 Species used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 3.1.1 Sea bream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.1.2 Seabass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.1.3 Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.2 Feeding trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2.1 Experimental conditions . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2.2 Growth determination . . . . . . . . . . . . . . . . . . . . . . . . . .23 3.3 Diet and feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.3.1 Live food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.3.1.1 Rotifers cultive . . . . . . . . . . . . . . . . . . . . .24 3.3.1.2 Rotifers enrichment . . . . . . . . . . . . . . . . . 24 3.3.1.3 Enrichment . . . . . . . . . . . . . . . . . . . . . . . .25 3.3.1.4 Brine shrimp cultive . . . . . . . . . . . . . . . . . 25 3.3.1.5 Enrichment . . . . . . . . . . . . . . . . . . . . . . . .26 3.3.2 Microdiets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 3.3.2.1 Microdiets preparation . . . . . . . . . . . . . . . 27 3.4 Behavioural studies in sea bream . . . . . . . . . . . . . . . . . . . . . . . . .29 Index II 3.4.1 Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.4.2 Stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.4.2.1 Sonorous stimuli . . . . . . . . . . . . . . . . . . . .29 3.4.2.2 Visual stimuli . . . . . . . . . . . . . . . . . . . . . . 30 3.4.3 Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 3.4.3.1 Cruise Swimming Speed . . . . . . . . . . . . . .31 3.4.3.2 Burst Swimming Speed . . . . . . . . . . . . . . 31 3.4.3.3 Burst Swimming Rate . . . . . . . . . . . . . . . .32 3.5 Zebrafish behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 3.5.1 Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 3.6 Biochemical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.6.1 Dry matter content . . . . . . . . . . . . . . . . . . . . . . . . . . . .34 3.6.2 Total lipids content . . . . . . . . . . . . . . . . . . . . . . . . . . . .35 3.6.3 Separation of polar lipids . . . . . . . . . . . . . . . . . . . . . . . .35 3.6.4 Fatty acid esters preparation and quantification . . . . . . 35 3.7 Histological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36 3.7.1 Sample collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.7.2 Processing and paraffin embedding . . . . . . . . . . . . . . . .37 3.7.2.1 Preparation of cuts . . . . . . . . . . . . . . . . . .38 3.7.2.2 Histological stains . . . . . . . . . . . . . . . . . . .38 3.7.2.2.1 Hematoxylin and eosin . . . . . . . . . . 38 3.7.2.2.2 Nissl staining . . . . . . . . . . . . . . . . . 39 3.7.2.3 Immunohistochemical techniques . . . . . . . 40 3.7.3 Preparation of samples for cryostat . . . . . . . . . . . . . . . 43 3.7.3.1 Immunofluorescence . . . . . . . . . . . . . . . . .44 3.7.4 Processing and inclusion in resin . . . . . . . . . . . . . . . . . . 45 3.7.4.1 Semithin sections . . . . . . . . . . . . . . . . . . .46 3.7.4.2 Ultra thin sections . . . . . . . . . . . . . . . . . . 48 3.8 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 4. Study I: Dietary DHA deficiency induce a reduced response in gilthead seabream ( Sparus aurata ) larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . .53 Index III 5. Study II: Increased Mauthner cells activity and escaping behaviour in sea bream ( Sparus aurata ) fed long chain polyunsaturated fatty acids . . 75 6. Study III: DHA but not EPA, enhances sound stimuli induced escaping behaviour and Mauthner cells activity in Sparus aurata . . . . . . . . . . . .93 7. Study IV: Dietary polyunsaturated fatty acids affect zebrafish ( Danio rerio ) behaviour and Mauthner cells . . . . . . . . . . . . . . . . . . . . . . . . .113 8. Study V: Use of calretinin (CR) and parvalbumin (PV) as Mauthner cells markers in sea bass ( Dicentrarchus labrax ) . . . . . . . . . . . . . . . . . . . .133 9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153 10. Spanish summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 11. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 12. Annexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Index IV XI L L Li i is s st t t o o of f f T T Ta a ab b bl l le e es s s 3.1 Main fatty acids of the major dietary lipid sources and deffated squid meal used in this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28 3.2 Histological samples manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.3 Antibodies tested in immunohistochemistry technique . . . . . . . . . . . . . .41 3.4 Primary antibody used in inmunofluorescence . . . . . . . . . . . . . . . . . . . . 45 4.1 Some fatty acids contents in total lipids from oils and enriched rotifers used to feed gilthead seabream larvae (% total determined fatty acids, n=3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.2 Fatty acids composition of initial 3 day-old larvae and 10 and 20 day-old larvae for each experimental group (n.d. ≤ 0.005) . . . . . . . . . . . . . . . . . 61 4.3 Some fatty acids contents in total lipids from brain and eyes in larvae after 20 days of feeding (% total determined fatty acids, n=3) . . . . . . . . . . . .62 5.1 Fatty acid profile of the experimental diets (% total identified fatty acids) used to feed gilthead sea bream larvae . . . . . . . . . . . . . . . . . . . . . . . .79 5.2 Fatty acids content (% total determined fatty acids, n=3) of 35 day-old sea bream larvae fed with fish oil microdiet and soybean oil microdiet (Mean values with their standard desviation) . . . . . . . . . . . . . . . . . . . .83 List of Tables XII 6.1 Lipid sources (% total ingredients) and crude lipid (% dry basis) content diets containing different proportions of EPA and DHA . . . . . . . . . . . . 97 6.2 Fatty acid profile of the experimental diets (% total identified fatty acids) used to feed gilthead sea bream larvae . . . . . . . . . . . . . . . . . . . . . . . .98 6.3 Main fatty acid composition of total lipids (% total identified fatty acids) from gilthead sea bream fed microdiets containing different EPA/DHA proportions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103 7.1 Some fatty acids contents in total lipids from Artemia fed different levels of DHA and used to feed zebrafish larvae (% d.w.) . . . . . . . . . . . . . . 118 8.1 Feeding protocol during the experimental trials . . . . . . . . . . . . . . . . . .137 8.2 Scores of the staining of M-cells in soma, dendrites and axon using PV and CR antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 L L Li i is s st t t o o of f f F F Fi i ig g gu u ur r re e es s s 1.1 Aquaculture production versus captures of gilthead sea bream in Europe from 1979 to 2007 (data elaborated from FAO FISHSTAT Plus) . . . . . . 2 1.2 Biosynthesis pathways of long-chain polyunsaturated fatty acids from C18 precursors, 18:3n-3 and 18:2n-6 . . . . . . . . . . . . . . . . . . . . . . . . . .5 1.3 Nerve growth without and with DHA supplementation (http://www.thevisualmd.com/health_centers/child_health/infant_nutriti on/dha_ara) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.4 Diagram of M-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 1.5 Sequential body orientations of a fish engaging in a tail-flip escape behaviour, what time progressing from left to right (Figure adapted from Eaton et al. , 2001) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 1.6 Myelinated axons from M-cells cross the midline to descend the length of the spinal cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 3.1 a) Sea bream larva. b) Sea bass larva. c) Zebra fish larva . . . . . . . . . . . .22 3.2 Application of the sonorous stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 3.3 Application of the visual stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 4.1 Development of growth in seabream larvae fed rotifers enriched with different types of lipids (n = 30, P<0.05) . . . . . . . . . . . . . . . . . . . . . . .63 4.2 (a) Development of cruise swimming speed (mm/s) under conditions of List of Figures XIV sound stimuli experiment (dark walls) in seabream larvae fed rotifers enriched with different types of lipids (n = 10, P<0.05). (b) Number of reacting seabream larvae for each experimental group after the sound stimuli. (c) Development of burst swimming speed (mm/s) after sound stimuli in seabream larvae fed rotifers enriched with different types of lipids (n = 10, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 4.3 (a) Development of cruise swimming speed (mm/s) under conditions of visual stimuli experiment (clear walls) in seabream larvae fed rotifers enriched with different types of lipids (n = 10, P<0.05). (b) Number of reacting seabream larvae for each experimental group after the sound stimuli. (c) Development of burst swimming speed (mm/s) after visual stimuli (clear walls) in seabream larvae fed rotifers enriched with different types of lipids (n = 10, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66 5.1 Larval reaction after the sound stimuli along larval development (a) Number (%) of reacting sea bream larvae for each experimental group after the sound stimuli fed diets containing fish oil and soybean oil. (b) Burst swimming speed (SL/s) in sea bream larvae fed microdiets enriched with different types of lipids: fish oil and soybean oil. a,b Mean values with unlike letters were significantly different between animals of same treatment (P<0.05), n=30. A,B Mean values with unlike letters were significantly different between animals of different treatment (n=30, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84 5.2 Quantification of green fluorescent intensity by confocal microscopy according to the immunopositive response of M-cells. * Mean values were significantly different between animals of different treatment (n=30, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85 5.3 Confocal microscopy image of the acetylcholine immunopositive response of M-cells longitudinal sections from larvae fed with fish oil microdiet (A) List of Figures XV and soybean oil microdiet (B). Scale bar 10 µm . . . . . . . . . . . . . . . . . .85 6.1. Larval reaction after the sound stimuli along larval development. Burst swimming speed (mm/s) in sea bream larvae fed microdiets enriched with different levels of EPA/DHA. * Mean values were significantly different between animals of different treatment (n=30, P<0.05) . . . . . . . . . . .104 6.2. Quantification of green fluorescent intensity by confocal microscopy according to the anti-choline acetyltransferase immunopositive response of M-cells. * Mean values were significantly different between animals of different treatment (n=30, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . .105 6.3 Confocal microscopy image of the acetylcholine immunopositive response of M-cells longitudinal sections from larvae fed with 1.5/9 microdiet (A) and 0.3/0.6 microdiet (B) (x1000) . . . . . . . . . . . . . . . . . . . . . . . . . . 105 7.1 Evolution of growth in zebrafish larvae fed Artemia enriched with different levels of DHA (n=12, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 7.2 (a) Peak angular speed (Degrees/msec) (b) Time to peak bend angle (msec) in zebrafish larvae fed with different levels of DHA. a, b Mean values with unlike letters were significantly different between animals of different treatment (n=6, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . .121 7.3 Immunopositive response (green fluorescence) observed by confocal microscopy in Mauthner cells from zebrafish fed different diets. (a) Zebrafish fed Diet L (0.06 DHA content). (b) Zebrafish fed Diet M (4.71 DHA content) (x1000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123 7.4 Quantification of green fluorescent intensity by confocal microscopy according to the anti-choline acetyltransferase immunopositive response of M-cells in zebrafish fed diets with different DHA levels. a, b Mean values List of Figures XVI with unlike letters were significantly different between animals of different treatment (n=30, P<0.05) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123 8.1 (a) Nissl body-like positive lump in the most external part of the cytoplasm of M-cells (black arrow). White arrow shows axon (x1000). (b) Basophilic staining present in soma (black arrow), dendrites, axon (white arrow) and axon cap (*) M-cells present in larvae of 10 days old. (›-‹) shows synaptic bed (x400). (c) Acidophilic stain in dendrites and axon and basophilic stain in soma (black arrow) in larvae of 13 days old (x1000). (d) Acidophilic stain in dendrites and axon and basophilic stain in soma (black arrow) in larvae of 17 days old (x1000). (e) Acidophilic stain in dendrites, axon (white arrow) and nucleus (pink arrow). Basophilic stain in soma (black arrow) in larvae of 28 days old. (›-‹) shows synaptic bed (x1000). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 8.2. (a) The soma, dendrites and axon of M-cells (black arrow) are PV immunonegative (x400). (b) PV immunostained in axon and dendrites (white arrows) of M-cells (black arrow) (x1000). (c) PV-containing soma, dendrites and axon of a M-cell (black arrow) (x1000). (d) PVimmunopositive Mauthner soma (black arrow), dendrites and axon (red arrow). PV-immunonegative Mauthner soma membrane (blue arrow) (x1000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143 8.3 (a) PV immunonegative nucleus (*) surrounded by PV immunostained cytoplasm of M-cells (black arrow) in sea bass larvae of 17 days old (x1000). (b) PV-immunopositive soma (white arrow) and axon (yellow arrow) of M-cells and PV-immunonegative nucleus (black arrow) in larvae of 24 days old (x1000) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 8.4 CR immunopositive in Mauthner axon cap (black arrow) (x1000) . . . . .145 XVII L L Li i is s st t t o o of f f a a ab b bb b br r re e ev v vi i ia a at t ti i io o on n ns s s ARA ARA Arachidonic acid (20:4n-6) CNS CNS Central nervous system CR CR Calretinin DAH DAH Day after hatching DHA DHA Docosahexaenoic acid (22:6n-3) EFA EFA Essential fatty acids EPA EPA Eicosapentaenoic acid (20:5n-3) FO FO Fish oil H&E H&E Hematoxilin and eosin HUFA HUFA Highly unsaturated fatty acids LA LA Linoleic acid (18: 2n-6) LNA LNA Linolenic acid (18:3n-3) LSO LSO Linseed oil LCPUFA LCPUFA Long chain polyunsaturated fatty acids M M- -cells cells Mauthner cells PBS PBS Phosphate bufferd saline PUFA PUFA Polyunsaturated fatty acids (equivalent to HUFA) PV PV Parvalbumin RSO RSO Rapseed oil SBO SBO Soybean oil SL SL Standar lenght List of abbreviations XVIII Introduction 1 1.1 Significance of fish lipid nutrition in larviculture Aquaculture has a long history of more than 4,000 years despite its significant contribution to global human food production is only very recent. It is one of the most rapid developing animal production sectors in the world, growing at a rate higher than 5% per year during the last decade (Izquierdo, 2005). Specifically, the contribution of world-wide farm production of fish, crustaceans and molluscs has increased from 3.9% of total fishing production in 1970 to 43% in 2008 (68,329280 million metric tons), being expected to reach 50% of total fishing production by the year 2025 (FAO, 2008) (Figure 1.1). According to FAO previsions, aquaculture is a feasible complement to wild captures fisheries to satisfy the high demand of marine products. One of the main challenges for the further development of aquaculture is to meet the demand on mass production of high quality fry (Izquierdo and Fernandez-Palacios, 1997). In marine fish species, larval stages are particularly sensitive, and sudden and uncontrolled mortalities or reduced growth constrain the further development of production of those species (Watanabe et al ., 1983; Yúfera and Pascual, 1984; Sargent et al . 1997; Izquierdo et al ., 2000). Larval rearing success is mostly affected by first feeding regimes and the nutritional quality of starter diets (Izquierdo et al ., 2000). Among the nutritional requirements of larvae, lipids are recognized as one of the most important nutrients affecting growth and survival of fish larvae (Watanabe et al ., 1983). The use of dietary lipids by the larvae, directly or indirectly, is affected by a sequence of morphological and physiological changes that occur during larval development. Thus, to ensure a better growth and a high survival rate, a reliable diet that meet the nutritional requirements of larvae, both qualitatively and quantitatively is essential (Kolkovski et al ., 1993; Sargent et al ., 1997). In the last years, more attention has been paid to study the importance of dietary lipids in marine fish larvae (Izquierdo et al ., 2003), since they Introduction 8 these data demonstrated that the brain is highly sensitive to a lack of PUFAs during maturation and that a chronically unbalanced early environment produced by deficient nutrition could lead to durable changes in cerebral function (Lauritzen et al. , 2001). Figure 1.3 Nerve growth without and with DHA supplementation (http://www.thevisualmd.com/health_centers/child_health/infant_nutrition/dha_ara). Other reports also implicate PUFAs in a number of seemingly unrelated neuropathologic conditions including depression, schizophrenia, hyperactivity disorder, and autism. In these patients, the plasma and erythrocytes membranes contain subnormal amounts of n -3 and n -6 PUFAs (Glen et al. , 1994; Edwards et al. , 1998; Maes et al. , 1999; Burgess et al. , 2000; Yao et al ., 2000; Vancassel et al. , 2001). Furthermore, intervention trials suggest that DHA, as well as its direct metabolic precursor, EPA, can be beneficial for treating some of these psychiatric diseases (Mellor et al. , 1996; Emsley et al. , 2002; Nemets et al ., 2002; Peet and Horrobin, 2002; Richardson and Puri, 2002). The brain is functionally and anatomically lateralized, with the hemispheres being specialized differently for cognitive and motor control. Deviations from the normal patterns of asymmetry appear to be related to cognitive behavioural deficits, Introduction 9 and anomalies in brain asymmetry have been widely documented in psychiatric disorders, such as schizophrenia, autism, and depression (Cowell et al ., 1999; Petty, 1999). Studies in rodents indicate that behavioural lateralization reflects inter hemispheric asymmetries involving complex genetic and environmental factors that are established during the prenatal and postnatal development of the brain (Glick and Cox, 1978; Carlson and Glick, 1996; Nielsen et al. , 1997). Early experiences and variables, such as prenatal stress and maternal ingestion of specific nutrients, cause long-lasting changes in the behavioural lateralization of the offspring (Rodríguez et al. , 1994; Alonso et al., 1997). In addition, differences in brain laterality appear to determine susceptibility to depression-like behaviours in rats exposed to stressors that cannot be controlled (Carlson and Glick, 1991; Alonso et al. , 1997). These data suggest that behavioural laterality is a labile process that can be modified by environmental stimuli, and these studies on n-3 PUFA deficiency and behavioural impairments led us to assume that a change in dietary n-3 PUFA supply from conception throughout life can lead to changes in specific lateralized behaviour by affecting structural or neuro chemical patterns of asymmetry in motor related brain structures, with potential consequences for cognition (Vancassel et al. , 2005). In fish, inadequate dietary EFA levels also result in poor feeding and swimming activities (Izquierdo, 1996) and altered fish larvae behaviour (Benítez-Santana et al. , 2007). Moreover, EFA deficient feeds delay the appearance of response to visual stimulus, in agreement with the reduction in DHA content in eyes and brains of these larvae and suggesting a delay in the functional development of brain and vision (Benítez-Santana et al. , 2007). In agreement with these results, reduction in DHA and EPA contents, reduces gilthead sea bream larvae eye diameter (Roo et al. , 1999; Izquierdo et al. , 2000). This fact, along with the higher density of cone photoreceptors, implies a significant improvement in larval visual potential (Roo et al. , submitted). Lower swimming and feeding activities in EFA-deficient larvae (Izquierdo et al ., 1989a; Rodríguez et al ., 1993; 1994) are frequently recognized by the larvae floating on the water surface denoting as well alterations in the functioning of swim bladder (Koven, 1991). Effects have also been reported on Introduction 10 swimming performance of Atlantic salmon ( Salmo salar ) in seawater when it is fed diets containing different supplemental oils (anchovy oil and poultry fat) depending on the different n-3 HUFA/saturated fatty acids ratios (Wagner et al. , 2004). Thus, n-3 HUFA have been recognised as a major limiting factor defining the nutritional value of larval diets for marine species, being indispensable through early stages of life (Watanabe and Kiron, 1994; Izquierdo, 1996; Sargent et al. , 1999b). Since very few studies have been aimed to determine the ontogeny of fish behaviour, the effect of dietary fatty acids on behaviour of this larva is also unknown. 