The white grunt, Haemulon plumierii (Lacepède, 1801) as paratenic and definitive host of two acanthocephalan species, with the description of a new species of Dollfusentis (Palaeacanthocephala: Leptohynchoididae) from the Yucatán Peninsula, Mexico

Abstract Acanthocephalans are a group of obligate endoparasites that alternate between vertebrates and invertebrates to complete their life cycles. Occasionally, the same individual host acts as a definitive or paratenic host for different acanthocephalan species. In this study, acanthocephalans were sampled in marine fish in three localities of the Yucatán Peninsula; adults and cystacanths were recovered from the intestine and body cavity, respectively, of Haemulon plumierii from off the coast of Sisal, Yucatán. Ribosomal DNA sequences (small and large subunits) were used to test the phylogenetic position of the species of the genus Dollfusentis, whereas the mtDNA gene cox 1 was used for assessing species delimitation. The cox 1 analysis revealed an independent genetic lineage, which is recognized herein as a new species, Dollfusentis mayae n. sp. The new species is morphologically distinguished from the other six congeners by having a cylindrical proboscis armed with 22–25 longitudinal rows bearing 12 hooks each. The cystacanths were morphologically identified as Gorgorhynchus medius by having a cylindrical trunk covered with tiny irregular spines on the anterior region, and a cylindrical proboscis armed with 17–18 longitudinal rows of 21 hooks each; small and large subunit phylogenetic analyses yielded G. medius within the family Isthomosacanthidae, suggesting that Gorgorhynchus should be transferred to this family from Rhadinorhynchidae where it is currently allocated.

Sixty-five species of acanthocephalans have been described as parasites of vertebrates, which represent the 5% of the diversity of helminths in Mexico (Pérez-Ponce de León et al. 2011).Twenty-three of them occur in estuarine and marine fishes across the Pacific, Gulf of Mexico, and Caribbean Sea (García-Prieto et al. 2010).In the Yucatán Peninsula (in southeastern Mexico), only 12 nominal species of acanthocephalans have been reported parasitizing 17 species of marine and estuarine fishes (García-Prieto et al. 2010).Two species of palaeacanthocephalans use a broad spectrum of definitive hosts.For example, Dollfusentis chandleri Golvan, 1969 is a leptorhynchoidid that has been reported from 10 fish species, i.e., Mayaheros urophthalmus (Günther), Lutjanus griseus (L.Chandler, 1934[Gorgorhynchus medius (Linton 1908) Chandler, 1934], were collected and morphologically identified from the intestines of the white grunt, Haemulon plumierii (Lacepède) and the striped mojarra, Eugerres plumieri from three localities.The main objectives of the current study were i) to characterize molecularly and morphologically the species Gorgorhynchus medius and Dollfusentis sp; ii) to test the phylogenetic position of these two species; iii) to analyze in further detail the interrelationships among species of Dollfusentis; and iv) to describe Dollfusentis sp., sampled from H. plumierii from off the coast of Sisal, Yucatán, Mexico.
followed by range in parentheses.For scanning electron microscopy (SEM), specimens were dehydrated through an ethanol series, critical point dried with CO 2 , sputter coated with gold, and examined with a Hitachi Stereoscan Model S-2469N scanning electron microscope operating at 10 kV at the Instituto de Biología, Universidad Nacional Autónoma de México (UNAM).
Newly generated sequences were aligned with published sequences for other acanthocephalans retrieved from the GenBank dataset.Alignments for each molecular marker (SSU, LSU, and cox 1) were constructed using the software Clustal W (Thompson et al. 1994).A nucleotide substitution model was selected for the dataset using jModelTest version 2.1.7(Posada, 2008).Phylogenetic analyses were inferred through maximum likelihood (ML) with the program RAxML version 7.0.4(Stamatakis 2006).A GTRGAM-MAI substitution model was used, and 10,000 bootstrap replicates were run to assess nodal support.In addition, a Bayesian analysis was carried out, using the program MrBayes 3.2.2(Ronquist et al. 2012) with two Markov chain Monte Carlo runs for 10 million generations, sampling every 1000 generations, a heating parameter value of 0.2 and a burn-in of 25%.The resulting phylogenetics trees were visualized and edited using FigTree v.1.4.4 (Rambaut & Drumond 2007).The genetic divergence among taxa was estimated using uncorrected "p" distances with the program MEGA version 11 (Kumar et al. 2016).

General
Sexual dimorphism evident, females larger than males.Trunk cylindrical, covered with spines on the anterior region of trunk, extending to level of 1/4 of proboscis receptacle in both sexes.Anterior spines larger than posterior spines.Proboscis long, cylindrical covered with 22-25 longitudinal rows with 12 hooks each, decreasing in size towards posterior end, with a transition to minute hooks and a ring of eight hooks in the posterior region of the proboscis.Neck smooth.Proboscis receptacle double-walled.Lemnisci elongate, one shorter than the other and both longer than proboscis receptacle.Gonopore terminal in both sexes.
Trunk cylindrical, swollen anteriorly, covered with tiny irregular spines on anterior region of trunk, reaching mid-level of proboscis receptacle in both sexes.Proboscis cylindrical armed with 17-18 longitudinal rows of 21 hooks each of different size; Apical hooks and basal hooks smaller.Neck short, conical, aspinose.Proboscis receptacle double-walled; cerebral ganglion sub-oval at posterior end; lemnisci tubular, longer than proboscis receptacle.Gonopore subterminal in both sexes.

