First report of Contracaecum jorgei (Nematoda: Anisakidae) in Centropomus armatus from a fish market in Panama City, Republic of Panama
Article Category: Research Note
Published Online: May 24, 2025
Page range: 77 - 82
Received: Jul 27, 2024
Accepted: Feb 21, 2025
DOI: https://doi.org/10.2478/helm-2025-0005
Keywords
© 2025 K. B. González-Serrano et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Among these species,
Fifteen specimens of
For morphological identification, labeled third stage larvae were cut into three pieces, the anterior and posterior tips were cleared with the ethanol/glycerine method (Seinhorst, 1962). The cuticular tooth, esophagus and cecum were observed in the anterior tip and the tail in the posterior tip with an optic microscope (Amscope, model B450.3MP) following dichotomous keys, used by Sardella
Total DNA was extracted from the excised midbody (approximately 0.5 mm) using a commercial kit DNeasy Blood & tissue (Qiagen), according to the manufacturer’s instructions. Two combinations of primers were used for the amplification of fragments. The first one, cytochrome oxidase 2 gene fragment (Cox-II) (Nadler & Hudspeth, 2000), was performed using the forward primer 211 (5′-TTTTCTAGTTATATAGATTGRTT TYAT-3′) and the reverse primer 210 (5′-CACCAACTCTTAAAATTATC-3′). Amplification was conducted with 5 μl of the DNA template, 12.5 μl of Taq PCR Master Mix Kit (Qiagen), 1 μl of each primer (10 pmol μl−1) and 5.5 μl of ddH2O, for a final volume of 25 μl; and processed using SimpliAmp (Applied biosystems by life technologies), with an initial denaturation at 94°C for 4 minutes, 25 cycles of denaturation at 94°C for 30 seconds, annealing at 45°C for 30 seconds, extension at 72°C for 50 seconds and a final extension step at 72°C for 5 minutes, followed by temperature on hold at 4°C.
For the small subunit of the mitochondrial ribosomal RNA gene (rrnS), primer MH3 (forward 5′-TTG TTC CAG AAT AAT CGG CTA GAC TT-3′) and MH4.5 (reverse 5′-TCT ACT TTA CTA CAA CTT ACT CC-3′) were used (D’Amelio et al. 2007). Amplification was conducted with 4 μl of the DNA template, 12.5 μl of Taq PCR Master Mix Kit (Qiagen), 1 μl of each primer (10 pmol μl−1), and 6.5 μl of ddH2O, for a final volume of 24 μl; and processed using SimpliAmp (Applied biosystems by life technologies), with initial denaturation at 95°C for 10 minutes, 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds and a final extension step at 72°C for 10 minutes followed by temperature on hold at 4°C.
PCR products were purified using Exosap-IT (Thermo Fisher Scientific), according to the manufacturer’s instructions, and directly sequenced in both directions using the Big Dye terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Waltham, MA, USA) and the 3130xl Genetic Analyzer (Applied Biosystems, Foster, Ca, USA). Sequences were edited using Sequencher version 5.4.6.
Partial Cox II and rrnS sequences were aligned with those available in GenBank. Alignments were performed with MAFFT version 1.5.0 (Katoh & Standley, 2013) and visualized using Geneious Prime 2024.0.1 (Kearse
The study received ethical exemption approval from the Research Ethics and Animal Welfare Committee of the University of Panama (CEIBA-UP) CEIBA-UP-021-2023.
The prevalence of parasites was 6 % (1/15), for
Morphological identification of the third stage larvae of
Phylogenetic trees of Cox II and rmS markers describe the relationship between the sequences generated in the study and the ones retrieved from the GenBank database. (Fig. 3 and Fig. 4). Notably, the bootstrapping analysis of Cox II phylogeny provided substantial support for the clade encompassing the present sequences and previously published
Phylogenetic relationship among different isolates of
Maximum likelihood tree of the two sequences of the rrnS fragment in the current study with other
The definitive host for
This study constitutes the first report of nematode larvae of the Anisakidae family, highlighting the need to continue research, in order to identify other parasites, especially from this family, considering their potential impact on public health.