1.3 Behaviour Exploration is a key animal behaviour in response to novelty (Kim et al. , 2005; Kliethermes and Crabbe, 2006; Kalueff and Zimbardo, 2007). Fishes are by far the most species-rich vertebrate taxon, and it is also the vertebrate group with the most strikingly diverse repertoire of behaviours and behavioural adaptations. As such, they provide us with many opportunities to explore the fascinating complexities of animal behaviour. Concern for the welfare of fish has increased during the last years (Huntingford et al. , 2006; Ashley, 2007; Arlinghaus et al. , 2007) to a large extent because of the possibility that fish can feel pain (Rose, 2002; Chandroo et al. , 2004). Stressful situations have been shown to affect the behaviour of fish (Sneddon et al. , 2003; Ashley, 2007) and recent studies suggests that their central processing of potentially painful stimuli leads to prioritization of Introduction 11 motivational drivers and modifies normal behaviour patterns (Ashley et al. , 2009). The physiological and behavioural mechanisms that result in pain and stress are evolved adaptations that enable animals to cope with natural challenges, such as the need to avoid unfavourable habitats, to find food or to avoid being killed by predators. Pain (or nociception as it is more properly called) allows animals to detect and avoid localised harmful stimuli. The stress response can be considered to be part of an adaptive strategy for coping with a perceived threat to homeostasis. Concern for the welfare of the animals that humans keep in captive conditions (for example, on farms or as pets) arises from the fact that these adaptive systems may be activated in contexts where they cannot bring about their natural outcome of either removing the stressor or removing the animal from the source of pain or stress. Much welfare research involves identifying signs that this is the case and finding ways of ameliorating the conditions responsible. From a physiological viewpoint, good welfare can be recognized by objective measurement of a range of biochemical and physiological indicators (Arlinghaus et al. , 2007). Exposure to some stressors and the resultant physiological response are not equivalent to suffering and may be beneficial, at least in the short term (Pickering, 1998; Huntingford et al. , 2006). Fish are able to adapt to stress for a period of time; they may look and act normal. However, energy reserves are eventually depleted and hormone imbalance occurs, suppressing their immune system and increasing their susceptibility to infectious diseases. Nevertheless, repeated exposure to acute stressors or prolonged exposure to poor conditions results in maladaptive responses (Wendelaar Bonga, 1997; Pickering, 1998), and monitoring of multiple components of the stress responses provides valuable insights into the welfare of fish (Turnbull et al. , 2005; Adams et al. , 2007). Fish have the same stress response and powers of nociception as mammals. Their behavioural responses to a variety of situations suggest a considerable ability for a higher level of neural processing, a level of consciousness equivalent perhaps to that attributed to mammals. Fish showing low blood concentrations of cortisol and normal values for parameters such as plasma osmolality and plasma concentrations Introduction 12 of glucose and lactate, as well as adequate growth and condition index are generally considered to be experiencing good health and welfare (Adams et al. , 1993; Turnbull et al. , 2005). Persistent disruption of homeostasis, results in a reduced appetite, poor growth and condition, and decreased immunocompetence (Adams et al. , 1993; Balm, 1997). In this sense, fish have the same stress response and powers of nociception as mammals and good welfare preserves or enhances fish health and well being (Arlinghaus et al. , 2007). Animals often express consistent individual differences in behaviour. Part of the individual variation in behaviour may be caused by random noise around an adaptive average and small variations in state or context, but in a variety of animal species individual behavioural differences are consistent across different social and environmental contexts and independent of sex, age and size (Sih et al. , 2004; Carere & Eens 2005; Bell, 2007; Reale et al. , 2007). For example, some individuals may be more aggressive or bolder than others. Such consistent individual differences are commonly termed personalities or temperament (Reale et al. , 2007). The fitness benefits associated to a specific behaviour in a particular context (e.g. aggression during a contest) may become costs in another (aggression during courtship). Therefore, different personalities may have different context-dependent fitness and the inherent trade-offs may, in general, hinder individuals from attaining theoretical fitness optima. There is little available information about the ecological and evolutionary implications of personalities, but there are indications that personalities may affect dispersal, antipredator behaviour, foraging and reproductive strategies and, therefore, play a part in population and community ecology (Sih et al. , 2004; Dingemanse & Reale, 2005; Reale et al. , 2007). Personalities have been found in various types of behaviours, mostly having to do with aggressiveness, boldness and reaction to novelty. Some of these include exchange of signals as basic components, but there are no direct investigations of the implications of personalities for communication. Reale et al. (2007) have suggested five behavioural categories for personalities: shyness–boldness; exploration–avoidance; activity; aggressiveness; sociability. Only the last two of these require the use of signals at least to some Introduction 13 degree, but the frequency of signal use and reception per se have not been investigated as specific personality traits. Yet the frequency of visual threat display has been used as an index of aggressiveness in fish, and the rate, frequency or timing of specific vocalizations could be used as an index of aggression or eagerness to mate as in birds (Matessi et al. , 2010). The spectacular array of behaviours exhibited by animals has been generated through the processes of evolution. Behavioural modifications are ultimately produced by differences in neural circuits. Probing the causes and consequences of nervous system evolution requires a tractable neural circuit and a group of animals that exhibit variation in this circuit (Tierney, 1996; Nishikawa, 1997; Katz and Harris-Warrick, 1999; Wright, 2000; Carr et al. , 2001; Rose, 2004). The circuitry that generates escape behaviour in teleost fishes presents such a system. The escape response, also known as the fast-start or startle response, is initiated by a pair of large, identifiable neurons in the hindbrain: the Mauthner cells (M-cells) (Figure 1.4) (Zottoli and Faber, 2000; Eaton et al. , 2001; Korn and Faber, 2005). Fast-starts in most teleost fish consist of a C-type fast-start (C-start), the first stage of which is characterized by a rapid unilateral contraction of trunk musculature leading to head and tail movement, which causes the fish to bend into a C-shape (Figure 1.5). Stage 1 is typically followed by subsequent movements, including a tail stroke that results in a forward propulsion of the centre of mass (stage 2), and either gliding or a burst swim (stage 3) (Eaton et al. , 1981; Foreman and Eaton, 1993; Domenici and Blake, 1997). M-cell activity precedes the C-start, and electrical stimulation of Mauthner axons can elicit a C-start (Zottoli, 1977; Nissanov et al. , 1990). Introduction 14 Figure 1.4 Diagram of M-cells. Figure 1.5 Sequential body orientations of a fish engaging in a tail-flip escape behaviour, what time progressing from left to right (Figure adapted from Eaton et al. , 2001). Introduction 15 1.4 Mauthner cells Fish can elude predatory attacks by producing a stereotyped escape behaviour, which is characterized by a rapid and powerful unilateral bending of the body and tail that involves most of its somatic musculature (Korn and Faber, 2005). This behaviour has a characteristic short latency when triggered by abrupt acoustic stimuli, and it is initiated by the activation of the M-cells. The M-cells are a pair of reticulospinal neurons located in the medulla of teleost fish (Beccari, 1907). These uncommonly large cells are anatomically and physiologically identifiable and have historically constituted a valuable preparation for the study of the cellular correlates of behaviour (Faber et al. , 1989; Korn and Faber, 2005). Their characteristic large myelinated axons, first noticed by Mauthner (1859), cross the midline to descend the length of the spinal cord (Figure 1.6), issuing axon collaterals that massively activate cranial and spinal motor systems via reliable (with high safety factor) chemical synapses (Faber et al. , 1989). Such an anatomical arrangement allows a single action potential in this cell to initiate an escape response by producing a tailflip. Reticulospinal neurons form one of the most important descending pathways in the vertebrate central nervous system. These neurons function as integration centers for sensory inputs and higher motor command in the brain and ultimately regulate motor functions in the spinal cord (Rovainen, 1967; Rovainen, 1982). Certain reticulospinal neurons, the Müller and M-cells, can be identified from animal to animal in anamniotic vertebrates. Because they are large cells and can be easily identified, these neurons have provided convenient experimental models to study neuronal properties and the neuronal basis of motor behaviours. Introduction 16 Figure 1.6 Myelinated axons from M-cells cross the midline to descend the length of the spinal cord. The M-cells are a pair of identifiable neurons found in the brain of teleost fishes and certain amphibians (Stefanelli, 1951). They have two major dendrites, both at least 500 µm in length; one extends laterally and the other either ventrally or medially. One of the more impressive features of the M-cells is the size of its dendrites, which allows repeated microelectrode penetrations along their entire length. The soma is located in the rostroventral medulla and the large lateral dendrite of each M-cell receives inputs mainly from the sensory neurons of the statoacoustic system. The club endings of the ipsilateral VIIIth nerve have been shown to make gap junctions with the lateral dendrite of the M-cells (Robertson et Introduction 17 al., 1963), a finding consistent with an electrically transmitting synaptic mechanism (Furshpan, 1964). The axon from the M-cells first traverses a group of interlacing nerve fibres, the so-called 'axon cap' (Bartelmez, 1915). It then crosses the midline, enters the spinal cord on the contralateral side as a myelinated giant nerve fibre and tapers towards its caudal end. Short collaterals project from the axon at regular intervals and contact interneurons and motoneurons. The medullary and spinals outputs of the M-cell have been well characterized electrophysiologically and morphologically (Hackett and Faber, 1983; Fetcho, 1991). Studies on Mauthner neurons have provided fundamental information on neuronal biochemistry, development, synaptic morphology and physiology, and control of behaviour than can be generalized to many central neurons throughout vertebrates (Faber and Korn, 1978; Nissanov and Eaton, 1989; Korn et al. , 1990). In fact, these identifiable pair of cells are the most studied in the central nervous system of vertebrates (Bullock, 1978). In fish, the shortest pathway for an escape in response to an acoustic input involves only four neurons the hair cells in the ear that connect to eighth nerve fibbers which in turn connect to the M-cell, which directly connects to motor neurons in spinal cord to activate the startle response. The speed of the pathways is further optimized by the presence of electrical synapses, which allow the direct current flow between one neuron and the next and reduce the delays associated with chemical neurotransmission. Finally, the startle circuits contain large, relatively fast conducting neurons in the pathways. These socalled giant neurons have relatively very large axons and, often, large cell bodies as well. Alberto Stefanelli’s publications on M-cells have helped establish these neurons as model system used to study the role of reticulospinal system in vertebrate motor control (Eaton et al. , 1981, Ward and Azizi, 2004) and the response of vertebrate central neurons to spinal cord injury (Zhang et al. , 2005). Stefanelli made many important contributions to neuroscience that include descriptions of the determination and differentiation of the M-cells in amphibian (Stefanelli, 1946; Stefanelli, 1951), axonal path finding of the Mauthner axon Material and Methods 22 Figure 3.1 a) Sea bream larva. b) Sea bass larva. c) Zebra fish larva. 3.1.3 Zebrafish Zebrafish ( Danio rerio ) larvae type Nacre (without pigments) (Figure 3.1c) were obtained from natural spawning from Department of Neurobiology and Behavior at Cornell University (Ithaca, USA) where these experients were carried out. Material and Methods 23 3.2 Feeding trials 3.2.1 Experimental conditions All tanks were supplied with sea water (about 37 ppm salinity) filtered by 50 μm mesh. Light intensity was kept at 1700 lux (digital Lux Tester YF1065, Powertech Rentals, Western Australia, Australia). Temperature and oxygen were daily measured by using an Oxy Guard-handy beta instrument (Zeigler Bros, Gardners, USA). Tanks were daily cleaned by hand between 18:00 and 20:00 pm with a hose by a siphon system. All tanks (170 L light grey colour cylinder fibreglass tanks) were supplied with filtered sea water previously stored in a 500 l tank for degasification. Tanks were subjected to an open circulation at different water flowing rates which were increasingly adjusted along the feeding trials. Water quality was daily tested and no deterioration was observed. Water was continuously aerated (125ml/min). Water temperature and dissolved oxygen were daily recorded at 15:00 (attaining 5-8 ppm) and saturation ranged between 60 and 80% in all experimental tanks. Photoperiod was kept at 12h light: 12h dark by fluorescent lights. 3.2.2 Growth determination Growth was determined by measuring dry body weight and total length of starved larvae. Whole body weight was determined by 4-3 replicates of 5-10 starved larvae washed with distilled water and dried in a glass slide at an oven at 110º C until constant weight, for approximately 24 h, followed by 1 h periods. Total or standard length of 20-30 anesthetised larvae from each tank were measured in a Profile Projector (V-12A Nikon, Nikon Co., Tokyo, Japan). Material and Methods 24 3.3 Diet and feed 3.3.1 Live food 3.3.1.1 Rotifers cultive The rotifer Brachionus plicatilis was used in larval rearing experiences; adult individuals had an average total length of 240μm, being classified as L morphotype. Rotifers production was carried out on cylinder conical fibre glass tanks with a total capacity of 1700 l filled with a mixture of fresh and seawater to attain a salinity of 25 ppt. Production cycles lasted 8 days. Initial rotifers density was 265 indv·ml-1; from day 4th of culture, harvest of 400 l volume was performed in alternative days, harvested volume being replaced by a mix of seawater and fresh water to reach a 25 ‰ salinity. The 8th day, the total volume was harvested and a new production cycle started. Routinely, after rotifers harvesting, a 1 min freshwater bath was applied to kill potential contaminants such as ciliates. Average total density and percentage of ovigerous females were daily calculated after individual counts (n=3) of 0.5 ml collected with a micropipette from a sample randomly collected from the culture tank. Similarly, oxygen and temperature were registered twice a day at 9:00 and 15:00 hours by means of a portable probe (Mod. Handy Polaris, OxyGuard; Birkerød, Denmark). Rotifers feeding consisted in lyophilized baker yeast ( Saccharomyces cerevisiae ) supplied at a dose of 0.4 g /106 rotifers. The first day of culture yeast was supplemented with lyophilized microalgae 0.1g /106 rotifers. Food was added manually at 09:00 and 15:00 hours, and automatically at 21h00 and 3h00. 3.3.1.2 Rotifers enrichment Rotifer Brachionus plicatilis reared under the, previously described protocols are deficient in n-3 LCPUFA and other nutrients, needing to be enriched previously to be fed to the larvae. Enrichment was performed in cylinder-conical fibre glass tanks 500 l capacity. Rotifers concentration was generally high (over 400 rotifers·ml-1), and an air diffuser was settled 15 cm from the tank bottom centre, to prevent sedimentation and to maintain the Material and Methods 25 oxygen levels. Along the different trials, rotifers enrichment with commercial or experimental products was performed. Generally, different enrichment products were used following manufacturer instructions, including 6 h enrichment time before harvesting. The enrichment product was spread in two separate doses (0h and 3 h). Thus, two tanks were daily used for rotifers enrichment; the first’s one was harvested at 8h30 and fed to the larvae. In this case the enrichment product was automatically added at 2h00 and 5h00. The second enrichment tank was harvested at 14h00, and the enrichment product was manually supplied at 8h00 and 11h00. After rotifers enrichment, they were harvested and cleaned to eliminate oil emulsions particles, concentrated in a 20 l basket and kept with air supply until larval feeding. A 5 ml sample was taken, diluted in 250 ml, homogenized and counted. Once calculated the needs for each larval tank, rotifer were fed to the larvae and the remaining rotifers discarded. Along the whole experimental period, different samples of the products and rotifers used were stored at -80ºC to study the biochemical composition and fatty acid profile. 3.3.1.3 Enrichment Emulsions were prepared with 2 g of oil, 5 g of soybean lecithin and 400 ml fresh water mixed in a blender for 2 min. This emulsion was added to the 30 l of rotifer culture at a concentration of 250 rotifers/ml during 12 h. After rotifers enrichment, they were harvested and cleaned to eliminate oil emulsions particles, concentrated in a 10 l basket and kept with air supply until larval feeding. A 5 ml sample was taken, diluted in 250 ml, homogenized and counted. Once calculated the needs for each larval tank, rotifer were fed to the larvae and the remaining rotifers discarded. Along the whole experimental period, different samples of the products and rotifers used were stored at - 80ºC to study the biochemical composition and fatty acid profile. 3.3.1.4 Brine shrimp cultive Nauplii of the brine shrimp Artemia constitute one of the basic foods items for marine larval rearing. For all the larval rearing experiences Artemia nauplii from two different origin were used, Artemia franciscana (AF Type; Material and Methods 26 INVE; Dendermode, Belgium) and Artemia salina (EG Type; INVE, Dendermode, Belgium). The decapsulation process to eliminate the external corion and to reduce the risk of external pathogens introduction was performed for all the Artemia batches. 3.3.1.5 Enrichment To improve nutritional quality of Artemia , an enrichment procedure was necessary. For this purpose, cylinder-conical fibre glass tanks of 1700 l total volume were employed. Strong aeration from the tank bottom and 24 h photoperiod were applied. Besides, an internal heater was used to maintain a culture temperature of 25-26º C. Just hatched out Artemia nauplii were introduced in the enrichment tank at a concentration of 250,000-300,000 nauplii·l-1. In most of the experimental trials the Easy DHA Selco (Inve, Dendermonde, Belgium) enrichment product was used. The enrichment last for 18-24 h with a concentration 0.6 gr·l-1, added in a single dose at the beginning of the enrichment process (h=0). Enriched nauplii were harvested in a sock net of 125 μm mesh size, washed with seawater to eliminate residual lipid particles and concentrated in a 20 l beaker to be counted and check the enrichment success. Enriched nauplii were added to the larval tank manually. 3.3.2 Microdiets Several isonitrogenous and isolipidic experimental microdiets (pellet size < 250 μm) having differing ratios of DHA, EPA were formulated in the different experiments using: EPA50 oil and DHA50 (CRODA, East Yorkshire, England, UK) oil in a triglycerides form as an sources of EPA and DHA. Vegetable oils were used in one of the experiments: fish oil (FO), soybean oil (SBO), lindseed oil (LSO) and rapeseed oil (RSO) (vegetal oils used were commercial food grade oil, Agroalimentary Commercial Oils; and FO was sardine oil, Croda, East Yorkshire, UK). The desired lipid content was completed with a non-essential fatty acid source, oleic acid (Merck, Darmstadt, Germany). The protein source used was squid meal (Riber & Son, Bergen, Norway) which was defatted (3 consecutive times with a chloroform:meal ratio of 3:1) to allow a better control of the microdiet fatty acid profile, except in one specific experiment. The Material and Methods 27 defatted squid meal (2.6% lipid content), DHA50 and EPA50 fatty acids profiles are shown in Table 3.1. 