The influence of diet on the early development of two seahorse species (H. guttulatus and H. reidi): Traditional and innovative approaches

Aquaculture 490 (2018) 75–90

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The influence of diet on the early development of two seahorse species (H. guttulatus and H. reidi): Traditional and innovative approaches

T

Randazzo B.a,1, Rolla L.a,b,1, Ofelio C.b, Planas M.b, Gioacchini G.a, Vargas A.a, Giorgini E.a, ⁎ Olivotto I.a, a

Dipartimento di Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy Departamento de Ecología y Recursos Marinos Instituto de Investigaciones Marinas - Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain

b

A R T I C L E I N F O

A B S T R A C T

Keywords: Copepods Artemia Fatty acids FPA-FTIR Imaging spectroscopy Seahorse Live prey Feeding

Larval nutrition plays a key role in the development of a sustainable aquaculture where fish development, health and wellness are of prime importance. For some species, satisfactory growth and survival rates are met providing exclusively enriched rotifers and Artemia; however, feeding on copepods during the larval period has been shown to improve growth in both larval and juvenile fish, including seahorses. For the first time, the effects of different diets (Artemia and copepods) on the early development of juvenile seahorses (H. guttulatus and H. reidi) development were analysed by combining biometry, traditional histology and FPA-FTIR Imaging spectroscopy. Survival and growth and biochemical composition on the liver in seahorse were significantly affected by the type of diet offered. The results achieved were related to differences in the digestion of the two types of live preys, mainly dependent on their biochemical composition and permeability of the exoskeleton.

1. Introduction The high demand of seahorses for the aquarium trade, the traditional Chinese medicine and as souvenirs, in addition to the destruction and degradation of their coastal habitats (seagrasses, coral reefs and mangroves), has raised many concerns over their long-term viability in nature (Vincent, 1996; Lourie et al., 1999; Vincent et al., 2011, Kumaravel et al., 2012). Currently, all seahorses species are included in Appendix II list of endangered species by CITES (Convention for the International Trade in Endangered Species), restricting the legal import and export of dead or alive seahorses. The ex-situ production of seahorses has the potentiality to represent a valid partial alternative to wild caught specimens but for most species such activity is still a relative new field that need the assessment of some technical challenges (Lourie et al., 1999; Foster and Vincent, 2004; Koldewey and Martin-Smith, 2010; Cohen et al., 2016; Planas et al., 2017). One of the critical bottlenecks that culturists have to face in seahorse rearing is the low survival in early developmental stages (Scaratt, 1995; Payne and Rippingale, 2000; Olivotto et al., 2008a). Such constrain is due to many factors related to environmental and zoothecnical conditions and feeding, among others (Woods, 2000; Chang and Southgate, 2001; Sheng et al., 2006; Olivotto et al., 2008b;

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Planas et al., 2017). Feeding and nutrition play a key role in the growth and survival of juveniles. Live prey such as rotifers, Artemia, Mysidiacea and copepods are required in the rearing of many seahorse species. In most cases, Artemia nauplii and rotifers (Brachionus sp.) are the first option because they can be easily cultured in large quantities at high densities (Olivotto et al., 2008c). However, rotifers and Artemia do not represent the natural food of seahorses, and do not always provide adequate fatty acid profiles. In addition, these prey do not always provide the sizes required by young seahorse (Faulk and Holt, 2005). Furthermore, difficulties are known regarding their digestion by early stages in some seahorse species (Olivotto et al., 2011; Blanco et al., 2015). Therefore, alternative prey, other than rotifers and Artemia, are explored with great interest by the scientific community (Calado et al., 2017). In the wild, adult seahorses feed on a variety of prey (mostly crustaceans) (Teixeira and Musick, 2001; Woods, 2002; Kendrick and Hyndes, 2005; Kitsos et al., 2008; Storero and González, 2008; Gurkan et al., 2011; Valladares et al., 2017), including copepods (Tipton and Bell, 1988; Franzoi et al., 1993) but the natural diet of young seahorses is still unknown. It has been reported that feeding young pipefish and seahorses on cultured copepods may significantly improve survival rates and growth (Payne et al., 1998; Payne and Rippingale, 2000; Olivotto et al., 2008a; Blanco

Corresponding author. E-mail address: [email protected] (I. Olivotto). The two authors equally contributed to this study.

https://doi.org/10.1016/j.aquaculture.2018.02.029 Received 21 December 2017; Received in revised form 13 February 2018; Accepted 19 February 2018 0044-8486/ © 2018 Elsevier B.V. All rights reserved.

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2.2. Breeding conditions

and Planas, 2015). Delbare et al. (1996) summarized the advantages of using copepods in larviculture, such as a wider size range, a typical movement, and a high and optimal content in essential highly unsaturated fatty acids (HUFAs). These fatty acids, particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n3), are extremely important for fish survival and growth, being essential in larval diets (Sargent et al., 1999). Any failure or limitation in nutrients or energy uptake may affect the adeguate development of organs and structures, and consequently, further growth and survival. Since the gastrointestinal tract and associated organs may account for up to 40% of an animal's metabolic rate (Cant et al., 1996), it is understandable that problems in digestion or periods of malnutrition could affect the digestive tract resulting in further serious functional disorders (Olivotto et al., 2011; Piccinetti et al., 2012). Therefore, degeneration and composition of tissues in some digestive organs such as intestine and liver are probably the best indicators for the identification of starvation conditions (Diamond and Hammond, 1992; Wang et al., 2006; Olivotto et al., 2011). Currently, there are several well established traditional analytical techniques and methods available for gut/liver analysis, including visual and microscopic observations as well as biochemical, molecular and proteome analysis. Those methodologies play a pivotal role in the analysis of fish gut/liver and some have been used as gold standards and standardized methods serving scientific researches due to their relative validity and accuracy. However, these techniques and methods are normally expensive, time-consuming, laborious and in some cases difficult to apply to seahorses. The present study investigates the effects of different diets on the composition of seahorses liver combining, for the first time, traditional histology and Focal Plane Array Fourier Transform Infra-Red (FPAFTIR) Imaging spectroscopy. FTIR spectroscopy is a fast, label-free analytical technique widely applied for investigating functional groups, bonding types, and molecular conformations of the most relevant biological molecules. This technique analyzes the vibrational transitions due to the interaction between matter and the infrared radiation and allows to obtain at the same time and on the same sample important information on the whole molecular composition of the analysed sample. FPA FT-IR Imaging spectroscopy couples the potential of visible microscopy, which permits a visual inspection of the sample, with innovative focal plane array detectors, which let simultaneously acquire small areas with a spatial resolution near to the diffraction. This analytical technique allows to perform the imaging analysis of nonhomogeneous biological samples in terms of composition and structure of the most relevant biomolecules (Gioacchini et al., 2014; Giorgini et al., 2015a, 2015b), and, hence, monitor the biochemical modifications, either induced or naturally occurring, in human and animal tissues and cells. In particular, it has been successfully applied to evaluate biochemical changes in liver of several fish species under different feeding conditions (Gioacchini et al., 2014; Carnevali et al., 2017; Forner-Piquer et al., 2017). On continuing to investigate this topic, in the present study, FPA-FTIR Imaging spectroscopy was applied, for the first time, to analyze the effects of different diets (rotifers, Artemia and copepods) on seahorses liver samples, with the aim to identify appropriate spectral biomarkers correlatable with the biochemical changes in tissues composition. The spectral data were also complemented with biometry and traditional histology outcomes.

