BackgroundGene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry.ResultsWe analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency.ConclusionOur results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-648) contains supplementary material, which is available to authorized users.

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.

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.

Summary: Four visual censuses targetingOctopus vulgaris living in dens on sandy bottoms were carried out from June to October 2013 in the National Park of the Atlantic Galician Islands (NW Spain). Censuses were undertaken by scuba diving between 5 and 21 m depth in daytime. The total area swept was 13.75 ha. There were no significant differences between octopus presence in dens during open and closed fishing seasons. Depth had a significant negative relationship with occupancy. The average number of dens per 1000 m 2 was 3.84±0.84 in June and 3.89 in October. The area per den was 260 m 2 . Den number estimations varied between 1586 and 2057. The largest number of dens (76.5%) was found between 5 and 10 m depth. Den distribution was clumped. No significant differences were found between octopus size classes (small, medium and large) and den diameter. Associate dens were observed. There were no significant differences in den diameter and shell types found around the middens. Many dens could be "permanent". Drilling bivalve shell behaviour is discussed. The surveyed area had around 1100 individuals, mainly small specimens. No significant differences were found between octopus size and depth. Substrate, den type and food abundance and availability (especially razors Ensis arcuatus) seem to be the main factors influencing dens and octopus density and distribution. Den availability does not appear to be a limiting factor in this case. Temperature, den availability, predators and fishing pressure influencing density and distribution are discussed. Rodas inlet may be a preferential habitat for O. vulgaris individuals ranging from 200 to 2000 g, but especially small specimens (≤1000 g).Keywords: den ecology; visual census; habitat selection; drilling behaviour; Octopus vulgaris; National Park of the Atlantic Galician Islands (NW Spain). Moradores de guaridas de fondos arenosos: rasgos ecológicos y de comportamiento de Octopus vulgarisResumen: Entre junio y octubre de 2013 se realizaron cuatro censos visuales enfocados a estudiar el pulpo común Octopus vulgaris que mora en guaridas en fondos arenosos. Los censos se realizaron en la ensenada de Rodas, ubicada en el archipiélago de Cíes dentro del Parque Nacional de las Islas Atlánticas de Galicia (NO España). Dichos censos se efectuaron con buceo autónomo entre 5 y 21 m de profundidad y durante el día. El área total barrida fue de 13.75 ha. No se encontraron diferencias significativas entre la presencia de pulpos en las guaridas durante la temporada de veda y la de pesca. La profundidad tuvo una significación negativa en relación con el grado de ocupación. El promedio de guaridas por cada 1000 m 2 fue de 3.84±0.84 en junio y 3.89 en octubre. El área por guarida fue de 260 m 2 . El número estimado de guaridas varió ente 1586 y 2057. El mayor número de madrigueras (76.5 %) se encontró entre 5 y 10 m de profundidad. La distribución de las guaridas era agregada. No se hallaron diferencias significativas entre el diámetro de los refugios y el tamaño de los pulpos. Tampoco hubo ...

6The first description of cannibalism in wild adults Octopus vulgaris is presented from three 7 observations made off the Ría de Vigo (NW Spain), which were filmed by scuba divers. Two 8 of these records occurred within the National Park of the Atlantic Islands of Galicia (NW 9 Atlantic) in December 2012 and November 2013, and another at the southern part off the Ría 10 de Vigo. These records, recorded with a Sony DV-Cam camcorder, allowed to obtain 11 common traits in cannibalistic behavior: i) it was an intercohort cannibalism; ii) attacks were 12 made by both males and females; iii) in two of the records, the preys were transported to the 13 den, which was covered with stones of different sizes; iv) the predator started to eat the tip of 14 the arms of its prey; v) predation on conspecifics occurred even if there were other abundant 15 and available preys (i.e. mussels); vi) facing an intruder (i.e. diver) the predator defense of its

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a b s t r a c tThe selection of spawning habitat of a population of Octopus vulgaris that is subject to a small-scale exploitation was studied in the Cíes Islands within the National Park of the Atlantic Islands of Galicia (NW Spain). The technique used was visual censuses by scuba diving. We conducted 93 visual censuses from April 2012 to April 2014. The total swept area was 123.69 ha. Habitat features (season, depth, zone, bottom temperature, swept area, bottom substrate type, and creels fishing impact) were evaluated as predictors of the presence/absence of spawning dens using GAM models. O. vulgaris has a noteworthy preference for spawning in areas with hard bottom substrate and moderate depth (approximately 20 m). The higher density of spawning dens (1.08 ha −1 ) was found in a surveyed area of 50.14 ha located in the northeastern part of the northern Cíes Island. We propose to protect the area comprised from Punta Escodelo to Punta Ferreiro between 5 and 30 m depth. This area has a surface of 158 ha equivalent to 5.98% of the total marine area of the Cíes islands. The strengths and weaknesses of a management strategy based on the protection of the species' spawning habitat are discussed.

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