3.3.2.1 Microdiets preparation The microdiet was prepared in the following manner: the squid powder was carefully mixed with the other hidrosoluble ingredients (attractants, minerals and hidrosoluble vitamins, Sigma-Aldrich, Madrid, Spain) in a mortar. In a separated mixture, oils and fat-soluble vitamins were combined to obtain a homogeneous mix which was afterwards put together with the powder mixture. Then, gelatin was dissolved in warm water and when its temperature was lower than 35º C, it was added to the rest of the previously mixed ingredients. The paste was then compressed pelleted (Severin, Suderm, Germany) and dried in an oven at 38º C for 24 h (Ako, Barcelona, Spain). Pellets were ground (Braun, Kronberg, Germany) and sieved (Filtra, Barcelona, Spain) to obtain the desired particle size. Diets were analyzed for proximote and fatty acid composition and each diet was tested in triplicate. Material and Methods 28 Table 3.1 Main fatty acids of the major dietary lipid sources and deffated squid meal used in this thesis. n.d.≤0.005 Fames DHA50 EPA50 Defatted squid meal 14:0 0.03 0.04 1.68 14:1n-5 0.20 0.24 0.04 14:1n-7 n.d. 0.01 0.04 15:0 n.d. 0.01 n.d. 15:1n-5 n.d. n.d. 0.04 16:0iso 0.01 0.02 0.11 16:0 1.21 0.36 23.80 16:1n-9 n.d. n.d. 0.02 16:1n-7 0.47 0.43 0.52 Me 16:0 0.01 0.01 0.10 16:1n-5 0.02 n.d. 0.16 16:2n-6 n.d. 0.05 0.06 16:2n-4 0.16 0.49 0.48 17:0 0.12 n.d. 0.06 16:3n-4 0.10 0.21 0.07 16:3n-1 0.06 0.14 0.12 16:4n-3 n.d. n.d. 0.38 16:4n-1 n.d. 0.33 0.03 18:0 2.32 0.27 3.64 18:1n-9 4.92 1.81 1.73 18:1n-7 0.87 0.55 0.94 18:1n-5 0.04 n.d. 0.24 18:2n-9 n.d. 0.44 n.d. 18:2n-6 0.59 2.16 0.24 18:2n-4 0.07 0.68 n.d. 18:3n-6 0.34 0.73 n.d. 18:3n-4 0.04 0.44 n.d. 18:3n-3 0.20 1.67 n.d. 18:3n-1 n.d. 0.32 n.d. 18:4n-3 0.31 10.44 0.07 18:4n-1 n.d. 1.01 n.d. 20:0 0.73 0.42 n.d. 20:1n9+n7 3.98 0.39 2.80 20:1n-5 n.d. n.d. n.d. 20:2n-9 0.07 0.41 n.d. 20:2n-6 0.54 0.13 0.12 20:3n-9 +7 0.21 0.72 n.d. 20:3n-6 n.d. n.d. n.d. 20:4n-6 2.46 3.49 0.49 20:3n-3 0.44 0.11 0.34 20:4n-3 0.92 2.49 0.06 20:5n-3 12.66 46.48 4.31 22:1n-11 2.29 0.31 0.04 22:1n-9 1.21 0.74 0.11 22:4n-6 2.84 0.49 0.13 22:5n-6 0.19 n.d n.d. 22:5n-3 5.53 3.21 n.d. 22:6n-3 53.80 17.72 9.40 Material and Methods 29 3.4 Behavioural studies in sea bream 3.4.1 Material The preparation is very similar in both stimuli. We used a 500 ml glass beaker (beaker experiment) with a seawater depth of 4 cm. Under the glass a chart paper was placed. The answers to each type of stimulus individual larvae were tested, using 10 larvae of each tank. Each larva was transferred from the feeding tanks to the experimental beaker and then video-recorded using a Sony digital video camera DCR-TRV27. The acclimation time for each larva in the glass beaker was 2 minutes. Different tests were done to determine this time. After recording for 90 s without disturbance, larva was scared by sound and visual stimuli to introduce a startle response. Stimuli were provided three times at 10 s intervals for each larva. To determinate the effects of diet on larval behaviour, different trials were performed throughout the experiments. In tests on day 1 after hatching (dah) and up to 6 dah larvae could scarcely been distinguish clearly in the digital camera and visual analysis had to be applied. In experiment 1, the escape behaviour was quantifed at 6, 10, 16 and 19 dah; in experiment 2 at 23, 27 and 34 dah; and in experiment 3 at 22, 25, 29 and 32 dah. These data were used for monitoring throughout the period of larval development and to acquire fluency in handling such sensitive animals with these techniques. Each larva used in the experiments was measured by a profile projector (V - 12ª Nikon, Tokyo, Japan). 3.4.2 Stimuli 3.4.2.1 Sonorous stimuli Consistent sound stimuli were produced using a steal nut (≈10 g) hung by a string (26 cm) that was released from a distance of 18 cm from the beaker wall (Figure 3.2). This beaker was covered with a black vinyl sheet only when sound stimuli was applied. The pendulum was located at a distance of 8 cm from beaker glass. Using a tape measure, the nut was carried at a distance Material and Methods 30 of 10 cm from the same pendulum, (the nut would be at a distance of 18 cm from the experimental beaker) and was launched without using force, just dropping it. The distance had to be very precise since an error of one centimeter may affect the performance parameters evaluation. Figure 3.2 Application of the sonorous stimuli. 3.4.2.2 Visual stimuli A flash of white light to simulate natural conditions was used as visual stimulus. The flash was located at 10 cm from the experimental beaker (Figure 3.3). Figure 3.3 Application of the visual stimuli. Material and Methods 31 3.4.3 Parameters As mentioned above, each larva was supplied three times the same stimulus with intervals of 10 s. In order to know if the larvae responds or not, that is to say, if the response is positive or negative, we had to observe with patience and dertermination because in the earliest days of sampling, the larvae could barely be clearly seen. The analysis began with the viewing of all the images to get an idea of which day the larvae will begin to react in front of the stumulus. 3.4.3.1 Cruise Swimming Speed Cruise swimming speed is defined as the natural larvae movement just before application of the stimulus (Masuda et al ., 2002). Video analysis was conducted frame by frame to calculate cruise swimming speed and burst swimming speed. Cruise swimming speed estimation was based on the 10 s video recording starting 30 s after the recording began. The movement of the fish was traced on an overhead projector transparency sheet, and this distance was divided by the time taken (10 s), obtaining the cruise swimming speed. The units used were mm/s and SL/s (SL, standard length), it cruise swimming speed was divided by the SL of each individual (Masuda et al. , 2002). This analysis was conducted only in larvae that had movement for both, the sound and the visual stimulus. 3.4.3.2 Burst Swimming Speed Burst swimming speed was calculated only when fish showed an obvious startle response. This obvious response could be a change of direction for visual and sonorus stimuli, and/or panic type for visual stimuli. After each stimulus, larval movement was traced for four consecutive frames, and the distance was divided by the time taken (4/30 s). Preliminary observations revealed that the faster movement appeared in any of the first four frames after providing a stimulus. Burst swimming speed was calculated as the average of the movement of four frames and defines it as the burst swimming speed. Material and Methods 38 3.7.2.1 Preparation of cuts Paraffin blocks were cut in the microtome (Mod. Jung Autocut 2055; Leica, Nussloch, Germany). Initially, gross cuts of 20-25 μm were performed until larvae tissues were reached and later definitive cuts of 3 μm were conducted. The cut series were introduced in a distilled water bath at 45-50º C. Brain tissue sections slides for immunohistochemistry studies were prepared with glue to increase the fixation of the tissues, preventing them off Poly-L (Sigma ®) (Annexe I) was applied in the slide. 3.7.2.2 Histological stains 3.7.2.2.1 Hematoxylin and eosin It is one of the most common techniques in histology laboratories. The aim of this technique was created to obtain a contrast between different parts of the cell for a proper assessment of different tissues under the microscope, using the different affinity of the tissues to absorb dyes. In this technique the dyes used were: - Hematoxylin: blue-purple coloration. Stains basic structures. - Eosin: Red-pink coloration. Stains acid structures. The samples were first dried in an oven (1h at 60º C). Then, samples were introduced in a xylol bath and re-hydrated in alcohol series of different gradation, finalizing with water: Xylene 2 min Xylene 2 min Alcohol 100º 2 min Alcohol 100º 2 min Alcohol 70° 2 min Distilled water 2 min Distilled water 2 min Distilled water 2 min Material and Methods 39 Then, the staining battery starts following the protocol modified from Matorja and Matorja-Pierson (1970) and Garcia del Moral (1993): Harris hematoxylin 15 min Rinse quickly in water Alcohol hydrochloric acid 4 quick passes Rinse quickly in water Ammonia water 15 passes Water 15 min Eosin 4 min Once staining was end, samples were dehydrated and clarified: Alcohol 96° 2 min Alcohol 96° 2 min Alcohol 100º 2 min Alcohol 100º 2 min Dehydration Xylene 2 min Xylene 2 min Xylene 2 min Clarification The preparations were mounted using DPX medium. 3.7.2.2.2 Nissl staining This method is used for the detection of Nissl body in the cytoplasm of neurons on paraformaldehyde or formalin-fixed, paraffin embedded tissue sections (Martoja and Martoja-Pierson, 1970). The Nissl body will be stained in purple-blue. This stain is commonly used for indentification of the basic neuronal structure in brain and spinal cord tissue. Material and Methods 40 Solutions a) Acetate buffer a. 2.71% sodium acetate. b. 1.20% acetic acetate. b) Cresyl extra solution a. Violet or thionin extra cresyl 0.25 g. b. Acetate buffer at pH = 3.8-4 100 ml. Procedure 1. Dehydrate and hydrate the samples. 2. Stain in cresyl violet solution for 10 min. 3. Rinse in acetate buffer. 4. Dehydrate, clear in xylene (no time) and mount with permanet mounting medium. 3.7.2.3 Immunohistochemical techniques To demonstrate the presence of different markers in the M-cells of fish larvae, we applied the avidin-biotin-peroxidase (ABC) immunohistochemical and immunofluorescence in method for simple markings. Immunohistochemistry is based on the application of a specific primary antibody in the tissues, usually created in rabbits (polyclonal) or hybridoma established in mouse spleen (monoclonal). The antibody is specific for the protein molecule studied. The method followed is described in the next paragraphs: 1. A blockade of endogenous peroxidase to avoid false positives. 2. An antigen unmasking process, used only when necessary to break the bonds created by formalin fixation. 3. Normal serum blocking. Normal serum should be long to the same species of which the secondary antibody is raised. Subsequently, a secondary serum is applied after the primary, serving as a Material and Methods 41 bridge between this and the following reaction amplifier complex called avidinbiotin-peroxidase (ABC). The development process can be carried out using two different chromogens, commonly called "red" (AEC, 3-amino-9 ethylcarbazole) or "brown " (3-3-diaminobenzidine) as the positive staining. Protocol Each antibody was tested for versioning operation and it is subject to variations to determine the concentrations and types of unmasking epitopes that maximize the positive. The antibodies tested were parvalbumin (PV) and calretinin (CR), both polyclonal (Table 3.3). Table 3.3 Antibodies tested in immunohistochemistry technique. Antibody Dilution Time to detection Company PV 1:700 10 min Swant CR 1:700 10 min Swant The steps of the ABC method protocol are as follows: 1. The samples were dried in an oven for at least 24 h at 37° C. 2. Wash 10 min and 2x5 min with xylene. 3. Wash 5 min in alcohol at 100º. 4. Inhibition of endogenous peroxidase by a solution of hydrogen peroxide 3% in methanol during 30 min with agitation. 5. Wash 5 min with alcohol at 100º. 6. Wash 5 min with alcohol at 96º. 7. Wash 5 with alcohol at 70º. 8. Wash 2x5 min with distilled water. Material and Methods 42 9. Wash 5 min with PBS (phosphate bufferd saline) in agitation. 10. Unmasking antigen epitopes using the enzyme treatment. This treatment involves applying a solution of the enzyme pronase in a proportion of 0.1 g per 100 ml of PBS buffer at room temperature for 7 min. 11. Wash 3x5 min with PBS buffer. 12. Application of normal goat serum for the following antibodies: parvalbumin and calretinin. Incubation in a humid chamber at room temperature with 10% normal goat serum (Vector ®) in PBS for 30 min. 13. Primary antibody: Incubate sections with primary antibodies. The antibodies were diluted in 1% normal goat serum in PBS. This process was conducted in a moist chamber at 4° C during 18 h. Tests were conducted with different concentrations of antibody to obtain the optimal dilution. 14. Remove the moist chamber of the refrigerator an hour before preceding the next steps. 15. Wash 3x5 min with PBS. 16. Secondary antibody: Incubation with secondary antibody diluted created in pig and normal rabbit serum 1% in PBS (concentration 1:250) for parvalbumin and rabbit anti-mouse calretinin (1:250). This process is conducted in a moist chamber at room temperature for 30 min. Note: Longer incubation time may produce higher background staining. 17. Wash 3x5 min with PBS. Note: Slides should be protected from light starting from this step until the end by covering slides with aluminum foil or a black box. Material and Methods 43 18. Incubation with streptavidin-biotin-peroxidase (ABC) (Dako ®) in a humid chamber in darkness room during 1h at room temperature. 19. Wash 2x5 min with PBS. 20. Wash 1x10 min with Tris (1 part Tris mother /9 parts 0.85% saline). 21. Reaction revealed by immersion in diaminobenzidine (DAB) prepared by dissolving with a stirrer 0.07 g DAB in 200 ml of Tris buffer. Dual role is filtered in a darkroom and 200 ul of hydrogen peroxide at 30% was added. Is revealed during approximately 2 min observing the positive control. 22. Wash 1x10 min with water. 23. Counterstain with Harris hematoxylin during 30 s. 24. Wash 1x10 min with water. 25. Dehydrated in alcohols and xylene (2 min in each one). 26. Coverslip with DPX mounting medium. 3.7.3 Preparation of samples for cryostat These techniques were learned at the Institute of Neurosciences of Castilla y León (INCYL) in Salamanca (Spain). Larvae were deeply anesthetized using an aqueous solution MS-222 and fixed in 1% (w./v.) paraformaldehyde and 15% (w./v.) picric acid in 0.1 M PBS, pH 7.4 during 4-6 hours (depending on the size of the sample) at room temperature. Some tests were performed using larvae fixed in 10% buffered formalin to improve the cut quality. Then, samples were washed 3 times with PBS during 5 min. Larvae heads were keeped in a freezer solution (Annexe I) at -20º C. After rinsing in PBS, tissue was cryoprotected with 30% (w./v.) sucrose in PBS during 12 hours at room temperature and embedded in a medium consisting of agar to 1.5% and 5% sucrose (w./v.). In each block was placed a single fish head and was oriented to have a horizontal cutting angle. Once cryoprotected, blocks were included in OCT 4583 (Middle of inclusion, Tissue-Tek, Miles), frozen with liquid nitrogen Material and Methods 44 and cut in cryostat at a temperature of -24º C. Gelatin-embedded larva blocks (Annexe I) were serially cut on Slee Mainz cryostat at 10 m. For immunofluorescence studies 30 larvae were collected and fixed in 10% buffered formalin. Each larva was included in a gelatin block horizontally orientated to obtain a better visualization of neuronal structures. Hematoxylineosin stains were tested to verify that sections right before the relevant evidence of study. Hematoxylin-eosin 1. Wash 3x15 min with PBS. 2. Wash 3x15 min with distilled water. 3. Hematoxylin: 1 min and 30 sec. 4. Wash in water during 30 min. 5. Wash in distilled water. 6. Dehydrated in alcohols and xylene (three steps of 5 min each). 7. Coverslip with DPX mounting medium. 3.7.3.1 Immunofluorescence This technique was setup in collaboration with the INCYL, which hosted a research visit. Subsequently, the samples of this thesis were processed in the IUSA. The sections previously cut in cryostat, were defrosted at room temperature during 1 h and washed in PBS to rehydrate the tissue, 3x10 min. This was followed by a preincubation with non-immune rabbit serum 5% (Sigma) and Triton X-100 (Probus SA) 0.2% in PB for 1 h at room temperature (species same as secondary antibody). With this procedure we get a better penetration of antibodies and also reduce nonspecific binding of the same tissue. Then sections were incubated at an appropiate dilution in primary antibody anti-Choline acetyltransferase (ChAT) (Table 3.4) in a humid chamber for 2 days at 4º C. After incubation sections were rinsed with PBS. Material and Methods 45 Table 3.4 Primary antibody used in inmunofluorescence. Antibody Dilution Company ChAt 1:500 Millipore After that, sections were incubated with the corresponding fluorescent secondary antibody anti-goat IgG (whole-molecule)_FITC (antibody made in rabbit, Sigma ®) diluted 1:250 for 1 h at room temperature in darkness. To mark the nuclei of all cells, incubation for 10 min in the dark with propidium iodide (PI, Sigma ®) (1:2000) in PB was performed. At the end, sections were washed with PB and mounted in a commercial medium Fluoromont ® (Sigma ®). To obtain and quantify the intensity of fluorescence, a Zeiss confocal microscope model LSM 510 of Department of Biochemistry (ULPGC) was used. To quantify the intensity of the sample a LSM 510 program was used by the next parameters: detector gain, amplifier offset and amplifier gain. 3.7.4 Processing and inclusion in resin This section describes the procedures for cutting and staining of semithin and ultrathin sections of resin-embedded samples. Larvae were anesthetized with MS-222 and fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH = 7.2) (Annexe I) for 1 h and stored in cacodylate buffer at 4ºC (Bancroft and Stevens, 1996). Samples were analyzed in the histological laboratory in IUSA. For this, samples were washed with washing liquid (Annexe I) for 24 h at 4º C. After this period, tissues were postfixed in 2% osmium tetroxide diluted in 1.5% ferrocyanide buffer (Annexe I) for 2 h. Then samples were dehydrated with acetone as follows: 30% acetone 10 min 50% acetone 10 min 70% acetone 10 min 80% acetone 10 min 90% acetone 10 min Material and Methods 46 Absolute acetone 10 min Then samples using were incubated in Durcupan ACM Fluka resin (Analitikal Fluka, Buchs, Switzerland): Resin/acetone; ½ (45 min or overnight) at 4° C Resin/acetone; 1/1 (2 h or overnight) at 4° C Resin/acetone; 2/1 (8 h or overnight) at 4° C Pure resin overnight at 4° C Afterward, samples were placed and oriented in the blocks with pure resin and kept overnight in an oven at 60º C. 3.7.4.1 Semithin sections The inclusion of samples in resin allows the production of sections much thinner than with paraffin. Although semithin sections were used as to select regions for further for transmission electron microscopy to obtained a higher resolution. Sections cut at 1 μm were floated in a water trough attached to the glass knife, and picked up with a shaved applicator stick. The sections were transferred to a drop of water on a clean microscope slide. The slide with floating sections was then placed on a hot plate prewarmed at 60° C. After few minutes, as the liquid warms, the sections spread and adhered to the slide. Without removing the slide from a hot plate, filtered toluidine blue in 1% sodium tetraborate is dropped on the sections. The slide was then heated on a hot plate at 60° C for 30 sec. After staining, rinse slide carefully with distilled water from wash bottle. The drain trained slide was blotted dry and mounted in DPX. The semi-thin section was then examined at the light microscope. Material and Methods 47 Procedure a. Block face was trimmed to approximately 1 mm2 and cutted off one corner so that orientation can be determined for later re-trimming. b. Semithin sections (approximately 0.5 μm thick) were cutted with a glass knife. A drop of distilled water was approximately placed at 1 cm in diameter on a clean glass slide. The sections were picked up with a wire loop. The loop was then invert to place 2-4 sections into the drop of water on the slide. c. The slide was label with the block number. d. The slide was put with the water droplet containing sections on a hot plate adjusted to approximately 60º C and wait until all the water evaporates. e. The sections were located on the slide and circle the bottom of the slide with a felt-tip marker to indicate their position. f. Few drops of 1% toluidine blue O in 1% sodium tetraborate were placed into the sections and the slide located on the hot plate. After the edges of the drop of stain began to turn golden (approximately 20-30 sec), the sections were quickly rinsed with a stream of distilled water to wash off the excess stain. Even if the stain dried down completely, the sections were useable. When washing, the stream was directed slightly above the sections with the slide tilted at about a 45º angle above a catch basin for the excess stain. When the stream was