Diel food intake and digestive enzyme production patterns in Solea senegalensis larvae

Aquaculture 435 (2015) 33–42

Contents lists available at ScienceDirect

Aquaculture journal homepage: www.elsevier.com/locate/aqua-online

Diel food intake and digestive enzyme production patterns in Solea senegalensis larvae Carmen Navarro-Guillén a,⁎, Francisco J. Moyano b, Manuel Yúfera a a b

Instituto de Ciencias Marinas de Andalucía (ICMAN-CSIC), Apartado Oficial, 11510 Puerto Real, Cádiz, Spain Dpto. Biología Aplicada, Escuela Politécnica Superior, Universidad de Almería, 04120 Almería, Spain

a r t i c l e

i n f o

Article history: Received 16 July 2014 Received in revised form 10 September 2014 Accepted 11 September 2014 Available online 19 September 2014 Keywords: Daily ingestion pattern Solea senegalensis Larval ontogeny Digestive enzyme activities

a b s t r a c t The aim of this study was to examine patterns of food intake and production of the main digestive enzymes of Senegalese sole larvae under a fixed 12 h light:12 h dark cycle. Daily gut content, soluble protein content and digestive enzyme activities were studied at different times of day in pelagic sole larvae (onset of feeding and 6 days post-hatch; dph) and in benthonic sole post-larvae (20 and 30 dph). The larvae displayed temporal changes in food intake throughout development. Pre-metamorphic larvae ceased feeding after lights were turned off. In contrast, post-metamorphic larvae continued to feed during the day, with a higher food intake occurring during dark phase. It was demonstrated that larval digestive enzymes, mainly lipases, were active before mouth opening. Tryptic activity varied with food intake at pre-metamorphic larvae, whereas its activity levels were kept along the day in post-metamorphic larvae. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Digestion mechanisms and digestive ontogeny in fish larvae have been particularly studied in the last decades with the overall objective of developing a formulated compound diet for replacing live preys in hatcheries (Babaei et al., 2011; Cahu and Zambonino-Infante, 2001; Gisbert et al., 2009; Lazo et al., 2000; Ribeiro et al., 2008; Zambonino-Infante and Cahu, 2001). Nevertheless, both the improvement of current feeding protocols of marine fish larvae based on live prey and the design of suitable artificial feeds require a good knowledge of the complexity of their feeding behavior and of factors modulating food processing (Rønnestad et al., 2013). Meal timing can interact with food utilization, and because the cost of feed is one of the factors that most influence the production cost of intensively cultured fish, Boujard and Leatherland (1992) suggested the need to consider feeding rhythms of domesticated fish species as a means to improve the production efficiency. Some other factors affecting net efficiency of the digestive process are the species, stage of development and diet composition, while on the other hand, feeding frequency and number of daily meals affect the absorption and assimilation of nutrients, and hence, fish larvae growth. Several studies evaluating larval gut content in fishes maintained under different photoperiods demonstrated that fish larvae, such as Japanese flounder Paralichthys olivaceus (Dou et al., 2000) and ayu Plecoglossus altivelis (Yamamoto et al., 2003), do not feed constantly under natural or laboratory conditions, but exhibit a “circadian-like” prandial pattern (Boujard and Leatherland, 1992; Kotani and Fushimi, ⁎ Corresponding author. Tel.: +34 956832612. E-mail address: [email protected] (C. Navarro-Guillén).

http://dx.doi.org/10.1016/j.aquaculture.2014.09.017 0044-8486/© 2014 Elsevier B.V. All rights reserved.

2011). Circadian rhythms of feeding activity have been also indentified in Dicentrarchus labrax (Sanchez-Vázquez et al., 1995), as food demand was strongly synchronized with the light:dark cycle, and fish exhibited both diurnal and nocturnal patterns. Nevertheless, with some exceptions (Tillner et al., 2013; Ueberschär, 1995), there are not studies assessing changes in pattern of production of digestive enzymes along a whole day linked to the feeding behavior of larvae. Senegalese sole is considered a promising candidate for marine aquaculture in the south of Europe since the nineties (Dinis et al., 1999; Imsland et al., 2003) due to its high price, market demand and high growth potential of the industry. Sole is a species with a complex metamorphosis phase, characterized by strong anatomical transformations. After metamorphosis, the settlement of larvae in the substratum and the change from the pelagic to benthic habitat implies important changes in feeding habits and digestive physiology (Fernández-Díaz et al., 2001). Senegalese sole larvae presents high capacity to digest live prey from the onset of exogenous feeding, this being reflected in high growth rates (Conceição et al., 2007). The enzymatic and histological development of the digestive system in Senegalese sole larvae based on just one daily sample has been previously studied (Conceição et al., 2007; Martínez et al., 1999; Padrós et al., 2011; Ribeiro et al., 1999a, 1999b), concluding that they exhibit digestive enzyme activity before mouth opening and a fast development of the digestive system between hatching and first feeding. Although the existence of daily feeding rhythms with an active nocturnal behavior has been reported in juveniles and adults of this species (Bayarri et al., 2004; Cañavate et al., 2006; Navarro et al., 2009), it is not clear when and how the feeding behavior turns diurnal to nocturnal habits. In addition, the impact of the daily ingestion pattern on the

