The great pipefish Syngnathus acus is one of the most representative European syngnathid, being highly associated with seagrass and macroalgal beds. Surprisingly, the ecology of this ovoviviparous marine fish has received scanty attention. The population inhabiting three sites on Cíes Archipelago (Atlantic Islands National Park, NW Spain) was monitored in 2017-2018 for spatial and temporal changes in abundances, reproduction traits, trophic niche occupancy and dietary regimes across reproduction states through an isotopic (δ13C and δ15N) approach. Abundances were highly variable across seasons and sites, decreasing significantly from mid-autumn. The population consisted almost exclusively of large adults that migrate by the end of the breeding season, which extended from mid-spring to summer. Operational sex ratios suggest that the species is sex-role reversed. S. acus is a secondary consumer (Trophic position= 3.36 ± 0.05) preferring amphipods but displaying annual and seasonal dietary plasticity. Mature fish were less selective than immatures (especially females) with a higher preference for amphipods (36-68%) in the former. The second most preferred prey were carideans, copepods or isopods, depending on the year and reproduction state. Overall, the wider trophic niches in females and immature specimens compared to males and mature fish would indicate a higher variability in both the use of prey resources and/or their origin. The present study highlights the trophic plasticity and unique features of S. acus population in Cíes Archipelago, especially regarding the outstanding size of the fish and the exceptional occurrence of breeders.

Relations between nutrient assimilation and growth rate in fishes may vary with abiotic factors such as temperature. The effects of feeding status, ontogeny and temperature regimes (15, 18 and 21 °C) on stable isotope (δ13C and δ15N) patterns were assayed and modelled in juveniles of the seahorse Hippocampus guttulatus. The use of effective day degrees (D°eff) and chronological time (age) were compared as development progress indices. Newborn seahorses were maintained at three temperature levels both deprived of food (5 days) or fed (30 days) on copepods or/and Artemia. Isotopic signatures in fed seahorses clearly differed from those in unfed juveniles. Temperature had a significant effect on δ13C values in fed juveniles throughout the experimental period. δ15N values also varied significantly with age, but not with temperature level. Faster growth and food assimilation in seahorses held at 18 and 21 °C were supported by faster variations in isotopic values. Our findings demonstrate that effective day degrees should be preferred over chronological time as index of developmental progress in temperature fluctuating scenarios or for comparative studies.

The initial development of seahorse juveniles is characterized by low digestion capabilities. Stable isotope analysis is an effective tool in studies of trophic food webs and animal feeding patterns. The present study provides new insights for the understanding of growth and food assimilation in early developing seahorses following a laboratory diet switch. The study was performed in early life stages of the seahorse Hippocampus reidi by assessing the influence of diet shift on changes and turnovers in carbon (δ13C) and nitrogen (δ15N) stable isotope in juveniles. Newborn seahorses were fed for 60 days following two feeding schedules (A6 and A11) based initially on copepods Acartia tonsa and subsequently on Artemia nauplii (since days 6 and 11, respectively). After prey shift, we determined δ13C and δ15N turnover rates as functions of change in either body mass (fitting model G) and days of development (fitting model D), contributions of metabolism and growth to those turnover rates, and diet-tissue discrimination factors. Survival, final dry weight and final standard length for diet A11 were higher compared to diet A6. The shift from copepods to Artemia lead to fast initial enrichments in δ13C and δ15N. Afterwards, the enrichment was gradually reduced until reaching the isotopic equilibrium with diet. In most cases, both fitting models performed similarly. The isotopic analysis revealed that 100% of tissue turnover was attributed to growth in diet A11, whereas 19-25% was endorsed to metabolism in diet A6. Diet-tissue discrimination factors were estimated for the first time in seahorse juveniles, resulting in higher estimates for diet A11 (2.9 ± 0.7‰ for δ13C; 2.5 ± 0.2‰ for δ15N) than in diet A6 (1.8 ± 0.1‰ for δ13C; 1.9 ± 0.1‰ for δ15N). This study highlights the relevance of feeding on copepods and their effect on isotopic patterns and discrimination factors in seahorse juveniles after a dietary shift. Regarding the application of the results achieved to feeding schedules in the rearing of H. reidi, a long period of feeding on copepods during the first days of development is highly recommended.

Isotopic stable analysis (SIA) is a powerful tool in the assessment of different types of ecological and physiological studies. For that, different preservative methods for the samples are commonly used prior to isotopic analysis. The effects of various preservative methods (drying, freezing, ethanol and formaldehyde) have been analyzed for C:N ratio, δ13C and δ15N on a variety of tissues including dorsal fins (three seahorse and two pipefish species), seahorse newborns (three seahorses species), and prey (copepods and different stages of Artemia) commonly used to feed the fishes in rearing conditions. The aims of the study were to: (i) evaluate isotopic effects of preservation methods across tissues; and (ii) construct the first conversion models available in syngnathid fishes. The preservation in ethanol and to a lesser extend in formaldehyde significantly affected δ13C values, whereas δ15N signatures were not affected significantly. Due to their low lipid content, the isotopic signals in fish fins were almost unaffected, supporting the suitability of dorsal fins as a convenient tool in isotopic studies on vulnerable species such as syngnathids. The regression equations provided permit the successful conversion of δ13C and δ15N values between preservative treatments. The conversion models can be applied to isotopic studies in the field and in the laboratory.

Pipefishes (Family Syngnathidae) are representative fishes in many European coastal areas asso-ciated with vegetal assemblages and other shallow habitats. The polyandrous pipefishes Syngnathus abaster and S. acus co-occur in Metruk Tuzla (Bargilya Wetland, Aegean Sea, Turkey), a protected but threatened wetland due to anthropogenic disturbances. Both species were sam-pled during the breeding season (April-2018 and June-2019) for the assessment of growth and trophic features revealed from stable isotope profiles (δ13C and δ15N) and gut content. Both species showed positive allometric growth, with similar length-weight relationships. The isotope approach indicated that both species occupy similar trophic niches, with mean δ13C and δ15N values of -17.0 ± 1.2 ‰ for δ13C and 13.2 ± 0.6 ‰ for δ15N in S. abaster, and -17.3 ± 1.2 ‰ for δ13C and 13.3 ± 0.7 ‰ for δ15N in S. acus. The niche area for all sex types (undifferentiated specimens, males and females) in S. acus was slightly broader than in S. abaster. The simulation analysis of the trophic position (TP) in both species agreed with the isotopic assessment (TP = 3.67 ± 0.61 in S. abaster and 3.46 ± 0.66 in S. acus). Despite the high trophic resemblance between S. abaster and S. acus, the gut content assessment revealed the large contribution of copepods (especially Calanipeda aquaedulcis) to the dietary regime in both pipefishes. However, while the diet in S. abaster seemed to be almost monospecific, cladocerans were also an important resource in S. acus. Hence, we suggest that potential inter-specific competition for dietary resources would be minimized likely due to the occupancy of different microhabitats, a hypothesis that needs to be confirmed by further research.