pointed directly at the sections, they may come off the slide from the water pressure. If the sections came off the slide too easily when washing, they were too thick or the hot plate temperature is too low. The washed slide was then replaced on the hot plate after gently drying the bottom of the slide with a piece of paper towel and wait for the residual water to evaporate from the slide surface. g. The slide was removed from the hot plate and add 1-2 drops of polymount Study I Dietary DHA deficiency induce a reduced response in gilthead seabream ( Spaurs aurata ) larvae This work has been published in: Benítez-Santana, T., Masuda, R., Juárez Carrillo, E., Ganuza, E., Valencia, A., Hernandéz-Cruz, C.M. and Izquierdo, M.S. Aquaculture (2007) 408-417. Abstract Developmental changes of swimming speed were analysed in the seabream ( Sparus aurata ). Four feeding regimes using live preys (rotifer Brachionus plicatilis ) enriched with fish oil, soybean oil, linseed oil and rapseed oil, differing in fatty acid profile were tested during the first weeks of larval life. There was an increase in burst swimming speed and cruise swimming speed during the visual stimulus experiment at day 16th of life in the present study in agreement with the better eye development in larvae of this age. Swimming activity before stimulus was significantly reduced rotifers enriched with vegetable oils. Larvae fed with rotifers enriched with fish oil reacted with a higher burst speed after a visual stimulus than after the sound stimulus (159.5 SL/s vs. 18.30 SL/s) denoting the importance of the vision during the period of the development not only for the predation but also for the burst. The reduction in dietary essential fatty acid contents, by the enrichment with vegetable oils, delays the appearance of response to visual stimulus, an agreement with the minor DHA content in eyes and brains of these larvae and suggesting a delay in the functional development of brain and vision. The results suggest that DHAdeficient fish show relatively slow burst swimming speed when visual stimuli were provided compared to those stimulated by sound stimuli. Keywords: Seabream larvae, essential fatty acids, behaviour, visual stimulus, sound stimulus, DHA, EPA, cruise swimming speed, burst swimming speed. Study 1 54 Introduction Sparus aurata is one of the most important products of the European aquaculture. Despite the good knowledge on the biology of this species, there is a lack of studies in areas like behaviour in intensive rearing. Description of the normal pattern of behaviour in fish larvae constitutes a powerful tool to study larval development since delays in the appearance of those patterns or deviations from the typical conduct in certain individuals or patches of larvae may constitute an effective non-invasive indicator of health, development and maturity of these fish. For instance, schooling behaviour is crucial to understand the great fluctuations from year to year which occur in wild fish stocks and hence numerous studies have focused the schooling behaviour of juvenile and adult fish (Pincher and Hart, 1982). However, only few studies focus on developmental aspects of behaviour in larvae. Despite numerous factors affect behavioural development, certain types of behaviour, such as schooling, seem to rely more on the proper development of central nervous system than in other causes such as alterations of sensorial organs or swimming capacity (Masuda and Tsukamoto, 1998). In turn deficiencies in certain nutrients, such as essentials fatty acids, markedly affect the normal development of the brain (Masuda et al. , 1999). Three very long chain polyunsaturated fatty acids, namely docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6) have a variety of very important functions in fish species, as in most vertebrates. Inadequate contents of those dietary essential fatty acids (EFA) give rise to several alterations such as poor feeding and swimming activities, poor growth and dropping mortality, fatty livers, abnormal pigmentation, disgregation of gill epithelia, immune-deficiency and raised basal cortisol levels (Izquierdo, 1996). Besides, an inappropriate dietary content of such fatty acids in diets for broodstock reduces fecundity and fertilization rates, originate embryo deformities and damage larval quality. In gilthead seabream larvae, DHA, EPA and in some extend ARA, have been also shown to be determinant of growth and survival performance (Izquierdo et al. , 2000, 2001). Thus, increasing dietary EFA either in live food or in microdiets Study 1 55 improves larval growth, survival and stress resistance (Koven et al. , 1990; Rodríguez et al. , 1994; Watanabe and Kiron, 1994; Izquierdo, 1996; Bessonart, 1997; Salhi, 1997; Sargent et al. , 1999). EFA, particularly DHA, are also necessary for the normal development of nervous system and sensory organs, larval eye and brain fatty acid composition clearly reflecting that of the diet (Navarro et al. , 1995). Despite variations in the dietary level of such fatty acids would markedly affect behaviour, few studies have been conducted to elucidate the effect of EFA on larval behaviour. For instance, in yellowtail larvae ( Seriola quinqueradiata ) dietary DHA has been shown to affect ontogeny of schooling behaviour as well as brain development (Ishizaki et al. , 2001). Since very few studies have been aimed to determine the ontogeny of behaviour in gilthead seabream, the effect of dietary fatty acids on behaviour of this larva is also unknown. At present, a stable high quality juveniles production is required to satisfy the constant increase in the production of gilthead seabream, a major species in Mediterranean aquaculture. Hence, finding of early noninvasive quality indicators such as behavioural patterns is of primary importance for commercial hatcheries. However, ontogeny of behaviour and its dependence on feeding is poorly understood in gilthead seabream. This study aimed to determine the parallelism between behavioural responses to different stimulus along the larval gilthead seabream development and the effect of distinct feeding regimes. Materials and Methods S. aurata eggs were obtained from natural spawning from broodstock of the ICCM (Instituto Canario de Ciencias Marinas, Las Palmas de Gran Canaria) and were distributed into sixteen 170 L fibreglass cylindrical tanks (100 eggs/L) filled with 50 μm filtered sea water at 21.28 °C±0.44 provided with constant aeration and water flow (0.5 L/min). From first feeding to day 4, water was stagnant, adding new water just to keep the water quality. Photoperiod of 12 h artificial light was kept constant during the experimental Study 1 56 period and no microalgae were added to the rearing tanks to obtain a better control of the EFA consumed by the larvae. From day 4th after hatching, larvae were fed with rotifers ( Brachionus plicatilis ) twice a day (at 900 h and 1500 h) for the following 20 days. In order to see the effect of different dietary fatty acids profiles, four types of rotifers were fed: “FO rotifers” enriched with fish oil, “SBO rotifers” enriched with soybean oil, “LSO rotifers” enriched with linseed oil and “RSO rotifers” enriched with rapeseed oil. Each type of rotifers was tested in four larval rearing tanks. Emulsions were prepared with 2 g of oil, 5 g of soybean lecithin and 400 ml fresh water mixed in a blender for 2 min. This emulsion was added to the 30 L of rotifer culture at a concentration of 250 rotifers/ml during 12 h. Rotifers concentrations in the larval tanks were kept at 5, 7, and 10 rotifers/ml until days 8, 15 and afterwards, respectively. Samples of rotifers were taken three times along the experimental period and stored at −80 °C until lipid analysis. Larval growth was assessed by determination of larval standard length at 1, 6, 10, 16 and 19 days after hatching by a profile projector (Nikon V-12A, Nikon, Tokyo, Japan). At day 10, larvae from one tank of each diet were sacrificed for analysis of their total lipid content and fatty acid composition. At the end of the experimental period, one hundred larvae were separated for eyes and brain dissection, whereas the remaining alive larvae were stored at −80 °C until lipid analysis. Methyl esters of fatty acids were obtained by transesterification with 1% sulfuric acid and methanol using heneicosanoic acid (10% of total lipids) as an internal standard. The fatty acid methyl esters obtained were separated by gas chromatography (ShimadzuGC-14 a, Kyoto, Japan) run at the operating conditions described previously by Izquierdo and Gil (1998), quantified by flame ionisation detectors (FID) and identified by comparison to well characterized external standards. Swimming speed of larvae from all groups was determined at 6, 10, 16 and 19 days after hatching, respectively, in a 1 L glass beaker (10 cm in diameter) with a water depth of 4 cm. This beaker was covered with a black vinyl sheet only when sound stimuli was applied. Each larva was transferred Study 1 57 from the feeding tanks to the experimental beaker and then video-recorded using a Sony digital video camera DCR-TRV27. After recording for 90 s without disturbance in order to determine cruise swimming speed, larvae was scared by sound and visual stimuli to introduce a startle response and determine cruise swimming speed. Consistent sound stimuli were produced using a steal nut (≈10 g) hung by a string (26 cm) that was released from a distance of 18 cm from the beaker wall. Sound stimuli were provided three times at 10 s intervals for each larva. Visual stimuli were produced using a flash from a distance of 10 cm from the beaker wall. After this operation, larvae standard length (SL) was measured by a profile projector (Nikon V-12A, Nikon, Tokyo, Japan). This procedure was repeated using 5 individuals of each rearing tank (Masuda et al. , 2002) for each stimulus. Video analysis was conducted frame by frame to calculate cruise swimming speed recording and burst swimming speed. Cruise swimming speed estimation was based on the 10 s video recording from 30 s after the recording was started. The movement of the fish was traced on an overhead projector transparency sheet, and this distance was divided by the time taken (10 s) obtaining the cruise swimming speed. To observe the response development to both stimuli, burst swimming rate was calculated by dividing the number of responses by the number of trials. Burst swimming speed was calculated only when fish showed an obvious startle response. After each stimulus, larval movement was traced for four consecutive frames, and the distance was divided by the time taken (4/30 s). Preliminary observations revealed that the faster movement appeared in any of the first four frames after providing a stimulus. Burst swimming speed was calculated as the average of the movement of four frames and defines it as the burst swimming speed. Both cruise swimming and burst swimming speeds were divided by the SL of each individual (Masuda et al. , 2002). Data were statistically analyzed with the software STATGRAPHICS PLUS for Windows 3.1 (Statistical Graphics Corp., Englewood Cliffs, NJ, USA) using one way analysis of variance (ANOVA) and Duncan test (P<0.05) for multiple comparison of means was applied. Study 1 58 Results Fatty acid analysis of prey, fish whole body, and the brain and eyes of experimental fish Analysis of the fatty acid composition of oils and rotifers showed that the fish oil used contained 17.11% n-3 HUFA as % of total fatty acids, with an EPA (20:5n-3)/ DHA (22:n-3) ratio of 1.03/1 (Table 4.1). This oil also showed the highest content of saturated fatty acids and was rich in 22:1n-11. Soybean oil was rich in fatty acids of the n-6 series, particularly 18:2n-6 and in a lesser extent oleic acid (18:1n-9), whereas linseed oil showed the greatest proportion of n-3 fatty acids, mainly linolenic acid (18:3n-3). Finally, rapeseed oil was characterized by a high proportion of n-9 fatty acids due to the high content in oleic acid. As expected, rotifers enriched with these oils reflected their particular enrichment oil fatty acid composition (Table 4.1). Hence, rotifers fed with vegetable oils showed a lower n-3 HUFA content than fish oil enriched ones. Although the highest DHA content was found in FO rotifers, EPA/DHA ratios were highest (1.2) for rotifers fed fish oil and rapeseed oil, and only 0.4 and 0.5 for rotifers fed soybean or linseed oils. Linoleic acid was high in all enriched rotifers, reflecting the use of soybean lecithin as an emulsifier, but it was about 50% higher in rotifers enriched with soybean and rapeseed oils. Regarding larval composition (Table 4.2), at the beginning the exogenous phase (3 day-old initial larvae), the main fatty acids of total lipids from larvae were 22:6n-3>16:0>18:1n-9>18:2n-6>16:1n-7>18:0>20:5n-3. Besides, EPA/DHA ratio in larval total lipids was very low (1/5.04). Larvae fed rotifers enriched with vegetable oils progressively reduced the n-3 HUFA proportion (both EPA and DHA in the same proportion), except for larvae fed FO rotifers. Hence, in FO larvae a slight reduction in n-3 HUFA proportion was found at day 10, followed by an increase in 20 d larvae, particularly due to the EPA increase. Nevertheless, the similar DHA contents between larvae fed rotifers enriched FO and the initial ones suggested the adequate level of this fatty acid in such rotifers to cover EFA requirements of seabream larvae. Compared to FO larvae, larvae fed rotifers enriched with vegetable oils showed lower proportion 22:1n-11, a fatty acid particularly rich in FO. Besides, higher Study 1 59 contents of certain Δ6 desaturase products such as 18:3n-9 in SBO larvae and 18:3n-6 in SBO and LSO were found, despite they were not detected in the rotifers. Table 4.3 shows the results of the lipid analyses for the determination of total lipids of brain and eye fatty acids in larvae of 20 days fed with the different diets. In FO larvae EPA/DHA ratio in eye samples was lower than in larval total lipids. Saturated fatty acids proportion was also higher in brain and eyes than in larval total lipids, although in eyes of SBO larvae the proportion was lower than in the rest of larvae. Both tissues fatty acids composition reflected to some extend that of the fed rotifers. Hence, linoleic acid was highly incorporated into brain and, in a higher extend, eye lipids of larvae fed rotifers enriched with rapeseed and, particularly, soybean oils. Linolenic acid was only slightly higher in brain and eyes of LSO larvae. Docosahexaenoic and, particularly, eicosapentaenoic acids were markedly reduced in larvae fed rotifers enriched with vegetable oils. Compared to FO larvae higher 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-3, 22:5n-3 and 22:6n-3 contents in eyes of larvae fed with rotifers enriched with linseed oil, rich in linolenic acid (18:3n-3) but low in those very long chain fatty acids was found. Study 1 60 Table 4.1 Some fatty acids contents in total lipids from oils and enriched rotifers used to feed gilthead seabream larvae (% total determined fatty acids, n=3). Fatty acids FO oil SBO oil LSO oil RSO oil FO rotifers SBO rotifers LSO rotifers RSO rotifers 14:0 0.28 0.08 0.07 0.02 6.98 1.45 0.06 1.38 14:1 0.34 n.d. n.d. n.d. 1.10 0.99 0.01 0.53 15:0 0.08 0.02 n.d. n.d. 0.74 0.28 0.03 0.31 16:0 13.23 11.18 6.95 4.88 18.99 8.99 5.39 10.89 16:1n-7 7.61 0.01 n.d. 0.46 30.62 9.57 0.12 15.49 16:1n-5 0.12 0.08 n.d. n.d. 0.79 0.36 0.01 0.03 17:0 0.48 0.07 n.d. n.d. 1.44 0.67 0.04 0.15 16:4n-4 0.45 0.05 n.d. n.d. 0.33 0.69 0.01 0.57 18:0 1.46 3.39 0.97 1.31 0.98 0.86 2.85 0.71 18:1 (n-9+ n-7) 12.53 27.13 3.21 63.37 31.10 24.23 19.64 71.86 18:2n-6 1.56 51.39 14.80 20.72 2030 35.42 14.89 33.52 18:3n-3 0.88 5.04 53.67 n.d. 3.52 3.76 56.42 9.51 18:4n-3 2.29 n.d. n.d. 8.03 3.33 0.13 n.d. 0.04 18:4n-1 0.15 n.d. n.d. n.d. 0.26 0.11 n.d. n.d. 20:0 0.13 0.36 n.d. n.d. 0.18 0.13 0.05 0.28 20:1n-9 12.92 n.d. 0.15 1.17 10.25 1.70 0.14 3.68 20:1n-7 n.d. 0.25 0.23 n.d. n,d, 0.43 n.d. n.d. 20:2n-9 0.20 n.d. n.d. n.d. 0.65 0.25 n.d. 0.11 20:2n-6 n.d. n.d. n.d. n.d. 0.52 0.74 n.d. 0.36 20:3n-6 0.09 0.03 n.d. n.d. 0.13 0.15 n.d. 0.07 20:4n-6 0.02 0.02 0.01 n.d. 0.69 0.61 0.01 0.11 20:3n-3 0.15 0.02 n.d. n.d. 0.15 0.09 0.02 0.13 20:4n-3 0.43 0.02 n.d. n.d. 1.16 0.15 0.02 0.19 20:5n-3 8.12 0.48 n.d. n.d. 13.47 0.90 0.02 0.36 22:1n-11 16.63 0.03 n.d. 0.01 11.25 0.70 0.02 1.62 22:3n-6 0.28 n.d. n.d. n.d. 0.45 0.35 0.01 0.07 22:4n-6 0.02 n.d. n.d. n.d. 0.07 0.29 n.d. n.d. 22:5n-6 0.11 0.05 n.d. n.d. 0.16 0.01 n.d. n.d. 22:4n-3 0.05 n.d. n.d. n.d. n.d. n.d. n.d. 0.10 22:5n-3 0.45 n.d. n.d. n.d. 0.86 0.29 0.02 0.08 22:6n-3 7.91 0.18 n.d. n.d. 11.19 2.16 0.04 0.31 Saturated 21.79 15.10 8.20 6.20 21.02 12.49 8.44 13.76 Monounsaturated 53.98 27.59 3.59 65.01 59.15 37.78 20.05 93.18 n-3 20.28 5.73 53.67 8.03 17.81 10.17 56.53 13.77 n-6 2.34 51.47 14.81 20.72 0.48 37.63 14.91 34.14 n-9 25.74 25.72 23.10 64.54 29.84 26.49 19.89 76.27 n-3HUFA 17.11 0.69 n.d. n.d. 17.81 3.59 0.11 1.17 AA/EPA n.d. 0.04 n.d. n.d. 0.05 0.68 0.55 0.32 EPA/DHA 1.03 0.04 n.d. n.d. 1.20 0.42 0.48 1.13 n-3 HUFA % dry wt 15.77 3.18 0.10 1.04 n.d. ≤ 0.005 Study 1 61 Table 4.2 Fatty acids composition of initial 3 day-old larvae and 10 and 20 day-old larvae for each experimental group (n.d. ≤ 0.005). Fatty acid 3 d old FO 10d SBO 10d LSO 10d RSO 10d FO 20d SBO 20d LSO 20d RSO 20d 14:0 2.56 1.41 1.09 1.17 0.91 1.82 0.86 0.48 0.52 14:1 0.08 0.86 0.35 0.38 0.27 0.48 0.49 0.02 0.33 15:0 0.30 0.15 0.40 0.44 0.51 0.81 0.60 0.09 0.13 16:0iso 0.43 0.46 0.38 0.41 0.34 0.36 0.26 0.11 0.19 16:0 20.50 10.32 13.44 14.45 16.27 17.72 15.15 7.09 10.40 16:1n-7 6.54 9.39 5.84 6.28 5.72 11.27 1.20 4.91 5.90 16:1n-5 0.08 0.46 0.36 0.39 0.31 0.69 4.32 0.48 0.17 16:2 0.48 0.95 0.57 0.61 0.70 1.36 0.78 0.52 0.72 17:0 0.38 0.53 0.65 0.67 0.66 1.21 0.32 0.17 0.74 16:4n4 n.d. 0.06 n.d. 0.18 0.42 0.89 0.33 0.20 0.31 16:4n-3 0.18 4.49 0.04 7.74 0.22 12.20 0.51 4.40 0.06 18:0 4.91 0.87 7.20 0.92 9.70 0.84 0.56 0.47 5.28 18:1(n-9+ n-7) 19.57 14.23 7.41 14.98 32.18 3.53 20.17 28.21 39.87 18:1n-5 0.29 0.99 n.d. 0.70 n.d. 0.32 0.33 0.07 n.d. 18:2n-9 0.20 n.d. 0.65 n.d. n.d. 0.32 0.78 0.19 0.13 18:2n-6 7.88 8.56 20.37 0.10 14.66 0.15 28.73 14.40 16.56 18:3n-9 0.07 0.10 1.26 n.d. n.d. 0,17 0.42 n.d. n.d. 18:3n-6 0.19 0.12 1.01 1.08 n.d. 1.13 0.51 0.31 n.d. 18:4n-6 n.d. 1.39 0,51 n.d. n.d. 0.21 n.d. n.d. n.d. 18:3n-3 0.13 0.06 n.d. 21.90 1.80 0.66 2.14 23.85 2.93 18:4n-3 1.39 0.51 0.09 0.07 n.d. 0.12 0.24 0.35 0.12 18:4n-1 0.43 0.05 0.12 n.d. n.d. 0.14 0.28 n.d. n.d. 20:0 0.15 0.10 0.06 0.21 0.09 0.26 0.37 0.13 0.35 20:1n-9 1.24 2.64 0.20 2.01 2.71 3.07 1.05 1.09 1.68 20:2n-9 0.06 0.43 n.d. 0.26 0.27 0.41 n.d. 0.10 0.29 20:2n-6 n.d. 0.63 0.24 1.01 0.28 0.85 1.11 0.56 1.18 20:3n-6 n.d. 0.19 0.93 0.24 0.16 0.21 0.56 0.11 0.43 20:4n-6 1.10 1.80 0.12 1.40 0.09 1.49 1.18 0.49 1.40 20:3n-3 0.10 0.12 1.31 0.14 1.14 0.11 0.28 1.36 0.33 20:4n-3 0.43 0.71 0.13 0.24 0.19 1.08 0.23 0.34 0.07 20:5n-3 4.59 6.73 0.22 2.20 2.25 9.12 1.67 1.51 1.91 22:1n-11 0.29 1.48 0.71 0.76 0.12 0.81 0.37 0.22 0.47 22:1n-9 0.11 n.d. 0.30 0.32 n.d. 0.66 0.20 0.34 n.d. 22:1n-7 n.d. n.d. 0.24 0.26 n.d. 0.44 n.d. 0.22 n.d. 22:3n-6 0.13 0.18 0.05 0.05 0.21 0.72 0.38 0.02 0.06 22:4n-6 0.08 0.13 0.15 n.d. n.d. 0.15 0.22 n.d. n.d. 22:5n-6 0.19 0.30 0.21 0.23 n.d. 0.29 0.44 0.14 n.d. 22:4n-3 0.04 n.d. 0.03 n.d. 0.22 0.13 0.21 0.22 0.24 22:5n-3 1.57 1.26 0.47 0.51 0.10 1.88 0.37 0.19 0.23 22:6n-3 23.15 18.38 9.88 10.63 7.50 21.92 6.99 6.66 7.06 Saturated 29.23 13.85 17.02 18.28 28.49 23.02 18.78 8.53 17.60 Monounsat. 28.30 38.54 16.51 32.74 41.0 20.58 28.54 35.08 48.24 n-3 31.58 32.27 20.19 21.62 13.42 47.23 12.65 38.88 12.93 n-6 9.57 13.30 23.64 25.93 15.40 5.19 33.13 16.03 19.62 n-9 21.25 17.41 30.32 17.57 35.16 6.05 22.62 23.56 41.97 n-3HUFA 29.88 27.21 12.78 13.71 11.39 34.25 9.76 10.28 9.83 AA/EPA 0.24 0.27 0.64 0.64 0.04 0.16 0.70 0.32 0.73 EPA/DHA 0.20 0.37 0.21 0.21 0.30 0.42 0.24 0.23 0.27 Total lipids % dry wt 14 17 15.30 16.5 12.2 19.17 16.42 20.75 17.96 Study 1 62 Table 4.3 Some fatty acids contents in total lipids from brain and eyes in larvae after 20 days of feeding (% total determined fatty acids, n=3). Fatty acids Brain FO Brain SBO Brain LSO Brain RSO Eyes FO Eyes SBO Eyes LSO Eyes RSO 12:0 5.37 9.12 6.41 9.01 4.29 3.95 6.08 7.13 14:0 3.47 6.25 4.24 4.75 3.21 3.42 3.47 2.71 14:01 1.57 2.42 0.95 n.d. 1.67 0.60 1.73 2.71 15:0 0.74 1.17 0.88 1.68 1.61 1.06 0.50 0.89 16:0iso 2.02 3.97 2.56 2.56 2.68 0.47 0.30 2.59 16:0 17.07 18.10 17.24 20.06 19.50 2.03 16.72 18.40 16:1 n-7 7.01 4.09 3.14 5.94 5.93 19.43 3.45 3.76 16:1n-5 n.d. 2.25 2.33 2.42 2.53 0.13 1.71 0.57 16:02 n.d. 0.54 n.d. n.d n.d. 0.26 n.d. n.d. 17:0 n.d 0.49 0.40 n.d n.d. 0.19 0.46 0.30 16:4n4 n.d 0.86 n.d n.d n.d. 0.71 n.d. 1.49 16:4 n-3 0.66 n.d 0.35 n.d n.d. 0.72 0.30 n.d. 18:0 10.35 8.30 13.66 9.84 9.27 10.52 10.63 9.99 18:1n-9 13.73 15.54 21.18 17.24 15.81 27.59 19.94 15.98 18:1n-7 3.79 2.05 2.62 2.35 2.47 1.55 2.46 2.48 18:1 n-5 n.d n.d 0.35 n.d n.d. 0.17 11.70 0.91 18:2n-9 0.84 n.d 0.42 n.d n.d. n.d. n.d. 0.61 18:2 n-6 7.34 10.94 7.56 13.95 12.29 20.16 3.27 10.90 18:3 n-9 n.d 0.47 n.d n.d n.d. 0.94 n.d. 2.32 18: 3n-6 n.d n.d 0.43 n.d n.d. 0.23 n.d. 0.47 18: 4 n-6 1.47 1.45 n.d 1.26 1.91 0.13 n.d. 1.67 18:3 n-3 n.d 0.70 3.48 n.d n.d. 0.47 0.85 0.56 18:4n-3 0.66 n.d 1.04 1.37 1.38 0.98 1.67 2.23 18:4n-1 n.d 1.27 n.d n.d n.d. n.d. 1.33 n.d. 20:0 n.d n.d 0.84 n.d 0.91 0.69 0.94 0.82 20:1n-9 1.58 0.63 0.83 1.26 0.95 0.67 0.52 1.46 20:1n-7 n.d 0.81 0.41 n.d n.d. 0.24 0.37 0.30 20:2n-9 n.d n.d n.d n.d n.d. 0.27 n.d. n.d. 20:2n-6 n.d n.d 0.36 n.d n.d. 0.16 n.d. 1.28 20:3n-6 n.d n.d 0.62 n.d n.d. 0.45 n.d. 0.17 20:4n-6 0.99 0.70 0.27 1.08 n.d. 0.17 0.63 0.82 20:3n-3 n.d n.d n.d n.d n.d. 0.29 0.84 n.d. 20:4n-3 0.87 0.74 1.24 n.d n.d. 0.49 0.84 1.00 20:5n-3 5.95 1.45 0.49 1.28 2.78 0.34 1.23 0.89 22:1n-11 n.d n.d 0.42 n.d n.d. n.d. 0.54 1.04 22:5n-6 0.66 n.d n.d n.d 1.38 n.d. n.d. n.d. 22:4n-3 n.d n.d n.d n.d n.d. n.d. 1.03 n.d. 22:5n-3 1.65 n.d 0.61 n.d n.d. 0.20 1.05 0.51 22:6n-3 12.21 5.69 4.66 3.97 9.42 0.34 4.81 3.04 Saturated 39.02 47.39 46.24 47.89 41.49 22.32 39.10 42.82 Monounsat. 