34

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

production of digestive enzymes remains unknown. The aim of this study was to get a better understanding on the changes of daily feeding behavior and digestive function in Senegalese sole during the first weeks of life in order to improve feeding protocols. 2. Materials and methods 2.1. Experimental procedure To reach the objectives four different developmental stages were considered, two of them before metamorphosis and two after metamorphosis: A) the onset of feeding (1, 2, 3 and 4 days post-hatch; dph); B) pelagic phase before the start of the metamorphosis (6 dph); C) just after eye-migration ended (20 dph); and D) benthonic postlarval phase (32 dph). At first feeding, sampling was carried out during three days in order to record all changes that may occur during the onset of feeding up to all larvae were actively eating. To achieve this objective, samplings were more frequent the hours between mouth opening and first feeding. In the posterior stages, sampling was performed each 3 h during a complete 24 h cycle, with the idea of evaluating the variations in feeding behavior in a whole day due to light/dark phases. In each sampling point, 10 larvae per tank were sampled for enzymatic analyses. In addition, for the determination of individual dry weight, 30 larvae were collected at 1, 2, 4, 6 and 10 dph and 15 larvae at 15, 21, 25, 29 and 32 dph, larvae were rinsed in distilled water and kept for 48 h at 80 °C. Animal treatments were carried out in compliance with the Guidelines of the European Union Council (2010/63/EU) and Spanish legislation for the use of laboratory animals, with approval of the Bioethics Committee of the Spanish National Research Council for project RIDIGEST (AGL2011-23722). 2.2. Larval rearing Larvae were obtained from The Oceanographic Institute of Santander (Santander, Spain) at 1 dph, with an average dry weight of 0.06 ± 0.05 mg (mean ± SD). Larvae were reared in three 200-L flat bottom trays with an initial density of 50 larvae L−1. Photoperiod was 12 h light: 12 h dark, with lights turned on and off at 9:00 and 21:00, respectively (UTC + 1). Water salinity was around 35 gL−1 and temperature 19.5 ± 0.10 °C. Larvae were fed on rotifers (Brachionus plicatilis) and Artemia nauplii and metanauplii according to the following protocol: 5 rotifers mL− 1 at first feeding, 10 rotifers mL−1 from 4 to 6 dph, 3 Artemia nauplii mL−1 from 7 to 19 dph, 5 Artemia metanauplii mL−1 from 20 to 25 dph and 7 Artemia metanauplii mL−1 at 26 dph to the end of the experiment. These concentrations were controlled by counting several times during the day and kept constant in order to ensure continuous prey availability to larvae. Rotifers were enriched with a mixture of microalgae (Nannochloropsis gaditana and Isochrysis sp.; 1:1 vol.) and Artemia metanauplii were enriched with Isochrysis sp. 2.3. Gut content In larvae fed rotifers, the number of preys in the gut was quantified by counting rotifers' mastax using a binocular microscope as described in Polo et al. (1992), due to low pigmentation in pre-metamorphic larvae it was possible to examine the digestive tract in whole larvae. Owing to the quick disintegration of Artemia after ingestion in postmetamorphic larvae, gut content was estimated by weighting the dissected gut. For this, the guts were put in slide covers previously weighted and kept during 24 h at 80 °C. After this, slide covers were weighted in a high precision scale (precision of 0.0001 mg; Mettler Toledo, España). The empty gut dry weight in post-metamorphic larvae (20 and 30 dph) was also determined; for this, 30 unfed larvae were transferred from the rearing tank to a small 3 L tank containing 2.5 L of seawater under identical conditions as the rearing tank for 24 h to

ensure the elimination of any gut content, gut dry weight was determined by dissection as described above. In this way, the dry weight of Artemia content in the larval gut was calculated by subtracting the empty gut dry weight to the gut dry weight of fed larvae. It is interesting to stand out the new methodology presented in this study for determining gut content in larvae fed Artemia. Regarding to visual techniques, identifying the number of rotifers consumed by the larvae might be easy by counting the mastax in the larval gut lumen, but the rapid disintegration of the Artemia after ingestion complicates this task. This technique allows to know the amount of biomass in the digestive and to estimate the number of prey by using the corresponding dry weight. 2.4. Enzyme activity analyses To prepare the enzyme extracts, larvae were previously freeze-dried and every single larva was manually homogenized in 200 μL distilled water. The homogenate was centrifuged for 10 min at 11,000 rpm, 4 °C to remove the tissue, then, the supernatant extract was used for the analysis of trypsin, amylase, and esterase/lipase activity. All samples were kept in ice during the process described above in order to avoid enzymes denaturation and/or damage. Enzyme extracts were kept at −20 °C until analysis. Concentration of soluble protein in extracts was determined by Bradford method (Bradford, 1976), using bovine serum albumin (1 mg mL−1) as a standard. There are previous studies assessing the variation in enzyme activities due to the pre-extraction preparation, concluding that freeze-drying samples before their utilization maintained normal enzymatic functions upon rehydration (Goodrich et al., 1992; Lau et al., 2013; Spigno et al., 2007). For trypsin analysis, the fluorogenic substrate Boc-Gln-Ala-Arg-7methylcoumarin hydrochloride (BOC-SIGMA B4153) was diluted in dimethyl sulfoxide (DMSO), to a final concentration of 20 μM. For analysis, 5 μL of this substrate, 190 μL of 50 mM Tris + 10 mM CaCl2 buffer (pH 8.5) and 15 μL of the larval homogenate were added to the microplate (Rotllant et al., 2008). Fluorescence was measured at 355 nm (excitation) and 460 nm (emission). Ultra Amylase Assay Kit (E33651) from Molecular Probes was used for amylase analysis. This kit contains a starch derivate labeled with a fluorophore dye as substrate. This substrate was diluted in 3-(N-morpholino)propanesulfonic acid (MOPS; pH 6.9) and substrate solvent (sodium acetate; pH 4.0), to a final concentration of 200 μg/mL. For analysis, 50 μL of the substrate solution and 15 μL of the larvae extract were added to the microplate. Fluorescence was measured at 485 nm (excitation) and 538 nm (emission). Esterase/lipase activity was assayed using 4-methylumbelliferyl butyrate (MUB-Fluka 19362), 4-methylumbelliferyl heptanoate (SigmaAldrich) and 4-methylumbelliferyl oleate (Sigma-Aldrich). The substrates were dissolved in phosphate buffer pH 7.0 to a final concentration of 0.4 mM, modified method from Rotllant et al. (2008), aliquoted and stored at − 20 °C. 15 μL of the larvae homogenate was added to the microplate and mixed with 250 μL of 0.4 mM substrate for the analysis. Fluorescence was measured at 355 nm (excitation) and 460 nm (emission). All enzyme activities were expressed as RFU (Relative Fluorescence Units) per mg larvae dry weight. 2.5. Changes in enzyme activity with age In order to study the changes of the three digestive enzymes: trypsin, amylase and lipase (the last one against three different length chain substrates), along the larval development, a comparison between enzyme profiles at three different ages (6, 20 and 32 dph) was carried out after normalization of the values. For this, average daily values (RFU/mg larvae) were calculated for each age and the value corresponding to the age with the highest enzymatic activity was considered 100%.

