Knowledge on seahorses is generally scarce but has been increasing in recent years due to their conservation status. Seahorse culture is a quite recent activity in the field of marine aquaculture in most countries attempting it, and captive breeding techniques are available only for some species. With the aim of contributing to the development of breeding in captivity for conservative purposes, broodstocks of the European long-snouted seahorse (Hippocampus guttulatus) were established with 32 wild seahorses captured in Galicia (NW Spain). This study describes the methodologies applied to the maintenance of the broodstocks, with special reference to aquaria design, feeding, growth and breeding. Procedures of seahorse identification (morphologically and genetically) as a tool for broodstock management are also considered. The results achieved during the first year demonstrate a rapid adaptation of wild seahorses to captive conditions. Seahorses were fed exclusively on enriched adult Artemia and displayed high growth rates. However, fatty acid analyses performed on unfertilised eggs of captive broodstock showed a progressive decrease with time in the content of essential fatty acids (DHA, EPA), suggesting the need for improvement in the nutritional quality of broodstock feed. during the first year demonstrate a rapid adaptation of wild seahorses to captive conditions. 30Seahorses were fed exclusively on enriched adult Artemia and displayed high growth rates. 31However, fatty acid analyses performed on unfertilised eggs of captive broodstock showed a 32 progressive decrease in the content of essential fatty acids (DHA, EPA) with time in captivity, 33suggesting the need for improvement in the nutritional quality of broodstock feed. 3435 36

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Syngnathids are vulnerable ovoviviparous fishes in which males undergo repeated brooding within a reproductive season. The isotopic effects of diet on both breeders (pooled sexes) and parent-egg transmission have been demonstrated in a few fish species but never in syngnathids. Quantifying isotopic changes due to diet is necessary to assess parent-newborn conversions and to estimate accurate trophic enrichment factors (TEF). We assessed the isotopic (δ13C and δ15N) effects of 3 experimental diets on TEFs in seahorse Hippocampus guttulatus breeders and isotopic inheritance. Our results suggest that H. guttulatus follows an income-capital continuum pattern for parent-egg transmission. The isotopic variability in diets for breeders and the resulting experimentally derived TEFs were compared with fixed TEFs from reviews to estimate their impact on the relative contribution of potential prey sources in syngnathids from the Cíes archipelago (Atlantic Islands National Park, NW Spain). We estimated source contributions using stable isotope mixing models (SIMMs) by combining prey sources into ecologically informative groups and incorporating informative priors. We demonstrate that (1) most frequently used TEFs from reviews might not be suitable for all fish species, particularly syngnathids, and (2) dietary source variability has a great effect on source contribution estimates. This study is also the first to provide specific TEFs for syngnathids.

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Background Syngnathid fishes (Actinopterygii, Syngnathidae) are flagship species strongly associated with seaweed and seagrass habitats. Seahorses and pipefishes are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently Data Deficient according to the IUCN (2019), requiring more biological and ecological research. This study provides the first insights into syngnathid populations in the two marine Spanish National Parks (PNIA—Atlantic- and PNAC—Mediterranean). Fishes were collected periodically, marked, morphologically identified, analysed for size, weight, sex and sexual maturity, and sampled for stable isotope and genetic identification. Due the scarcity of previous information, habitat characteristics were also assessed in PNIA. Results Syngnathid diversity and abundance were low, with two species identified in PNIA (Hippocampus guttulatus and Syngnathus acus) and four in PNAC (S. abaster, S. acus, S. typhle and Nerophis maculatus). Syngnathids from both National Parks (NP) differed isotopically, with much lower δ15N in PNAC than in PNIA. The dominant species were S. abaster in PNAC and S. acus in PNIA. Syngnathids preferred less exposed sites in macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. The occurrence of very large specimens, the absence of small-medium sizes and the isotopic comparison with a nearby population suggest that the population of Syngnathus acus (the dominant syngnathid in PNIA) mainly comprised breeders that migrate seasonally. Mitochondrial cytochrome b sequence variants were detected for H. guttulatus, S. acus, and S. abaster, and a novel 16S rDNA haplotype was obtained in N. maculatus. Our data suggest the presence of a cryptic divergent mitochondrial lineage of Syngnathus abaster species in PNAC. Conclusions This is the first multidisciplinary approach to the study of syngnathids in Spanish marine NPs. Habitat preferences and population characteristics in both NPs differed. Further studies are needed to assess the occurrence of a species complex for S. abaster, discarding potential misidentifications of genus Syngnathus in PNAC, and evaluate migratory events in PNIA. We propose several preferential sites in both NPs for future monitoring of syngnathid populations and some recommendations for their conservation.

Although breeding conditions are rather well established for some seahorse species 10 (Hippocampus spp.), zootechnics and the effect of non-biological factors are still unknown for 11 many species. The present study is focussed on the effects of aquarium type/design, 12 photoperiod regime and aeration level on the early performance (growth and survival) of 13 Hippocampus guttulatus juveniles. Three aquarium types were evaluated: pseudoKreisel, 14 spherical and rectangular. Juveniles grown in pseudoKreisel aquaria showed lower growth 15 16 (DAR) when compared to either spherical (30 ± 22%) or rectangular (16 ± 12%) aquaria.

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Breeding in captivity for research or exhibition (e.g. in aquaria) can replace the capture of wild specimens of endangered species and allow controlled reinforcement of wild populations. With this aim, diet analysis and establishing the adequate prey are determinant factors for breeding success. However, non-invasive approaches such as faecal DNA analysis are advisable for analysing the diet of these species. Therefore, the aim of the present study was to demonstrate the usefulness of faecal DNA analysis by specific PCR amplification of prey DNA for assessing the diet of the seahorse Hippocampus guttulatus. In a comparison of the suitability of different genes (COI, 18SrRNA and 28SrRNA), 18SrRNA was found to be the most suitable for designing specific primers for the prey types fed to seahorses (Artemia, Palaemonetes and Mysidae). The technique was assessed in feeding experiments in which prey ingestion was recorded daily, and faeces were collected for DNA extraction and presence/absence PCR analysis. Amplification of the prey DNA in faeces was consistent with the sequence of prey supplied (prey eaten the day before was always detected). Differences in the time between feeding and detection in faeces suggested prey-specific gut passage times, which were shorter for Palaemonetes than for Mysidae. This fact highlights the importance of feeding trials to avoid overestimating the consumption of prey with long gut retention when PCR-based methods are used. This molecular technique is thus applicable for studying the feeding behaviour of captive seahorses and could be adapted for use in other marine species.