27.68 25.55 29.91 26.79 26.83 50.25 41.37 28.66 n-3 22.01 8.58 11.87 6.62 13.58 3.82 12.60 8.23 n-6 10.46 13.09 9.23 16.29 15.57 21.30 3.90 15.30 n-9 16.14 16.64 22.43 18.50 16.76 29.47 21.12 20.38 n-3HUFA 20.68 7.88 6.99 5.25 12.20 1.65 9.79 5.44 EPA/DHA 0.49 0.25 0.10 0.32 0.30 1.00 0.25 0.29 n.d. ≤ 0.005 Study 1 69 Reaction after a visual stimulus appeared later, at day 10 after hatching, being about 10 times that of cruise speed, but increased from day 16 being from there always higher than that after sonorous stimulus, in agreement with the better eye development at this age (Roo, 1999). The appearance of the cruise swimming speed obtained in the sonorous stimulus by larvae fed with rotifers rich in fish oil presents similarities with other species, such as club mackerel (Masuda et al. , 2002) but with inferior absolute values. The same happens with the burst swimming speed. The moment which it begins to detect answer before the stimulus is very similar but the results are totally opposed, since in mackerel they are increased throughout all the experiment. Swimming activity before stimulus was significantly reduced by feeding rotifers enriched with vegetable oils. Despite reaction against sonorous stimulus was not affected by feeding vegetable oils, appearance of reaction after visual stimulus was delayed to day 19th in larvae fed LSO rotifers and it was also delayed and significantly reduced by feeding the other vegetable oils. Higher burst swimming speed in larvae fed LSO is in agreement with the higher response to acute stress found in juveniles of the same species fed with linseed oil compared to fish oil (Montero et al. , 2003). Besides, larvae fed with rotifers enriched with fish oil reacted with a higher burst speed after a visual stimulus than after the sound stimulus, denoting the importance of the vision during this period of the development not only for the predation but also for the burst. However, the reduction in dietary essential fatty acid contents, by the enrichment with vegetable oils, delays the appearance of response to visual stimulus, in agreement with the minor DHA content in eyes and brains of these larvae and suggesting a delay in the functional development of brain and vision. In addition, larvae fed with rotifers enriched with fish oil, with levels of n-3 HUFAs sufficient to cover their requirements denoted a greater cruise swimming speed than larvae fed rotifers enriched with vegetable oils with low essential fatty acid content. Lower cruising speed may also be related with lower prey capture efficiency. Study 1 70 Regarding the burst swimming speed, the greatest movement of the burst swimming speed took place in the four first sequences after the performance of the stimulus, in agreement with findings in other fish species (Masuda et al. , 2002). Burst swimming speed after visual stimulus was much higher than that obtained after sonorous stimulus. These results indicate once more the importance of visual stimulus and visual sharpness during this period of larval development, which is faster transmitted through seawater and is essential for prey finding and flee from predators. Besides, dietary changes did not affected larval reaction to the sound stimuli, whereas it significantly affected reaction to the visual stimuli. Indeed, higher burst and cruise swimming speed in the larvae fed rotifers enriched with fish oil and linseed oil, would be related to the higher levels of DHA in larval eyes and brain, since this fatty acid is involved in several neural tissue related functions such as neurocytes myelination and synapse construction, both functions being sensitive to nutritional deficiencies (Krigman and Hogan, 1976). A histological study comparison of DHA, EPA, and oleic acid enriched juveniles striped jack ( Longirostris delicatissimus ) demonstrated the DHA enriched juveniles have a more developed superficial white and gray zones on their optic tectum than to the other two groups (Masuda, 1995). Conclusion Reduction in the rotifer EFA content by enrichment with vegetable oils, affects larval normal behaviour, reducing cruise swimming speed, and particularly delaying the appearance of the response to visual stimulus, suggesting a delay in the functional development of brain and vision, in agreement with the minor EPA and DHA found in eyes and brains of these larvae. Acknowledgements This study was partially supported by an INNOVA grant from Fundación Canaria Universitaria de Las Palmas to Tibiabin Benítez-Santana. Study 1 71 References Bell, M.V., Batty, R.S., Dick, J.R.., Fretwell, K., Navarro, J.C., Sargent, J.R., 1995. Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring ( Clupea harengus L.). Lipids 30, 443–449. Bessonart, M.G., 1997. Efecto de las relaciones EPA/AA en el cultivo larvario de dorada ( Sparus aurata ). Tesis doctoral pp. 181. Ishizaki, Y., Masuda, R., Uematsu, K., Shimizu, K., Arimoto, M., Takeuchi, T., 2001. 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Study 1 74 Study II Increased Mauthner cells activity and escaping behaviour in sea bream ( Spaurs aurata ) fed long chain polyunsaturated fatty acids This work is been published in: Benítez-Santana, T., Juárez Carrillo, E., Betancor M.B., Torrecillas, S., Caballero, M.J. and Izquierdo, M.S., 2011 (in press) British Journal of Nutrition. Abstract There is limited information on the specific effects of long chain polyunsaturated fatty acids (LCPUFA) on neuron development and functioning. Deficiency of those essential fatty acids impairs escape and avoidance behaviour in fish, where Mauthner cells play a particularly important role to initiate this response. Gilthead seabream larvae fed two different LCPUFA profiles were challenged with a sonorous stimulus. Feeding Omega-3 (n-3) LCPUFA increased the content of these fatty acids in fish tissues and caused a higher number of larvae to react to the stimulus and a faster burst swimming speed response. This faster startle response in fish fed n-3 LCPUFA was also associated to an increased immunepositive neural response, particularly in M-cells, denoting a higher production of acetylcholine. The present study shows the first evidence of the effect of n-3 LCPUFA on functioning of particular neurons in fish, the Mauthner cells, and the behaviour response that they modulate to escape from a sound stimulus. Keywords: Burst swimming behaviour, DHA, fish larvae behaviour, Mauthner cells. Study 2 76 Introduction Despite Omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFA) have been long recognized as being important for brain development and function, little is known on their specific effects on neuron activity in relation to behaviour. N-3 LCPUFA play important roles in neural growth, development of synaptic processing of neural cell interaction, and expression of genes regulating cell differentiation and growth (Uauy and Dangour, 2006). Essential fatty acid metabolism can influence many aspects of brain development, including neuronal migration, axonal and dendritic growth, and the creation, remodelling and pruning of synaptic connections (Guesnet and Alessandri, 2010; Robson et al. , 2010). Animal studies have shown that both neural integrity and function can be permanently disrupted by deficits of n-6 and n-3 fatty acids during foetal and neonatal development (Yamamoto et al. , 1988; Bourre et al. , 1989). While both n-6 and n-3 fatty acids are required, the n-3 fatty acids such as docosahexaenoic acid (DHA, 22:6n-3) appear to play a special role in highly active sites such as synapses and photoreceptors, and deficiencies have particularly been linked to visual and cognitive deficits (Neuringer et al. , 1986; Neuringer et al. , 1994). In marine fish, n-3 LCPUFA are essential and play very important physiological roles (Izquierdo et al. , 1989; Izquierdo, 1996). Specifically, DHA and eicosapentaenoic (EPA, 20:5n-3) acids must be supplied in the diet and function as critical structural and physiological components of the cell membranes of most tissues, being necessary for fish growth, welfare, survival and development (Watanabe and Kiron, 1994; Izquierdo et al. , 2000). In particular, DHA has been found to be required for normal development of the nervous system and sensory organs, such that larval brain and eye fatty acid compositions reflect the diet (Masuda et al. , 1999; Benítez-Santana et al. , 2007). Moreover, DHA deficiency impairs vision in juvenile herring ( Clupea harengus ) (Bell et al. , 1995). Therefore, dietary fatty acid contents could potentially affect behavior, stress reactions or pain and comfort, despite the lack of studies on this subject in fish. Recently, dietary fatty acids have been Study 2 77 found to affect escape and avoidance behavior in fish larvae after a sound or visual stimulus (Masuda et al. , 1999; Benítez-Santana et al. , 2007). Fish can elude predatory attacks by producing a stereotyped escape behaviour, which is characterized by a rapid and powerful unilateral bending of the body and caudal fin that involves most of its somatic musculature (Korn and Faber, 2005). This behaviour is initiated by the activation of the Mauthner cells (Mcells). M-cells integrate diverse sensory inputs (Faber et al. , 1989; Eaton et al., 2001) and are able to reset swimming rhythms in the course of its initiation of escape behaviours (Svoboda and Fetcho, 1996). In chronic recordings of freely swimming intact fish, the M-cells have been shown to fire an action potential at the initiation of C-start responses (Zottoli, 1977; Eaton et al. , 1988; Canfield and Rose, 1996; Weiss et al. , 2004). Because the axons of each M-cell decussate, the firing of one leads to a contraction of trunk musculature that is contralateral to the cell soma (Zottoli, 1977; Foreman and Eaton, 1993). To date there is no evidence of an effect of essential fatty acids on M-cells activity. Therefore, the aim of this study was to better understand the effect of dietary n-3 LCPUFA on fish escape and avoidance behaviour and neural function. For that purpose the effect of two different lipid sources, with different n-3 LCPUFA content, fed to gilthead sea bream ( Sparus aurata ) during early larval and brain development on the fish reaction to a sonorous stimulus and M-cells activity was investigated. M-cells activity was determined by choline acetytransferase (ChAt) distribution by immunolabelling as a marker of cholinergic neuron density, since the cholinergic neurotransmission system has been found to be sensitive to dietary n-3 PUFA in rats (Aïd et al. , 2003). Study 2 84 Figure 5.1 Larval reaction after the sound stimuli along larval development (a) Number (%) of reacting sea bream larvae for each experimental group after the sound stimuli fed diets containing fish oil and soybean oil. (b) Burst swimming speed (SL/s) in sea bream larvae fed microdiets enriched with different types of lipids: fish oil and soybean oil. a,b Mean values with unlike letters were significantly different between animals of same treatment (P<0.05), n=30. A,B Mean values with unlike letters were significantly different between animals of different treatment (n=30, P<0.05). b a Aa b a b a b b B b b Study 2 85 Mauthner cells activity Confocal microscopy analysis showed a greater acetylcholine immunopositive response (green fluorescence) in larvae fed the fish oil microdiet than in those fed the soybean oil microdiet (Figure 5.2). Fluorescence quantification showed a significantly higher immunoposititve response in larvae fed fish oil than in larvae fed the soybean oil microdiet (Figure 5.3). Figure 5.2 Quantification of green fluorescent intensity by confocal microscopy according to the immunopositive response of M-cells. * Mean values were significantly different between animals of different treatment (n=30, P<0.05). Figure 5.3. Confocal microscopy image of the acetylcholine immunopositive response of M-cells longitudinal sections from larvae fed with fish oil microdiet (A) and soybean oil microdiet (B). Scale bar 10 µm. Study 2 86 Discussion In the present study, the reduction in dietary n-3 LCPUFA caused a significant reduction in the contents of these fatty acids in the fish. In gilthead Sea bream, brain fatty acid composition is also modified by the n-3 LCPUFA content of the diet (Benítez-Santana et al. , 2007), and therefore, being necessary for the normal development of nervous system and sensory organs, these fatty acids could affect physiological functions in the brain. In fact, a diet unbalanced in n-3 PUFA may cause changes in cell permeability and synaptic membrane fluidity (Yehuda et al. , 2005), or modifications in the number and affinity of receptors, in the function of ion channels and on the activity of neurotransmitters (Jump, 2002). Thus, alterations in brain fatty acid composition could potentially affect behaviour. An important behaviour to escape from predation is the startle response, which is initiated by a sudden stimulus and results in a rapid reaction. In the present study, substitution of soybean oil by fish oil in microdiets for larval gilthead Sea bream markedly increased n-3 LCPUFA content in fish tissues and affected fish behaviour in terms of the startle response to a sonorous stimulus. Thus, during fish development, a higher but not statistically different number of larvae reacted to the stimulus when they were fed fish oil, rich in n-3 LC-PUFA. Moreover, a n-3 LC-PUFA increase in the diet also led to a faster swimming speed burst in those larvae that reacted to the sonorous stimulus at 32 dph. Therefore, dietary reduction in n-3 LC-PUFA impaired fish larval response to the stressor, reducing the escape behaviour in larvae with a lower content of n-3 LC-PUFA in their body tissues. These results highlight the important role of these fatty acids in the response to a sonorous stimulus, in agreement with their importance for sensory organs functioning (Izquierdo, 2005). In contrast with the present study, younger and less developed gilthead sea bream burst swimming response to a sound stimulus was not affected by dietary n-3 LCPUFA, whereas they had a high burst swimming speed after a light stimulus (Benítez-Santana et al. , 2007). This suggests that despite the neural and Study 2 87 muscular responses were well developed in those young larvae since they were able to react to a visual stimulus, the reception to the sound stimulus was not sufficiently developed to produce a consistent response according to dietary differences. The mechanoreceptive neuromast cells associated with the lateral line system and the inner ear (auditory nerve) are the major receptors for external vibrational and gravitational stimuli in fish. The lateral line system of teleost fish typically consists of a row of pores along the tail, body and head, leading into an underlying fluid-filled lateral line canal. The neural impulses from these receptors are transmitted along the anterior and posterior lateral line nerves to the octavolateralis area of the medulla (Bleckmann et al. , 1987). In the fish used in the present study the lateral line was better developed, whereas in the former trial, conducted with younger larvae, the lateral line had not started to appear until the end of the experiment (15-20d). A further development of the sensorial organs in the larvae of the present study would allow a different perception of the stimulus by larvae fed different n-3 LC-PUFA suggesting the importance of these fatty acids for the normal functioning of the lateral line. The Mauthner neurons are known to receive sensory information not only from the auditory nerve (Faber et al. , 1991; Zottoli et al. , 1995), but also from the optic tectum (Canfield, 2003), from the lateral line mechanosensory system (Zottoli and Danielson, 1989), from somatosensory channels (Chang et al. , 1987), and via the electrosensory system in weakly electric fish (Zottoli et al. , 1995). Thus a variety of sensory modalities could modulate the relative excitation or inhibition of the M-cells prior to a startling stimulus driving it beyond threshold levels. Escaping behaviour in fish has been particularly related to M-cells. This pair of reticulospinal neurons initiates fast startle responses in fishes and amphibians and constitute an important model system in studies of vertebrate neurons and their control of behaviour (Korn and Faber, 2005). In the present study, faster startle response in fish fed n-3 LCPUFA was also associated with an increased immunopositive neural response, particularly in M-cells, denoting a higher production of acetylcholine. Acetylcholine release in the hippocampus has been found to be Study 2 88 reduced under neuronal activation in rats receiving a chronically n-3 PUFA deficient diet (Aïd et al. , 2003; Kodas et al. , 2004). N-3 LC-PUFA, and particularly DHA, markedly affect membrane fluidity and functioning and have been found to be important for neurocyte myelination and synapse construction, with both functions being sensitive to nutritional deficiencies (Krigman and Hogan, 1976). Moreover, these fatty acids are nutritional antioxidants that prevent the formation of cerebral lipid peroxides (ChoinKwon et al., 2004), stabilizing the oxidant/antioxidant status of membrane structures in brain (Sarsilmaz et al. , 2003), and protecting from several neurological and neuropsychiatric disorders (Black et al. , 2004). Most of DHA accumulation occurs during late prenatal and early postnatal development, coinciding with the formation of synapses (Green et al. , 1999). Similarly, DHA accumulates in brain and sensorial organs of fish during larval development and it is retained in neural tissues even during periods of starvation (Izquierdo et al. , 2001; Izquierdo, 2005). Adequate dietary availability of DHA during this period is essential for optimal central nervous system development and functioning. Inadequate intake of DHA is thus associated with impaired attention and learning performance as well as modifications in emotional status including elevated behavioural indices of anxiety, aggression and depression (Fedorova and Salem, 2006). The present study shows the first evidence of the importance of n-3 LCPUFA for the adequate functioning of particular neurons, the M-cells, and, subsequently for the behaviour response that they modulate to escape from a sound stimulus. Further studies are being conducted to understand the role of these essential fatty acids on neural development and functioning. Study 2 89 References Aïd, S., Vancassel, S., Poumes-Ballihaut, C., Chalon, S., Guesnet, P. and Lavialle, M., 2003. Effect of a diet-induced n-3 PUFA depletion on cholinergic parameters in the rat hippocampus. J. Lipid. Res. 44, 1545–1551. Atalah, E., Hernández-Cruz, C.M., Benítez-Santana, T., Ganga, R., Roo J. and Izquierdo M.S., 2010. 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Incorporation of dietary docosahexaenoic acid into the central nervous system of the yellowtail Seriola quinqueradiata. Brain Behav. Evol., 53, 173–179. Neuringer, M., Connor, W.E., Lin, D.S., Barstad, L. and Luck, S., 1986. Biochemical and functional effects of prenatal and post natal n-3 fatty acid deficiency on retina and brain in rhesus monkey. Proc. Nati. Acad. Sci. U.S.A. 83, 4021-4025. Neuringer, M., Reisbeck, S. and Janowsky, J., 1994. The role of n-3 fatty acids in visual and cognitive development: current evidence and methods of assessment. Pediatr. 125 (Suppl), 39-47. Robson, L.G., Dyall, S., Sidloff, D. and Michael-Titus, AT., 2010. Omega-3 polyunsaturated fatty acids increase the neurite outgrowth of rat sensory neurones throughout development and in aged animals. Neurobiology of Aging, 31 (4), 678-687. Study 2 92 Sarsilmaz, M., Songur, A., Ozyurt, H., Kus, I., Ozen, O.A., Ozyurt, B., Sogut, S. and Akyol, O., 2003. Potential role of dietary omega-3 essential fatty acids on some oxidant/antioxidant parameters in rat’s corpus striatum. Prostaglandins Leukot Essent Fatty Acids 69, 253-259. Svoboda, K.R. and Fetcho, J.R., 1996. Interactions between the neural networks for escape and swimming in goldfish. J. Neurosci. 