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The determination of in vitro digestibility of acid proteases in the digestive gland (DG) of adult O. vulgaris (>1,2 Kg) was performed, using four different preys (Mackrel, Scomber scombrus; Green crab, Carcinus maenas; Swimming crab, Necora puber; Shortfin squid, Illex coindetti). For each prey, four treatments were applied: 1) Frozen, 2) boiled at 100ºC for 1 minute, 3) Lyophilized and 4) Oven dried at 70ºC. A pH-Stat was used to determine in vitro digestibility at pH 3. Results showed that digestibility was higher (p0.05), compared to the other two treatments that suffered heating (boiled and oven dried), regardless of the diets tested. Regarding acid proteases metabolism, which is the most important for O. vulgaris, results indicate that heat treatments close and/or higher than 70ºC promote changes in the different diets that prevent their assimilation by the DG of O. vulgaris.

This study analyzes the effect of low salinity on survival and daily food ingestion of octopus subjected to three different water salinities (20, 25, and 30 psu) for 3 days. During the 3-day experiment, octopuses at 20 psu immediately stopped eating, and all died during the first day; at 25 and 30 psu, the octopuses gradually reduced their feed intake and stopped eating after 2 days, but all survived, and when normal salinity conditions were reestablished (34–35 psu), they ingested 30–40 (~27.5% of the initial food intake) and 70–90 g of food/day (~63% of the initial food intake) respectively over the 5-day recovery period. Water salinity of 30 psu could be considered as the recommended lower limit below which actions, such as transferring the commercial floating cages to another location or submerging them, should be taken in order to prevent the negative effects of low salinities on farmed octopuses., 0,0000

6 pages, 3 figures, 2 tables, The common octopus, Octopus vulgaris is a new candidate species for aquaculture. However, rearing of octopus paralarvae is hampered by high mortality and poor growth rates that impede its entire culture. The study of genes involved in the octopus development and immune response capability could help to understand the key of paralarvae survival and thus, to complete the octopus life cycle. Quantitative real-time PCR (RT-qPCR) is the most frequently tool used to quantify the gene expression because of specificity and sensitivity. However, reliability of RT-qPCR requires the selection of appropriate normalization genes whose expression must be stable across the different experimental conditions of the study. Hence, the aim of the present work is to evaluate the stability of six candidate genes: β-actin (ACT), elongation factor 1-α (EF), ubiquitin (UBI), β-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GADPH) and ribosomal RNA 18 (18S) in order to select the best reference gene. The stability of gene expression was analyzed using geNorm, NormFinder and Bestkeeper, in octopus paralarvae of seven developmental stages (embryo, paralarvae of 0, 10, 15, 20, 30 and 34 days) and paralarvae of 20 days after challenge with Vibrio lentus and Vibrio splendidus. The results were validated by measuring the expression of PGRP, a stimuli-specific gene. Our results showed UBI, EF and 18S as the most suitable reference genes during development of octopus paralarvae, and UBI, ACT and 18S for bacterial infection. These results provide a basis for further studies exploring molecular mechanism of their development and innate immune defense, This work was funded by the “Xunta de Galicia” – Spain 10PXIB402116PR research project, and “AGL20134910-C02-2R” from the Spanish Ministerio de Economia y Competitividad. S. Castellanos-Martínez thanks “CONACYT” – Mexico for her awarded scholarship, and P. García-Fernández thanks “Xunta de Galicia” for his predoctoral fellowship

Octopus vulgaris farming at large scale can only be attained using live prey during the paralarvae stage. Presently, only Artemia complies with this requirement. Nevertheless, its sole use delivers poor paralarvae growth and survival. Some species of marine zooplankton are better prey for marine fish larvae compared to Artemia, since its composition is richer in several important nutritional components. Among these are phospholipids and specific fatty acids, namely docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid. During the present experiment, octopus paralarvae were fed a co-feeding scheme of spider crab (Maja brachydactyla) zoeae and Artemia (1:2, Artemia/Crab zoeae). The use of spider crab zoeae was justified by their availability in commercial facilities, where adult spider crabs are maintained to be sold to the public. There, fecund and spawning females are present in large numbers, and zoeae availability is often high and implies no production or zoeae collection costs. O. vulgaris paralarvae fed on Artemia and crab zoeae grew larger (3.00 ± 0.56 mg dw−1) after 30 days, compared to previous published papers. Also, the paralarvae lipid content was substantially enhanced in highly unsaturated fatty acids and phospholipids. However, survival after 30 days was still very low (1.75 % after 30 days) and needs to be greatly improved. Publicado 1

