Tubulovesicula lindbergi (Layman, 1930) (Digenea: Hemiuridae) in the Southwestern Atlantic Ocean: A Morphological and Phylogenetic Study Based on Specimens Found in Nebris microps (Actinopterygii: Sciaenidae) off the Brazilian Coast

: This study presents the first record of T . lindbergi from the southwestern Atlantic Ocean, based on specimens collected from the smalleye croaker Nebris microps Cuvier (Sciaenidae), off the coast of Maranh ã o Island, State of Maranh ã o, Brazil. Our approach included a morphological analysis complemented by DNA sequencing (28S, ITS2 rDNA, and cox 1 mtDNA). Our phylogenetic analysis revealed the affinity of T . lindbergi to its congener T . laticaudi Parukhin, 1969, a digenean parasite commonly found in hydrophiine snakes inhabiting the Pacific Ocean. The interspecific divergence between T . lindbergi and T . laticaudi measures 3.80% for 28S, 7.49–7.64% for ITS2, and 16.29–16.70% for cox 1. Our findings expand the documented geographic range of T . lindbergi into the southwestern Atlantic Ocean, report a novel host record, and increase the number of hemiurids known from Brazil to 30 species. Additionally, this study represents the initial documentation of a marine digenean fish within the North Brazil Shelf.

The synonymity of some species has increased the complexity associated with T. lindbergi; therefore, the taxonomic history of this species needs to be reevaluated.Tubulovesicula lindbergi is reported mostly from the Pacific, but also in the Indian and Atlantic Oceans.The species purportedly has a broad spectrum of host species, including fish belonging to at least 29 families and 15 orders (Table 1).Given the complexity of the genus Tubulovesicula, molecular taxonomy can be a valuable tool for enhancing our understanding of its true diversity and distribution.
Numerous hemiurids have been documented in Brazil; nevertheless, presently, no Tubulovesicula species have been documented in the coastal waters of the southwestern Atlantic Ocean.However, the Hemiuridae family is well represented along the coast of the country, with 29 species in 10 genera, making it the second richest family of digeneans in marine fishes [13,14].
The demersal sciaenid fish studied, Nebris microps Cuvier, commonly known as the smalleye croaker, is found in the western Atlantic Ocean, ranging from Costa Rica to Brazil [15].This fish is an important local fishery resource [16] and holds significant ecological value.However, the potential role of the smalleye croaker in parasite life cycles remains underexplored.To date, the only digenean reported in this host is the acanthocolpid Pleorchis americanus Lühe, 1906 (=Pleorchis mollis), collected off Macaé, Rio de Janeiro, Brazil, southwestern Atlantic Ocean, by Vicente and Santos [17].Other studies have reported nematodes [Anisakis sp., Procamallanus (S.) pereirai (Annereaux, 1946), Raphidascaris (I.) vicentei Santos, 1970, and Raphidascaris sp.] and one monogenean (Rhamnocercus micros Chero, Cruces, Sáez & Luque, 2022) [18].As part of our ongoing investigation of the Brazilian fish trematode fauna, we conducted a morphological and molecular analysis of hemiurid trematodes collected from the stomach of N. microps off Maranhão Island, North Brazil Shelf, Maranhão, Brazil.Our data represent the first record of T. lindbergi in the southwestern Atlantic Ocean, a novel host record, and the first assessment of its phylogenetic position.