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

Using this relation, percentages corresponding to the other two ages were calculated for each enzyme. This normalization allowed comparison between results obtained for different enzyme activities measured with different substrates. 2.6. Statistical analysis Differences in gut content, soluble protein concentration and activity of enzymes between sampling moments were evaluated using a oneway ANOVA after assessing equality of variances by a Levene's test. Post hoc multiple comparisons were carried out using Tukey's test (P N 0.05). All statistical analyses were performed with SPSS 15.0 software (IBM, New York, USA). Results are given as means and standard deviations (SD). 3. Results

35

period and to decrease after light were turned off (Fig. 2B). Significantly higher values of trypsin activity were detected at 22:00 h (ZT-13) in larvae 3 dph (249.99 RFU/mg larvae) and at 20:00 h (ZT-11) in larvae 4 dph (237.57 RFU/mg larvae). In contrast, the pattern of lipase activity was not directly connected to the presence of food in the gut of larvae, although important differences, both qualitative and quantitative, were detected when comparing the lipase activities toward the three different substrates (Fig. 2C, D, E). Then, higher values of activity were measured toward butyrate and heptanoate, which ranged between 10.89–73.62 and 9.67–53.00 RFU/mg larvae, respectively, while much lower activity was measured toward oleate (between 0 and 15.91 RFU/mg larvae). In addition, while this latter activity was present before larvae started exogenous feeding and almost disappeared from 2 dph onwards, an opposite pattern was detected in the previous ones, characterized by a net increase and a more or less maintained production, irrespective of the amount of gut contents. No amylase activity was detected in larvae at this age.

3.1. Daily food intake pattern Larvae exhibited linear growth during the period of study (Fig. 1). Exogenous feeding started at 2 dph (Fig. 2) in Senegalese sole larvae. At that age, 70% of larvae showed preys within the gut at 22:00 h (ZT-13). Since 3 dph at 16:00 h (ZT-7), gut content was observed in 100% of larvae. Feeding pattern at first stages (2, 3 and 4 dph) showed an increase in gut content during the light hours, peaking just after lights were turned off. During dark hours gut content showed a significant decrease. At 6 dph (Fig. 2) the feeding pattern was kept similar to the above described, with a gradual increase in gut content during light hours, a peak between 20:50 and 22:00 h (ZT-12 and ZT-13), and a further emptying of the gut during darkness. In post-metamorphic stages (Fig. 3), significant differences in gut content were not found, although a slight trend to a higher gut content at night was detected, being this trend more pronounced at 32 dph than at 20 dph. 3.2. Daily activity of digestive enzymes 3.2.1. Very young larvae (1–4 dph) In these larvae the pattern of trypsin activity was closely related to the presence of gut content; it showed a trend to increase during light

3.2.2. Six dph larvae In these larvae trypsin activity was maintained in close relation to the presence of food in the gut (Fig. 2B), showing an increase during the light hours that reached a maximum at 03:00 h (ZT-18) (305.56 RFU/mg larvae) and decreasing from that moment onwards. In contrast to what described for younger larvae, the activity patterns of lipase toward the 3 different substrates showed to be much more similar (Fig. 2C, D, E), being the more noticeable features the progressive increase in the activity of lipase toward heptanoate and oleate from the onset of feeding and the maintained activity toward butyrate irrespective of the hour or the presence of food in the gut. Amylase activity was detected at this age either. 3.2.3. Twenty dph larvae Again, the activity of trypsin was closely connected to the presence of food in the gut and showed almost absence of variations along the day (average value during the day: 331.33 RFU/mg larvae; Fig. 3B), with the exception of the significantly lower values measured at the moment of turning the lights on. In contrast to what described for younger larvae, amylase activity was clearly detected in 20 dph larvae and showed a pattern of activation from the beginning of sampling followed by high values during the whole period (from 10.90 to 20.73 RFU/mg larvae) and a certain, but not significant trend to increase during

Fig. 1. Growth of S. senegalensis: dry weight (solid line) and total length (dashed line). Values are represented as means ± SD (n = 10).

36

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

Fig. 2. Gut content (-•-) and percentage of larvae with gut content (-○-) (A), trypsin (B), 4C-like lipase (C), 7C-like lipase (D) and 18C-like lipase activities (E) in pre-metamorphic Senegalese sole larvae (first stages of life and 6 dph). Activity values are expressed as RFU/mg larval dry weight. Results are represented as means ± SD (n = 10). Letters mean significant differences between hours. Gray areas represent dark periods.

