Background: Gene 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. Results: We 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. Conclusion: Our 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.

Four visual censuses targeting Octopus 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 m2 was 3.84±0.84 in June and 3.89 in October. The area per den was 260 m2. 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).

11 páginas, 5 figuras, 5 tablasWe evaluated specific habitat features (bottom substrate type, depth, temperature, season and latitude-longitude) at random locations in the Cíes Islands (Galician Atlantic Islands National Park, Northwest Spain) to determine their impact on Sepia officinalis spawning habitat use. We performed underwater visual transects via scuba diving. In total, 94 transects were conducted between April 30, 2012 and July 17, 2015. Habitat features were evaluated as predictors of the presence/absence of egg clusters using Generalised Additive Models. Statistical analyses revealed the following: (a) the probability of finding eggs was significantly higher on hard bottom substrate type containing sea fans and sea tube worms; (b) the sectors where the egg presence was highest were the most shelters zones of Bajo de Viños and Piedra del Borrón, two hard bottom shoals covered by sea fans and sea worms and located in the central Cíes Islands or Faro Island; (c) The preferential depth ranged from 8 to 13 m; (d) as temperatures rose, the probability of finding eggs decreased, which was a sign of the seasonality of the spawning event of the species; and (e) eggs were most common in the winter. A total of 18 additional non-random underwater visual transects were then performed in the Viños and Borrón shoals between December 10, 2014 and August 20, 2015. We calculated the number of egg clusters found in two fenced areas (0.04 ha) located in Viños and Borrón, which showed that Viños was a most suitable spawning habitat than Borrón. The number of eggs per cluster varied from 4 to 82. The spawning period for this species lasted from the beginning of December until the end of June, when bottom temperature ranged from 12.5 °C to 14.75 °C. These microhabitat data reveal indicators of common cuttlefish spawning habitat utilisation and should help identify targets for habitat improvement projects and ecosystem management approaches within this national park and elsewhereM. Sestelo’s research was supported by research grant SFRH/BPD/93928/2013 of “Fundação para a Ciência e a Tecnologia” (FCT) and by FEDER Funds through “Programa Operacional Factores de Competitividade—COMPETE” and by Portuguese Funds through FCT, in the form of grant PEst-OE/MAT/UI0013/2014. Project financial support was provided by the Organismo Autónomo de Parques Naturales de España of the Ministerio de Agricultura, Alimentación y Medio Ambiente (CEFAPARQUES, Project number: 458/2011)Peer reviewe

Syngnathids are considered as flagship species for marine conservation. Seahorses and pipefish are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently considered Data Deficient by IUCN, requiring more biological and ecological research. Although syngnathids are well known for their unusual breeding biology, some aspects on the ecology of this family have rarely received attention. The knowledge on the factors governing syngnathids distribution is limited to some species and geographical regions. The present study is the first approach to predict syngnathid habitat preference in Spanish coasts, particularly in a marine National Park. In this study, Species Distribution Models (SDMs) were implemented to investigate the preferential habitat and distribution of the pipefish Syngnathus acus in Cíes Archipelago (Atlantic Islands of Galicia National Park, PNIA). Occurrence data of the species obtained from 2016 to 2018 surveys in PNIA were modeled as a function of bathymetric (depth, slope), substrate (sediment texture) and oceanographic (waves exposure) variables, using GAM, Random Forest and Maxent algorithms. From those SDMs, prediction models were built and the ensemble map of predictions was performed. The variables that most determined the distribution of the species were depth and wave exposure. The results of this study provide information on (1) habitat preference in the most dominant species in PNIA, the pipefish S. acus, towards sustainable management of this species in the National Park, and (2) predictive statistical tools for proper spatial conservation plans of this syngnathid species.