Computational protocol: Species of Pseudorhabdosynochus (Monogenea, Diplectanidae) from Groupers (Mycteroperca spp., Epinephelidae) in the Mediterranean and Eastern Atlantic Ocean, with Special Reference to the ‘Beverleyburtonae Group’ and Description of Two New Species

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Protocol publication

[…] We used the QIAamp DNA Mini Kit (Qiagen), as per the manufacturer’s instructions, to perform DNA extraction. The 5′ region of the cytochrome oxidase I (COI) mitochondrial gene was amplified with the primers FishF1 (5′-TCAACCAACCACAAAGACATTGGCAC-3′) and FishR1 (5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′) []. PCR reactions were performed in 20 μl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 66 μM of each dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). The amplification protocol was 4 min at 94°C, followed by 40 cycles at 94°C for 30 sec, 48°C for 40 sec, and 72°C for 50 sec, with a final extension at 72°C for 7 min. PCR products were purified and sequenced in both directions on a 3730xl DNA Analyzer 96-capillary sequencer (Applied Biosystems). We used CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA) to edit sequences, compared them to the GenBank database content with BLAST, and deposited them in GenBank under accession numbers KX255747 –KX255751 (). Species identification was confirmed with the BOLD identification engine []. Details about molecular identification of host fish are provided briefly in the description of the monogenean species. [...] Diplectanids collected from fish gills were prepared by three methods: a) mounted in ammonium picrate-glycerine [] (designated as ‘p’); b) mounted in Berlese fluid (designated as ‘b’); and c) dehydrated in an ethanol series, stained with carmine and permanently mounted in Canada balsam (designated as ‘c’) []. Specimens were drawn using an Olympus BH2 microscope equipped with drawing apparatus and DIC optics. The terminology for the sclerotised parts, i.e. the male quadriloculate organ and the vagina follows Justine (2007) []. Measurements, in micrometres, were taken with the help of a custom-made transparent rule and are expressed as the mean followed in parentheses by the range, the standard deviation when n≥30, and (n) the number of observations; measurements were taken as in in Chaabane et al. (2015) []. The measurements of the right-hand haptoral hard-parts and left-hand equivalents were pooled. The measurements of the holotype are separated and indicated by ‘h’. Drawings were scanned and redrawn on a computer using Adobe Illustrator. The museum abbreviation used is as follows: MNHN, Muséum National d’Histoire Naturelle, Paris. [...] We used a QIAmp DNA Micro Kit (Qiagen) to extract DNA from whole monogenean specimens. The specific primers COI-ASmit1 (forward 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and COI-ASmit2 (reverse 5′-TAAAGAAAGAACATAATGAAAATG-3′) were used to amplify a fragment of 424 bp of the COI gene []. The PCR reaction was performed in 20 μl, containing 1 ng of DNA, 1× CoralLoad PCR buffer, 3 mM MgCl2, 0.25 mM dNTP, 0.15 μM of each primer, and 0.5 units of Taq DNA polymerase (Qiagen). Thermocycles consisted of an initial denaturation step at 94°C for 2 min, followed by 37 cycles of denaturation at 94°C for 30 sec, annealing at 48°C for 40 sec, and extension at 72°C for 50 sec. The final extension was conducted at 72°C for 5 min. Sequences were edited with CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA), compared to the GenBank database content with BLAST, and deposited in GenBank under accession numbers KX255741 –KX255746 ().Pairwise nucleotide distances were assessed using the Kimura 2-parameter (K2P) model [] in MEGA 7. The phylogenetic tree was constructed using the Neighbour Joining (NJ) method based on the Kimura 2-parameter (K2P) model in MEGA 7 []; all codon positions were used. […]

Pipeline specifications

Software tools FISHR, CodonCode Aligner, Adobe Illustrator, MEGA
Applications Miscellaneous, Phylogenetics, Population genetic analysis
Organisms Pseudotriton ruber, Danio rerio, Nitrosomonas sp., Homo sapiens