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

darkness hours (Fig. 3C). The activity of lipase toward 7 and 18 carbon substrates showed a similar pattern, with maintained levels of activity during the day that increased at night, while lipase activity toward 4 carbon substrate did not show significant differences in activity between hours (Fig. 3D, E, F). 3.2.4. Thirty-two dph larvae In these larvae, trypsin activity increased at first hours in the morning and kept maintained along the day, with a trend to decrease at late night (from 03:00 h, ZT-18, onwards; Fig. 3B). In the case of amylase not significant differences in activity were detected between hours, although three peaks corresponding to 12:00, 20:50 and 06:00 h were detected (ZT-3, 12 and 21, respectively; Fig. 3C). The pattern obtained for lipases was greatly coincident to that described for 20 dph larvae; a constant production of lipase toward butyrate during the whole day, but a clear increase of the activities toward heptanoate and oleate during the darkness period (Fig. 3D, E, F). 3.3. Soluble protein concentration It was not possible to analyze the concentration of soluble protein in the youngest larvae (1, 2, 3 and 4 dph), due to lack of sensitivity of the Bradford method to such a low amounts. The concentration of soluble protein in extracts from 6 dph larvae ranged between 0.0019 and 0.0160 mg soluble protein/mg larvae dry weight (Fig. 4A). The values tended to increase during the light period, peaking at 22:00 h (ZT-13) with a subsequent trend to decrease. At 20 dph (Fig. 4B) the lowest values in soluble protein concentration were found at 03:00 (ZT-18) and 06:00 h (ZT-21) (0.109 and 0.103 mg soluble protein/mg larvae DW, respectively), while the highest protein content was recorded at 09:10 + 1 h (ZT-24) (0.254 mg soluble protein/mg larvae DW); there were no significant differences between the rest of samples. In Senegalese sole larvae of 32 dph (Fig. 4C), soluble protein concentration showed a trend to decrease during the light and increase during the dark period, achieving the maximum value at 06:00 h (ZT-21) (0.422 mg soluble protein/mg larvae DW). 3.4. Comparative enzyme production between ages The comparison of the relative development of the different enzyme activities throughout larval development resumed in Fig. 5 revealed a different pattern for each one. While trypsin and amylase increased in activity with age, the relative production of the lipase activity toward butyrate maintained constant while that toward substrates of a longer chain was significantly reduced. 4. Discussion 4.1. Daily food intake pattern The results obtained in the present study showed a close relation between feeding activity and the light/dark cycle in Senegalese sole larvae. The larvae started to eat quickly when compared with other marine fish (Yúfera, 2011) and 80% of the population was able to ingest rotifers several hours after the opening of the mouth. The second day of feeding the whole population was able to eat only some hours after the lights turned on. While pre-metamorphic, fish mostly consumed food during the light period, post-metamorphic larvae showed a continuous feeding pattern along the whole day, with a trend of higher gut content during the dark hours at the end of the experimental period. It seems that sole larvae change from visual consumers in the pre-metamorphic stage, with a clear dependency from light, to chemical and mechanical consumers after metamorphosis. Food is detected via a wide range of chemical, visual and mechanical stimuli. Sensory organ development and prey detection since larvae hatching have been broadly studied

37

in marine fish larvae (Blaxter, 1986; Evans and Browman, 2004; Fukuhara, 1985; Osse and van den Boogaart, 1999; Padrós et al., 2011; Pankhurst, 2008; Pavlov and Kasumyan, 1990; Rønnestad et al., 2013). In general, vision plays the most important role in prey detection in the larvae of most fish species, although some species are capable of eating and growing independent of light or in situations with low light availability (Chesney, 1989; Downing and Litvak, 2001; Mukai et al., 2008; Rønnestad et al., 2013). These species use the help of other sensory organs for food detection. In haddock larvae, feeding success was evaluated using different light-spectrums and intensities. In the above mentioned study, significantly greater feeding success was recorded at intermediate light intensity and when exposed to blue light, which indicate that artificial light conditions for optimal feeding in haddock larvae were those that resembled open ocean nursery grounds (Downing and Litvak, 2001). Similar results have been described for European sea bass larvae (Villamizar et al., 2009), where the best feeding performance was achieved under the light conditions that best approached those of their natural aquatic environment (blue light, 12 light:12 dark). Light may also make conditional to feeding anticipatory activity (FAA) due to a synchronization of activity rhythms to light, as seen in goldfish where it has been revealed that FAA is driven by an endogenous timing system that may be trained by periodic feeding and influenced by the light regime (Aranda et al., 2001). In the case of Senegalese sole, recent research has revealed that spawning and hatching rhythms, larval development, and growth performance are strongly influenced by lighting conditions (Blanco-Vives et al., 2012). Also, the impact of different photo-cycles of distinct wavelengths has been investigated (Blanco-Vives et al., 2010, 2012). Results showed again that the best performance, fastest development and lowest degree of deformity were achieved in larvae reared under conditions similar to their natural aquatic environment (blue light, 12 light:12 dark), and under this photoperiod regime larval activity changed from diurnal to nocturnal on days 9 to 10 dph, and had the highest prey capture success rate. The effect on feeding of two different photoperiods (14 L:10D and 24 L:0D) have been also analyzed in sole larvae (Cañavate et al., 2006), where it was observed that pre-metamorphic larvae depended on light to capture rotifers and from 10 dph onwards larvae were feeding both during light and dark phases. These results agree with our findings because they suggest that from 10 dph others stimuli than the visual one may help larvae to catch food, allowing feeding during both light and dark periods. The daily feeding activity linked to protein metabolism in Senegalese sole post-larvae (35 dph) has been analyzed using radiolabeled Artemia (Navarro-Guillén et al., 2014). In accordance to the present study, our previous results (Navarro-Guillén et al., 2014) revealed that the development of sole larvae is tightly controlled by light characteristics, and suggested the existence of a feeding rhythmicity in Senegalese sole post-larvae with a broad feeding peak after the onset of the light and another one during the dark phase. This change in daily feeding pattern through development has been also described in Tongue sole (Cynoglossus semilaevis) (Ma et al., 2006), in which different feeding rhythms in the pre- and post-metamorphosis stages were found, with the highest feeding activity in the day-time during the larval planktonic stage, and at night during the juvenile benthic stage. Therefore, our present results suggest that one month might not be enough to completely cover the transition period from diurnal to nocturnal behavior in Senegalese sole larvae, since, mainly nocturnal habits have been identified in sole juveniles (Bayarri et al., 2004). 4.2. Daily activity of digestive enzymes In the present study, different digestive enzyme activities were analyzed during a whole day and at four different developmental stages of Senegal sole larvae. Significant differences in the studied enzyme activities were found in relation to time of the day, light conditions, feed gut content and fish size. Trypsin activity at pre-metamorphic stages was

