A new deep-sea sponge Fibulia occiensis sp. nov. (Poecilosclerida: Dendoricellidae) from the 25° South Oceanic Core Complex in the Central Indian Ocean Ridge

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Authors describe a new deep-sea sponge species, Fibulia occiensis sp. nov., from the Central Indian Ocean Ridge (CIOR) at Rodrigues Triple Junction (RTJ). The sequences of Fibulia occiensis sp. nov. were submitted to NCBI GenBank and received the accession numbers: 28S MW810076 and 18S MW810071. The 28S is 716 bp, while the 18S is 1705 bp. 

The total genomic DNA was extracted from a small part of tissue around ~100 mg using a DNEasy Blood & Tissue Kit (Qiagen), eluted in 100 μL of sterile distilled water, and stored at a temperature of −20°C. The 18S rRNA gene was amplified by PCR using primers 18S1 and 18S2 (Borchiellini et al. 2001; Belinky et al. 2012) and 28S rRNA markers of the C1–D2 region using Ep1b and D2 primers (Chombard et al. 1997). Polymerase Chain Reaction (PCR) was performed in a reaction of 25 μL of TaKaRa Ex Premier DNA Polymerase (1X) (Takara) including 1 μL of DNA template (avg. conc. ≈70-80 ng/1 μL), 1μL of each (forward and reverse) primers (10 mM), and sterile distilled H2 O to a total volume of 25 μL. The PCR protocol for 18S gene amplification was: 3 minutes at 95°C (initial denaturation step), plus 34 cycles of 95°C for 40 seconds; 54°C for 40 seconds and 72°C for 1 minute, and final extension of 5 minutes at 72°C. A different annealing temperature was used for the amplification of the 28S fragment: one cycle of 3 minutes at 95°C, plus 34 cycles of 95°C for 40 seconds; 60°C for 40 seconds and 72°C for 1 minute, and final cycle of 5 minutes at 72°C.