Newborn seahorses were obtained from broodstock of the seahores Hippocampus reidi (tropical species) and Hippocampus guttulatus (temperate species). Breeders were maintained in four aquaria units connected to a recirculation system and under moderate aeration (Planas et al., 2008). Each husbandry aquaria consisted of three subunits of 160 L each (85 height × 75 length × 50 wide cm) working as an autonomous closed system. Water quality was checked periodically for NO2, NO3 and NH4/NH3 content (0 mg L−1). Salinity and pH levels were 37 ± 2 and 8.0 ± 0.2, respectively for both species. Temperature levels and photoperiods were adapted according to the needs of each species. A water temperature of 19 ± 0.5 °C and a 16L:8D photoperiod regime were applied for H. guttulatus during the breeding season (Planas et al., 2013), whereas a temperature of 26 ± 0.5 °C and a constant 14L:10D photoperiod (Olivotto et al., 2008c) were applied to H. reidi. Adult seahorses were fed twice daily on a diet consisting on long time enriched adult Artemia sp. (EG, AF, MC450; Iberfrost®, Spain; 40–70 Artemia seahorse−1 dose−1) (Planas et al., 2017) and frozen Mysis (3F®-Frozen Fish Food, Iberfrost, Spain). When available, a single daily dose of wild-captured Mysidacea (15–20 Leptomysis sp. and/or Siriella sp.) was also provided. Wastes and uneaten food were removed daily by siphoning the bottom of aquaria. 2.3. Rearing system Seahorses broodstocks were monitored several times a day to check for newborn release from male's pouch. Newly released juveniles were carefully collected by siphoning and transferred (5 juveniles L−1) to 30 L pseudo-Kreisel aquaria connected to a semi-opened recirculation. Temperatures and photoperiod were same as for breeders. The aquaria were filled with seawater filtered by a series of filter-cartridges (20, 10, 5 and 1 μm) and UV treated (76w; 16 L min−1) (JR1/50). The rearing system included a degasifying column and two 50 L chambers including mechanical (up to 20 μm) and biological filters (perforated plastic bioballs) and aerators. From the biofilter unit, the seawater was pumped to 36w UV units (AquaMedic®, Germany) and then to a 50 L reservoir aquarium, being finally routed by gravity towards the rearing aquaria (Illuminati et al., 2010; Planas et al., 2012). 2.4. Live prey culture Microalgae (Phaeodactylum tricornutum, Rhodomonas lens and Isochrysis galbana) were cultured at 22 ± 1 °C in 80 L plastic bags containing sterilized seawater supplemented with F2P (100 g L−1) media (VarAqua©). Additionally, silicates were added to P. tricornutum cultures, and 200 μL F2P media to the R. lens culture flasks. Artemia cysts (AF®, Inve, Spain) were hatched at 28 °C for 20 h in 20 L units, and the hatched nauplii gently rinsed with tap-water, collected on a 125 μm mesh, rinsed and transferred to 5 L buckets for metanauplii production (100 Artemia mL−1). Metanauplii of several ages and sizes (24 h, 48 h, 72 h and 96 h; Fig. 3.4) were enriched twice daily on a mixture consisting on live microalgae (P. tricornutum 107 cells mL−1), Red Pepper (0.015 g L−1), Bernaqua®, Belgium) and dried Spirulina (0.03 g L−1, Iberfrost®, Spain). Artemia nauplii and enriched metanauplii were used for feeding seahorse juveniles. Adult Artemia was produced for the feeding of adult seahorses. For that, the nauplii (EG, AF, MC450; Iberfrost®, Spain) were grown in 100 L units, at 26–28 °C with gentle aeration and constant light. A longtime enrichment (3–6 days) was carried out in Artemia from day 16 onwards on a mixture consisting on live microalgae P. tricornutum and I. galbana (107 cells mL−1), Red Pepper (0.015 g L−1, Bernaqua®, Belgium) and dried Spirulina (0.03 g L−1, Iberfrost®, Spain) (Planas et al., 2017). Copepods (Acartia tonsa) were cultivated for the early feeding of seahorse juveniles in 700 L tanks at 26–27 °C and 38 salinity, at an initial density of 1 copepod mL−1. Copepods were fed every two

2. Material and methods 2.1. Ethics Animal capture, handling and sampling were conducted in compliance with all bioethics standards on animal experimentation of the Spanish Government (Real Decreto 1201/2005, 10th October) and the Regional Government Xunta de Galicia (Ref. REGA ES 36057020 2001/ 16/FUN/BIOL.AN/MPO02). 76

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Fig. 1. Survivals in H. guttulatus (a) and H. reidi (b) juveniles fed on diets C, A and CA. Data are provided as mean ± standard deviation.