38

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

Fig. 3. Gut content (A), trypsin (B), amylase (C), 4C-like lipase (D), 7C-like lipase (E) and 18C-like lipase activities (F) in post-metamosphic Senegalese sole larvae (20 and 32 dph). Activity values are expressed as RFU/mg larval dry weight. Results are represented as means ± SD (n = 10). Letters mean significant differences between hours. Gray areas represent dark periods.

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

closely related to the amount of food present in the digestive tract while in post-metamorphic larvae the activity kept more or less constant values during the whole day, with the exception of first and last hours in the day, when activity levels were lower. At 6 dph, decrease in trypsin activity was recorded when gut was almost empty. This decrease from 03:00 h (ZT-18) was also recorded in 20 and 32 dph larvae, independently of gut content. In larvae at first stages (pre-metamorphic larvae), the postprandial tryptic response might be expected in order to save energy and nutrients, being the enzymes produced only when needed in very fast growing stages where processes and organs are not yet fully developed. The absence of the indicated correlation between trypsin activity and food contents in the gut found in 32 dph larvae as the darkness period advanced (from 03:00 h onwards, ZT-18) has been also described in turbot larvae fed three times a day with rotifers (Rønnestad et al., 2013). In this study the larvae reacted to the first two meals with immediate trypsinogen secretion, but there was no response to the third meal in the afternoon. These results revealed a limited digestive ability of the larvae to maintain a continuous production of certain enzymes or the result of a negative feedback between the satiety factor cholecystokinin (CCK) and the peptidase enzyme trypsin, already described in Atlantic cod larvae (Tillner et al., 2013). Additional studies about the role of CCK on appetite regulation in Senegalese sole larvae would be necessary to confirm this hypothesis. In the present study, amylase activity was not detected in very young larvae, being measured only in 20 and 32 dph larvae. These results do not agree with previous studies where amylase activity was identified in sole larvae during the first developmental stages (Martínez et al., 1999; Ribeiro et al., 1999b). This might be due to the different methodologies used; in the present study enzyme activities were analyzed using a single larva and fluorescent substrates, while the aforementioned works used pool of larvae and spectrophotometry for activity assays. Regarding lipase, considering the great differences in the total values and daily patterns observed (Figs. 1C, 2C, 3D and 4D) clearly the activities measured toward the different substrates interestingly seem to correspond to different types of enzymes. Unlike trypsin, lipase activity was not linked to the amount of food present in the gut. In very young larvae, the lipase activity toward oleate was high prior the onset of exogenous feeding and decreased after mouth opening when larvae started exogenous feeding activity. This may be related to the presence in the yolk sac of Senegalese sole of glycoproteins and long chain neutral lipids (Ribeiro et al., 1999a). The presence of specific lipases related to yolk-sac absorption which are different to those related to digestion of exogenous lipids has been previously pointed out by Oozeki and Bailey (1995). 4.3. Soluble protein concentration Although the expression of activities in relation to soluble protein is the most common way to represent ontogenic changes in digestive enzymes (Babaei et al., 2011; Gisbert et al., 2009; Martínez et al., 1999; O'Brien-MacDonald et al., 2006; Ribeiro et al., 1999b) it provides limited information on their real significance, since in fast growing organisms like fish larvae or juveniles, it is affected by the changing proportion of soluble protein in relation to total body components. Also, in the present study it was supposed that soluble food proteins still present within the digestive tract at the moment of sampling should result in a high bias. As shown in Fig. 4, it was demonstrated that the relative contribution of this pool of soluble protein to the total soluble protein in samples changes with age as while in 6 dph larvae, a great correlation between changes in gut content and values of soluble protein was found; such correlation (and hence the suggested relevance of the suggested effect) was not so clear in older larvae. This probably means that the importance of dietary proteins within the protein pool in the larvae at early stages, were still higher than functional and structural proteins. Our

