Scaphanocephalus spp. (Trematoda: Opisthorchiidae) in intermediate and definitive hosts of the Yucatán Peninsula, Mexico, with a re-description of Scaphanocephalus expansus

Summary Scaphanocephalus is a small trematode genus belonging to the family Opistorchiidae. The genus currently contains only three species associated with marine fish as intermediate hosts and fish-eating birds as definitive hosts. Here, specimens of Scaphanocephalus were collected from the Osprey, Pandion haliaetus, and the White mullet, Mugil curema in the Yucatán Peninsula, Mexico. We report for the first-time DNA sequences of adult specimens of Scaphanocephalus, particularly S. expansus, as well as a sequence of a different species sampled as metacercaria. Morphological comparisons of Scaphanocephalus expansus confirmed the identity of the adult specimens, with minor morphological variations; Scanning electron photomicrographs were included, and the species was re-described. Phylogenetic analysis based on 28S rDNA sequences showed that Scaphanocephalus is monophyletic within Opisthorchiidae and consists of three independent lineages. Sequences of adults are identical to those of S. expansus. Instead, the sequence of the metacercaria sampled from the mesentery of Mugil curema nested with specimens reported as Scaphanocephalus sp. from a labrid fish in the Mediterranean Sea, herein named it as Scaphanocephalus sp. 2.


Introduction
The opisthorchiid trematode genus Scaphanocephalus Jägerskiöld, 1904 comprises only three species which exhibit a complex life cycle involving marine snails as their first intermediate hosts, marine fishes as second intermediate hosts, and fish-eating birds from the order Accipitriformes as definitive hosts (Kohl et al. 2019;Sokolov et al. 2021).They are characterized by a distinctive T-shaped form of the body resembling a cross-section of a mushroom (Pearson, 2008;Katahira et al. 2021).
Scaphanocephalus expansus (Creplin 1842) [type-species] was originally described as Monostomum expansum from the intestine of the Osprey, Pandion haliaetus (L.) (Pandionidae) in Europe.Subsequently, Jägerskiöld (1904) reassigned those specimens and new material collected from the same host in the Red Sea, to the genus Scaphanocephalus.Adult specimens of this species seem to be highly host-specific since they have been reported in the Red Sea, Mediterranean Sea, Gulf of California, Gulf of Mexico, and Canary Archipelago, only parasitizing the Osprey, P. haliaetus (Creplin, 1842;Jägerskiöld, 1904;Hoffman et al. 1953;Dubois, 1960;Schmidt & Huber, 1985;Kinsella et al. 1996;Foronda et al. 2009).In contrast, larval stages of S. expansus have been observed on fins and scales of 17 species of reef fish allocated in 12 families in the Western Pacific, Caribbean Sea, and Gulf of Mexico (Yamaguti, 1942;Kohl et al. 2019;Montoya-Mendoza et al. 2021).The metacercariae of Scaphanocephalus causes the black-spot syndrome (Elmer et al. 2019;Kohl et al. 2019;Cohen-Sánchez et al. 2023).
Until now, 28S rDNA sequences have been obtained only for the metacercarial stages of Scaphanocephalus spp.We collected metacercariae of Scaphanocephalus from the body cavity of one out of 67 specimens of White mullet, Mugil curema (Mugilidae) sampled in three coastal lagoons of Yucatán.Additionally, adult specimens of Scaphanocephalus spp.were collected from the intestine of one specimen of the Osprey, Pandion haliaetus from Champotón, Campeche, also in the Yucatán Peninsula.Adults were morphologically identified as Scaphanocephalus expansus.Thus, the main objective of this study was two-fold: to corroborate molecularly the identification of the adults, and to establish a molecular link between the metacercariae and adults from the same geographical region.