Fig. 2. Wet weights (mg) (a) and standard length (mm) (b) in H. guttulatus juveniles fed on diets C, A and CA. Data are provided as mean ± standard deviation.

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Fig. 3. Wet weights (mg) (a) and standard length (mm) (b) in H. reidi juveniles fed on diets C, A and CA. Data are provided as mean ± standard deviation.

days on the microalgae R. lens (103 cells mL−1). Siphoning of the culture tanks and water renewals (10% of the total volume) were carried out three times per week.

aquaria (N = 15 per aquarium) at 0, 5, 10, 20 and 30 DAR. Seahorses were anesthetized with Tricaine MS-222 (0.1 g L−1, Sigma Aldrich), transferred to Petri dishes and photographed for further length measurement. Standard lengths (SL) were obtained by means of an image processing software (NIS, Nikon, Spain) as the sum of head, trunk and tail length (curved measurement) (Lourie, 2003). Wet weight was analysed on a Sartorious microbalance MC210P ( ± 0.01 mg). For histological analysis, 5 seahorses of both species were also euthanized for each experimental group (C, A, and CA diets) at 0, 5, 10, 20 and 30 DAR and processed as described in Histology section. For FTIR analysis, additional 5 seahorses of both species were euthanized for each experimental group (C, A, and CA diets) at 10 and 30 DAR. From all euthanized fish, livers were completely removed under a stereomicroscope (Optech LFZ) and cryopreserved at −80 °C before cutting. Final survivals at 30 DAR were calculated considering dead and sampled individuals.

2.5. Experimental design Each batch of newborn seahorses was distributed in three experimental groups (n = 150 per group) in triplicate and fed on different prey during the first 10 days after release (DAR) as follows: - Group C: two daily doses of Acartia tonsa (0.67 copepods mL−1 dose−1). - Group A: two daily doses of Artemia nauplii (1 Artemia mL−1 dose−1). - Group CA: two daily doses of Acartia tonsa (0.67 copepods mL−1 dose−1) until 5 DAR and two daily doses of Artemia nauplii (1 Artemia mL−1 dose−1) from 6 to 10 DAR. From 11 DAR, all experimental groups were fed on the same diet, consisting on two daily doses of enriched Artemia metanauplii (1 Artemia mL−1). Each treatment was run in triplicate. Dead seahorses were daily removed (8:00 am and 15:00 pm) and counted. The same experimental design was used for both seahorse species. Chemical physical conditions were the same of brood stock (for details see Section 2.2).

2.7. Lipid content and fatty acids in prey Lipids from live prey (10–20 mg dry weight per sample) were extracted according to Bligh and Dyer (Bligh and Dyer, 1959). Aliquots of total lipid extracts with known lipid content were centrifuged, resuspended with 0.5 M ammonium formate solution, freeze-dried and stored at −80 °C until further analyses. Total lipid content was quantified gravimetrically (Herbes and Allen, 1983). Fatty acid (FA) composition of lipids was analysed by gas-chromatography (GC) according to Christie (1982). Lipids were transmethylated (Lepage and Roy, 1986) and fatty acids analysed by GC (Perkin Elmer, Clarus 500 gas chromatograph) as described in Planas et al. (2010). Samples were analysed in duplicate.

2.6. Sampling Live preys (Acartia tonsa and Artemia nauplii and metanauplii) were sampled in duplicate on different days, freeze dried and maintained at −80 °C until further lipids and fatty acids analysis. Seahorse juveniles were randomly sampled from each experimental 78

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Table 1 Fatty acids composition (relative percentage and mg per gram of dry weight) of prey (Artemia nauplii and metanauplii and Acartia tonsa copepods). Fatty acids

14:0 15:0 16:0 16:l n-7 17:0 18:0 18:l n-9 18:l n-7 18:2 n-6 18:3 n-6 20:0 18:3 n-3 20:l n-9 18:4 n-3 20:2 n-6 20:3 n-6 20:4 n-6 20:4 n-3 20:5 n-3 24:0 22:4 n-6 24:l n-9 22:5 n-6 22:5 n-3 22:6 n-3 Total Saturates Monounsaturates Polyunsaturates n-3 n-6 n-3 HUFA DHA/EPA DHA/ARA n-3/n-6

mg g−1 DW

Total fatty acids percentages Nauplii

Metanauplii

Acartia

Nauplii

Metanauplii

Acartia

2,34 ± 0,13 1,26 ± 1,20 16,4 ± 0,58 11,4 ± 3,13 2,45 ± 0,83 6,19 ± 1,00 13,6 ± 1,33 10,3 ± 1,99 5,91 ± 2,31 1,1 ± 0,22 0, 78 ± 0,44 6,33 ± 6,47 0, 74 ± 0,22 0,19 ± 0,02 0,47 ± 0,21 0,4 ± 0,00 3,3 ± 0,61 0,4 ± 0,01 12,4 ± 3,27 0,17 ± 0,00 0,04 ± 0,06 0 ± 0,00 1 ± 0,87 0,27 ± 0,19 2,57 ± 2,19 100 29,6 ± 1,0 36 ± 6,7 34,4 ± 7,7 22,2 ± 5,2 12,2 ± 2,5 15,6 ± 1,3 0,24 ± 0,24 0,85 ± 0,82 1,81 ± 0,05

2,21 ± 0,15 0,89 ± 0,62 17,38 ± 0,88 10,29 ± 1,51 2,29 ± 0,67 6,92 ± 0,09 12,43 ± 0,23 10,12 ± 1,68 6,81 ± 0,95 1,31 ± 0,08 0,65 ± 0,24 6,17 ± 6,47 0,70 ± 0,15 0,21 ± 0,04 0,41 ± 0,11 0,43 ± 0,02 3,23 ± 0,52 0,38 ± 0,07 12,47 ± 3,36 0,18 ± 0,02 0,00 ± 0,00 0,00 ± 0,00 1,19 ± 0,63 0,16 ± 0,01 3,18 ± 1,46 100 30,5 ± 2,3 33,5 ± 3,1 36,0 ± 5,4 22,6 ± 4,6 13,4 ± 0,9 16,2 ± 1,8 0,28 ± 0,19 1,03 ± 0,62 1,68 ± 0,24

