Morphology, DNA barcoding and seasonal occurrence of Ergasilus lizae Krøyer, 1863 (Copepoda: Ergasilidae) parasitizing mullets from northwestern Mexico

Ergasilus lizae Krøyer, 1863 is a parasitic copepod known to infect mullets (Mugilidae) in different parts of the world. It was originally reported from the east coast of North America, but the original description lacks enough detail, making identification with this information difficult. In this study, we provide a redescription of E. lizae found on Mugil curema Valenciennes and M. cephalus Linnaeus, caught in two coastal lagoons of northwestern Mexico during two climatic seasons: warm/rainy and cold/dry. The prevalence of this parasite was higher in the warm season than in the cold season. To facilitate the species identification, new sequences of the barcoding gene (COI mtDNA) of E. lizae were generated and compared against unpublished sequences of E. lizae available in the Barcode of Life Database (BOLD). Our results suggest that the sequences of BOLD possibly belong to a species misidentified as E. lizae.

In northwestern Mexico, E. lizae was reported by Causey (1960) from M. cephalus collected in Mazatlán, Sinaloa state and San Blas, Nayarit state, but comments related to the species identification were not provided.As far as we know, the presence of E. lizae in Mexican fishes has not been confirmed in previous studies other than Causey (1960).In fact, ergasilids remain largely unstudied in Central America and Mexico and many reports are referred to as Ergasilus sp.(Suárez-Morales & Santana-Piñeros, 2008;Jiménez-García & Suárez-Morales, 2017).
The identification of ergasilids could be more precise with the integration of morphological and molecular data, particularly the barcoding gene, cytochrome c oxidase subunit I (COI mtDNA).However, the number of COI sequences is currently limited to very few species of Ergasilus, in part due to difficulties in the amplification process (Míč et al., 2023).Recently, a comparative analysis of COI sequences of ergasilids could only include sequences for three known species of Ergasilus due to the unavailability of verifiable published sequences (Fikiye et al., 2023).One of those three species was E. lizae whose unpublished sequences are available in the Barcode of Life Database (BOLD).These sequences were generated from specimens found in Fundulus diaphanus (Lesueur) collected in Richelieu River, Quebec, Canada.
In the present study, we provide morphological data of specimens of E. lizae collected from M. curema and M. cephalus from two coastal lagoons in Sinaloa state, northwestern Mexico.Newly generated COI sequences of E. lizae were compared with sequences retrieved from BOLD.Additionally, the prevalence and intensity of the infection in warm and cold seasons were assessed.

Fish and parasite collection
Ninety-three specimens of M. curema were purchased from local fishermen in two seasons.These fish were caught during August 2022 (warm/rainy season, with an average temperature of 32 °C) and January 2023 (cold/dry season with an average temperature of 19 °C) in Urías estuary and Huizache-Caimanero coastal lagoon, southern Sinaloa state, northwestern Mexico (Figure 1, Table 1).Additionally, 21 specimens of M. cephalus were purchased from fisherman, caught during August 2023 in Huizache-Caimanero.
Both lagoons have multiple anthropogenic stressors (Martínez-Salcido et al., 2018).Urías estuary has been strongly impacted by sewage and industrial effluents from the Mazatlán harbor, whereas Huizache-Caimanero lagoon has mostly been impacted by agriculture.The geographical distance between these lagoons is approximately 45 km.
In the laboratory, fish were identified to species level following Froese and Pauly (2023).The total length (cm) of each fish was recorded.Gills were removed and examined for the presence of parasitic copepods with the aid of a dissecting microscope (Motic, Richmond, BC, Canada).Copepods were counted, removed using fine needles and preserved in 96% ethanol.

Morphological analysis
Copepods were cleared in 85% lactic acid for a few minutes and then examined under a Leica DMLB microscope.The total body length, from the anterior margin of the prosome to the posterior margin of caudal rami (excluding caudal setae), was measured using an ocular micrometer.Drawings of the entire body and dissected appendages, temporarily mounted in slides with lactic acid, were made with the aid of a drawing tube.Final illustrations were digitally inked using INKSCAPE 1.0.The voucher specimens were deposited in the Copepoda collection of the Instituto de Ciencias del Mar y Limnología, Unidad Académica Mazatlán (ICML-EMUCOP), Sinaloa, Mexico.