Specimen collection
Adult specimens of Scaphanocephalus sp. were recovered from the intestine of one specimen of Pandion haliaetus collected in Champotón, Campeche.Additionally, we collected 67 specimens of the White mullet, Mugil curema (Valenciennes) in four coastal lagoons of northern Yucatán, namely La Carbonera, Dzilam de Bravo, Ría Lagartos, and Celestún (Fig. 1).Only one fish was found to be infected in the mesentery with an individual of Scaphanocephalus sp. from Celestún.All Scaphanocephalus specimens were fixed in nearly boiling distilled water and preserved in 100% ethanol for DNA analyses.Some adult specimens were fixed in hot 4% formalin solution for morphological studies and Scanning Electron Microscopy (SEM) studies.

Alignments and phylogenetic analyses
The newly generated sequences of Scaphanocephalus were aligned with other Opisthorchiidae species downloaded from GenBank, along with 14 members of Heterophyidae used as outgroups.Alignment was performed using the software SeaView version 4 (Gouy et al. 2010) and adjusted with the Mesquite program (Maddison & Maddison, 2011).A nucleotide substitution model was selected using jModelTest v2.1.7 (Posada, 2008) and applying the Akaike information criterion, with the best nucleotide substitution model for data being GTR + G + I.
Phylogenetic analyses were conducted using Bayesian inference (BI) and maximum likelihood (ML) methods through the online interface Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway v3.3 (Miller et al. 2010).The BI analysis was inferred with MrBayes v.3.2.7 (Ronquist et al. 2012), with two simultaneous runs of the Markov Chain Monte Carlo (MCMC) for 10 million generations, sampled every 1000 generations, using a heating parameter value of 0.2 and the first 25% of the sampled trees were discarded.The ML analysis was carried out with RAxML v.7.0.4 (Silvestro & Michalak, 2011), and 1000 bootstrap replicates were run to assess nodal support.Phylogenetic trees were drawn in FigTree v.1.3.1 (Rambaut, 2012).Genetic divergence among taxa was estimated using uncorrected 'p' distances with MEGA version 6 (Tamura et al. 2013).
Testes two, deeply lobated, in tandem, in posterior third of body.Posterior testis larger.Seminal vesicle tubular, winding, dorsally to uterus.Ovary deeply lobated, post-ecuatorial, pretesticular.Mehli´s gland sinistral, at ovary level.Seminal receptacle postovarian, pretesticular.Laurer's canal between ovary and Mehli's gland.Vitelline glands in the lateral fields, from caecal bifurcation to posterior body end; confluent in post-testicular area.Vitelloducts at ovary level.Uterus pretesticular, deeply coiled, between ventrogenital complex region and ovary, opening to genital pore in ventrogenital complex.Eggs small, oval to round shaped.Excretory vesicle Y-shaped, extending sinuously to reach anterior of body.

Remarks
Our specimens were identified as Scaphanocephalus expansus by having features consistent with the diagnosis of the original description and recent re-descriptions (Creplin, 1842;1904;Dubois, 1960;Pearson, 2008;Foronda et al. 2009).For instance, our specimens possess an anterior body with wing-like expansions, testes deeply lobated located in tandem in the posterior third of body, ovary lobated, pretesticular, and tegument covered with spines.In addition, metrical data of our specimens are like those reported in previous studies (Table 1).We noticed, however, some slight morphological differences, such as the variation in body length; the newly sampled specimens are, on average, smaller than those reported in previous studies (2256À5063 vs 2812À5000).The ovary and testes are also smaller (Ovary: 105À263 vs 160À380; anterior testis: 221À465 vs 400À690; posterior testis: 276À495 vs 548À840) (Table 1).In addition, SEM images allowed to show that S. expansus presented two types of spines, i.e., pectinate and arrowlike (Figs.3D, 3E).Dome-like papillae surrounding the aperture of the oral sucker are described for the first time (Fig. 3C).
We re-examined the specimens deposited of the metacercariae of Scaphanocephalus expansus by Montoya-Mendoza et al. (2021) in the Colección Nacional de Helmintos, México City (CNHE No. 11508) reported encysted on the fins of the labrid Halichoeres radiatus in the Veracruz coral reef, Mexico; and noticed that they are morphologically very similar to our adult specimens, both with multilobulated testes.However, DNA sequences are required of these larvae stages to corroborate the status and complete the life cycle of the species.