5,67 ± 0,91 0,28 ± 0,01 20,38 ± 3,10 0,62 ± 0,04 2,38 ± 0,69 4,98 ± 0,65 2,76 ± 0,84 2,52 ± 0,00 1,70 ± 0,56 0,06 ± 0,08 0,17 ± 0,03 17,78 ± 7,25 0,24 ± 0,04 0,21 ± 0,06 0,13 ± 0,00 0,00 ± 0,00 0,55 ± 0,05 1,46 ± 0,09 15,45 ± 2,99 0,21 ± 0,04 0,23 ± 0,12 0,32 ± 0,01 0,34 ± 0,12 0,47 ± 0,18 21,10 ± 7,54 100 34,0 ± 2,6 6,5 ± 0,8 59,5 ± 3,4 56,5 ± 3,6 3,0 ± 0,2 38,5 ± 10,8 1,34 ± 0,23 38,08 ± 10,12 18,82 ± 2,32

3,45 ± 1,41 2,15 ± 2,41 23,71 ± 7,62 17,43 ± 10,47 3, 79 ± 2,49 8, 76 ± 1, 73 20,16 ± 8,98 15,59 ± 8,25 8,03 ± 0,30 1,55 ± 0,26 1,25 ± 1,04 7,56 _ 6,17 1,14 _ 0, 70 0,29 ± 0,13 0, 74 ± 0,55 0,58 ± 0,20 4,98 ± 2,60 0,59 ± 0,20 18,94 ± 11,20 0,25 ± 0,09 0,08 ± 0,11 0,00 ± 0,00 1,24 ± O, 75 0,44 ± 0,42 3,19 ± 1,86 145,9 ± 51,8 43,4 ± 16,8 54,3 ± 28,4 48,2 ± 6,6 31,0 ± 3,9 17,2 ± 2,7 23,2 ± 10,0

2,33 ± 0,27 0,93 ± 0,61 18,34 ± 0,08 10,83 ± 1,09 2,40 ± 0,59 7,31 ± 0,24 13,14 ± 0,85 10,65 ± 1,28 7,22 ± 1,34 1,38 _ 0,02 0,68 _ 0,23 6,67 ± 7,14 0, 73 ± 0,13 0,22 ± 0,03 0,43 ± 0,10 0,46 ± 0,00 3,40 ± 0,39 0,40 ± 0,09 13,09 ± 2,94 0,19 ± 0,03 0,00 ± 0,00 0,00 ± 0,00 1,27 ± 0,73 0,16 ± 0,00 3,40 ± 1,70 105,6 ± 4,9 32,2 ± 1,0 35,3 ± 1,6 38,1 ± 7,5 24,0 ± 6,0 14,2 ± 1,6 17,1 ± 1,2

5,39 ± 3,02 0,26 ± 0,10 19,37 ± 10, 72 0,58 ± 0,28 2,05 ± 0,28 4,46 ± 1,33 2,71 ± 1,84 2,32 ± 0,97 1,68 ± 1,17 0,04 ± 0,05 0,15 ± 0,04 17,78 ± 13,54 0,22 ± 0,06 0,19 ± 0,02 0,12 ± 0,05 0,00 ± 0,00 0,49 ± 0,16 1,33 ± 0,48 13,66 ± 3,21 0,18 ± 0,04 0,19 ± 0,02 0,29 ± 0,12 0,29 ± 0,02 0,40 ± 0,02 17,99 ± 1,20 92,1 ± 38,6 31,9 ± 15,5 6,1 ± 3,3 54,1 ± 19,8 51,3 ± 18,5 2,8 ± 1,3 33,4 ± 4,9

VERTEX 70 interferometer coupled to a Hyperion 3000 Vis-IR microscope equipped with a liquid nitrogen cooled bidimensional FPA detector (detector area size 64 × 64 pixels; Bruker Optics GmbH, Germany; IR beamline SISSI, ELETTRA-Synchrotron, Trieste). By using a 15× condenser/objective, the visible image of each section was achieved to identify the areas of interest, on which the IR maps (164 × 164 μm2, pixel resolution ~2.56 μm) were acquired in transmission mode in the spectral range 4000–700 cm−1 (4096 spectra; 256 scans; spectral resolution 4 cm−1). Background spectra were acquired on clean portions of CaF2 windows. IR maps were pre-processed to correct the atmospheric contributions of carbon dioxide and water vapor, and vector normalized for avoiding artefacts induced by local thickness variations (OPUS 7.1 software package). The topographical distribution of lipids, phospholipids, glycogen and proteins was retrieved by integrating IR maps of each experimental group under the following spectral ranges: 3050–2800 cm−1 (asymmetric and symmetric stretching modes of CH2 and CH3 mainly in lipids alkyl chains, representative of overall lipids, named Lipid) (Giorgini et al., 2015a); 1780–1720 cm−1 (carbonyl ester moiety of phospholipids, named Phospholipid); 1180–900 cm−1 (COH vibrational modes of glycogen, named Glycogen); 1720–1480 cm−1 (AmideIand Amide II bands of proteins, named Protein). An arbitrary color scale was used, white color indicating the highest absorbance value, while blue color representing the lowest one.

2.8. Histology Seahorses were euthanized with Tricaine MS-222 (0.1 mg L−1, Sigma Aldrich). For each experimental group, individuals (n = 5) were taken at each sampling time and fixed in 4% buffered formaldehyde (pH 7.2). Subsequently, each specimen was transferred into a single cassette and washed overnight with tap water for formaldehyde residues removal. Decalcification was performed by plunging seahorses for five days in a bath of 10% formic acid. Subsequently, the individuals were washed overnight with tap water, dehydrated in graded series of ethanol and embedded in paraffin wax. Tissues were cut in 5 μm section by a rotary microtome and stained with haematoxilyn-eosin (H&E).