Commercial culture of octopus has not yet been achieved due to the high mortality recorded during the first weeks of life. Several researchers have pointed out nutrition as the key factor of influence on paralarval mortality in culture (Iglesias, Sanchez, Bersano, Carrasco, Dhont, Fuentes, Linares, Munoz, Okumura, Roo, van der Meeren, Vidal & Villanueva 2007; Villanueva, Escudero, Deulofeu, Bozzano & Casoliva 2009; Seixas, Rey-Mendez, Valente & Otero 2010; Seixas, Otero, Valente, Dias & Rey-Mendez 2010; Fuentes, Sanchez, Lago, Iglesias, Pazos and Linares 2011). Nevertheless, several other factors, such as culture volume, can influence the success in paralarvae rearing. The lack of a standardized culture technique has meant that some authors have reared paralarvae in relatively large tank volumes: from 20 000 L used by Hamazaki, Fukunaga, Yoshida and Maruyama (1991), 9000 L (Moxica, Linares, Otero, Iglesias & Sanchez 2002), 6000 L (De Wolf, Lenzi & Lenzi 2011) and 1000 L (Iglesias, Otero, Moxica, Fuentes & Sanchez 2004), whereas others have worked with smaller volumes: Villanueva, Koueta, Riba and BoucaudCamou (2002) used 25 L tanks, Carrasco, Arronte & Rodriguez 2006; 30 L and Seixas (2009) 50 L. This study analyses tank volume effect on growth and survival of octopus paralarvae in their first month of life, using enriched artemia as live diet. Protein and lipid composition of paralarvae were also analysed to have an approach to paralarvae condition. Materials and methods

Marine protected areas (MPAs) are becoming increasingly important tools in the conservation of cetaceans. This has led to an interest in the use of species distribution models to predict where cetacean species are likely to occur based on the distribution of environmental variables. However, relationships between cetacean distribution and environmental variables are generally assumed to reflect the environmental preferences of their prey. Thus, understanding the distribution of prey may increase our ability to identify important areas for cetaceans. Here, we describe the diet of Risso’s dolphin (Grampus griseus) by analysing stomach contents of individuals stranded in Scotland over the last twenty years. Next, we use habitat modelling to test whether Rissos’s dolphin distribution in Scottish waters, as inferred from sightings, is related to the distribution of its main prey, the octopus Eledone cirrhosa. While good models of the relationship between the distribution of E. cirrhosa and environmental variables were obtained, there was no evidence of a relationship between modelled octopus distribution and the occurrence of Risso’s dolphins. These results suggest that identifying key areas for its main prey species is unlikely to help identify potential MPAs for Risso’s dolphin, at least at the spatial resolution used in this study.

Octopus maya is a carnivorous species and protein is the main energy source. During the present study, two different dietary protein levels (40 and 60% CP) were offered to octopuses as specifically designed artificial diets, to determine protein needs and the effects on metabolism. Frozen crab (Callinectes spp.) was used as control. Results obtained demonstrated that crab remains as one of the best diets for O. maya. The artificial diet with 60% CP produced a low but positive growth rate, and at times, a physiological response similar to that observed in octopuses fed crabs. The present results show the capacity of O. maya juveniles to adjust their digestive enzymes to different types of food and protein level, and this appears to be well correlated with octopus growth. General proteases and trypsin from the pancreas were well correlated with growth rates. A low activity was observed in octopuses fed 40% PC diet (negative growth rate), while a high activity was present in octopuses fed 60% CP diet and crabs (low and high growth rate, respectively). In contrast, these same enzymes were inducted in the salivary glands of octopuses fed with the diet that promoted weight loss (40% CP diet), while a reduced activity was observed in octopuses fed crabs. Energy budget indicates that the animals ingested more than 1,000 kJ week−1 kg−1; with such energy, octopuses should satisfy their physiological demands such as was observed when animals were fed crab (I = 1,300 kJ week−1 kg−1; P = 834 kJ week−1 kg−1). However, a very low digested energy was observed in octopuses the fed artificial diets, indicating that these could have a factor that limits digestibility., 1

The purpoase of this research was to test the effects of three culture densities on the growth and survival of Octopus vulgaris. A total of 141 sub-adult octopuses (1,175.4 ± 194.9 g) were randomly distributed in nine tanks of 2,000 l each (3.6 m × 1.1 m, and 50 cm water depth). Three tanks were stocked with a low initial density of 4 kg/m3, while three other tanks were stocked with an initial density of 8 kg/m3, and the remaining three tanks were stocked at an initial density of 15 kg/m3. Octopuses were all fed frozen squid (Loligo gahi) at 5% body weight per day (%BW/day). The experiment lasted for 70 days. Water temperature varied between 20 ± 2°C, and salinity varied between 36 ± 1 ppt. During the entire experiment, dissolved oxygen was always >75%, and ammonia was always lower than 0.1 mg/l. No differences in growth or growth rates (between 0.9 and 1.1%BW/day for the three densities) were found. Nevertheless, mortality was significantly lower for the low density compared to the other two densities tested. Maximum densities in the culture tanks (>25 kg/m3) were attained in the higher culture densities after 56 days of the experiment., 1

