Computational protocol: Presence of the tunicate Asterocarpa humilis on ship hulls and aquaculture facilities in the coast of the Biobío Region, south central Chile

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Protocol publication

[…] Between December 2014 and October 2015, surveys and samplings were done on the hulls of three international vessels arrived into Talcahuano port (36°43′S, 73°07′W). The shipping routes of these vessels were traced back and the type of antifouling used (paint or silicone) was also recorded (). Samples of the biofouling community were collected by scraping in the following areas of the hull: bilge keel, sea chest, propeller/rope guard and rudder.For comparative purposes with the surveys made on the hulls, 18 settlement panels (10 cm × 10 cm) made of polypropylene were suspended at mid water depth in an area close to the sampling area of ships in Talcahuano bay, between April 16 and June 16, 2015. In addition, in 2016–2017, surveys and samplings of the subtidal hard bottom communities were made by scuba diving in seven localities along approximately 100 km of shoreline (): Talcahuano port, Coronel (37°1′49″S, 73°9′14″W), Chome (36°46′25″S, 73°12′49″W), San Vicente (36°45′33″S, 73°9′18″W), Lirquén (36°42′36″S, 72°58′57″W), Coliumo (36°32′16″S, 72°57′26″W) and El Manzo yacht club (a locality 1 km apart from the Talcahuano port). Finally, two aquaculture facilities (capture-based floating longlines) located nearby Llico (37°9′15″S 73°34′8″W), c.a. 70 km southward Talcahuano port, and nearby Coliumo (c.a. 20 km northward) were surveyed ().During these surveys, ascidian specimens were collected in plastic bags and transported back to the laboratory of the Faculty of Sciences at the Universidad Católica de la Santísima Concepción for further species identification. The specimens were first characterized under binocular according to morphological traits, as described notably in the online resource 1 and 2 in and by , which provide comprehensive recent morphological descriptions and references.A piece of branchial basket tissue was preserved in ethanol 95% for subsequent molecular analyses (performed on specimens collected prior 2017). We indeed ascertain the species identification by a molecular DNA barcoding approach following the method detailed in . Combining morphological and molecular data, these authors validated the use of COI to distinguish A. humilis and provided reference data for COI and 18S genes for this species. Briefly, total DNA was extracted using the “Nucleospin 96 Tissue core kit” (Macherey-Nagel, Düren, Germany) following the manufacturer’s protocol with a final elution in 100 µl. Two markers were used, the mitochondrial gene cytochrome c oxidase subunit I (COI) and the ribosomal RNA gene 18S. For the COI gene, PCR amplification was performed with the specific primer pair Ah-COIF (5′-CTAATTCGTACTGAGCTTTC-3′) and Ah-COIR (5′-GTTACTAATACCGTCCAACA-3′) developed by and which produces a fragment of 467 base pairs (bp). For 18S, we used two primer pairs: 18S1 (5′-CCTGGTTGATCCTGCCAG-3′) and 18S4 (5′-GATTAAAGAAAACATTCTTGGC-3′) (), and 18S-A (5′-CAGCAGCCGCGGTAATTCCAGCTC-3′) and 18S-B (5′-AAAGGGCAGGGACGTAATCAACG-3′) (). The two overlapping fragments of the 18S gene were amplified over a total length of 1640 bp. PCR conditions for the two genes are described in the online resource 1 in . PCR products were visualized by electrophoresis in a 1.5% agarose gel. Direct Sanger sequencing was performed in both directions at Eurofins Genomics (Berlin, Germany).Sequences were checked with the software CodonCodeAligner 5.1.4 (CodonCode Corporation, Dedham, MA, USA) and aligned using BioEdit (). Finally, sequences were analyzed by BLAST in GenBank ( which includes the sequences reported in for European and New Zealand samples of A. humilis, as well as samples from northern Chile (Coquimbo; ). Note that we did not use the BOLD database (Barcoding of Life Database) as the two A. humilis sequences deposited in BOLD are those from , already obtained from GenBank.Besides computing similarity indices, for displaying the results (i.e., similarities with previous published results) in a graphical way, we constructed a neighbor-joining tree of the Styelidae family, using MEGA v 6.06 (). For the nuclear 18S gene, a 583 base-pair fragment was considered for tree reconstruction to allow comparison with a large number of sequences available in the GenBank dataset, including particularly the sequence for Cnemidocarpa humilis (actually Asterocarpa humilis; see ) reported in (GenBank Accession No. FM244859) and the haplotype named Ah-H1 found in all A. humilis European samples examined by (GenBank Accession No. JX312280.1). Similarly, a neighbor-joining tree was constructed for the COI fragment, considering 394 base pairs and including the two haplotypes previously identified on European samples by (GenBank Accession No. JX312278.1, JX312279.1) and (GenBank Accession No. KU299758.1, KU299759.1). GenBank Accession numbers of the sequences used are indicated on and . […]

Pipeline specifications

Software tools BioEdit, MEGA-V
Applications Phylogenetics, GWAS
Organisms Homo sapiens
Diseases Cytochrome-c Oxidase Deficiency