2.9. FPA-FTIR Imaging analysis FPA-FTIR Imaging analysis was performed on liver samples to retrieve qualitative information on its macromolecular composition. For this purpose, from each liver sample, three 10 μm thick sections were cut at intervals of ~100 μm with a cryomicrotome. Sections were immediately deposited, without any fixation process, onto CaF2 optical windows (1 mm thick, 13 mm diameter), and air-dried for 30 min for further infrared analysis (Giorgini et al., 2015a, 2015b). For both fish species, a total of 15 sections were analysed for each experimental group (C, A, and CA diets) at 10 and 30 DAR. Samples acquisition was carried out within 24 h from cutting. Previous experiments carried out in our laboratories evidenced that liver sections as above prepared, showed a good stability in time, providing homogeneous and reliable data sets. FPA-FTIR measurements were carried out by mean of a Bruker

2.10. Statistical analysis Data on survival, standard length (SL) and wet weight (WW) were submitted to one or two-way analysis of variance followed by Tukey's 79

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until 12 DAR among the 3 experimental groups (p > 0.05) (Fig. 1a). From 13 DAR, a sharp decrease was observed in survival rates of groups A (fed on Artemia) and CA (fed on copepods and Artemia), which lead to a significantly lower final survival at 30 DAR than in group C (79.8% ± 4.6, fed on copepods, Fig. 1a). Final survivals in groups A (52.2% ± 5.3) and CA (55% ± 9.0) did not differ significantly (p > 0.05). In H. reidi, no significant differences (p > 0.05) among treatments were observed in survival until 6 DAR (Fig. 1b). However, survival from 7 DAR until the end of the experiment (30 DAR) was significantly lower (p < 0.05) in group A (20.8% ± 6.3) with respect to groups C (66.7% ± 17.2) and CA (59.0% ± 7.0). Survivals in group C and A differed significantly (p < 0.05) from 17 DAR (Fig. 1b).

3.2. Weight and length Significant differences (p < 0.05) in wet weight (Fig. 2a) and standard length (Fig. 2b) among the different experimental groups of H. guttulatus were detected from 10 DAR. Wet weights and standard lengths in group C were significantly higher (p < 0.05) than in groups A and CA, which performed similarly (p > 0.05) for the whole experiment. At the end of the experiment (30 DAR) no significant differences in wet weight and standard length were observed among treatments, reaching values in group C, A and CA of 53.8 ± 5.7, 37.4 ± 24.3 and 30.7 ± 3.6 mg and 36.5 ± 2.2, 28.4 ± 8.3 and 29.1 ± 6.0 mm, respectively. Significant differences (p < 0.05) in weight (Fig. 3a) and length (Fig. 3b) among experimental groups of H. reidi were also detected from 10 DAR. Weight and length in groups C and CA were significantly higher (p < 0.05) than in group A. Seahorses from groups A and CA performed similarly (p > 0.05) during the whole experiment. Significant differences (p < 0.05) were observed in weight and length at the end of the experiment (30 DAR), reaching values of 56.48 ± 8.3, 31.19 ± 1.55 and 44.66 ± 11.3 mg, and 30.53 ± 0.6, 25.51 ± 0,3 and 30,47 ± 2,4 mm in groups C, A and CA, respectively. Fig. 4. Micrograph of a sagittal histological section of a 10 DAR seahorse (H. guttulatus) stained with hematoxylin and eosin. Asterisk (*) indicates the portion of the mid intestine were the histological analysis were performed for all sampled specimens. In the insert a cross section of H. guttulatus showing intestinal chambers. Iv = ileocecal valve. Scale bar = whole body micrograph: 200 μm; insert: 100 μm.

3.3. Live prey composition Total lipids in A. tonsa and Artemia nauplii and metanauplii (24 h enriched) accounted for 18.23 ± 6.59, 25.30 ± 6.97 and 19.35 ± 0.68% dry weight, respectively. Highly unsaturated fatty acids from the series n-3 (HUFA n-3), particularly 22:6n-3, were noticeably high in Acartia compared to Artemia, which was reflected in high DHA/EPA, DHA/ARA and n-3/n-6 ratios (Table 1). In contrast, monounsaturated fatty acids (16:1n-7, 18:1n-9 and 18:1n-7) were notably high in Artemia nauplii and metanauplii. Protein and carbohydrates in Artemia (ca 43% DW and 6.8–11.6% DW, respectively) were notoriously higher than in copepods (38.4% DW and 3.3% DW, respectively) (data from Blanco et al., 2014).

post-comparisons test (Prism5, GraphPadSoftware®, San Diego, CA, USA). Significance level was set at p < 0.05. 3. Results 3.1. Survival No significant differences in survivals were observed in H. guttulatus

Fig. 5. Sagittal histological section of H. guttulatus (H&E), DAR 5. (a) intestine of seahorses feed with Artemia. Arrow mark the undigested Artemia, scale bar = 100 μm; (b) intestinal folds with abundant supranuclear vescicles (arrow) and thick brush border in seahorses fed on copepods (asterisk mark the partially digested copepod. BB, brush border; LP, lamina propria. Scale bar 20 μm.

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Fig. 6. Sagittal histological section of H. guttulatus (H&E), DAR 5–10. (a) intestine of 5 DAR seahorses feed with copepods (b) intestine of 5 DAR seahorses feed with Artemia (c) intestine of 5 DAR seahorses fed on copepods and Artemia (d) intestine of 10 DAR seahorses fed on copepods (e) intestine of 10 DAR seahorses fed on Artemia (f) intestine of 10 DAR seahorses fed on copepods and Artemia. Arrowheads indicate supranuclear vescicles; asterisks (*) indicate undigested or partially digested Artemia metanauplii. Scale bar = 20 μm.

Fig. 7. Sagittal histological section of H. reidi and H. guttulatus (H&E), DAR 5. (a) undigested Artemia (asterisk) in the intestine of H. reidi fed on Artemia. Mucous cells indicated by arrows (b). Supranuclear vescicles (arrowheads) are associated with digested matters in the intestine of H. guttulatus fed on copepods. Scale bar = 20 μm.

species. Considering H. guttulatus, at 10 DAR, supranuclear vesicles were abundant in groups C (Fig. 6d) and CA (Fig. 6f) while smaller vesicles were also observed in group A (Fig. 6e). In at 10 DAR H. reidi juveniles individuals from group A did not show supranuclear vesicles (Fig. 8e) although they were present in juveniles from group C (Fig. 8d) and CA (Fig. 8f). At 20 and 30 DAR, no differences among the three experimental groups were observed for both species and inflammatory evidences were not detected.