39

results in the present study indicate that soluble protein analyzed came principally from food proteins, and depending on the feeding behavior of the species the protein concentration will change along the day. For this reason, the expression of activities in relation to dry weight of larvae used in the present work was considered more accurate, taking into account that each extract was prepared using a single complete individual. In this way, the activity values were linked to the larval condition, and not to dietary protein content this allowing making comparisons between different larval states. 4.4. Comparative enzyme production between ages Previous results about enzymatic ontogeny throughout larval development have been presented, mainly, as specific activity. A typical pattern for trypsin and amylase activities has been described at first stages in various species of fish larvae, characterized by an increase of specific enzymatic activity during the first 10 days post-hatching, followed by a decrease (Gamboa-Delgado et al., 2011; Kolkovski, 2001; Rønnestad et al., 2013; Zambonino-Infante and Cahu, 2001). This pattern has been also identified in Senegalese sole larvae (Conceição et al., 2007; Martínez et al., 1999; Ribeiro et al., 1999b). This decline in specific enzyme activity is not due to a diminution in enzyme synthesis but is the result of an increase in tissue proteins (Zambonino-Infante and Cahu, 2001). In the present study, the ontogeny of the digestive capacity has been also analyzed as relative activity (Fig. 5). In this comparison, the decline of an enzyme activity along the ontogeny would indicate that this enzyme is not as important as other enzymes whose activity levels remained stable or increase. Specifically, the ability of Senegalese sole to digest proteins and carbohydrates increases sharply whereas that of their ability to digest medium and long-chain lipids decreases along larvae development, although the ability to digest shortchain lipids is maintained stable. Accordingly, recently, a gradual increase in trypsin and amylase transcript levels throughout ontogeny has been identified in common sole (Solea solea) (Parma et al., 2013). In a previous study which measured lipase activity along Senegalese sole larval development, the highest values were achieved at ages corresponding to the metamorphic process. These higher values were associated to a possible decrease in feed intake and the need to hydrolyze stored lipids during this period (Martínez et al., 1999). It is known that some flatfish species stop feeding during metamorphosis (Tanaka et al., 1996). In the case of Senegalese sole, subsequent studies identified that this species continues to eat during metamorphosis, although the total daily amount ingested remains almost constant during this stage in spite of the fact that the body continues to grow. This factor entails a decrease in specific ingestion rate, which might be compensated with an increase in the hydrolysis of stored lipids (Parra and Yúfera, 2001). In the present study, the enzymatic activity during the metamorphic period was not studied. It could be expected an increase in lipase activity throughout development due to the capacity to digest complex substrates increase with age, but this decrease in activity might be due to 6 dph larvae still have high quantity of lipases for yolk-sac absorption and once that it is completely absorbed lipases activity were stabilized. In conclusion, this study helps to understand Senegalese sole larval daily feeding behavior and digestive enzyme activity. Sole larvae showed changes in their feeding pattern throughout development. Pre-metamorphic Senegalese sole larvae showed a close relation between feeding activity and illumination cycle, ceasing the food consumption after lights were turned off. In contrast, post-metamorphic larvae showed a continuous feeding pattern along the whole day, with a trend of higher gut content during the dark period. Regarding the digestive enzyme activity, sole larvae appear to have a good digestive capacity, exhibiting digestive enzyme activity before mouth opening, mainly lipase activity. Tryptic activity was related to gut content at pre-metamorphic stages, whereas activity levels were kept along the day in post-metamorphic larvae. Although lipase activity was not linked

40

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

Fig. 4. Protein content (second axis; A—6 dph; B—20 dph; C—32 dph) in Senegalese sole larvae. Area graphs represent gut content at each age (first axis). Results are represented as means ± SD (n = 10). Letters mean significant differences in larval protein content between hours. Gray areas represent dark periods.

to the amount of food present in the gut, 18-carbon like lipase seems to be the digestive enzyme more synchronized with the presence of gut content in Senegalese sole larvae.

The results offered about the ontogenetic changes in digestive enzymes of Senegalese sole larvae might be useful to understand the kind of nutrients that might be mainly supplied to the larvae along

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

Fig. 5. Relative activity of digestive enzymes in Senegalese sole larvae at 6, 20 and 32 dph. Data set shown for trypsin (-•-), amylase (..○..), 4-carbon (-▼-), 7-carbon (-Δ-) and 18-carbon (-■-) like lipases. Results are represented as relative activity (RFU/mg fish DW) normalized to percentage. Letters and numbers mean significant differences between ages.

their development. This point, together with a better understanding of Senegalese sole larvae daily feeding pattern, may allow the improvement of current feeding protocols of the species, being important for both research and industry. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgments The authors wish to thank J.A. Mata for practical assistance. This research was funded by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) by project RIDIGEST (AGL2011-23722). C. Navarro-Guillén was supported by a doctoral fellowship (BES-2012051956) from MINECO (Spain). References Aranda, A., Madrid, J.A., Sánchez-Vázquez, F.J., 2001. Influence of light on feeding anticipatory activity in Goldfish. J. Biol. Rhythm. 16, 50–57. Babaei, S.S., Abedian Kenari, A., Nazari, R., Gisbert, E., 2011. Developmental changes of digestive enzymes in Persian sturgeon (Acipenser persicus) during larval ontogeny. Aquaculture 318, 138–144. Bayarri, M.J., Muñoz-Cueto, J.A., López-Olmeda, J.F., Vera, L.M., Rol de Lama, M.A., Madrid, J.A., Sánchez-Vázquez, F.J., 2004. Daily locomotor activity and melatonin rhythms in Senegal sole (Solea senegalensis). Physiol. Behav. 81, 577–583. Blanco-Vives, B., Villamizar, N., Ramos, J., Bayarri, M.J., Chereguini, O., SánchezVázquez, F.J., 2010. Effect of daily thermo- and photo-cycles of different light spectrum on the development of Senegal sole (Solea senegalensis) larvae. Aquaculture 306, 137–145. Blanco-Vives, B., Aliaga-Guerrero, M., Cañavate, J.P., García-Mateos, G., Martín-Robles, A.J., Herrera-Pérez, P., Muñoz-Cueto, J.A., Sánchez-Vázquez, F.J., 2012. Metamorphosis induces a light-dependent switch in Senegalese sole (Solea senegalensis) from diurnal to nocturnal behavior. J. Biol. Rhythm. 27, 135–144. Blaxter, J.H.S., 1986. Development of sense organs and behaviour in teleost larvae with special reference to feeding and predation avoidance. Trans. Am. Fish. Soc. 115, 98–114. Boujard, T., Leatherland, J., 1992. Circadian rhythms and feeding time in fishes. Environ. Biol. Fish 35, 109–131. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cahu, C., Zambonino-Infante, J., 2001. Substitution of live food by formulated diets in marine fish larvae. Aquaculture 200, 161–180. Cañavate, J.P., Zerolo, R., Fernández-Díaz, C., 2006. Feeding and development of Senegal sole (Solea senegalensis) larvae reared in different photoperiods. Aquaculture 258, 368–377.