Materials and Methods
Twelve specimens of Nebris microps Cuvier, 1830 (Sciaenidae), were obtained in October 2022 from artisanal fishermen off the Maranhão Island (2 • 24 ′ 29 ′′ S, 44 • 05 ′ 52 ′′ W), near the municipality of Raposa, State of Maranhão, Brazil.The specimens were recently deceased.The fish were examined for the presence of infection with helminth parasites.Trematode individuals collected from the examined fish were washed in 0.9% saline and fixed in 80% ethanol.A small piece of the ecsoma of each specimen selected for molecular analyses was excised and used for DNA extraction, and the remaining piece was used for morphological analysis (hologenophore, see Pleijel et al. [53]).Hologenophores and remaining specimens (paragenophores) were stained in Mayer's hydrochloric carmine solution, dehydrated in ethanol, cleared in clove oil, and mounted in Canada balsam, and thereafter used for morphological evaluation.The fish identification was determined according to Marceniuk et al. [16].Drawings were made using a drawing tube attached to a light microscope Olympus CH-2 and then digitised.Measurements were taken using Leica Application Suite software (LAZ EZ), v.2.0.software adapted to the Leica DM 750 optical microscope (Leica Microsystems, Wetzlar, Germany) and were given in micrometres (µm).Voucher material was deposited in the Helminthological Collection of the Oswaldo Cruz Institute, CHIOC (CHIOC-40430a,b; CHIOC-40431a-e), Rio de Janeiro, and in the Collection of Trematodes of the Federal University of Minas Gerais, UFMG (UFMG-TRE137), Belo Horizonte, Brazil.
Our specimens agree with the original description of T. lindbergi (=Lecithaster lindbergi) provided by Layman [8] and collected from a variety of fishes (most Pleuronectiformes) in the Peter Great Bay, Russia, Pacific Ocean, particularly in body shape (maximum width at level of ventral sucker or at posterior body extremity), in having soma longer than ecsoma, in having testes and ovary smaller than ventral sucker, in position of pars prostatica (between sinus-sac and posterior margin of ventral sucker or anterior hindbody), in sucker ratio (ventral sucker approximately two times larger than oral sucker) and in extension of intestinal caeca (extending into ecsoma).However, our specimens differ from the material of Layman [8] by having larger dimensions, except for the soma length, soma width, testes and ovary, where the dimensions overlap, and in having smaller sinus-sac (Table 2).Later, Yamaguti [7] described Tubulovesicula spari Yamaguti, 1934 from the Inland Sea, Japan, Pacific Ocean, which was considered identical, except for the egg size (slightly smaller in material of Layman [8]), with T. lindbergi by Sogandares-Bengal [9] who put T. spari as its synonym.In comparison with the material of Yamaguti [7], our specimens possess larger dimensions, except for body (soma) length, for ovary width and egg length, where our specimens are smaller, as well as for the preoral lobe length and for testes, where the dimensions overlap (Table 2).
McFarlane [37] described Dinurus nanaimoensis MacFarlane, 1936, which was transferred to the genus Tubulovesicula by Manter [11] and later synonymised with T. lindbergi by McCauley [12].In comparison with material of McFarlane [37], our specimens differ in having larger dimensions, except in sinus sac length and ovary length, for which the dimensions overlap, and in having smaller eggs (Table 2).Park [34] described T. californica, (1936) which was later considered synonymous of T. lindbergi by McCauley [12].In comparison with material of Park [34], the maxima for most internal organs of our specimens are higher, except for the body length, posterior testis length, sinus sac length, ovary length, and egg width, for which the dimensions overlap (Table 2).Nigrelli [1] described T. madurensis Nigrelli (1940), and then the species was synonymised with T. lindbergi by Manter [11].In comparison with material of Nigrelli [6], our specimens differ mainly in having smaller total length, smaller testes, larger oral sucker, larger pharynx length, larger ventral sucker, and longer eggs (Table 2).In comparison with material of McCauley [12], our possess larger dimensions except for body length and eggs where the dimensions overlap.The dimensions of our material overlap within those of Shen [24], except for in oral sucker, pharynx length, ventral sucker, and in pars prostatica length, which our dimensions are higher (Table 2).
Tubulovesicula muraenesocis Yamaguti, 1934, and T. pseudorhombi Yamaguti, 1938, were considered synonyms of T. lindbergi by Manter [11].Bray [2], in a detailed taxonomic review, synonymised both species with T. angusticauda.Later, Madhavi and Bray [4] listed these two species as synonymous of T. lindbergi.Checking the original descriptions, we agree with Bray [2] because the lengths of pars prostatica of T. muraenesocis and T. pseudorhombi are more related to the description of T. angusticauda.Sogandares-Bernal [9] considered T. anguillae Yamaguti, 1934, a synonym of T. lindbergi.Later, Bray [2] considered T. anguillae a synonym of T. angusticauda.However, we express doubt about the interpretation of Bray [2] regarding the length of the pars prostatica in T. anguillae.Although Yamaguti [7] did not provide a detail description of the terminal genitalia, the figure provided by him, in our interpretation, seems to show that the pars prostatica is starting from the posterior margin of the ventral sucker.
According to the WoRMS Editorial Board [10], Tubulovesicula has been reported to comprise a total of 25 species.To investigate the taxonomic history of T. lindbergi, a review of the literature was conducted.Through our examination, we were able to assess the validity and synonymies associated with this species.Previously, McCauley [12] synonymised T. californica and T. madurensis with T. lindbergi, while Sogandares-Bernal [9] concluded that the type species T. spari is a synonym of T. lindbergi.