Phylogenetic analyses
The LSU dataset comprised 36 sequences with 1,247 characters.The alignment was trimmed to the shortest and included the four newly generated sequences of Scaphanocephalus, and six sequences previously identified either as Scaphanocephalus sp., or S. expansus.In addition, the alignment included 11 sequences of members of Opisthorchiidae, plus 14 sequences of Heterophyidae.Phylogenetic analyses conducted through BI and ML methods recovered similar topologies (Fig. 4).Analyses showed Opisthorchiidae as monophyletic with strong nodal support (1/100); Cryptocotylinae was also recovered as monophyletic, including Cryptocotyle spp.and Scaphanocephalus spp.The genus Scaphanocephalus was divided in three highly supported clades (1/100).The first clade herein referred as Scaphanocephalus sp. 1 (MN160569 and MT461356) included two sequences from a siganid and acanthurid from the Arabian Gulf and the Caribbean Sea, respectively.This clade was resolved as the sister group of the other two clades of Scaphanocephalus.The second clade was formed by three of the newly generated sequences of S. expansus (from the definitive host, P. haliaetus), plus one sequence previously identified as S. expansus (MK680936) sampled from a scarid fish, and one sequence identified as Scaphanocephalus sp.(MN160570) from an acanthurid fish, both from the Caribbean Sea.Finally, the third clade was yielded as the sister group of S. expansus and is formed by four sequences, the specimen from Mugil curema (Fig. 2D; CNHE 12842), and those previously identified as Scaphanocephalus sp. from a labrid in the Mediterranean Sea, herein referred as Scaphanocephalus sp. 2.