16, 843–852. Uauy, R. and Dangour, A.D., 2006. Nutrition in brain development and aging: role of essentials fatty acids. Nutr. Rev. 64 (5 Pt 2), S24-33. Watanabe, T. and Kiron, V., 1994. Prospects in larval fish dietetics. Aquaculture 124, 223–251. Weiss, S.A., Zottoli, S.J., Faber, D.S. and Preuss, T., 2004. Chronic medullary recordings from freely swimming fish during the C-start escape. Soc Neurosci Abstr No. 672.4. Yamamoto, N., Hashimoto, A., Takemoto, Y., Okuyama, H., Nomura, M., Kitajima, R., Togashi, T. and Tamai, Y., 1988. Effect of dietary alpha-linolenate/linoleate balance on brain lipid compositions and learning ability of rats. II: Discrimination process, extinction process, and glycolipid compositions. J. Lipid Res. 29, 1013–1021. Yehuda, S., Rabinovitz, S. and Mostofsky, D.I., 2005. Essential fatty acids and the brain: From infancy to aging. Neurobiology of Aging 26, 98–102. Zottoli, S.J., 1977. Correlation of the startle reflex and Mauthner cell auditory responses in unrestrained goldfish. J. Exp. Biol. 66, 243–254. Zottoli, S.J., Bentley, A.P., Prendergast, B.J. and Rieff, H.I., 1995. Comparative studies on the Mauthner cell of teleost fish in relation to sensory input. Brain Behav. Evol. 46, 151–164. Zottoli, S.J. and Danielson, P.D., 1989. The lateral line afferent and efferent systems of the goldfish with special reference to the Mauthner cell. In The mechanosensory lateral line: neurobiology and evolution (Coombs S, Görner P, Münz H, eds), pp 461–478. New York: Springer. Study 3 99 All larvae of each tank were washed with distilled water and kept at -80º C for biochemical composition after 16 h of starvation at the end of the trials. Moisture (A.O.A.C., 1995) and crude lipid (Folch et al. , 1957) contents of larvae and diets were analyzed. To determine fatty acid profiles, methyl esters of fatty acids were obtained by transesterification with 1% sulfuric acid and methanol using heneicosanoic acid (10% of total lipids) as an internal standard. The fatty acid methyl esters obtained were separated by gas chromatography (ShimadzuGC-14a, Kyoto, Japan) run at the operating conditions described previously (Izquierdo et al. , 1990), quantified by flame ionisation detectors (FID) and identified by comparison to well characterized external standards. Swimming speed behaviour Swimming speed of larvae from all dietary groups was determined at 22 and 29 DAH, respectively, in a 1 L cylindrical black glass container (10 cm in diameter) with a water depth of 4 cm. Each larva was transferred from the feeding tanks to the experimental beaker and then video-recorded using a Sony digital video camera DCR-TRV27. After recording for 30 s, the larva was auditorially stimulated to induce a startle response and determine burst swimming speed. Consistent sound stimuli were produced using a steel nut (≈10 g) hung by a string (26 cm) that was released from a distance of 18 cm from the beaker wall. The sound was produced by swinging the nut like a pendulum to tap the side wall of the tank. Sound stimuli were provided three times at 10 s intervals for each larva. After this experiment, larva standard length (SL) was measured by a profile projector (Nikon V-12A, Nikon, Tokyo, Japan). This procedure was repeated using 10 individuals of each rearing tank following the procedure described in (Masuda et al. , 2002; Benítez-Santana et al. , 2007). Frame by frame video analysis was conducted to calculate burst swimming speed. To observe the response development to sound stimuli, burst swimming speed rate was calculated by dividing the number of responses by number of trials. Burst swimming speed was analyzed only when the larva showed an obvious startle response. The fish movement was traced for four consecutive Study 3 100 frames, and the distance was divided by the time taken (4/30 s). Preliminary observations in very young larvae (Benítez-Santana et al. , 2007) revealed that the fastest movement appeared in any of the first frames after providing a stimulus. In the present research, later larval stages provided the same results, where the fastest movement appeared in the first frames. Burst swimming speed was calculated as the average of the movement of four frames (Masuda et al. , 2002; Benítez-Santana et al. , 2007). Immunofluorescence study Thirty larvae per tank ( n =60) were collected and fixed in 10% buffered formalin at the end of the experiment. Each larva was mounted in a gelatin block in horizontal orientation to obtain better visualization of neuronal structures. Gelatinembedded larva blocks were serially cut on a Slee Mainz cryostat at 10 m. The antibody was tested to determine the optimal working concentration and quality of the signal, and sea bream larvae were processed for the demonstration of immunoreactivity. Each section for immunofluorescence slides was collected on gelatin-coated slides. The slides were covered with 5% rabbit serum and 0.2% Triton in PBS for 1 h prior to incubation with the primary antibody for 48 h at 4º C. When the primary polyclonal antibody was used, anti-choline acetyltransferase (Millipore, Billerica, USA) diluted 1:250 in PBS was applied for 1 hour as secondary (anti-goat IgG FITC conjugate; antibody developed in rabbit affinity isolated antigen specific antibody; Sigma) reagent in a dark room at room temperature. A propidium iodide complex diluted 1 mg in 250 ml PBS was applied for 5-10 min at room temperature to detect the different substrates. A Zeiss LSM 510 confocal system (Zeiss, Thornwood, NY, USA) was used to visualize M-cell green immunofluorescence. To quantify the intensity of the sample, a LSM 510 program was used, which measured the laser ray intensity required to excite the neurons to the same threshold level. Confocal images were collected at x1000. Study 3 101 Statistic analysis Statistical analysis was performed using the software SPSS (SPSS 11.5 for Windows, SPSS Inc, Chicago, IL, USA) using one-way analysis of variance (ANOVA) following the general linear model: Y ij = m + D i + e ij where Y ij is the mean value of the tank, m is the mean population, D i is the fixed effect of the diet and e ij is the residual error. Means of fatty acid levels and behavioural studies were compared by Duncan’s test (P<0.05). Results Feeding trial All experimental microdiets were well accepted by the larvae. Lowest survival rate (14.6%) was obtained in larvae fed the lowest dietary LCPUFA content (diet 0.3/0.6), whereas there were no significant differences among larvae fed the other diets (1.5/9 = 18.9%; 4/1 = 25.67%; 4/3 = 27.59%). Since only the biggest larvae survived in treatment with the lowest dietary LCPUFA content (diet 0.3/0.6), no significant differences were found in larval growth. Average lipid content of whole larval body did not differ significantly among larvae fed the different diets. By contrast, fatty acid composition of larval whole body lipids reflected the dietary fatty acid profile (Table 6.3). Thus, the elevation of dietary oleic acid from 0.92 to 10.15 (g/100g diet) induced a significantly correlated (r2=0.94) increase in larval oleic acid content. In addition, dietary elevation of either EPA (r2= 0.82) or DHA markedly increased the incorporation of these fatty acids into larval tissues. Therefore, larvae fed with 0.3/0.6 microdiet showed the lowest content of DHA and EPA. Despite low levels of dietary saturated fatty acids, particularly for palmitic acid (16:0), they were high in larval lipids, and particularly in larvae fed the 0.3/0.6 diet which had the lowest n-3 LCPUFA contents. In these larvae, oleic acid (18:1 n-9) and linoleic acid (18:2n-6) were also very high. Thus, in general, the larvae content of linoleic acid was not correlated with that of the diet, but with the dietary n- Study 3 102 6/ARA (r2=0.92), denoting a higher incorporation of linoleic acid into larval tissues when dietary ARA was lower. Behavioural performance For the startle response assay, there was no significant difference in percentage of larvae that reacted to the stimulus (n=30 larvae/diet/challenge). The results of the behavioural trial showed that after only one week of feeding (22 days after hatching, DAH) larvae fed the highest DHA (1.5/9 diet) level tended to show a higher burst swimming speed as an escape response to the auditory stimulus. After two weeks of feeding, 29 DAH larvae fed the highest level of DHA (1.5/9 diet) showed significantly (P<0.05) the highest burst swimming speed followed by larvae fed the second highest dietary DHA level (4/3 diet) (Figure 1). The lowest burst swimming speed was found in larvae fed the lowest DHA and EPA content (0.3/0.6 diet), whereas elevation of dietary EPA (4/1) did not significantly increase burst swimming speed. Thus, burst swimming speed was significantly correlated to DHA dietary levels (r2=0.89), but not with dietary EPA. Similarly, the increase in burst swimming speed was significantly correlated to whole body DHA contents (r2=0.98), but not to EPA content in whole body lipids. About the percentage of larvae not shown a startle response 29 DAH larvae fed the lowest level of DHA (0.3/0.6 diet) significantly (P<0.05) showed the higher percentage of not startle response. Larvae fed the lowest level of DHA (1.5/9 diet) showed significantly (P<0.05) the lowest percentage of response. Study 3 103 Table 6.3 Fatty acids content (% total determined fatty acids, n=3) of 32 day-old sea bream larvae fed with different EPA/DHA microdiets. Diet EPA/DHA 0.3/0.6 1.5/9 4/1 4/3 Lipid w.b. 2.22±0.20a 2.26±0.28a 2.86±0.59a 2.5±0.30a 14:0 0.70 0.57 0.77 1.08 14:1n-5 0.02 0.02 0.01 n.d. 14:1n-7 0.10 0.23 0.12 0.21 15:0 0.42 0.44 0.40 0.43 15:1n-5 0.16 0.16 0.13 0.16 16:0iso 0.44 0.37 0.44 0.33 16:0 21.62 16.10 18.54 15.76 16:1n-9 n.d. 0.66 0.80 0.78 16:1n-7 1.37 2.89 1.80 3.58 Me16:0 0.18 0.07 0.17 0.13 16:1n-5 0.32 0.59 0.32 0.51 16:2n-6 0.52 0.21 0.42 0.29 16:2n-4 0.81 0.96 0.83 0.83 16:3n-4 1.06 0.72 0.81 0.65 16:3n-3 n.d. 0.04 n.d. 0.06 16:3n-1 n.d. n.d. n.d. 0.06 16:4n-3 0.95 1.06 0.93 0.99 16:4n-1 0.88 0.50 1.00 0.69 18:0 9.23 9.80 8.50 8.48 18:1n-9 30.37 11.84 22.76 17.17 18:1n-7 4.74 3.47 4.12 4.25 18:1n-5 0.22 0.18 0.19 0.28 18:2n-9 0.15 0.68 0.26 0.73 18:2n-6 6.59 3.07 3.96 3.28 18:2n-4 n.d. 0.11 n.d. 0.06 18:3n-6 0.14 0.26 n.d. 0.09 18:3n-4 0.10 n.d. 0.12 0.06 18:3n-3 n.d. 0.23 0.29 0.31 18:3n-1 n.d. 0.09 n.d. 0.05 18:4n-3 n.d. n.d. 0.32 0.36 18:4n-1 0.28 0.40 0.28 0.27 20:1n-9 n.d. n.d. n.d. 0.09 20:1n-7 0.67 1.00 0.54 0.84 20:2n-9 0.06 0.33 n.d. 0.24 20:2n-6 n.d. 0.19 n.d. 0.14 20:3n-9 0.19 0.17 n.d. 0.14 20:3n-6 0.16 0.09 0.15 0.12 20:3n-3 n.d. 0.13 n.d. 0.04 20:4n-6 2.52 2.75 3.18 2.73 20:4n-3 n.d. 0.09 0.34 0.36 20:5n3 3.27 3.98 10.67 8.68 22:1n-11 n.d. 0.34 0.11 0.46 22:1n-9 n.d. 0.09 0.08 0.10 22:4n-6 0.80 1.34 0.42 0.70 22:5n-3 1.05 1.44 2.09 1.86 22:6n-3 9.92 32.33 14.16 21.56 Saturated 31.99 26.84 28.25 25.65 Unsaturated 33.03 15.08 26.23 20.39 Monoenoics 36 16.92 27.80 23.19 Polyunsaturated 29.26 51 40.23 45.21 n-3 14.14 37.86 26.71 32.36 n-6 10.73 7.91 8.13 7.35 n-9 31.25 13.94 23.64 19.17 n-3 LCUFA 13.19 36.53 25.17 30.64 AA/EPA 0.77 0.69 0.30 0.31 EPA/DHA 3.03 8.12 1.33 2.48 Study 3 104 Figure 6.1. Larval reaction after the sound stimuli along larval development. Burst swimming speed (mm/s) in sea bream larvae fed with EPA/DHA microdiets. * Mean values were significantly different between animals of different treatment (n=30, P<0.05). Mauthner cells activity Observation of M-cell activity by confocal microscopy showed higher choline acetyltransferase activity in larvae fed the highest dietary DHA levels (1.5/9 diet) and lowest in larvae fed lowest dietary LCPUFA (0.3/0.6 diet) (Figure 2). Quantification of the amount of green fluorescence produced by the antibody reaction to choline acetyltransferase showed a significantly (P<0.05) higher immunopositive response in larvae fed the highest DHA levels (1.5/9 diet) than in larvae fed the lowest content of these essential fatty acids (Figure 6.3). 0 200 400 600 800 1000 1200 1400 1600 1800 22 29 Burst swimming speed (mm/s) Days (d) 4/1. 4/3. 1.5/9 0.3/0.6 * Study 3 105 Figure 6.2 Quantification of green fluorescent intensity by confocal microscopy according to the anti-choline acetyltransferase immunopositive response of M-cells. * Mean values were significantly different between animals of different treatment (n=30, P<0.05). Figure 6.3 Confocal microscopy image of the acetylcholine immunopositive response of Mcells longitudinal sections from larvae fed with 1.5/9 microdiet (A) and 0.3/0.6 microdiet (B) (x1000). Study 3 106 Discussion Feeding the lowest n-3 LCPUFA levels lead to a lower escape response after an auditory stimuli in gilthead sea bream larvae, in agreement with the reduced response behaviour to visual stimulus found in our previous studies in younger larvae of this species (Benítez-Santana et al. , 2007). These results are in agreement with the great relevance of dietary n-3 LCPUFA for behaviour and brain health known in mammals (Marszalek and Lodish, 2005). Elevation of dietary DHA correlated with a higher burst swimming speed in response to a sound stimulus, denoting the higher importance of this n-3 LCPUFA in escaping behaviour in comparison to EPA. In agreement, dietary DHA has been found to improve development of normal behaviour in yellowtail (Masuda and Tsukamoto, 1998). Incorporation of DHA into gilthead sea bream larval tissues was proportional to dietary levels of this fatty acid and correlated well with the burst swimming activity in response to sound stimuli. In this species, a previous study to determine brain and eyes fatty acid profile composition showed that DHA is the main LCPUFA in tissues of these organs in quantities dependant on its dietary levels (Benítez-Santana et al. , 2007). In turbot, DHA is selectively assimilated in phosphatidyl ethanolamine, the largest phospholipid class in neural tissue (Mourente and Tocher, 1992). In other fish species, DHA has been found to accumulate in olfactory nerve and photoreceptors (Bell et al. , 1995), being associated with sensory organ function (Izquierdo, 2005). However, in the present study, increase in dietary EPA did not significantly improve escaping response of fish, as will be discussed later on. Neural regulation of escaping behaviour in fish is based on the activation of M-cells to initiate the startle response. Startle response is a fast primary sensorimotor reaction to avoid predators initiated by simple and fast neural circuits, the large Mauthner neurons. In the present study, increased M-cell activity, denoted by the higher production of acetylcholine, was found in larvae fed and containing in their tissues the highest levels of DHA and showing the fastest startle response. Several pathways may be involved in the effect of DHA on neural activity. On one hand, in mammals, DHA promotes outgrowth of neurites, through the enhancement of phospholipids synthesis, particularly phosphatidyl ethanolamine, in the Study 3 107 membranes needed for neurite elongation (Ikemoto et al. , 1997). On the other hand, DHA content in neural membrane affects Na+ and K + channels as well as neurotransmitter receptors (Bowen and Clandinin, 2002; Farkas et al. , 2002; Levant et al. , 2004), interfering with eicosanoid production and transcription factors (Samadi et al. , 2006). Particularly, acetylcholine levels are restored in hippocampus when dietary DHA increases, improving learning performance (Horrocks and Yeo, 1999). In agreement, dietary DHA apparently ameliorates the learning performance failure caused by cholinergic dysfunction (Minami et al. , 1997). Finally, another mechanism for the neuronal protective role of DHA could be the inhibition of apoptosis induced by sphingosine (Horrocks and Yeo, 1999). In fish, EPA also plays an important role in modulation of eicosanoid synthesis and transcription factors (Ganga et al. , 2005), but this fatty acid is much less efficient than DHA in regulation of membrane fluidity and incorporation into phosphatidyl ethanolamine (the main phospholipid in neural tissues), what could explain its lower ability to initiate a startle response. Moreover, increased dietary EPA reduces incorporation of dietary DHA into marine fish larvae phospholipids (Izquierdo et al. , 2000). The results of the present study have shown that DHA, rather than EPA, boosts escape behaviour in gilthead sea bream, and this effect may be at least partly mediated by the increase in neural activity in M-cells. Nevertheless, this reaction could also be related to a faster response against a stressor (auditory stimuli) since it has been recently shown that DHA has the ability to regulate cortisol release by ACTH induced interrenal cells of gilthead sea bream (Ganga et al. , 2006). Reduced dietary supply of DHA during early development may have longlasting effects on brain function and cytoarchitecture of the developing brain (Koletzko et al. , 2008). Early detection of insufficient DHA contents in neural tissues of marine fish larvae by simple behaviour observations may constitute a noninvasive tool to allow the prevention of posterior neural disorders and improve marine fry production by reducing mortality. Study 3 108 References A.O.A.C., 1995. Official Methods of Analysis of the Association Analytical Chemist. U.S.A., pp. 1018. Atalah, E., Hernández-Cruz, C.M., Ganuza, E., Benítez-Santana, T., Ganga, R., Roo, J., Montero, D. and Izquierdo, M.S., 2010. Importance of dietary arachidonic acid for the growth, survival and stress resistance of larval European sea bass ( Dicentrarchus labrax ) fed high dietary docosahexaenoic and eicosapentaenoic acids. Aquaculture Research. 1-8 doi:10.1111/j.1365-2109.2010.02714.x. Atalah, E., Hernandez-Cruz, C.M., Izquierdo, M.S., Rosenlund, G., Caballero, M.J., Valencia, A. and Robaina, L., 2007. Two mircoalgae Crypthecodinium cohnii and Phaeodactylum tricornutum as alternative source of essential fatty acids in starter feeds for seabream ( Sparus aurata ). Aquaculture 270, 178-185. Bell, M.V., Batty, R.S., Dick, J.R. Fretwell, K., Navarro, J.C. and Sargent, J.R., 1995. Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring ( Clupea harengus L.). Lipids 30, 443–449. Benítez-Santana, T., Masuda, R., Juárez-Carrillo, E., Ganuza, E., Valencia, A., Hernández-Cruz, C.M. and Izquierdo, M.S., 2007. Dietary n-3 HUFA deficiency induces a reduced visual response in gilthead seabream Sparus aurata larvae. Aquaculture 264, 408–417. Bourre, JM., 2004. Roles of unsaturated fatty acids (especially omega-3 fatty acids) in the brain at various ages and during aging. J Nutr Health Aging 8, 163-174. Bowen, R.A.R. and Clandinin, M.T., 2002. Dietary low linolenic acid compared with docosahexaenoic acid alter synaptic plasma membrane phospholipids fatty acid composition and sodium-potassium ATPase kinetics in developing rats. J. Neurochem. 83, 764–774. Brinkmeyer, R. and Holt, G.J., 1998. Highly unsaturated fatty acids in diets for Red drum ( Sciaenops ocellatus ) larvae. Aquaculture 161, 253-268. Copeman L.A., Parrish, C.C., Brown, J.A. and Harel, M. 2002. Effects of docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth, survival, lipid composition and pigmentation of yellowtail flounder ( Limanda ferruginea ): a live food enrichment experiment. Aquaculture 210, 285-304. Eaton, R.C., Lee, R.K.K. and Foreman, M.B., 2001. The Mauthner cell and other identified neurons of the brainstem escape network of fish. Progress in Neurobiology. 