Remarks
The genus Gorgorhynchus includes 12 valid species as parasites of teleost marine fishes and elasmobranchs distributed in tropical and subtropical waters (Amin & Van Ha, 2011;Smales et al. 2019).In the Americas, five species of Gorgorhynchus have been described, three in South America, i.e., G. clavatus Van Cleave, 1940 (13-15 longitudinal rows of hooks); G. lepidus Van Cleave 1940 (14-16 longitudinal rows of hooks), and G. trachinotus Noronha, Vicente, Pinto & Fabio, 1986 (14 longitudinal rows with 11-12 hooks per row); one in North America (G.gibber Chandler, 1934) (24 longitudinal rows with 18 hooks per row); and one in the Caribbean Sea (G.medius) (22 longitudinal rows with 20 hooks per row); representing 41.6% of the species described in the genus.The acanthocephalans recovered from the white grunt, H. plumierii from off Sisal, Yucatán, show similar morphological characteristics compared to those assigned to G. medius including i) an elongated cylindrical trunk covered with tiny irregular spines on the anterior region of trunk, ii) neck short, conical, iii) a cylindrical proboscis, iv) proboscis hooks arranged in 17-18 longitudinal rows of 21 hooks per row, v) a double-walled proboscis receptacle, and vi) four long tubular cement glands very long in males (Figs.4-5).

Nuclear ribosomal markers
The alignment of the SSU consisted of 93 taxa and 2219 sites, whereas the LSU alignment included 48 taxa with 3077 sites.The phylogenetic trees inferred with the SSU dataset recovered Echinorhynchida as paraphyletic with the highest bootstrap (100%) and Bayesian posterior probability support values (1.0) (Fig. 6).On the one hand, Dollfusentis spp.were recovered as members of Leptorhynchoididae, which   The phylogenetic trees inferred with the LSU dataset yielded different topology than the SSU trees.Taxa representation for both molecular markers is somewhat different, although both analyses agreed on the systematic position of the taxa under study.For instance, D. bravoae, D. salgadoi, and D. mayae n. sp. were placed together within a clade formed by species of the genera Pseudoleptorhynchoides, Leptorhynchoides, Metacanthocephalus, Illiosentis, and Koronacantha, with strong bootstrap (100%) and Bayesian posterior probability support values (1.0) (Fig. 7).LSU sequences of D. lenti are available in GenBank but they are very short; hence, they were not included in the present analysis.The new species was not clearly distinguished from the other two congeners using this molecular marker.Additionally, the LSU sequence of G. medius nested as the sister taxa of two isolates of Serrasentis sagitiffer (Linton 1889) Linton 1932 and these two as the sister group of three species of Gorgorhynchoides, i.e., G. gnathanodontos Smales 2014 and G. bullocki + G. pseudocarangis Huston & Smales 2021, all of them members of Isthomosacanthidae.These relationships were highly supported by bootstrap and Bayesian posterior probability support values (Fig. 7).In summary, the phylogenetic trees inferred with each nuclear ribosomal marker consistently placed D. bravoae, D. salgadoi, and D. mayae n. sp.inside Leptorhynchoididae (order Echinorhynchida), and the species G. medius inside Isthomosacanthidae (order Polymorphida), both clades highly supported by bootstrap and Bayesian posterior probability support values (Figs.6-7).     it inside Polymorphida and not in Echinorhynchida as previously classified.Our phylogenetic analyses inferred with the same molecular markers support the conclusion of Huston et al. (2020).
In the current study, we also generated cox 1 sequences for two samples of G. medius.The alignment included sequences from the genera Gorgorhynchoides, Serrasentis, and Gorgorhynchus (Fig. 8B).The two isolates of G. medius were nested with an unidentified species of Gorgorhynchus from the dusky flounder (S. papillosum) from the Yucatán Peninsula forming a clade (Fig. 8B).Interestingly, these sequences nested within Serrasentis, a genus that seems to need a deep revision.The intraspecific genetic divergence among the three samples of Gorgorhynchus ranged from 0.07% to 2.0%, which is similar to that found in other genera of Isthomosacanthidae.For instance, the divergence among three isolates of Gorgorhynchoides queenslandensis from Australia ranged from 0.03% to 0.06%; between two isolates of G. pseudocarangis, divergence was 0.01%, and between two sequences of S. nadakali, it was 0.05%.It seems that the sequences from the dusky flounder (S. papillosum) reported by Vídal-Martínez et al. (2019) are conspecific with G. medius.
Finally, the life cycle of acanthocephalans is very complex and the inclusion of paratenic hosts is considered an ecological trait that has been conserved across the phylum (Kennedy, 2006).Since the same individual host can act alternatively as definitive and paratenic host, such as the white grunt, H. plumierii.

Figure 6 .
Figure 6.Maximum likelihood tree and consensus Bayesian Inference trees inferred with small subunit from 18S rDNA; numbers near internal nodes show posterior probabilities (BI) and ML bootstrap values.Sequences in bold were generated in this study.

Figure 7 .
Figure 7. Maximum likelihood tree and consensus Bayesian Inference trees inferred with large subunit from 28S rDNA; numbers near internal nodes show posterior probabilities (BI) and ML bootstrap values.Sequences in bold were generated in this study.

Figure 8 .
Figure 8. Maximum likelihood tree and consensus Bayesian Inference trees inferred with cytochrome c oxidase subunit 1 from the mitochondrial DNA of Dollfusentis spp., (A); members of the family Isthomosacanthidae (B).Numbers near internal nodes show posterior probabilities (BI) and ML bootstrap values.Sequences in bold were generated in this study.