Discussion
The genus Scaphanocephalus was originally considered a member of Heterophyidae (Pearson, 2008;Dennis et al. 2019).However, Sokolov et al. (2021) recently transferred Scaphanocephalus to the subfamily Cryptocotylinae within Opisthorchiidae using molecular and morphological data.Our LSU phylogenetic analyses unequivocally corroborated that Scaphanocephalus is monophyletic and belongs to Opisthorchiidae.Dennis et al. (2019) performed the first molecular phylogenetic analysis including two lineages of Scaphanocephalus, recognizing to separate species as Scaphanocephalus sp. 1 and Scaphanocephalus sp. 2. The latter was nested with S. expansus reported by Kohl et al. (2019)  nested with Scaphanocephalus sp. 1 from the Caribbean Sea reported by Dennis et al. (2019).
Moreover, the sequence reported as Scaphanocephalus sp. 2 (MN160570) by Dennis et al. (2019) from the acanthurid A. chirurgus in the Caribbean Sea nested as sister taxon of S. expansus from the scarid S. chrysopterum in the same geographical region as reported by Kohl et al. (2019).Our sequences from adult specimens of S. expansus from the definitive host were also nested within this clade.Based on this evidence, we consider that Scaphanocephalus sp. 2 of Dennis et al. (2019) as S. expansus.Furthermore, Cohen-Sánchez et al. (2023) recently reported sequences of Scaphanocephalus sp. from the labrid X. novacula, in the Mediterranean Sea.These authors made no further taxonomic consideration because their study was focused on determining the effect of the black spot disease produced by Scaphanocephalus on the oxidative stress of the host.We included these sequences in our analyses and found that they nested with the newly sequenced individual from M. curema from Yucatán, with null genetic variation, suggesting this lineage is widely distributed across the Atlantic Ocean parasitizing marine fishes.This clade is herein considered as Scaphanocephalus sp. 2 (Fig. 4).In addition, M. curema represent a new species of intermediate hosts for Scaphanocephalus.Interestingly, only one specimen of Scaphanocephalus was recovered from the mesentery of 1 out of 67 individuals of M. curema studied for parasites in four localities of northern Yucatán.The infection site (mesentery) and the low prevalence of infection suggest that the presence of the parasite in M. curema may represent an accidental infection.The metacercariae of Scaphanocephalus have been reported as ectoparasites encysted on the fins and under the scales of their hosts (Dennis et al. 2019;Elmer et al. 2019;Kohl et al. 2019;Shimose et al. 2020;Katahira et al. 2021;Montoya-Mendoza et al. 2021;Cohen-Sánchez et al. 2023).
The LSU genetic divergence observed among the three clades of Scaphanocephalus was relatively low (1.06 to 1.73%).However, these genetic divergence values are like those reported for other  Up to the present, only three species have been included in the genus Scaphanocephalus, namely S. expansus, S. australis and S. adamsi.The first two were described from their definitive hosts, S. expansus from P. haliaetus, and S. australis from H. leucogaster.In contrast, the description of S. adamsi was based only on the metacercarial stage from the labrid B. mesothorax (Tubangui 1933).
In the present study, two unidentified Scaphanocephalus (sp.1 and sp.2) represent larval stages.Whether or not they represent the two species of Scaphanocephalus not yet sequenced needs to be tested once molecular studies are conducted on these species.Interestingly, Scaphanocephalus sp. 2 contain specimens sampled from M. curema in Mexico and, more importantly, from the labrid X. novacula from Spain (Cohen-Sánchez et al. 2023).The latter represents the same host-type (Labridae) of S. adamsi, although the species was described from B. mesothorax in the Philippines.Two pieces of information are missing to test the hypothesis that Scaphanocephalus sp. 2 represents in fact S. adamsi, sampling and sequencing metacercariae from the type locality and, ideally, sampling adult forms from Accipitriformes.Furthermore, the only published record of S. australis was from adults collected from the accipitrid H. leucogaster in Australia (Johnston 1917), and their larval forms have not been reported from marine fishes in the area.The White bellied sea eagle, H. leucogaster has an extensive geographic distribution, extending from the Indian west coast, China, to all over South-East Asia, including Indonesia, Papua New Guinea, and Australia (Shephard et al. 2005).It is imperative to collect new material of S. australis to determine whether or not Scaphanocephalus sp. 1 or 2 belong in that species, or if they represent a separate species whose adults have not been found in Accipitriformes.
The osprey, Pandion haliaetus has a cosmopolitan distribution which probably has a key role in the distribution of S. expansus across the world.In the Caribbean two subspecies have been reported, the North American osprey, P. haliaetus carolinensis, which is migratory, and the non-migratory osprey, P. haliaetus ridgwayi (Wiley et al. 2014) This could explain the presence of the two lineages of Scaphanocephalus in the Caribbean, S. expansus and Scaphanocephalus sp. 1.Still, gathering new samples of adults and metacercariae from different host species is necessary to test the hypothesis on the potential link between larval forms and adults described from accipitriformes, and to understand the evolution and biogeographic history, as well as the interrelationships and host-parasite interactions of this enigmatic group of digeneans.
. Al-Salem et al. (2021) and Cohen-Sánchez et al. (2023) obtained sequences of Scaphanocephalus although without a molecular phylogenetic analysis.Unexpectedly, our phylogenetic analysis showed that the sequence of Scaphanocephalus from the Arabian Gulf (MT461356) reported by Al-Salem et al. (2021)

Figure 4 .
Figure 4. Consensus Bayesian inference (BI) tree and Maximum likelihood (ML) tree inferred from the large subunit from nuclear ribosomal DNA.Numbers on internal nodes show posterior probabilities (BI) and ML bootstrap clade frequencies.Sequences generated in this study in bold.Grey: specimens from Arabian Gulf.Blue: specimens from the Caribbean Sea.Green: specimens from Yucatán Peninsula, Mexico.Red: specimens from the Mediterranean Sea.Scale bar shows the number substitutions per site.

Table 1 .
Comparative morphometric data for Scaphanocephalus expansus Tatonova & Besprozvannykh (2019)or instance,Tatonova & Besprozvannykh (2019)reported a genetic divergence of 2% between Cryptocotyle lingua(Creplin, 1825)and C. lata Tatonova and Besprozvannykh, 2019.Interestingly, in our study we found no genetic divergence within each lineage of Scaphanocephalus.These data suggest that definitive and intermediate hosts are playing an important role for the distribution of each lineage of Scaphanocephalus.