3.4. Histological observations 3.4.1. Intestine The intestine of newly released seahorses was rather similar in both species, showing short folding of the intestinal mucosae and anterior and distal regions separated by an ileoceacal valve. A well characterized subdivision in chambers is shown in Fig. 4. At 5 DAR, the intestine appeared as a simple and straight tube. A columnar epithelium characterized both anterior and distal portions, with the mucosa layer lined by a thick lamina propria and marked brush border (Fig. 5b). An increase in length was observed at 10 DAR, where the tubular shape of the incipient intestine of both species started to accommodate the intestinal pack into a compacted structure and form the first intestinal loop in H. reidi. At 5 DAR, a large amounts of undigested Artemia were observed both in H. guttulatus and H. reidi intestines of juveniles fed on diet A (Figs. 5a, 7a). Juveniles from groups CA and C showed partially digested copepods (Figs. 5b, 7b). At that stage, abundant supranuclear vesicles were detected in group C (Figs. 5b, 6a, 7b, 8a), whereas their occurrence was reduced in group CA (Figs. 6c, 8c) and absent in group A (Figs. 6b, 8b) for both

3.4.2. Liver In both species, the liver was characterized by packed hepatocytes with central nucleus, organized in a chord-like pattern (Figs. 9, 10). A network of biliary canaliculi and sinusoid capillaries (Figs. 9b, 10c) were interdispersed into the liver parenchyma, with presence of some lipid deposit in the cytoplasm. In H. guttulatus, no differences were observed among the groups at 5DAR and steatotic events were not detectable. At 10 DAR, remarkable differences were observed in lipid content between treatments. Juveniles from group C showed a compact parenchyma without an 81

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Fig. 8. Sagittal histological section of H. reidi (H&E), DAR 5-10. (a) intestine of 5 DAR seahorses fed on copepods (b) intestine of 5 DAR seahorses fed on Artemia (c) intestine of 5 DAR seahorses fed on copepods and Artemia (d) intestine of 10 DAR seahorses fed on copepods (e) intestine of 10 DAR seahorses fed on Artemia (f) intestine of 10 DAR seahorses feed with copepods and Artemia. Arrowheads indicate supranuclear vescicles; circle indicate acidophilic inclusions, arrow indicate mucous cells, asterisks (*) indicate undigested or partially digested Artemia metanauplii. Scale bar = 20 μm. Fig. 9. Sagittal histological section of liver in H. guttulatus (H&E), DAR 10-30. (a) liver of 10 DAR seahorses fed on copepods (b) liver of 10 DAR seahorses fed on Artemia (c) liver of 10 DAR seahorses fed on copepods and Artemia (d) liver of 30 DAR seahorses fed on copepods (e) liver of 30 DAR seahorses fed on Artemia (f) liver of 30 DAR seahorses fed on copepods and Artemia. Arrow indicate hepatocyte, asterisk indicate lipid deposit, arrowhead indicate sinusoid capillary. Scale bar = 10 μm.

that of group C (Fig. 10d). Considering 5 DAR juveniles of H. reidi no differences were observed in liver morphology. However, remarkable differences were observed in lipid accumulation from 10 DAR until the end of the experiment (30

appreciable occurrence of lipids accumulation in the hepatocytes (Fig. 9a) while groups A and CA showed a large quantity of lipid vacuoles (Fig. 9b, c). Finally, at 30 DAR a lipid reduction appeared also in groups A and CA (Fig. 10e, f) showing a liver morphology similar to

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Fig. 10. Sagittal histological section of liver in H. reidi (H&E), DAR 10–30. (a) liver of 10 DAR seahorses fed on copepods (b) liver of 10 DAR seahorses fed on Artemia (c) liver of 10 DAR seahorses fed on copepods and Artemia (d) liver of 30 DAR seahorses fed on copepods (e) liver of 30 DAR seahorses fed on Artemia (f) liver of 30 DAR seahorses fed on copepods and Artemia. Arrow indicate hepatocyte, asterisk indicate lipid deposit, arrowhead indicate sinusoid capillary. Scale bar = 10 μm.

considerably higher in group C (Fig. 14d); glycogen absorbance values showed slightly higher values in group A (Fig. 14h), while proteins did not shown differences among the experimental groups (Fig. 14l, m, n).

DAR) among the 3 experimental groups. Group C did not show any sign of hepatic steatosis (Fig. 10a, d), but a conspicuous lipid accumulation was observed in livers from group A (Fig. 10b, e) and CA (Fig. 10c, f) both at 10 and 30 DAR.

4. Discussion 3.5. FPA-FTIR Imaging analysis The results achieved in the present study demonstrate the survival and growth rates of H. guttulatus and H. reidi were enhanced by using copepods as first prey. Larval nutrition plays a key role in the development of a sustainable aquaculture where fish development, health and wellness are of prime importance (Lall and Tibbetts, 2009; Olivotto et al., 2017; Piccinetti et al., 2017). Since malnutrition has recently been included in the stressors list, nutritional requirements needed for optimal larval development and growth are of great relevance (Cahu et al., 2003). Fatty acids and amino acids are some of the essential nutrients affecting fish development, along with carotenoids, vitamins, minerals and energy-binding macronutrients such as protein, lipids and carbohydrates (Lall and Tibbetts, 2009; Karlsen et al., 2015). For some species, satisfactory growth and survival rates are met providing exclusively enriched rotifers and Artemia (O'Brien-MacDonald et al., 2006; Park et al., 2006; Garcia et al., 2008a, 2008b). However, feeding on copepods during the larval period has been shown to improve growth in both larval and juvenile fish, including seahorses (Støttrup, 2000; Imsland et al., 2006; Olivotto et al., 2008b, 2010, 2011; Koedijk et al., 2010; Blanco et al., 2015). Hence, copepods can be considered an important complement to rotifers and Artemia as live feed in first feeding marine fish larvae (Olivotto et al., 2008c; Olivotto et al., 2008b). In fact, copepods are the preferred natural prey in most marine fishes during the early developmental phase (Payne and Rippingale, 2001; Drillet et al., 2011). A number of beneficial effects have been linked to the biochemical composition of copepods in early larval nutrition. In particular, emphasis has been directed towards lipid composition and