41

Chesney Jr., E.J., 1989. Estimating the food requirements of striped bass larvae Morone saxatilis: effects of light, turbidity and turbulence. Mar. Ecol. Prog. Ser. 53, 191–200. Conceição, L.E.C., Ribeiro, L., Engrola, S., Aragão, C., Morais, S., Lacuisse, M., Soares, F., Dinis, M.T., 2007. Nutritional physiology during development of Senegalese sole (Solea senegalensis). Aquaculture 268, 64–81. Dinis, M.T., Ribeiro, L., Soares, F., Sarasquete, C., 1999. A review on the cultivation potential of Solea senegalensis in Spain and in Portugal. Aquaculture 176, 27–38. Dou, S., Seikai, T., Tsukamoto, K., 2000. Feeding behaviour of Japanese flounder larvae under laboratory conditions. J. Fish Biol. 56, 654–666. Downing, G., Litvak, M.K., 2001. The effect of light intensity and spectrum on the incidence of first feeding by larval haddock. J. Fish Biol. 59, 1566–1578. Evans, B.I., Browman, H.I., 2004. Variation in the development of fish retina. Am. Fish. Soc. Symp. 40, 145–166. Fernández-Díaz, C., Yúfera, M., Cañavate, J.P., Moyano, F.J., Alarcón, F.J., Díaz, M., 2001. Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory. J. Fish Biol. 58, 1086–1097. Fukuhara, O., 1985. Functional morphology and behavior of early life stages of Red Sea bream. Nippon Suisan Gakk. 51, 731–743. Gamboa-Delgado, J., Le Vay, L., Fernandez-Diaz, C., Canavate, P., Ponce, M., Zerolo, R., Manchado, M., 2011. Effect of different diets on proteolytic enzyme activity, trypsinogen gene expression and dietary carbon assimilation in Senegalese sole (Solea senegalensis) larvae. Comp. Biochem. Physiol. B 158, 251–258. Gisbert, E., Giménez, G., Fernández, I., Kotzamanis, Y., Estévez, A., 2009. Development of digestive enzymes in common dentex Dentex dentex during early ontogeny. Aquaculture 287, 381–387. Goodrich, R.P., Sowemimo-Coker, S.O., Zerez, C.R., Tanaka, K.R., 1992. Preservation of metabolic activity in lyophilized human erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 89, 967–971. Imsland, A.K., Foss, A., Conceição, L.E.C., Dinis, M.T., Delbare, D., Schram, E., Kamstra, A., Rema, P., White, P., 2003. A review of the culture potential of Solea solea and S. senegalensis. Rev. Fish Biol. Fish. 13, 379–408. Kolkovski, S., 2001. Digestive enzymes in fish larvae and juveniles—implications and applications to formulated diets. Aquaculture 200, 181–201. Kotani, T., Fushimi, H., 2011. Determination of appropriate feeding schedules from diel feeding rhythms in finfish larviculture. Aquaculture 315, 104–113. Lau, C.C., Abdullah, N., Aminudin, N., Shuib, A.S., 2013. Effect of freeze-drying process on the property of angiotensin I-converting enzyme inhibitory peptides in grey oyster mushrooms. Dry. Technol. 31, 1693–1700. Lazo, J.P., Holt, G.J., Arnold, C.R., 2000. Ontogeny of pancreatic enzymes in larval red drum Sciaenops ocellatus. Aquacult. Nutr. 6, 183–192. Ma, A.J., Liu, X.Z., Xu, Y.J., Liang, Y., Zhuang, Z.M., 2006. Feeding rhythm and growth of the tongue sole, Cynoglossus semilaevis Günther, during its early life stages. Aquac. Res. 37, 586–593. Martínez, I., Moyano, F.J., Fernández-Díaz, C., Yúfera, M., 1999. Digestive enzyme activity during larval development of the Senegal sole (Solea senegalensis). Fish Physiol. Biochem. 21, 317–323. Mukai, Y., Tuzan, A.D., Lim, L.S., Wahid, N., Sitti Raehanah, M.S., Senoo, S., 2008. Development of sensory organs in larvae of African catfish Clarias gariepinus. J. Fish Biol. 73, 1648–1661. Navarro, D.B., Rubio, V.C., Luz, R.K., Madrid, J.A., Sánchez-Vázquez, F.J., 2009. Daily feeding rhythms of Senegalese sole under laboratory and farming conditions using selffeeding systems. Aquaculture 291, 130–135. Navarro-Guillén, C., Yúfera, M., Engrola, S., 2014. Senegalese sole post-larvae exhibit daily rhythmicity of feed intake and protein metabolism. Aquaculture Europe 2014. San Sebastián, Spain. October 14–17. O'Brien-MacDonald, K., Brown, J.A., Parrish, C.C., 2006. Growth, behaviour, and digestive enzyme activity in larval Atlantic cod (Gadus morhua) in relation to rotifer lipid. ICES J. Mar. Sci. 63, 275–284. Oozeki, Y., Bailey, K.M., 1995. Ontogenetic development of digestive enzyme activities in larval walleye pollock, Theragra chalcogramma. Mar. Biol. 122, 177–186. Osse, J.W.M., van den Boogaart, J.G.M., 1999. Dynamic morphology of fish larvae, structural implications of friction forces in swimming, feeding and ventilation. J. Fish Biol. 55, 156–174. Padrós, F., Villalta, M., Gisbert, E., Estevez, A., 2011. Morphological and histological study of larval development of the Senegal sole Solea senegalensis: an integrative study. J. Fish Biol. 79, 3–32. Pankhurst, P.M., 2008. Mechanoreception. In: Finn, R.N., B.G., K. (Eds.), Fish Larval Physiology. Science Publisher, Enfield, NH, pp. 305–329. Parma, L., Bonaldo, A., Massi, P., Yúfera, M., Martínez-Rodríguez, G., Gatta, P.P., 2013. Different early weaning protocols in common sole (Solea solea L.) larvae: implications on the performances and molecular ontogeny of digestive enzyme precursors. Aquaculture 414–415, 26–35. Parra, G., Yúfera, M., 2001. Comparative energetics during early development of two marine fish species, Solea senegalensis (Kaup) and Sparus aurata (L.). J. Exp. Biol. 204, 2175–2183. Pavlov, D.V., Kasumyan, A.O., 1990. Study of fish behavior and sensory systems in Russia. J. Ichthyol. 34, 1–26. Polo, A., Yúfera, M., Pascual, E., 1992. Feeding and growth of gilthead seabream (Sparus aurata, L.) larvae in relation to the size of the rotifer strain used as food. Aquaculture 103, 45–54. Ribeiro, L., Sarasquete, C., Dinis, M.T., 1999a. Histological and histochemical development of the digestive system of Solea senegalensis (Kaup, 1858) larvae. Aquaculture 171, 293–308. Ribeiro, L., Zambonino-Infante, J.L., Cahu, C., Dinis, M.T., 1999b. Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858. Aquaculture 179, 465–473.