The effects of two alternative prey (crayfish and hake) were tested on growth and survival of both juveniles and adults of Octopus vulgaris in two experiments. Octopuses fed the control (squid) were larger (3.0 ± 0.7 kg) than those fed crayfish (2.4 ± 0.6 kg) at the end of experiment I. Similarly, overall growth rates were higher for octopuses fed squid (1.7 ± 0.3 and 1.2 ± 0.2 %BW day−1, respectively). Average feeding rates for the experiment were not different, being 6.5 ± 0.9 and 7.5 ± 0.9 %BW day−1, respectively, for octopuses fed either squid or crayfish. Nevertheless, food conversions for the experiment were higher (42.4 ± 2.7%) for octopuses fed squid compared to the ones fed crayfish (23.9 ± 1.9 g). For experiment II, hake and crayfish were compared to squid; the final weight of octopuses fed squid, hake or crayfish was 1,183.0 ± 242.7 g, 1,175.6 ± 240.1 g and 922.3 ± 160.1 g, respectively. Overall growth rates for the experiment were 1.9 ± 0.2 %BW day−1, 1.9 ± 0.3 %BW day−1 and 1.1 ± 0.3 g, respectively. Final weight and growth rates were never different (P > 0.05) between octopuses fed squid and hake, but were always higher (P < 0.05) compared to the ones fed crayfish. Average feeding rates for experiment II were similar for the three diets, and of 4.6 ± 1.5, 4.2 ± 1.3 %BW day−1 and 5.1 ± 0.9 %BW day−1, respectively, for octopuses fed squid, hake or crayfish. Food conversions for experiment II were of 41.0 ± 9.6%, 40.5 ± 9.9% and 21.3 ± 7.4 g, respectively, for octopuses fed squid, hake or crayfish, and were always higher for octopuses fed squid and hake compared to crayfish. The results indicate that crayfish is not an adequate replacement for the usual prey to fatten octopus, even considering its much lower market price., 1

15 pages, 2 figures, 2 tables Due to the high mortality rates and poor growth generally observed in Octopus vulgaris paralarval rearing experiments, it was decided to organize a working group in order to formulate recommendations to tackle this problem. Over a dozen scientists representing the most active current research groups related to this subject attended the meeting in Vigo, Spain, in November 2005. The aim of this working group was to determine the bottlenecks that prevent success in paralarval rearing, define the most appropriate rearing conditions, and identify required future research. This paper describes rearing techniques for the O. vulgaris paralarvae used by the different research participant teams, with regard to tank systems, feeding environment, and diets (Artemia, crustacean zoeae, sandeel flakes, copepods, etc.). Additionally, it includes other related themes such as the culture of Artemia and copepods, organisms that are commonly used in paralarval rearing. When embarking on O. vulgaris rearing it is advised to use prey rich in DHA (docosaenoic acid, 22:6n-3) and EPA (eicosapentaenoic acid, 20:5n-3), and with high DHA/EPA ratio. Such prey could be enriched Artemia, accompanied or not by crustacean zoeae or any microdiet. It is also recommended that, in future studies, values of growth and survival rates are recorded at the beginning of the benthic phase, in order to compare them to successful previous studies. Dry weight and DHA/EPA ratio of paralarvae may also be good criteria to define paralarval viability and evaluate success of the rearing system.It is further concluded that the nutritional aspect is the most important factor influencing larval mortality. Certain lipids (phospholipids, cholesterol, and polyunsaturated fatty acids), amino acids (lysine, leucine and arginine), and essential elements (e.g. copper) play a relevant role in the larval nutrition. It is believed that the PUFA content, especially DHA and EPA, constitutes one of the basic nutritional requirements. Regarding new research lines, no standardized system for paralarval rearing exists, and it is essential to make progress on this issue. Research on nutritional requirements is considered an area of highest priority, especially the development of a specific enrichment for Artemia, the search for alternative live preys, and the development of suitable formulated diets. ThisWorking Group has been supported by the General Fisheries Secretariat (Spanish Ministry of Agriculture, Fishery and Food). Roger Villanueva was supported by the Centre de Referència de Recerca i Desenvolupament en Aqüicultura, CIRIT, Generalitat de Catalunya. Érica A. G. Vidal was supported by the Brazilian National Research Council (CNPq-PROFIX).

The effect of a dry pelleted diet on growth of the Yucatan octopus (Octopus maya) was determined and compared with crab diet (Callinectes spp). Two groups of 15 wild collected animals were used. Octopuses were placed in isolation, in a flow-through system composed of 30 circular tanks (80 L), with a bottom of 40 cm in diameter and a water depth of 80 cm. Experiment lasted 40 days, and octopuses were weighed every 10 days to determine growth rates. Octopuses were fed between 7% and 10% body weight (BW) per day, twice a day at 9:00 h and 17:00 h. Uneaten food was removed after 4 h in the tanks and weighed, to determine food ingestion and conversion. No mortality as a result of natural causes was observed. The 15 octopuses fed on frozen crabs and the 15 fed on the artificial diet had similar weights (P > 0.05) at the start of the experiment, of 486.2 ± 214.8 and 472.5 ± 228.3 g, respectively. At the end of the experiment, octopuses fed on frozen crabs weighed 1466.5 ± 484.0 g, and were significantly larger (P, 2