63, 467– 485. Study 4 114 Introduction Over the past twenty years, the zebrafish ( Danio rerio ) has emerged as a great vertebrate model system for screening of therapeutic drugs (Penberthy et al. , 2002; Sumanasa and Lin, 2004) and to better understand the neural basis of behaviour (Zottoli and Faber, 2000; Eaton et al. , 2001; Kohashi and Oda, 2008; Fetcho and MacLean, 2010; MacLean and Fetcho, 2010). Behaviours like the escape response, also known as the fast-start or startle response, are initiated by a pair of large, identifiable neurons in the hindbrain: the Mauthner or M-cells (Zottoli and Faber, 2000; Eaton et al. , 2001; Korn and Faber, 2005). The M-cells are a pair of reticulospinal neurons located in the medulla teleost fish (Beccari, 1907). They are characterised by the large size of their somata and their myelinated axons that cross the body midline to descend the length of the spinal cord on the opposite side of the body from its soma, issuing axon collaterals that massively activate cranial and spinal motor systems (Faber et al. , 1989). The presence of two distinct dendrites, the association of the lateral dendrite with the VIIIth cranial nerves, and the presence of a unique structure, the so-called axon cap (Triller and Korn, 1980), around the cell’s initial segment, convey a morphological identifiably to this pair of cells in different animals like the otophysan fish (Zotoli, 1978, Bierman et al. , 2009). During sudden stimuli, fish perform an escape that starts with a rapid bend of the body to one side. M-cells play an important role in initiating this escape response: a single spike in one of the M-cells triggers an escape toward the contralateral side (Eaton et al., 2001; Korn and Faber, 2005). During Mauthner-triggered escapes, motoneurons on the side of the escape bend are activated, while those on the opposite side are almost simultaneously inhibited (Yasargil and Diamond, 1968). Fast-starts in most teleost fish consist of a C-type fast-start (C-start), the first stage of which is characterized by a rapid unilateral contraction of trunk musculature leading to head and tail movement, which causes the fish to bend into a C-shape. Subsequent movements typically follow stage 1, including a tail stroke that results in a forward propulsion of the centre of mass (stage 2), leading to gliding or a burst swim (stage 3) (Foreman and Eaton, 1993; Domenici and Blake, 1997). M-cells activity precedes the C-start and electrical stimulation of Mauthner axons can elicit a C-start (Nissanov Study 4 115 et al. , 1990). Several studies have used zebrafish as a model to study M-cells functioning (Eaton and Nissanov, 1985; Fetcho and Liu, 1999; Lorent et al. , 2001; Hale, 2002). Several factors seem to affect M-cells performance (Chang et al. , 1987; Zottoli and Danielson, 1989; Canfield and Rose, 1993; Zottoli et al. , 1995) but little is known on the effect of nutritional factors. N-3 long chain fatty acids (LCPUFA), and particularly docosahexaenoic acid (DHA, 22:6n-3), are essential components of cellular membranes and they are particularly accumulated in neural tissues and sensorial organs (Bell and Tocher, 1989; Bell and Dick, 1991; Mourente et al. , 1991; Lauritzen et al. , 2001; Benítez-Santana et al. , 2007). LCPUFA are essential compounds that play key roles in numerous metabolic and physiological processes ensuring normal cellular function. In fish, DHA and eicosapentaenoic (EPA, 20:5n-3) acids, or their precursor, linolenic acid (LNA, 18:3n-3), must be supplied in the diet, and function as critical structural and physiological components of the cell membranes, being necessary for fish growth, welfare, survival and development (Watanabe and Kiron, 1994; Izquierdo, 1996; Sargent et al. , 1999; Izquierdo et al. , 2000). Inadequate contents of those dietary essential fatty acids (EFA) in fish diets give rise to several alterations such as poor feeding and swimming activities (Masuda et al. , 2002; Benitez-Santana et al. , 2007), poor growth and dropping mortality (Hernández-Cruz et al. , 1999), fatty livers, abnormal pigmentation, disgregation of gill epithelia, immune-deficiency (Izquierdo, 1996) and raised basal cortisol levels (Montero et al. , 2003). Our previous studies (Benítez-Santana et al. , in press) showed the first evidences that DHA, rather than EPA, boosts escaping behaviour in gilthead sea bream ( Sparus aurata ) and suggest that this effect is at least partly mediated by the increase in neural activity, in particular, of M-cells. Dietary fatty acids have been found to affect escape behaviour in fish larvae after a sound or visual stimulus (Masuda et al. , 2002; Benítez-Santana et al. , 2007). As in mammals, inadequate intake of DHA is associated with impaired attention and learning performance as well as modifications in emotional status including elevated behavioural indices of anxiety, aggression and depression (Fedorova and Salem, 2006). Study 4 116 Therefore, the aim of this study was to determine the effect of dietary n-3 LCPUFA levels on M-cells functioning in zebrafish in relation to escape behaviour and neural function. Material and Methods All experiments conformed to NIH guidelines regarding animal care and were approved by Cornell University’s Institutional Animal Care and Use Committee. Feeding conditions and growth The nacre type zebrafish larvae used were spawned at the Department of Neurobiology and Behaviour in Cornell University (Mudd Hall, Ithaca, USA). Twelve larvae were individually distributed into 12 aquariums filled with filtered water at 28.5 °C and submitted to a 14 h/10 h light /dark photoperiod. From 4-12 days after hatching (dah) larvae were fed Shrimp Larva Diet (Miami Aqua-culture, Inc. Florida). From 12 dah larvae were fed Artemia twice a day (at 9:00 h and 15:00 h) for 25 days. Three different lots of Artemia were fed with three levels of DHA: “Diet L” (low content of n-3 LCPUFA), “Diet M” (medium content of n-3 LCPUFA), supplemented with 1 capsule of MorDHA (241 mg DHA and 33 mg EPA, Minami Nutrition, Belgium) and “Diet H” (high content of n-3 LCPUFA) enriched with 2 capsules of MorDHA. Larval growth was evaluated by determining larval total length at 5 and 30 dah under the light microscope. Biochemical analyses Diets were analyzed for crude lipid content and fatty acid composition (Table 7.1). Diets were sampled for lipid and fatty acid composition of total lipids. Lipids were extracted by chloroform:methanol (Folch et al. , 1957). Methyl esters of fatty acids were prepared by transesterification with 1% sulfuric acid and methanol using hexaenoicosanoic acid (10% of total lipids) as an internal standard (Christie, 1982). The fatty acid methyl esters obtained were separated by gas chromatography (ShimadzuGC-14a, Kyoto, Japan) run at the operating conditions previously Study 4 117 described (Izquierdo et al. , 1992), quantified by flame ionisation detectors (FID) and identified by comparison to well characterized external standards. Behavioural studies Response to a vibrational stimulus was studied determining the peak angle of escape bend, the peak angular speed of the escape turn and time to respond in challenged zebra fish larvae (6 larvae per diet, 10 stimulus per larva) at 19 dah, after one week of feeding Artemia containing different n-3 fatty acid levels. The trials were recorded by a high-speed camera that digitally captures images at 1000 frames/s (EG&G Reticon, Sunnyvale, CA) using the software called Photron Fastcam Viewer (San Diego, CA, USA). Data were analyzed using a custom written image analysis program focusing on the performance of the initial turn in the fish escape, since the Mauthner array is essential for generating the high-performance movements during this turn. Mauthner cells studies In order to study Mauthner cells development and morphology these neurons were labelled via electroporation in 6 larvae per treatment at 4 dah prior to the feeding trial. At this age, nacre zebrafish larvae are transparent with sparse pigmentation, that allows the identification and picturing of neurons in the brain and spinal cord of the intact animal. For electroporation, larval zebrafish were anesthetized with 0.02% 3-aminobenzoic acid ethyl ester (MS222, Sigma Aldrich, St. Louis, MO, USA) and positioned straight in 1.2% agarose in 0.3’ phosphate buffered saline (PBS) on a glass slide. A 10% solution of rhodamine (3000 molecular weight or MW; Molecular Probes, Eugene, OR) in 10% Hanks solution was electroporated into the Mauthner cell (Bhatt et al. , 2004). After injection, the animals were given 24 h to allow for recovery. Study 4 118 Table 7.1 Some fatty acids contents in total lipids from Artemia fed different levels of DHA and used to feed zebrafish larvae (% d.w.). Fatty acids Diet L Diet M Diet H 14:00 0.08 0.22 0.31 14:1n-7 0.10 0.27 0.41 14:1n-5 0.05 0.13 0.21 15:00 0.02 0.04 0.07 15:1n-5 0.01 0.05 0.06 16:0ISO 0.05 0.14 0.21 16:00 1.11 3.35 4.51 16:1n-7 0.23 0.63 0.95 16:1n-5 0.07 0.18 0.25 16:2n-6 n.d. 0.03 0.05 16:2n-4 0.08 0.22 0.28 17:00 0.06 0.17 0.25 16:3n-4 0.05 0.17 0.24 16:3n-3 0.04 0.17 0.25 16:3n-1 0.01 0.04 0.06 16:4n-3 n.d. 0.01 0.02 18:00 0.60 1.71 2.14 18:1n-9 1.98 6.13 8.00 18:1n-7 0.64 1.62 2.51 18:2n-9 0.04 0.12 0.18 18:2n-6 0.61 3.15 3.49 18:2n-4 n.d. 0.02 0.01 18:3n-6 0.03 0.08 0.13 18:3n-3 2.40 7.23 9.90 18:4n-3 0.40 1.03 1.47 18:4n-1 n.d. n.d. n.d. 20:00 0.01 0.06 0.08 20:1n-9+n-7 0.05 0.27 0.37 20:1n-5 0.01 0.05 0.06 20:2n-9 0.01 0.01 0.02 20:2n-6 0.03 0.09 0.11 20:3n-6 n.d. 0.02 0.03 20:4n-6 0.05 0.15 0.33 20:3n-3 0.08 0.29 0.38 20:4n-3 0.13 0.41 0.46 20:5n-3 0.17 2.19 3.05 22:1n-11 0.08 0.18 0.23 22:1n-9 0.07 0.45 0.62 22:4n-6 0.01 0.01 0.02 22:5n-6 n.d. 0.10 0.11 22:5n-3 0.03 0.71 0.96 22:6n-3 0.06 4.71 6.39 Saturated 1.85 5.49 7.26 Monoenoics 2.85 8.74 11.86 Polyunsaturated 4.23 21.02 27.97 n-3 3.30 16.73 22.86 n-6 0.72 3.63 4.26 n-3 LCPUFA 0.46 8.30 11.25 AA/EPA 0.28 0.07 0.11 EPA/DHA 2.86 0.46 0.48 n.d.≤0.005 Study 4 119 At the end of the experiment all the fish larvae were briefly anesthetized, embedded on their backs in soft agar on a cover glass in a petri dish (Eaton et al. , 1984) and then rinsed with 10% Hanks solution to allow recovery from the anesthetic. Confocal images were obtained by looking into the head of the intact fish using a Zeiss LSM 510 confocal microscope (Fetcho and O’Malley, 1995; O’Malley et al. , 1996). The fish transparency allowed not only to clearly visualize neurons inside the living animal but also to monitor fish viability by observing the heartbeat and blood flow. To confirm the identity of the cells studied physiologically, stacks of images showing the morphology in successive confocal sections were acquired. Signal averaging was used when acquiring this morphological data. Maximum projections were made from stacks of these sections. The image stacks were also reconstructed in three-dimensions using the Zeiss software or Imaris, allowing us to examine the details of the dendritic morphology and axonal projections of each cell. After getting the images, larvae were collected and fixed in 10% buffered formalin. Each larva head was mounted in a gelatin block in horizontal orientation to obtain better visualization of neuronal structures. Gelatin-embedded larva head blocks were cryoprotected in 30% sucrose and serially cut on a Slee Mainz cryostat at 10 m. Each gelatin section for immunofluorescence slides was collected on gelatin-coated slides. The antibody was tested to determine the optimal working concentration and quality of the signal and zebrafish larvae were processed for the demonstration of immunoreactivity. The slides were covered with 5% rabbit serum and 0.2% Triton in phosphate-buffered saline (PBS) for 1 h. Incubations with anticholine acetyltransferase primary antibody (1:250, Millipore, Billerica, USA) was carried out for 48 h at 4º C in a 0.1 M (PBS) solution with 0.1% Triton X-100 and 2% goat serum (Vector). As secondary antibody we used anti-goat IgG FITC conjugate (1:250, antibody developed in rabbit affinity isolated antigen specific antibody; Sigma) diluted in PBS and applied for 1 hour in a dark room at room temperature. A propidium iodide complex at concentration of 1:104 was applied for 5-10 min at room temperature and was used for nuclei contrast staining. In order to see the M-cells green immunofluorescence a Zeiss LSM 510 confocal system (Zeiss, Thornwood, NY) was used. In order to prevent fluorescence data were not Study 4 120 compromised, in each batch of samples processed always all treatments were included. Negative controls were run by replacing each primary antibody by PBS to test for the specificity of an antibody involved. Statistical treatment of data Data were statistically analyzed with the software SPSS (SPSS 11.5 for Windows, SPSS Inc, Chicago, IL, USA) using one-way analysis of variance (ANOVA) following the general linear model: Y ij = m + D i + e ij where Y ij is the mean value of the tank, m is the mean population, D i is the fixed effect of the diet and e ij is the residual error. Means of fatty acid levels and behavioural studies were compared by Duncan’s test (P<0.05). Results Biochemical analysis of non-enriched Artemia (Diet L) and Artemia enriched with one (Diet M) or two (Diet H) capsules of DHA showed a progressive increase in total lipids content (9.5, 36.8 and 49.3% lipids in dry basis of Artemia , respectively for diets L, M and H). As expected, enrichment with DHA capsules markedly changed the fatty acid profile of Artemia . Thus, major fatty acids in Diet L (non enriched Artemia ) were 18:3n-3, 18:1n-9 and 16:0, whereas in diets M and H ( Artemia enriched with DHA) were 18:3n-3, 18:1n-9 and DHA, followed by 18:2n-6 and EPA. Enrichment with DHA capsules progressively increased DHA in larvae, together with other fatty acids such as 18:2n-6, 18:3n-3 and 20:5n-3. After 17 days of feeding, zebra fish fed Artemia not enriched with DHA capsules, containing the lowest polyunsaturated fatty acid contents (Diet L), showed significantly lower growth than fish fed with Artemia enriched with one capsule of DHA (Diet M) (Figure 7.1). However, further enrichment with two DHA capsules (Diet H) significantly reduced final fish total length. Study 4 121 Figure 7.1 Evolution of growth in zebrafish larvae fed Artemia enriched with different levels of DHA (n=12, P<0.05). Figure 7.2 (a) Peak angular speed (Degrees/msec) (b) Time to peak bend angle (msec) in zebrafish larvae fed with different levels of DHA. a, b Mean values with unlike letters were significantly different between animals of different treatment (n=6, P<0.05). 5 6 7 8 9 10 11 5d 30d Total lenght (mm) Days (d) Diet L Diet M Diet H * Study 4 122 Behavioural performance At the beginning of the study there was a good feeding activity in the fish larvae, regardless the type of feed. After one week of feeding Artemia the different diets, zebrafish fed non-enriched Artemia (Diet L) containing the lowest EPA and DHA (0.17 & 0.06, respectively) showed a lower response to the vibration stimulus, whereas a significantly highest response was found in fish fed Diet M. Thus, fish fed Diet M showed a significantly higher peak angle and speed angle after sound stimuli (Figure 7.2a, b). However, fish fed with Artemia enriched with two DHA capsules (Diet L) showed lower values of peak angle and speed angle than fish fed Diet M, and only slightly higher than those of fish fed Diet L. Mauthner cells activity In agreement with the lower response after the vibration stimulus in fish fed Diet L, confocal microscopy analysis showed a low anti-choline acetyltransferase signal (green fluorescence) in M-cells from this fish, whereas enrichment of Artemia with one DHA capsule (Diet M) increased fluorescence suggesting a perhaps a healthier neuron (Figure 7.3). On the contrary, further enrichment with two DHA capsules (Diet H) reduced the immunopositive response (Figure 7.3). Analysing the amount of green fluorescence by confocal microscopy we found a significantly highest signal in fish fed Diet M, denoting a higher neuron activity than fish fed Diets L or H (Figure 7.4). a Study 4 123 Figure 7.3 Immunopositive response (green fluorescence) observed by confocal microscopy in Mauthner cells from zebrafish fed different diets. (a) Zebrafish fed Diet L (0.06 DHA content). (b) Zebrafish fed Diet M (4.71 DHA content) (x1000). Figure 7.4 Quantification of green fluorescent intensity by confocal microscopy according to the anti-choline acetyltransferase immunopositive response of M-cells in zebrafish fed diets with different DHA levels. a, b Mean values with unlike letters were significantly different between animals of different treatment (n=30, P<0.05). b a Annex I 10% buffered formalin 100 ml 40% concentrated formaldehyde 900 ml distilled water 4 g NaH2PO4 · H2O 6 g Na2HPO4 Poli L 0.1 g Poli-L in 1000 ml milli-Q water 0.01 g Poli-L in 100 ml of water a) Wash slides in this solution during 5 min - 2 water baths during 5 min - Dry and use b) Wash slides in this solution during 5 min c) Dry and use Freezing mixture This mixture serves to preserve the tissues in the freezer without destroying, and preventing the formation of crystals. It also has a controlled pH. - Material Sodium dihydrogen phosphate, 2-hydrate. NaH2PO4 2H2O p.m.= 156.01 di-Sodium hydrogen-Na2HPO4 p.m. = 141.96 Polyethylene glycol 400. HO (C2H40) p.m. = 380-420 98% glycerol. CH2OH CHOH CH2OH · (use pure) Distilled water Annex I 222 - Formula 30% v / v glycerol 30% v / v ethylene glycol 0.1M phosphate buffer pH = 7.3 - Preparation 0.5 M phosphate buffer: 7.8 g NaH2PO4 in 100 ml H20 (A) 14.2 g Na2HPO4 in 200 ml H20 (B) Mix slowly (A on B) pH = 7.3-7.4. For 1l of solution, mix 200 ml of 0.5 M phosphate buffer (to make it a final concentration of 0.1 M), 300 ml glycerin, 300 ml of ethylene glycol and adjusting the volume to 1l with H2O. Mixing thoroughly. Gelatinized slides Mix 4.5 g of gelatin powder with 900 ml of water and dissolve by heating to 80° C. Add 0.44 g of chromium III and potassium sulphate. Insert the clean slides in the filtered solution at 70° C for 5 min and allow to dry in an oven overnight. The operation is repeated, and stored in a cool place. Reactives Electro Microscopy - Lead citrate o 1.33 g of lead nitrate o 1.77 g of sodium citrate o 30 ml H2=  Shake vigorously for 10 min  Add 8 ml of 1N NaOH Vol  Turn slowly - 4% glutaraldehyde o 84 ml of 0.1 M cacodylate buffer (or 42 ml of distilled water and 42 ml of 0.2 M cacodylate buffer) Annex I 223 o 16 ml of 25% glutaraldehyde - 0.1 M cacodylate buffer o sodium cacodylate (PM 214.02), 2.1402 g o 100 ml distilled water o cacodylate is dissolved in water adjusting pH (7.2-7.4) with 0.1 N hydrochloric acid - 0.2 M cacodylate buffer o 4.28 g sodium cacodylate o 100 ml distilled water o Adjust pH (7.2-7.4) with 0.1 N hydrochloric acid - Washer liquid (cacodylate/sucrose) o 0.2 M cacodylate buffer 50 ml o 50 ml distilled water o 5 g sucrose Formvar-coated slot grids Dissolve 1.1 g Formvar in 100 ml chloroform in a glass-stoppered erlenmeyer flask while stirring. Transfer the Formvar solution gently into a coplin jar. Lead citrate Working solution: 0.1 g lead citrate in 50 ml 0.1 N NaOH Procedure: - Lead citrate solution 1 min. - 3 times in ultrapure water. - Dry the grils with whatman filter paper. Annex I 224 Annex 2 ARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSA 62 The incorporation of new marine species in aquaculture is essencial for the development of this activity. The recent advances on the establishment of techniques to produce red porgy (Pagrus pagrus), allow us to consider it a strong potencial to aquaculture, being predictable its implement in the Canary Archipel. Nevertheless, it has been not possible so far to produce it comercially, due to difficulties in assuring a continual supply of larvae and fry. Knowing this, it is crucial to continue studying the production techniques of larvae and the feeding system during this period. The aim of the work is then to optimize production techniques of larvae and fry of the red porgy according to the environmental conditions of the Canary Archipel. The work is based on a novel japanese technique, which intents to establish the feeding system in accordance with larvae behaviour, as to complete the larval cycle of Pagrus pagrus. The observed behaviour will be compared with the seabream one (Sparus aurata), one of the most studied species due to its importance in European aquaculture in the past years. In the light of these analyses a quality diet criteria based on the padrons of larval behaviour will be established. DESARROLLO DE LAS TÉCNICAS DE PRODUCCIÓN DE CRÍAS DE BOCINEGRO (PAGRUS PAGRUS) EN CANARIAS La incorporación de nuevas especies de peces marinos a la acuicultura, es un reto para el futuro desarrollo de esta actividad. Los avances realizados en el establecimiento de las técnicas de cultivo de bocinegro (pargo, Pagrus pagrus), permiten considerarlo como una especie con un fuerte potencial para la acuicultura siendo previsible que su cultivo experimente un impulso importante en el archipiélago canario. Sin embargo, las investigaciones han permitido identificar varios problemas que deben solventarse antes de que el bocinegro pueda cultivarse comercialmente, siendo una de las necesidades más acuciantes la de completar las técnicas de producción de larvas y alevines, y estudiar detenidamente la alimentación a suministrar durante todo el ciclo larvario. Por ello, el trabajo que se presenta tiene como objetivo principal tratar de optimizar las técnicas de producción de larvas y alevines de bocinegro en las condiciones ambientales del archipiélago canario. Esto se realizará a partir de una técnica japonesa muy novedosa, para establecer según el comportamiento larvario, las pautas de alimentación a seguir para completar el ciclo larvario del bocinegro. El comportamiento obtenido se comparará con el de la dorada, ya que es una de las especies más estudiadas debido a la importancia adquirida en la acuicultura europea de los últimos años. Con todo esto, se construirá un criterio de calidad de la dieta a partir de las pautas de comportamiento establecidas. PRESENTACIÓN Los avances realizados en el establecimiento de las técnicas de cultivo de bocinegro (pargo, Pagrus pagrus), permiten considerarlo como una especie con un fuerte potencial para la Acuicultura, siendo previsible que su cultivo experimente un impulso importante tanto en áreas del Mediterráneo, como en zonas templadas