The spectroscopic imaging analysis of liver samples from H. guttulatus and H. reidi seahorses at 10 DAR and 30 DAR fed on A, C, and CA diets was performed. For H. guttulus liver samples, the imaging spectroscopic analysis (Figs. 11 and 12, respectively), evidenced that: (i) at 10 DAR, the absorbance related to lipids resulted higher in groups A and CA (Fig. 11b, c); phospholipids were more represented in group C and CA (Fig. 11d, f); higher glycogen absorbance values were recorded in group A (Fig. 11h), and, finally, proteins showed the same absorbance values in all experimental groups, with no appreciable differences among them (Fig. 11i, m, n); (ii) at 30 DAR, the three experimental groups showed similar lipid levels (Fig. 12a, b, c), always lower with respect to those detected at 10 DAR; phospholipid absorbance values resulted higher in group C (Fig. 12g); glycogen absorbance values were higher in group A and CA (Fig. 12h, i), while proteins did not shown different absorbance values among the three experimental groups (Fig. 12i, m, n). For H. reidi liver samples, the analysis of the distribution of lipids, phospholipids, glycogen and proteins (Figs. 13 and 14, respectively), evidenced that: (i) at 10 DAR, lipid absorbance values resulted remarkably higher in group A (Fig. 13b); phospholipid absorbance values were higher in group C with a packed appearance (Fig. 13d); glycogen absorbance values were higher in group A (Fig. 13h), while protein absorbance values appeared very similar among the three experimental groups (Fig. 13l, m, n); (ii) at 30 DAR, lipid absorption values were higher in group A (Fig. 14b); phospholipids absorbance values were 83

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Fig. 11. Representative FPA-FTIR Imaging analysis of liver sections from H. guttulatus seahorses at 10 DAR fed C, A and CA diets. IR maps describe the topographical distribution of the following macromolecules: (a-c) Lipids (3050–2800 cm−1); (d-f) Phospholipids (1780–1720 cm−1); (g-i) Glycogen (1180–900 cm−1), and (l-n) Proteins (1720–1480 cm−1). White color indicates higher absorbance values, while blue color indicates lower ones. At 10 DAR, the absorbance related to lipids resulted higher in groups A and CA (Fig. 11b, c); phospholipids were more represented in group C and CA (Fig. 11d, f); higher glycogen absorbance values were recorded in group A (Fig. 11h), and, finally, proteins showed the same absorbance values in all experimental groups, with no appreciable differences among them (Fig. 11i,m,n). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

developmental stages due to an inadequate nutritional profile (Estévez et al., 1999; Sargent et al., 1999; Applebaum and Holt, 2003; Olivotto et al., 2003; Faulk and Holt, 2005). In seahorses, several studies showed that including copepods in the diet may significantly improve growth and survival of juveniles (Palma et al., 2014; Blanco and Planas, 2015). Also, copepods are preferred to Artemia nauplii during the first days of development in H. guttulatus (Blanco and Planas, 2015). Differences in the digestion efficiency of live preys mainly depends on their biochemical composition (e.g. nutritional profile) and permeability of the exoskeleton, their digestion and on the developmental stage of fishes (Rainuzzo et al., 1997; Bodin et al., 2012). Previous studies suggested that Artemia is hardly digested by early

the content and ratio of highly unsaturated fatty acids (HUFA), particularly docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA) (Scott and Middleton, 1979; Seikai, 1985; Kanazawa, 1993; Reitan et al., 1994, 1997; Nanton and Castell, 1998; Venizelos and Benetti, 1999; Bell et al., 2003; Tocher, 2003). The composition of lipid classes and distribution of certain fatty acids in neutral and polar lipids are also important characteristics of copepods (Olsen et al., 1991; Coutteau et al., 1997; Geurden et al., 1998; McEvoy et al., 1998; Sargent et al., 1999). Currently, many seahorse species are still reared using Artemia nauplii (Olivotto et al., 2008a; Planas et al., 2017). Unfortunately, Artemia does not always promote optimal fish growth at early 84

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Fig. 12. Representative FPA-FTIR Imaging analysis of liver sections from H. guttulatus seahorses at 30 DAR fed C, A and CA diets. IR maps describe the topographical distribution of the following macromolecules: (a–c) Lipids (3050–2800 cm−1); (d–f) Phospholipids (1780–1720 cm−1); (g–i) Glycogen (1180–900 cm−1), and (l–n) Proteins (1720–1480 cm−1). White color indicates higher absorbance values, while blue color indicates lower ones. At 30 DAR, the three experimental groups showed similar lipid levels (Fig. 12a, b, c); phospholipid absorbance values resulted higher in group C (Fig. 12g); glycogen absorbance values were higher in group A and CA (Fig. 12h, i), while proteins did not shown different absorbance values among the three experimental groups (Fig. 12i, m, n). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

life stages of fish, including seahorses (Olivotto et al., 2011; Planas et al., 2012; Blanco et al., 2015). The absence of pharyngeal teeth associated with the lack of a stomach in seahorses suggests that prey digestion is mainly achieved by enzymatic processes occurring in the midgut and hindgut. However, mechanical trituration of prey resulting in the breaking of exoskeletons seems to be an important feature in facilitating digestion and assimilation in seahorses. The present study pointed out the occurrence of undigested Artemia nauplii in the seahorse juveniles guts during the first feeding days. In H. guttulatus juveniles fed on Artemia nauplii, the low efficiency in food digestion/ assimilation of nauplii during the first 2–3 days implies an initial negative energetic balance (Blanco et al., 2011) and, in consequence, an increase in mortalities. This inefficiency may also be a result of a

different degree in development of the two seahorse species because of differences in the optimal rearing temperature (temperate vs tropical). In accordance with a previous study (Novelli et al., 2015) a faster appearance of the first intestinal loop was observed in H. reidi (8 DAR) respect to H. guttulatus (DAR 15).These findings are in agreement with other studies suggesting that Artemia passes largely indigested through the gut, whereas copepods are more efficiently digested and assimilated during the early development of seahorses juveniles (Shields et al., 1999). Artemia and copepods differ in the characteristics of their exoskeleton (Bresciani, 1986). Copepod cuticle consists of a protein matrix containing lipids and chitin rods (Bouligand and Neville, 1973). Such cuticle is are segmentated, thin and easy to break by mechanical impact. Conversely, Artemia cuticle is thicker and difficult to brake, 85