42

C. Navarro-Guillén et al. / Aquaculture 435 (2015) 33–42

Ribeiro, L., Couto, A., Olmedo, M., Álvarez-Blázquez, B., Linares, F., Valente, L.M.P., 2008. Digestive enzyme activity at different developmental stages of blackspot seabream, Pagellus bogaraveo (Brunnich 1768). Aquac. Res. 39, 339–346. Rønnestad, I., Yúfera, M., Ueberschär, B., Ribeiro, L., Sæle, Ø., Boglione, C., 2013. Feeding behaviour and digestive physiology in larval fish: current knowledge, and gaps and bottlenecks in research. Rev. Aquac. 5, S59–S98. Rotllant, G., Moyano, F.J., Andrés, M., Díaz, M., Estévez, A., Gisbert, E., 2008. Evaluation of fluorogenic substrates in the assessment of digestive enzymes in a decapod crustacean Maja brachydactyla larvae. Aquaculture 282, 90–96. Sanchez-Vázquez, F.J., Madrid, J.A., Zamora, S., 1995. Circadian rhythms of feeding activity in sea bass, Dicentrarchus labrax L.: dual phasing capacity of diel demand-feeding pattern. J. Biol. Rhythm. 10, 256–266. Spigno, G., Tramelli, L., De Faveri, D.M., 2007. Effects of extraction time, temperature and solvent on concentration and antioxidant activity of grape marc phenolics. J. Food Eng. 81, 200–208. Tanaka, M., Kawai, S., Seikai, T., Burke, J.S., 1996. Development of the digestive organ system in Japanese flounder in relation to metamorphosis and settlement. Mar. Freshw. Behav. Physiol. 28, 19–31.

Tillner, R., Rønnestad, I., Harboe, T., Ueberschär, B., 2013. Hormonal control of tryptic enzyme activity in Atlantic cod larvae (Gadus morhua): involvement of cholecystokinin during ontogeny and diurnal rhythm. Aquaculture 402–403, 133–140. Ueberschär, B., 1995. The use of tryptic enzyme activity measurement as a nutritional condition index: laboratory calibration data and field application. In: Pittman, K.B., R.S., Verreth, J. (Eds.), Mass Rearing of Juvenile Fish: a symposium held in Bergen, 21–23 June 1993ICES Marine Science Symposia. ICES, Kopenhagen, pp. 119–129. Villamizar, N., García-Alcazar, A., Sánchez-Vázquez, F.J., 2009. Effect of light spectrum and photoperiod on the growth, development and survival of European sea bass (Dicentrarchus labrax) larvae. Aquaculture 292, 80–86. Yamamoto, S., Fujii, Y., Murata, M., 2003. Developmental changes in diurnal feeding activity and daily food consumption in Ayu, Plecoglossus altivelis larvae. Suisan Zoshoku 51, 73–80. Yúfera, M., 2011. Feeding behavior in larval fish. In: Holt, G.J. (Ed.), Larval Fish Nutrition. Wiley-Blackwell, Ames, Iowa, pp. 285–305. Zambonino-Infante, J.L., Cahu, C.L., 2001. Ontogeny of the gastrointestinal tract of marine fish larvae. Comp. Biochem. Physiol. C 130, 477–487.