Molecular analysis
Total DNA was extracted using the Jena Bioscience kit, following the manufacturer's instructions (Jena Bioscience, Jena, Germany).Amplification and sequencing of the COI gene were carried out using invertebrate universal "Folmer" primers LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′).Thermal cycling conditions for amplification reactions were 94 °C for 2 min, 30 cycles at 94 °C for 1 min, 48 °C for 40 s, 72 °C for 2 min and a final extension at 72 °C for 7 min.PCR products were sequenced using an ABI 3730xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at the Laboratorio Nacional de la Biodiversidad, Instituto de Biología, Universidad Nacional Autónoma de México.
Forward and reverse sequences were assembled using Geneious 5.1.5(Biomatters Ltd.Auckland, New Zealand).The newly generated sequences were compared against sequences of E. lizae retrieved from BOLD and the sequences of other 10 species of Ergasilus available in GenBank (Table 2).Sequences were aligned using MUSCLE 3.8 (Edgar, 2004) with default parameters, and trimmed using trimAL 1.2 (Capella-Gutiérrez et al., 2009) with the parameter gappyout.The best evolutive model, GTRGAMMA, was selected with the program ModelFinder (Kalyaanamoorthy et al., 2017).A phylogenetic tree was constructed under Maximum likelihood (ML) with RAxML 8.2.12 (Stamatakis, 2014).Nodal support for the tree was assessed through bootstrap analysis with 1000 replicates.Lernaea cyprinacea (Linnaeus, 1758) (Copepoda: Cyclopoida: Lernaeidae) was used as the outgroup.Pairwise sequence similarity was estimated with Clustal-Omega 2.1 (Sievers et al., 2011).Gen-Bank accessions of the COI sequences generated for E. lizae of the present study are given in Table 2.

Occurrence
The prevalence and median intensity of infection (sensu Bush et al., 1997;Reiczigel et al., 2019), with confidence intervals (CI) at 95%, were calculated and compared between seasons with Fisher's exact test and bootstrap t-test (with 1000 replications), respectively, using the software Quantitative Parasitology (Reiczigel et al., 2019).
Mouth parts (Figure 3B) comprising mandible, maxillule and maxilla.Mandible consisting of 3 blades of which anterior blade smallest and with teeth on anterior margin, middle blade wide with teeth anteriorly and posteriorly, posterior blade elongated with teeth on posterior margin.Maxillule lobate, with 3 unequal setae.Maxilla 2-segmented; distal segment bearing sharp teeth anteriorly and 1 spinulate seta.
Swimming legs 1 to 4 (Figs.4A, B, 5A, B) biramous.All rami 3-segmented, except for 2-segmented exopod of leg 4 (Figure 5B).All legs with row of spinules on outer margin of both rami and setules on inner margin of first exopodal segments.Second leg basis with spiniform process between insertion of endopodal and exopodal rami.Interpodal plates with rows of spinules on posterior margin (Figure 3C).Armature of legs as follows (Roman and Arabic numerals for spines and setae, respectively).

Occurrence
The prevalence of E. lizae on M. curema was significantly higher in the warm than in the cold season in both coastal lagoons.In Urías estuary, the prevalence was 62% (CI 43-78%) and 12.5% (CI 3.5-30%) in the warm and the cold season, respectively, whereas in Huizache-Caimanero lagoon it was 55% (CI 32-76%) and 12% (CI 2-34%) in the warm and the cold season, respectively, respectively (P < 0.05).In both lagoons, the median intensity was 2 and 4 (CI 1-6) copepods per fish in the warm and cold season, respectively.This difference was not significant (P > 0.05).This analysis was not performed for M. cephalus because samples were not obtained during the cold season.

