Paralarvae were collected from a wild female that was spawning at Ria de Vigo in summer 2008. One thousand recently hatched paralarvae were transferred into a 30 L tank provided with filtered seawater (1 m). Tank was rectangular with black walls and white bottom. Mean water temperature was 19.3ºC (17.9-20.2), salinity 35‰ (34.4-35.6) and a 24 h light cycle. Live diet consisted of adult Artemia salina (2-9 mm total length) at a concentration of 0.05-0.1 ind. ml-1. During the experiment, nine paralarvae that fed on Artemia were fixed immediately in ethanol 70% and stored at -20ºC. Genomic DNA was extracted from a single paralarvae using the Qiagen tissue extraction kit (QIAGEN) following instructions from the supplier with minor modifications. A 658 pb fragment of the mitochondrial cytochrome oxidase I (COI) gene was amplified using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994). COI amplifications were set up in a 25 μl volume composed of 100 ng of genomic DNA from each Octopus, Artemia and the octopus that fed on Artemia, 2.5 μl 10X PCR reaction buffer, 0.5 μl dNTPs, 0.75 μl each primer and 0.025 U μl-1 Taq polymerase (Roche). Thermal cycling used a TGradient (Biometra). It began with an initial denaturation at 94º for 2 min, followed by 40 cycles of 15 sec at 94º, 30 sec at 48º and 45 sec at 70º and a final cycle of 7 min at 70 º.Due to the low amounts of Artemia inside Octopus paralarvae, specific primers were designed (using PRIMER3, Rozen and Skaletsky, 2000) for nested PCR in COI. A 250 pb fragments inside the COI gene was amplified using the specific primers ArteF: 5’-CTCCTCCTGGCCAGCTCTATG-3’ and ArteR: 5’-GGACGGCTGTAATTCCGACTG-3’. Amplifications were set up following the same procedure, but more specific primers were added: 0.8 μl and templates ranged from 1-3 μl COI product. The PCR thermal regime consisted of an initial denaturation at 94º for 3 min, followed by 40 cycles of 94º for 15 sec, 56º for 35 sec, 70º for 45 sec and a final step at 70º for 5 min. All PCR products were separated in 1.75% agarosa gel and visualized with ethidium bromide under UV light. The Artemia band of 250 Pb was detected into the nested PCR of the paralarvae that fed on Artemia, but no Artemia signal was detected into the nuclear extract of the paralarvae. Negative control made with Octopus paralarvae has no signal while the positive control (Artemia extract) presented the 250 pb band. PCR products were extracted from the gel, purified with QIAEX II Gel Extraction Kit and sequenced to check that the Artemia’s band was in fact from Artemia salina and no from contamination

Cephalopod International Advisory Council Symposium. Vigo (Spain), 3-11 September 2009

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