Sequence Analysis Of 5.8S Rdna And Internal Transcribed Spacers Of The Ribosomal Region Of Americonuphis Reesei (Polychaeta: Onuphidae) In The Republic Of Panama. First Report.

摘要

Specimens of the polychaete Americonuphis reesei from two locations in the Pacific Ocean off the coast of the Republic of Panama were examined by sequencing of the internal transcribed spacers (ITS1 and ITS2). The complete sequence of the 5.8S ribosomal DNA (rDNA) was obtained and the 3’ end of the 18S rDNA and 5’ end of the 28S rDNA were also determined. Neighbor-joining analysis of ITS variability indicates a close relationship between the two populations of Americonuphis reesei. Introduction Americonuphis reesei belongs to the Onuphidae family of the phylum Annelida. This species consists of individuals that live in a thick mud-walled tube; they are carnivorous and extend out of the tube to trap their prey (Villalaz, 1997). A. reesei is harvested and used locally in Panama to provide feed for the culture systems of penaeid shrimp (D’Croz et al., 1988, Spadafora, 1994). Because these shrimp are a species of key economic importance in Panama, extensive studies of A. reesei concerning their ecology, embryology and physiology have been performed (Luna et al., 2001; Villalaz et al 2001).A. reesei is found in two locations in the Gulf of Panama, Republic of Panama: one in muddy bottoms at a depth of 27 meters and the other in the sandy bottoms of an intertidal region (Fauchald, 1973). Two populations from the intertidal region have been selected for phylogenetic comparison in part on the basis of a different morphology, namely their reproductive period (Luna et al., 2001).A. reesei has previously been characterized using morphological markers (Fauchald, 1973; Luna et al., 2001 ) and phylogenetic studies of this species have not yet been reported. However, phylogenetic studies of other species of annelids belonging to the families Tubificidae, Dorvilleidae, Syllidae, Eunicidae, Onuphidae and Hartemanniellidae have been conducted using molecular markers such as 18S ribosomal DNA (rDNA), as well as 16S mitochondrial rDNA (Dahlgren et al. 2001; Struck et al. 2002; Nygren y Sundberg 2003). Additionally, randomly amplified polymorphic DNA (RAPDs) and internal transcribed spacers (ITS1 and ITS2) of rDNA have been used to genetically characterize cryptic species of annelids, such as Petitia oculta (Westheide and Hass-Cordes, 2001; Meyer et al. 2008).In this study we analyzed the sequence of the region of rDNA spanning from the 3’end of the 18S rDNA, including ITS1, ITS2, and 5.8 rDNA and ending at the 5’end of the 28S rDNA of A. reesei from two localities off the Pacific coast of the Republic of Panama. MATERIAL AND METHODS Sampling:The polychaetes were collected by hand from Agallito beach in Herrera Province and El Salao beach in Coclé Province, which are both located on the Pacific side of the Republic of Panamá.A total of 25 A. reesei specimens, 15 from the Salao population and 10 from the Agallito population were examined by ITS analysis.DNA extraction and Phylogenetic analysisGenomic DNA was extracted from the polychaetes as previously described, with small modifications (Chen and Yu, 2000 and Chen et al., 2002). Specifically small pieces of tissue were transferred to lysis buffer (0.4 M NaCl, 200mM EDTA, pH 8.0) and then incubated at 65°C for 30 minutes. Proteinase K was then added to the sample at a final concentration of 0.1 mg/ml, and samples were incubated at 55°C for 3-4 hours. After centrifugation at 12000 rpm for 10 minutes, the supernatant was extracted with equal volumes of phenol/chloroform, and DNA was precipitated overnight at -20°C with absolute ethanol and 0.3 M sodium acetate. The samples were centrifuged at 12000 rpm for 15 minutes, washed with 70% ethanol, resuspended with distilled water and stored at -20°C.ITS rDNA amplification and sequencing:PCR reactions were set up as previously described (Chen et al., 2002). The forward primer, 5’-GGTACCCTTT GTACA-CACCG-CCCGTCGCT3’ annealed to the 3’ end of the 18S rDNA and the reverse primer, 5’GCTTTGGGCT-GCAGTCCCAAG-CAACCCGACTC-3’ to the 5’ end of the 28S rDNA. PCR produc