Molecular Results
In the present study, four novel sequences were generated for two isolates of T. lindbergi (2 = 28S; 1 = ITS2; 1 = cox1).Phylogenetic analyses were performed using the 28S rDNA alignment (1150 pb), and the resulting tree provided insights into the phylogenetic relationships of T. lindbergi within the Hemiuridae (Figure 2).The newly obtained 28S rDNA sequences of T. lindbergi from N. microps were closely related to T. laticaudi (OR209733), collected from Hydrophis cyanocinctus Daudin (Elapidae), in Sri Lanka.This relationship was supported by high nodal values (Figure 2).The intraspecific divergence between the two generated sequences (28S) was null; therefore, we deposited only one sequence (PP889615) and the interspecific divergence between T. lindbergi and T. laticaudi was 3.80% (42 bp), which is of the same order of magnitude as other congeneric species of the family included in the analysis.The other sequences utilised for the phylogenetic analysis displayed a difference of more than 12% compared to those generated in the present study.Additionally, pairwise genetic distances were calculated between T. lindbergi and T. laticaudi for ITS2 and cox1.The interspecific divergence between T. lindbergi (PP889614) and T. laticaudi (OR209735 and OR209736) based on an ITS2 comparison was 7.49−7.64%(42−43 bp).The interspecific divergence between both species (PP891444; T. lindbergi) and (OR221151, OR221153 and OR221154, T. laticaudi) based on a cox1 comparison was 16.29−16.70%(79−81 bp).
From our perspective, the accuracy of some of these reports may be revaluated.Our opinion is primarily based on poorly written descriptions provided in many studies.Additionally, it is worth noting that molecular data have been provided for only one species until the present study (see Martin et al. [5]).However, the euryoxenous nature of these species might be true.Low host specificity has been recorded from hemiuroids species (see Miller et al. [76]), and some studies have been demonstrated a euryoxenous nature within hemiurids through molecular data [14,77], including, recently, in the genus Tubulovesicula [5].However, a critical analysis of the host specificity of these species of Tubulovesicula will be possible when further studies including integrative taxonomy approaches will be available.
The genus Tubulovesicula comprises 22 species, many of which have been insufficiently documented in terms of their descriptions.Inadequate detail, i.e., incomplete information regarding the terminal genitalia, coupled with the lack of distinctive morphological characteristics, makes it difficult to distinguish species.Certain species within the genus, like T. diacopae and T. hebrae, do not exhibit typical Tubulovesicula traits as observed by Bray [2], such as the post-equatorial ventral sucker and the presence of plications in the body.This raises doubts about whether these species should be classified within the genus.Previous studies have suggested synonyms to simplify the taxonomy of poorly understood forms within the genus, particularly for T. angusticauda and T. lindbergi.However, a comprehensive revision remains necessary.DNA sequencing can provide valuable additional information for accurate identification, especially for species like T. lindbergi, which exhibits widespread occurrence and slight morphometric differences across diverse hosts and geographic regions.A comprehensive taxonomic revision of T. lindbergi should be conducted when more data are accumulated, including DNA sequences and life cycle information.Our findings, with careful consideration due to the absence of molecular data from outside Brazilian waters, suggest that the geographical distribution of T. lindbergi is even broader.Therefore, it is important to revisit the Indo-West Pacific and examine the hosts from which T. lindbergi (primarily Pleuronectiformes) was described to conduct an integrative taxonomy study to add confidence to the identification of the new material.
Our findings also show, molecularly, that the species of the genus Tubulovesicula distributed in fishes and sea snakes are closely related.Recently, Martin et al. [5] provided the first molecular information for the only species of the genus known from non-fish hosts, T. laticaudi, collected from elapid sea snakes.Their findings demonstrated the polyphyly of the subfamily Dinurinae Looss, 1907, proposing an alternative classification primarily based on the nature of the sinus organ and on the molecular information of the family available.The resurrection of the subfamily Mecoderinae aimed to relocate species possessing a temporary sinus organ, including members of Tubulovesicula, Mecoderus Manter, 1940, Allostomachicola Yamaguti, 1958, and Stomachicola Yamaguti, 1934.The authors propose restricting the Dinurinae to accommodate species with a permanent sinus organ, such as members of Dinurus Looss, 1907, Ectenurus Looss, 1907, Erilepturus Woolcock, 1935, Paradinurus Vigueras, 1958, and Qadriana Bilqees, 1971.However, Ghanei-Motlagh et al. [79] provided the phylogenetic position of Stomachicola muraenesocis, revealing that this species is not closely related to T. laticaudi.The recent findings and previous studies indicate that further investigation into the molecular data of the Hemiuridae is necessary to propose a new subfamilial classification and determine which morphological characters hold taxonomic value for this classification.

Conclusions
Using morphological and genetic analyses, we were able to identify T. lindbergi and report this species in the Southeastern Atlantic Ocean for the first time.This discovery also represents the first observation of a marine fish digenean within the "North Brazil Shelf".Our study highlights the importance of investigating this rich and poorly known region in terms of marine diversity.Consequently, our findings contribute to expanding the number of hemiurid species identified off the Brazilian coast to 30 species.

Figure 2 .
Figure 2. Phylogram from maximum likelihood (ML) analysis based on the 28S rDNA sequences of the Hemiuridae.Nodal support values is given as ML/BI (Bayesian inference).Support values lower than 70 (ML) and 0.70 (BI) are not shown.The scale bar indicates the expected number of substitutions per site.The newly generated sequence is highlighted in bold.