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Fig. 13. Representative FPA-FTIR Imaging analysis of liver sections from H. reidi seahorses at 10 DAR fed C, A and CA diets. IR maps describe the topographical distribution of the following macromolecules: (a–c) Lipids (3050–2800 cm−1); (d–f) Phospholipids (1780–1720 cm−1); (g–i) Glycogen (1180–900 cm−1), and (l–n) Proteins (1720–1480 cm−1). White color indicates higher absorbance values, while blue color indicates lower ones. At 10 DAR, lipid absorbance values resulted remarkably higher in group A (Fig. 13b); phospholipid absorbance values were higher in group C with a packed appearance (Fig. 13d); glycogen absorbance values were higher in group A (Fig. 13h), while protein absorbance values appeared very similar among the three experimental groups (Fig. 13l, m, n). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

with previous studies on growth, stress response and deformities occurrence in larvae of other fish species (Koven et al., 1998; Cahu and Zambonino Infante, 2001; Mai and Loebmann, 2009). The dietary requirements in n-3 HUFAs in marine fish larvae has been largely studied and assessed for many species (Sargent et al., 1997, 1999; Tocher et al., 2003; Olivotto et al., 2005). In fish, lipids and their constituent fatty acids (FAs) play essential roles in maintaining optimum growth, survival, feed efficiency, health, neural and visual development, and response to stressors in addition to generally being the main energy source (Sargent et al., 1989, 2002; Tocher, 2003, 2010). As a consequence, the higher growth rates and survivals observed in the present study in seahorse juveniles fed on copepods are very likely due to a higher HUFAs content/ratio in copepods with respect to Artemia.

representing an important barrier for the digestion and assimilation of live prey content (Blanco et al., 2015). Nutritional deficiencies of Artemia are well documented (Sargent et al., 1997). Ash and protein content were similar in the preys used in the present study, but the proportion of carbohydrates and total lipid was higher in Artemia nauplii compared to copepods. Regarding lipid sources, Artemia is richer than copepods in triacylglycerols whereas the latter is richer in phospholipids (Sargent et al., 1989; Morais et al., 2006). When marine fish larvae are fed on preys rich in phosphoacylglycerols, containing high n-3 HUFA levels, both digestion and lipid transport result enhanced, promoting larval growth (Izquierdo et al., 2000). A beneficial effect of feeding on copepods (richer in phosphoacylglycerols) was evidenced in the present study, being in accordance 86

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Fig. 14. Representative FPA-FTIR Imaging analysis of liver sections from H. reidi seahorses at 30 DAR fed C, A and CA diets. IR maps describe the topographical distribution of the following macromolecules: (a–c) Lipids (3050–2800 cm−1); (d–f) Phospholipids (1780–1720 cm−1); (g–i) Glycogen (1180–900 cm−1), and (l–n) Proteins (1720–1480 cm−1). White color indicates higher absorbance values, while blue color indicates lower ones. At 30 DAR, lipid absorption values were higher in group A (Fig. 14b); phospholipids absorbance values were considerably higher in group C (Fig. 14d); glycogen absorbance values showed slightly higher values in group A (Fig. 14h), while proteins did not shown significant differences among the experimental groups (Fig. 14l, m, n). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Imbalanced HUFAs levels in diets negatively affect fish survival and growth during larval development (Sargent et al., 1997). The regulation of lipid homeostasis in fish is a complex balance between lipid uptake, transport, storage, energy utilization, and biosynthesis. Such processes are controlled independently but also in conjunction with others (Tocher, 2003; Leaver et al., 2008). As in adults, the liver plays a key role as energy reservoir in fish larvae and its macromolecular composition may depend on both the diet provided and the digestive capabilities of fish. Analyzing of the macromolecular composition of liver would help to better understand which macromolecules are assimilated and stored by the fish during a certain period of its life history and how such molecules may contribute in the development. The present study evidenced

the potential role of FPA-FTIR technique for the analysis of liver macromolecules, in agreement with previous studies on the liver macromolecular composition of zebrafish (Gioacchini et al., 2014; FornerPiquer et al., 2017) and seabream juveniles (Carnevali et al., 2017). The results achieved through FPA-FTIR analysis evidenced changes in liver biochemical composition depending on the type of diet, suggesting an important role of diet in early developing seahorses. In particular, phospholipids increased with copepods administration whereas Artemia mainly caused higher content in carbohydrates and lipids. These data are in agreement with the proximal biochemical composition of prey (Blanco, 2014). In addition, the histological analysis of liver samples showed a higher lipid accumulation in early developmental stages of both H. guttulatus and H. reidi juveniles when fed on 87

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Artemia, which is supported by the higher lipid (particularly triacylglycerols) content of Artemia compared to copepods. The reduction observed in hepatic fat for H. guttulatus at 30 DAR suggested that the capacity of lipid absorption and its location in the hepatic epithelium is species-specific (Izquierdo et al., 2000). Finally, the absence of supranuclear vesicles in the intestine of both H. guttulatus and H. reidi juveniles fed exclusively on Artemia confirmed differences in the digestion of the two types of live preys, confirming the importance of an appropriate diet in the early life stages of seahorses. In conclusion, the present study highlighted the importance of copepods in the early rearing of seahorses. For the first time, the effects of different diets on seahorses gut development were analysed by combining biometry, traditional histology and an innovative technique, the Fourier Transform Infra-Red (FTIR) spectroscopy.

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