Morphology
The morphological characteristics of the specimens examined in the present study agree with those of E. lizae as redescribed by El-Rashidy (1999), who examined the lectotype material (CRU-7091) deposited in Copenhagen Zoological Museum.We agree with El-Rashidy (1999) that the material redescribed by Kabata (1992) is not conspecific with E. lizae.Kabata's material lacks the depression between the cephalosome and first pedigerous somite, the spinulate seta on the basis of the maxilla, the surface ornamentation on the interpodal plates, and the outer spine on the second exopodal segment of leg 1, all of which are typical for E. lizae.Additionally, the maxillule has 3 outer setae and 1 medial process in E. lizae but only 2 outer setae are present in Kabata's material.Also, the second segment of leg 5 has 3 setae in E. lizae but only 2 in Kabata's material.Regarding the habitat, our specimens occurred on fish caught in marine waters, whereas the Australian specimens were taken in freshwater habitats.According to Jiménez-García and Suárez-Morales (2017), E. lizae seems to be restricted to marine habitats.
Ergasilus lizae morphologically resembles E. arthrosis Roberts, 1969, E. atafonensis Amado & Rocha, 1996, and E. parabahiensis El-Rashidy & Boxshall, 1999(El-Rashidy, 1999).Compared with E. arthrosis, E. lizae has a shorter antennary claw and a distal segment of the antenna with 8 setae instead of 5. Additionally, E. arthrosis appears to be restricted to freshwater habitats (Jiménez-García & Suárez-Morales, 2017).Ergasilus atafonensis lacks of spinulate seta of the maxilla and spiniform process on the basis of the second leg.In E. parabahiensis, the antennal segments are shorter and the spiniform process on the basis of the second leg is absent.According to El-Rashidy (1999), E. lizae also resembles E. tissensis D'yachenko, 1969; however, the mouth parts of this species were not described.The separation between the newly generated sequences and the sequences of E. lizae from Canada indicated by the ML tree suggests that these belong to different species.As stated above, E. lizae seems to be restricted to marine habitats.However, the Canadian specimens were isolated from a freshwater fish, F. diaphanus.The Canadian specimens were sequenced by Dr. Sean Locke (currently at the University of Puerto Rico).According to Dr. Locke (pers.comm.), the specimens that he sequenced possibly were misidentified as E. lizae since there was no rigorous morphological examination.The limited number of published of COI sequences for Ergasilus hinders the ability to perform a comprehensive phylogenetic analysis.Despite these limitations, the sequences provided herein represent a valuable contribution to the genetic database of Ergasilus species and lay the groundwork for future research.Expanding the dataset and incorporating additional genetic information will enhance our understanding of the evolutionary relationships and diversity of this important group of parasitic copepods.

Occurrence
The higher prevalence of E. lizae observed during the warm season agrees with the seasonal occurrence reported for other ergasilids.For instance, studies of Ergasilus sieboldi von Nordmann, 1832 on pikeperch, and Sinergasilus major (Markevich, 1940) and S. polycolpus (Markevich, 1940) on farmed carp reported higher infection levels during summer (Molnár & Székely, 1997;Nie & Yao, 2000).This was probably due to the slower formation of eggs with the decrease of water temperature (Paperna & Zwerner, 1976).As typically observed in marine ectotherms, within an optimal range, water temperature has a negative relationship with development times, body size, and reproductive outputs of parasitic copepods (Samsing et al., 2016).Thus, generation times are shorter at high temperatures, which can be reflected in higher infection levels during warm seasons; nonetheless, females are bigger and produce more eggs at low temperatures, a situation that also can result in high infection levels during cold seasons.For instance, Hogue et al. (1993) observed that E. nerkae Roberts, 1963 was more prevalent on longnose sucker in winter.

Conclusions
Based on a detailed morphological analysis, this study confirms the presence of the parasitic copepod E. lizae on mullets (M.cephalus and M. curema) from northwestern Mexico.Additionally, COI sequences for E. lizae are provided for the first time.Given that E. lizae is a cosmopolitan species, our findings could enhance the precision of species identification in future studies on ergasilid diversity worldwide.

Conflict of interest
The authors declare no competing interests.

Ethical approval Not applicable.
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Fig. 6
Fig. 6 Maximum likelihood tree based on COI sequences of copepods of the genus Ergasilus.The branch labeled as Northwestern Mexico contains the newly generated sequences for E. lizae.Bootstrap support is displayed at the nodes.The cyclopoid copepod Lernaea cyprinacea (Linnaeus, 1758) was used as the outgroup Technology of Mexico (CONAHCYT) provided a postgraduate student scholarship to S.C.-Z.The Instituto de Ciencias del Mar y Limnología, UNAM, financed the filed work.Sequencing was funded by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT, UNAM) through Project IN215722, awarded to Alejandro Oceguera Figueroa.Data availability Voucher material deposited in the Copepoda collection of the Instituto de Ciencias del Mar y Limnología, Unidad Académica Mazatlán (ICML-EMUCOP), Sinaloa, Mexico.Sequence data have been deposited in Gen-Bank (accession coded PP239046, PP239047, PP239048).

Table 1
Samples of Mugil curema and M. cephalus obtained from Urias Estuary (UE) and Huizache-Caimanero lagoon (HC) during two seasons.TL = Mean ± SD of the fish total length.

Table 2
Species of Ergasilus included in the comparative analysis of COI sequences.All the accession numbers correspond to GenBank records, except for those of E. lizae from Canada, which correspond to BOLD records (https:// www.bolds ystems.org/ accessed on November 30, 2023).