del Atlántico, incluyendo el archipiélago canario, ya que esta especie constituye un plato fundamental en la gastronomía típica de las Islas. Sin embargo, las investigaciones han permitido identificar varios problemas que deben resolverse T. Benítez-Santana R. Masuda A. Valencia M.S. Izquierdo RTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOS ARTÍCULOS 63 antes de que el bocinegro pueda cultivarse comercialmente, siendo una de las necesidades más acuciantes la de completar las técnicas de producción de larvas y alevines. INTRODUCCIÓN Importancia del cultivo larvario en el desarrollo de la acuicultura Hoy en día, la producción larvaria y la obtención de alevines de buena calidad, siguen representando el cuello de botella para el desarrollo de la acuicultura, sobre todo en ciertas especies marinas como el bocinegro. Esto es debido a que las larvas de peces marinos no están completamente desarrolladas cuando eclosionan. Sus tejidos y órganos, así como el sistema nervioso, no están del todo formados y experimentan importantes cambios de tipo morfológico, funcional y fisiológico durante la maduración. Por esta razón, durante estas primeras etapas de desarrollo de estos organismos, se necesitará un manejo más delicado. Una solución a este inconveniente, sería optimizar las condiciones de manejo de las diferentes especies marinas que actualmente se cultivan. Dentro de este contexto, son muy importantes los estudios referentes a la nutrición y selección genética de reproductores, requerimientos nutricionales de las larvas, mejora de las técnicas de cultivo, estudios patológicos,... Pero todavía existen diferentes áreas de estudio que no han sido prácticamente abordadas, como el estudio del comportamiento de larvas y alevines. El estudio del comportamiento de las larvas a lo largo de su desarrollo permitiría establecer los momentos de aparición de determinadas pautas típicas en animales sanos. Posibles desviaciones en el tipo de comportamiento o retrasos en la aparición de esas pautas, constituirían indicadores del estado de desarrollo, madurez y salud del animal. Así, estos estudios se revelan como excelentes indicadores del estado de bienestar de las larvas, y por lo tanto, del éxito del cultivo larvario. La llave del desarrollo del comportamiento de escuelas (formación de cardúmenes) implica en mayor medida al sistema nervioso central que a los órganos sensoriales y de natación (Masuda y Tsukamoto, 1998). El DHA (Ácido docosahexaenoico) en la dieta influye en el desarrollo del comportamiento de escuelas y cerebro en larvas de seriola (Seriola quinqueradiata) (Ishizaki, 2001). Determinados autores, trabajando con larvas de dorada, como con otras especies de peces marinos, han demostrado que al aumentar el porcentaje de n-3 HUFA tanto en alimento vivo como en microdietas, se obtienen larvas con un mejor crecimiento y una supervivencia más alta (Salhi, 1997; Sargent et al., 1999). También se ha descrito que cuando se suministran rotíferos o microdietas deficientes en n-3 HUFA se produce además una mayor sensibilidad al estrés para algunas especies marinas (Izquierdo, 1996). La composición de ácidos grasos de los tejidos como ojo y cerebro se muestra como un claro reflejo de la dieta de la larva, así, Navarro et al. (1993) señalan que la cantidad de DHA en los ácidos grasos de la fosfatidiletanolamida de los ojos es particularmente susceptible a la deficiencia de los mismos en la dieta. La importancia de altos niveles de DHA para el correcto desarrollo neural en larvas ha sido demostrada. RTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOS El estudio del comportamiento de las larvas a lo largo de su desarrollo permitiría establecer los momentos de aparición de determinadas pautas típicas en animales sanos Bocinegro (Pagrus pagrus) ARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSA 64 mar cubierto por una funda negra. Las larvas fueron grabadas con una cámara de video digital Sony DCRTRV27. Después de 90 s de grabación, se suministró un estímulo (sonoro o visual) (Masuda et al., 2002). Se utilizaron 5 larvas de cada replicado. Se realizó un análisis de imagen para calcular la velocidad de crucero y la velocidad de huída. Los datos fueron analizados estadísticamente con el programa STATGRAPHICS PLUS para Windows 3.1 (Stadistical Graphics Corp., Englewood Cliffs, NJ, USA) usando un análisis de una sóla vía (ANOVA) y Duncan (P < 0.05) para una comparación de medias. RESULTADOS Composición de ácidos grasos obtenida en las muestras de dorada En el presente apartado se hace una valoración comparada de la composición lipídica que presentan los rotíferos cuando son enriquecidos con aceites que contienen distintos porcentajes de n-3 HUFA. En la tabla I se presentan los resultados de los análisis lipídicos para la determinación de los lípidos totales y ácidos grasos de los aceites y presas utilizados en el experimento. Examinando los ácidos grasos de los aceites enriquecedores cabe destacar algunas diferencias importantes. El aceite de pescado posee un 17.11% de n-3 HUFA, de los cuales el EPA (20:5n-3) tiene una representación ligeramente superior al DHA (22:6n-3), siendo la relación entre ambas de 1.04/1. El contenido en ácidos grasos saturados es elevado, sobre todo en el ácido palmítico (16:0), 13.22%. También es bastante alto el contenido en ácidos monoenoicos, principalmente en 22:1n-11 con un 16.63%. El aceite de soja se caracteriza por presentar una mayor cantidad en ácidos grasos de la familia n6, 52.77%. También es rico en ácido oleico (18:1n-9) con un porcentaje de Este trabajo tiene como objetivo principal establecer el momento de aparición de ciertas respuestas del comportamiento a lo largo de los primeros días de vida del bocinegro, determinar el efecto de la alimentación con diferentes tipos de fuentes lipídicas y distintos niveles de DHA sobre dichas pautas y construir un criterio de calidad de la dieta a partir de las pautas de comportamiento establecidas. El comportamiento obtenido se compará con el de la dorada, que es una de las especies más estudiadas debido a la importancia en la acuicultura mediterránea de los últimos años. MATERIAL Y MÉTODOS El presente trabajo se llevó a cabo en el Instituto Canario de Ciencias Marinas (ICCM). Los huevos de dorada utilizados procedían del "stock" de reproductores existente en la propia planta de cultivos del ICCM, mientras que los huevos de bocinegro procedían de un "stock" de reproductores de la planta de cultivos del Centro de Maricultura de Calheta (Madeira). Estos fueron distribuidos en dieciséis tanques de fibra de vidrio de 170 l de capacidad (100 huevos/l). Las larvas de bocinegro y dorada fueron alimentadas con rotíferos enriquecidos con aceite de pescado (FO), aceite de soja (SBO), aceite de lino (LSO) y aceite de colza (RSO). Al día 10 de experimentación, un tanque de cada dieta de dorada fue sacrificado para realizar análisis del contenido total de lípidos y de la composición de ácidos grasos. Al final del periodo experimental, 100 larvas de dorada fueron separadas para la extracción de cerebro y ojos, y el resto de larvas supervivientes se guardaron en bolsas de plástico a -80ºC para realizar análisis bioquímicos. La velocidad de natación en larvas de bocinegro fue medida los días 9, 10, 12 y 13 de vida, y en dorada los días 6, 10, 16 y 19. Se utilizó un vaso precipitado de 500 ml con agua de El DHA en la dieta influye en el desarrollo del comportamiento de escuelas y cerebro en larvas ARTÍCULOS 65 RTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOS Reconociendo las muestras de rotíferos enriquecidos con los distintos aceites, se puede comprobar que los ácidos grasos presentes en los aceites, comentados anteriormente, están presentes en éstos. Hay que señalar que en la muestra de rotíferos enriquecidos con aceite de pescado, la relación EPA/DHAes mayor a la del aceite de pescado. Esta relación es de 1.2/1. Para el 26.91. El aceite de lino posee una mayor proporción de ácidos grasos de la familia n-3, sobre todo linolénico (18:3n-3) con un 53.68%. Por otro lado, el aceite de colza se caracteriza por mostrar una proporción alta en ácidos grasos de la familia n-9, sobre todo en ácido oleico (63.37%), y también en monoenoicos. La cantidad de n-3 HUFA presente en los aceites vegetales es inapreciable. Ácidos ACEITE ACEITE ACEITE ACEITE Rot. Rot. Rot. Rot. Grasos FO SBO LSO RSO FO SBO LSO RSO 14:0 14:1 15:0 16:0 16:1n-7 16:1n-5 17:0 16:4n-4 18:0 18:1 (n-9+ n-7) 18:2n-6 18:3n-3 18:4n-3 18:4n-1 20:0 20:1n-9 20:1n-7 20:2n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1n-11 22:3n-6 22:4n-6 22:5n-6 22:4n-3 22:5n-3 22:6n-3 Saturados Monoinsaturados n-3 n-6 n-9 n-3HUFA AA/EPA EPA/DHA n-3 HUFA% peso seco 0,28 0,34 0,08 13,23 7,61 0,12 0,48 0,45 1,46 12,53 1,56 0,88 2,29 0,15 0,13 12,92 n.d. 0,20 n.d. 0,09 0,02 0,15 0,43 8,12 16,63 0,28 0,02 0,11 0,05 0,45 7,91 21,79 53,98 20,28 2,34 25,74 17,11 n.d. 1,03 0.51 0.01 0.18 14.86 0.31 0.04 0.08 0.11 2.96 15.12 27.43 2.73 n.d. 0.04 0.18 1.55 0.05 n.d. 0.11 n.d. 0.31 0,2 0.05 4.42 0.25 0.06 0.06 n.d. n.d. 0.12 0.12 19.02 17.90 34.66 27.97 16.70 16.81 14.09 2.72 0,07 n.d. n.d. 6,95 n.d. n.d. n.d. n.d. 0,97 3,21 14,80 53,67 n.d. n.d. n.d. 0,15 0,23 n.d. n.d. n.d. 0.01 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 8,20 3,59 53,67 14,81 23,10 n.d. n.d. n.d. 0,02 n.d. n.d. 4,88 0,46 n.d. n.d. n.d. 1,31 63,37 20,72 n.d. 8,03 n.d. n.d. 1,17 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0,01 n.d. n.d. n.d. n.d. n.d. n.d. 6,20 65,01 8,03 20,72 64,54 n.d. n.d. n.d. 6,98 1,10 0,74 18,99 30,62 0,79 1,44 0,33 0,98 31,10 20,30 3,52 3,33 0,26 0,18 10,25 n,d, 0,65 0,52 0,13 0,69 0,15 1,16 13,47 11,25 0,45 0,07 0,16 n.d. 0,86 11,19 21,02 59,15 17,81 0,48 29,84 17,81 0,05 1,20 15.77 Tabla I. Principales ácidos grasos encontrados en las muestras n.d. _ 0.005 1,45 0,99 0,28 8,99 9,57 0,36 0,67 0,69 0,86 24,23 35,42 3,76 0,13 0,11 0,13 1,70 0,43 0,25 0,74 0,15 0,61 0,09 0,15 0,90 0,70 0,35 0,29 0,01 n.d. 0,29 2,16 12,49 37,78 10,17 37,63 26,49 3,59 0,68 0,42 3.18 1,38 0,53 0,31 10,89 15,49 0,03 0,15 0,57 0,71 71,86 33,52 9,51 0,04 n.d. 0,28 3,68 n.d. 0,11 0,36 0,07 0,11 0,13 0,19 0,36 1,62 0,07 n.d. n.d. 0,10 0,08 0,31 13,76 93,18 13,77 34,14 76,27 1,17 0,32 1,13 1.04 0,06 0,01 0,03 5,39 0,12 0,01 0,04 0,01 2,85 19,64 14,89 56,42 n.d. n.d. 0,05 0,14 n.d. n.d. n.d. n.d. 0,01 0,02 0,02 0,02 0,02 0,01 n.d. n.d. n.d. 0,02 0,04 8,44 20,05 56,53 14,91 19,89 0,11 0,55 0,48 0.10 < ARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSA 66 Ácidos L3d FO 10d SBO 10d LSO 10d RSO 10d FO 20d SBO 20d LSO 20d RSO 20d Grasos 14:0 14:1 15:0 16:0iso 16:0 16:1n-7 16:1n-5 16:2 17:0 16:4n4 16:4n-3 18:0 18:1(n-9+ n-7) 18:1n-5 18:2n-9 18:2n-6 18:3n-9 18:3n-6 18:4n-6 18:3n-3 18:4n-3 18:4n-1 20:0 20:1n-9 20:2n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1n-11 22:1n-9 22:1n-7 22:3n-6 22:4n-6 22:5n-6 22:4n-3 22:5n-3 22:6n-3 Saturados Monoinsaturados n-3 n-6 n-9 n-3HUFA AA/EPA EPA/DHA Lípidos totales % Peso seco 2,56 0,08 0,30 0,43 20,50 6,54 0,08 0,48 0,38 n.d. 0,18 4,91 19,57 0,29 0,20 7,88 0,07 0,19 n.d. 0,13 1,39 0,43 0,15 1,24 0,06 n.d. n.d. 1,10 0,10 0,43 4,59 0,29 0,11 n.d. 0,13 0,08 0,19 0,04 1,57 23,15 29,23 28,30 31,58 9,57 21,25 29,88 0,24 0,20 14±0.0a 1,41 0,86 0,15 0,46 10,32 9,39 0,46 0,95 0,53 0,06 4,49 0,87 14,23 0,99 n.d. 8,56 0,10 0,12 1,39 0,06 0,51 0,05 0,10 2,64 0,43 0,63 0,19 1,80 0,12 0,71 6,73 1,48 n.d. n.d. 0,18 0,13 0,30 n.d. 1,26 18,38 13,85 38,54 32,27 13,30 17,41 27,21 0,27 0,37 17±0.0a 1,09 0,35 0,40 0,38 13,44 5,84 0,36 0,57 0,65 n.d. 0,04 7,20 7,41 n.d. 0,65 20,37 20,37 1,01 0,51 n.d. 0,09 0,12 0,06 0,20 n.d. 0,24 0,93 0,12 1,31 0,13 0,22 0,71 0,30 0,24 0,05 0,15 0,21 0,03 0,47 9,88 17,02 16,51 20,19 23,64 30,32 12,78 0.64 0.21 15.30±0.0a 1,17 0,38 0,44 0,41 14,45 6,28 0,39 0,61 0,67 0,18 7,74 0,92 14,98 0,70 n.d. 0,10 n.d. 1,08 n.d. 21,90 0,07 n.d. 0,21 2,01 0,26 1,01 0,24 1,40 0,14 0,24 2,20 0,76 0,32 0,26 0,05 n.d. 0,23 n.d. 0,51 10,63 18,28 32,74 21,62 25,93 17,57 13,71 0,64 0,21 16.58±0.0a 0,91 0,27 0,51 0,34 16,27 5,72 0,31 0,70 0,66 0,42 0,22 9,70 32,18 n.d. n.d. 14,66 n.d. n.d. n.d. 1,80 n.d. n.d. 0,09 2,71 0,27 0,28 0,16 0,09 1,14 0,19 2,25 0,12 n.d. n.d. 0,21 n.d. n.d. 0,22 0,10 7,50 28,49 41,00 13,42 15,40 35,16 11,39 0,04 0,30 12.21±0.0a n.d. _ 0.005 1,82 0,48 0,81 0,36 17,72 11,27 0,69 1,36 1,21 0,89 12,20 0,84 3,53 0,32 0,32 0,15 0,17 1,13 0,21 0,66 0,12 0,14 0,26 3,07 0,41 0,85 0,21 1,49 0,11 1,08 9,12 0,81 0,66 0,44 0,72 0,15 0,29 0,13 1,88 21,92 23,02 20,58 47,23 5,19 6,05 34,25 0,16 0,42 19.17±0.0a 0,86 0,49 0,60 0,26 15,15 1,20 4,32 0,78 0,32 0,33 0,51 0,56 20,17 0,33 0,78 28,73 0,42 0,51 n.d. 2,14 0,24 0,28 0,37 1,05 n.d. 1,11 0,56 1,18 0,28 0,23 1,67 0,37 0,20 n.d. 0,38 0,22 0,44 0,21 0,37 6,99 18,78 28,54 12,65 33,13 22,62 9,76 0,70 0,24 16.42±0.0a 0,48 0,02 0,09 0,11 7,09 4,91 0,48 0,52 0,17 0,20 4,40 0,47 28,21 0,07 0,19 14,40 n.d. 0,31 n.d. 23,85 0,35 n.d. 0,13 1,09 0,10 0,56 0,11 0,49 1,36 0,34 1,51 0,22 0,34 0,22 0,02 n.d. 0,14 0,22 0,19 6,66 8,53 35,08 38,88 16,03 23,56 10,28 0,32 0,23 20.75±0.0a 0,52 0,33 0,13 0,19 10,40 5,90 0,17 0,72 0,74 0,31 0,06 5,28 39,87 n.d. 0,13 16,56 n.d. n.d. n.d. 2,93 0,12 n.d. 0,35 1,68 0,29 1,18 0,43 1,40 0,33 0,07 1,91 0,47 n.d. n.d. 0,06 n.d. n.d. 0,24 0,23 7,06 17,60 48,24 12,93 19,62 41,97 9,83 0,73 0,27 17.96±0.0a < Tabla II. Ácidos grasos presentes en lípidos totales de larvas de 72 horas (Ld3), larvas de 10 y 20 días de dorada alimentadas con las diferentes dietas ARTÍCULOS 73 sa la aparición del estímulo visual, lo cual sugiere un retraso en el desarrollo funcional del cerebro y la visión y estaría de acuerdo con el menor contenido de DHA en los ojos y cerebros de estas larvas. Además, las larvas que son alimentadas con rotíferos enriquecidos con aceite de pescado, con niveles de n-3 HUFAs adecuados para cubrir sus requerimientos para el desarrollo del cerebro, presentan una mayor velocidad de crucero que las larvas donde los requerimientos y en especial el DHA, no han sido cubiertos. Estos resultados concuerdan con los cambios anatómicos de los componentes del cerebro de seriola encontrados por Ogawa (1967), y en los que el desarrollo notable del cerebelo coincide con la mejora de los cambios de natación, demostrándose una íntima relación entre las modificaciones del comportamiento y los cambios morfológicos de algunos componentes del cerebro. Así, el presente estudio demuestra como al no suministrarse las cantidades idóneas de DHA para que se desarrolle el cerebro con toda normalidad, la natación de esta especie, el movimiento natural de la larva, y su velocidad de crucero se verán afectadas. Esto va a tener consecuencias tróficas, puesto que variaciones en el comportamiento como es la velocidad de natación, van a repercutir en la estrategia de captura de presas. Con lo que larvas "más lentas" van a presentar menores capturas y menores tasas de crecimiento, sin perjuicio de que las muchas otras funciones que los ácidos grasos esenciales realizan para el correcto crecimiento de la larva se vean también afectadas. En cuanto a la velocidad de huída es interesante resaltar que el mayor movimiento de la velocidad de huída se produce en las cuatro primeras secuencias tras la actuación del estímulo. Este mismo hecho se ha encontrado en otras especies de peces (Masuda et al., 2002). Por otro lado, en larvas de bocinegro este mayor movimiento se produce entre las siete y diez primeras secuencias tras la ejecución del estímulo. Los resultados obtenidos en velocidad de huída para el estímulo visual en larvas de dorada y bocinegro fueron mucho mayores que los obtenidos en el estímulo sonoro. De nuevo estos resultados indican la mayor importancia del estímulo visual y la agudeza del mismo durante este periodo del desarrollo larvario. Hay que resaltar que en su medio natural el estímulo visual se transmite en el agua mucho más rápido que el sonoro, y que posiblemente el estímulo visual sea más vital para la larva a la hora de situarse, encontrar alimento, huir de predadores, etc. Así, mientras con el estímulo sonoro no se encontraron diferencias significativas en la respuesta de las larvas de alimentadas con diferentes dietas ante el estímulo visual, las larvas de dorada presentaron una mayor velocidad de huída cuando habían sido alimentadas con rotíferos enriquecidos con aceite de pescado y lino que las alimentadas con aceite de soja y colza. Comparando el DHA retenido por cada una de las larvas, se observa que las larvas alimentadas con rotíferos ricos en aceite de pescado y lino, presentaron las mayores concentraciones de este ácido graso en ojo. Por lo que se puede relacionar, la velocidad de huída ante un estímulo visual con la cantidad de DHA acumulada en ojo. Es interesante denotar por otra parte, que la inclusión de aceite de lino en los piensos para dorada origina una reacción más rápida y mayor, expresada en niveles de cortisol plasmático, frente a un estrés agudo (Montero et al., 2003). Si este hecho sucediese igualmente en los estadíos larvarios de esta especie contribuiría a explicar la mayor velocidad de huída de las larvas alimentadas con lino como una mayor y más rápida respuesta ante el estrés que supone el estímulo visual. Así, la mayor velocidad de huída y de crucero en las larvas de dorada RTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOS ARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSARTÍCULOSA 74 alimentadas con aceite de pescado, está relacionada con los niveles de DHA en el ojo y cerebro de las larvas, dadas las importantes funciones que este ácido graso cumple en dichos tejidos. Así por ejemplo, parte de las posibles funciones del DHA en el cerebro implican la mielinación de los neurocistes y la construcción de la sinapsis; ambas funciones consideradas esenciales para la formación de la red neural. Además, ambas funciones son presentadas por ser sensibles a una deficiencia nutritiva u hormonal (Krigman y Hoga, 1976). Una comparación histológica del jurel (Longirostris delicatissimus) mantenido con dietas enriquecidas con DHA, EPA y ácido oleico demuestran que los juveniles alimentados con dietas ricas en DHA presentaron un mejor desarrollo superficial de la zona blanca y gris sobre el tectum óptico que con los otros dos grupos (Masuda, 1995). Considerando que la zona superficial blanca y gris está principalmente compuesta de axones neurales y dendrites (Northcutt, 1983), el DHA puede estar involucrado en la composición de la red neural, algunos de los cuales están implicados en muchos comportamientos complejos, como el de formación de escuela (Masuda et al., 1999). Las regiones del cerebro tectum óptico y cerebelo influyen en la habilidad visual y en el funcionamiento de natación en seriola (Ogawa, 1967) En conclusión, los resultados del presente estudio demuestran que la dieta va a repercutir en la recepción y reacción de estímulos externos tales como luz y sonido, y en la capacidad de natación de las larvas y por ello en la habilidad para la predación y la huída y finalmente en el crecimiento. Deficiencias en DHA van a dar lugar a un inadecuado desarrollo del sistema nervioso central y ojo, con lo que va a inducir a un incorrecto desarrollo del comportamiento, y más concretamente el comportamiento de alimentación. Esto sugiere que los requerimientos de DHA en la dieta pueden estar relacionados con los cambios fisiológicos y de comportamiento durante el desarrollo larvario de estas especies. Son necesarios futuros experimentos para establecer el efecto del DHA de la dieta con el crecimiento volumétrico de distintas regiones del cerebro de las larvas de dorada y bocinegro, y enlazarlo con el desarrollo del comportamiento en la vida de estas especies. Estos resultados revelan la importancia de la alimentación en el comportamiento larvario. Este estudio sobre el comportamiento larvario del bocinegro podrá servir a futuros experimentos para mejorar la calidad de la dieta así como optimizar tanto las condiciones de manejo como las técnicas de producción de larvas y alevines de esta especie. TIBIÁBIN BENÍTEZ SANTANA Licenciada en Ciencias del Mar por la Universidad de Las Palmas de Gran Canaria (ULPGC). Posee el Master Internacional de Acuicultura impartido por la ULPGC, el Instituto Canario de Ciencias Marinas (ICCM) y el Centro Internacional de Altos Estudios Agronómicos Mediterráneos (CIHEAM). Actualmente esta realizando el doctorado Acuicultura: Producción controlada de Animales Acuáticos en la Universidad de Las Palmas de Gran Canaria y ha participado en jornadas internacionales sobre acuicultura. email: [email protected] BIOGRAFÍA La mayor velocidad de huída y de crucero en las larvas de dorada alimentadas con aceite de pescado, está relacionada con los niveles de DHA en el ojo y cerebro de las larvas ARTÍCULOS 75 BIBLIOGRAFÍA BELL, M.V., BATTY, R.S., DICK, J.R., FRETWELL, K., NAVARRO, J.C. y SARGENT, J.R. 1995. Dietary deficiency of docosahexaenoic acid impairs vision at low light intensities in juvenile herring (Clupea harengus L.). Lipids. 30, 443-449. BLAXTER, J.H.S. 1986. 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