The marine ornamental fish examined in this study were donated by a commercial aquarium in Merida, Yucatán between January 2018 to December 2020, a total sample of 348 ornamental fish (Table 1). Most of the fish were originally captured from the natural environment of the Indo-Pacific region, although the exact capture locations were not available to the importer. The ornamental fish were imported from their centre of origin and distributed to several regions of Mexico (specifically, until their point of sale) or transferred to the market Morelos in Mexico City, which represents one of the main commercialization and distribution centres for ornamental fish, before being dispersed throughout the country (e.g. Merida, Yucatán). The imported fish were transported in isolated plastic bags with artificial aeration. Once at their point of sale (i.e. the aquarium in Merida), the dead or dying fish were separated and kept in coolers, posteriorly donated and transported to the Aquatic Pathology Laboratory at CINVESTAV-IPN Unidad Mérida for parasitological examination. Once at the laboratory, fish was measured to obtain its total length (TL, cm), standard length (SL, cm) and total weight (W, g). The surface of the skin and eyes, gills, scales from the lateral line and fins were examined under a stereomicroscope (Stemi 305 Carl Zeiss) for ectoparasites. Whenever parasites were found, they were counted, preliminarily identified to the genus level and fixed depending on the taxonomic group (Whittington 2004). Capsalid monogeneans were isolated, counted in situ, cleaned with physiological saline and preserved in 4% formalin or 96% alcohol labelled vials for subsequent morphological or molecular studies, respectively (Brazenor et al. 2018a, b). They were removed with fine paintbrushes, stained with ammonium picrate and identified to the species level according to suitable literature (e.g., Whittington and Kearn 1993; Hargis 1995; Ogawa et al. 2006). Infection parameters, such as prevalence, mean abundance and mean intensity were those proposed by Bush et al. (1997). Standard measurements were made using an OLYMPUS BX50 compound microscope (Olympus, Tokyo, Japan) and ImageJ software (Wayne Rasband Scientific Software, Kensington, Maryland, USA). Drawings were prepared using Adobe Illustrator software (Adobe Inc., San Jose, California, USA). The following features were measured for morphological and morphometric description: body length and width; pair of anterior attachment organs, length by width; haptor, length; anterior hamuli, length, posterior hamuli, length, accessory sclerites, length; pair of testes, length by width; ovary, length by width; egg, length by width (Whittington and Kearn 1993; Whittington 2004) (Table 2). All measurements are given below in millimetres (mm), with the range followed by the mean in parentheses (Table 2).
Dna Amplification, Sequencing And Phylogenetic Analyses
For the genetic study, genomic DNA was extracted from each specimen of Neobenedenia using a DNeasy TM Blood & Tissue Kit (Qiagen, Hilden, Germany) following the standard manufacturer’s protocol. Specimens of different host species were chosen for extraction. Given that the 28S ribosomal gene has been used in other studies to identify species of Neobenedenia (Brazenor et al. 2018a, b), we also amplified the D1, D2 and D3 regions of this gene. The amplification was carried out with the primers 391 (Nadler and Hudspeth 1998) and 536 (García-Varela and Nadler 2005), and the conditions of the polymerase chain reaction (PCR) amplification were: 94°C for 5 min, 35 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min and a post-amplification extension at 72°C for 10 min. For sequencing, the two amplification primers plus 503 (Stock et al. 2001) and 504 (García-Varela and Nadler 2005) were used. Sequencing was carried out in GENEWIZ (South Plainfield, NJ, USA). The sequences obtained from each primer were read, edited and assembled into a consensus sequence for each extracted specimen using Geneious Pro 4.8.4® (Biomatters Ltd.). The new sequences were submitted to GenBank for publication and public access. For phylogenetic analyses, the new sequences were aligned with other 28S sequences from Neobenedenia available in GenBank. The alignment was performed using ClustalW (Thompson and Gibson 1994), implemented in http://www.genome.jp/tools/clustalw/ (“SLOW/ACCURATE” and “CLUSTALW (for DNA)”). The nucleotide evolution model was estimated in jModelTest v.2 (Darriba et al. 2012). A maximum likelihood analysis (ML) was performed to obtain the phylogenetic tree with RAxML v. 7.0.4 (Stamatakis 2006) and 1,000 bootstrap repetitions (bt) were implemented. The ML tree was visualized in FigTree v.1.4.3. (Rambaut 2000). The genetic distances of 28S gene, were calculated using uncorrected p-value (p-distances) in MEGA v.6.0 (Tamura et al. 2013).