Digenean trematodes infecting the frigate tuna Auxis thazard (Scombriformes, Scombridae) off the Rio de Janeiro coast, Brazil, including molecular data

Although some parasitological efforts have focused on the frigate tuna Auxis thazard (Lacepède) (Scombriformes, Scombridae) in Brazil, its digenean fauna remains poorly known. Combining morphological and molecular methods, we investigated the diversity of digenean trematodes of A. thazard collected from the coastal waters off the state of Rio de Janeiro, Brazil in 2021. Six species belonging to four families were recorded: the bucephalid Rhipidocotyle cf. angusticolle Chandler, 1941, the didymozoid Didymocystis sp. 6 sensu Louvard et al. (2022), the fellodistomid Tergestia sp., and three hemiurids, Dinurus euthynni Yamaguti, 1934, Lecithochirium floridense (Manter, 1934), and L. synodi Manter, 1931. The current study brings the total number of digenean trematode species parasitising A. thazard in Brazil up to eight, with hemiuroid trematodes being most diverse. Auxis thazard is a new host record for L. floridense, L. synodi and potentially for R. angusticolle. The geographic distribution of several species found in our study appeared to be wider than previously known. Our study is the first to apply a DNA-based approach to digenean diversity in marine fishes in Brazil and we believe that both morphological descriptions and molecular sequence data provided in our study will aid future research assessing the diversity of digenean trematodes of A. thazard and other marine fishes in Brazil.


Introduction
The Brazilian marine fish fauna is exceptionally diverse and encompasses over 1200 species which comprise nearly 7% of the world's marine fish species [30,77]. These fishes certainly host a rich diversity of parasites. However, literature suggests that our knowledge on this diversity is fragmentary and incomplete due to differential study efforts and uneven geographical coverage; most studies have been conducted off the southern coast in contrast to the north-eastern Atlantic coast [9]. Although helminth parasites have only been reported from a small portion of fishes (less than 20%) in Brazilian marine ecosystems, existing records suggest that digenean trematodes are one of the most diverse groups of parasites [41,52]. To date, there are reports of 184 species from 28 families of digenean trematodes, with the families Didymozoidae Poche, 1907, Hemiuridae Looss, 1899, and Opecoelidae Osaki, 1925 being the most diverse in the region [6,21,38,41]. The most investigated fish family is the Carangidae followed by the Sciaenidae and Scombridae. All previous records are based on morphology and to the best of our knowledge, no attempts applying a molecular methodology have been made to address the taxonomic diversity of marine digenean trematodes in Brazil. Thus, it can be expected that many unknown digenean trematode species await their discovery, and many known species are yet to be genetically characterised.
The present study was carried out within a short-term project aiming to discover and morphologically and molecularly characterise the diversity of trematode species of marine fishes off the Brazilian Atlantic coastline. The current paper reports on the findings of a survey on digenean trematodes parasitizing the scombrid Auxis thazard (Lacépède) collected from the coastal waters off the state of Rio de Janeiro. Commonly known as frigate tuna, this pelagic fish is an important component of marine food webs and has high economic importance [25,29,30]. Auxis thazard is a widespread species found in the Atlantic, Indian, and Pacific (Western and Central) oceans [30] and, due to its ubiquity, numerous studies investigating trematodes of this fish have been conducted around the world (Table 1). These studies recorded digenean trematodes from at least six families, of which the Didymozoidae is by far the most speciose. Most records of the trematode fauna of A. thazard derive from the Indo-west Pacific region (the South China Sea and Hawaii). The digenean trematode fauna of A. thazard in Brazil is yet to be comprehensively assessed; however, two species have been reported: the bucephalid Rhipidocotyle pentagonum (Ozaki, 1924) [24] and the didymozoid Melanocystis kawakawa Yamaguti, 1970 [38]. In Brazil, A. thazard is also known as a host for one species of acanthocephalan [Rhadinorhynchus pristis (Rudolphi, 1802)], six species of monogeneans [Allopseudaxine macrova (Unnithan, 1957), Capsala magronum (=Caballerocotyle lenti) (Ishii, 1936), Churavera triangula (Mamaev, 1967), Hexostoma thynni (=Hexostoma auxisi) (Delaroche, 1811), H. keokeo Yamaguti, 1968 and Sibitrema sp.] and three species of nematodes [Anisakis typica Diesing, 1860, An. physetesis (Baylis, 1923), Oncophora melanocephala (Rudolphi, 1819)] [21,36,39,51,61,64,66,72]. Our study further explores the diversity of trematodes and suggests that A. thazard off the Brazilian Atlantic coastline is infected by at least eight species from four families. Details of morphology, novel DNA sequence data, and host and geographical records are presented.

Ethics statement
All applicable institutional, national and international guidelines for the ethical handling of animals were followed. According to Brazilian laws, species registration for scientific research purposes was carried out at SisGen (Number AFB3925).

Sampling and morphological evaluation
Three specimens of A. thazard (total length 30-40 cm) were obtained from local fisherman in Cabo Frio coastal zone (22°52 0 46 00 S, 42°01 0 07 00 W), State of Rio de Janeiro, Brazil in January 2021. The fish host nomenclature follows Froese and Pauly [30]. Fish were dissected fresh and examined for the presence of helminths. Trematodes were rinsed in 0.9% saline and fixed in 4% hot formalin (paragenophores according to Pleijel et al. [71]) and in molecular grade 96% ethanol. Cysts containing trematodes were removed from the stomach tissue of fish using needles. Specimens selected for molecular genetic study, i.e., hologenophores were processed as described in Faltýnková et al. [23]. Hologenophores and formalin-fixed specimens transferred to 70% ethanol were stained with Mayer's hydrochloric carmine solution, dehydrated in an ascending ethanol series, cleared with eugenol (clove oil), mounted in Canada balsam and thereafter, used for morphological study. Drawings were made using a drawing tube attached to an Olympus BX 51 microscope. Measurements were taken using QuickPHOTO CAMERA 2.3 image analysis software adapted to an Olympus BX51 and are given in micrometres unless otherwise stated. The voucher specimens were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC).
The Basic Local Alignment Search Tool (BLAST) (http:// www.ncbi.nlm.nih.gov/blast) was used to compare sequences obtained in the present study to those available in GenBank. Four alignments including novel and previously published sequences were built using ClustalW implemented in Geneious v. 11. All four alignments were used for comparative sequence analysis (p-distance and nucleotide (nt) difference) and Alignment 4 was further used for Bayesian inference (BI) and maximum likelihood (ML) phylogenetic analyses. Alignment 1 (1220 nt) included 28S rDNA sequences of nine Rhipidocotyle spp.; one sequence generated in the present study. Alignment 2 (598 nt) included six ITS2 sequences of Rhipidocotyle spp.; one sequence generated in the present study. Alignment 3 (978 nt) included 12 28S rDNA sequences of the subfamily Didymozoinae; two sequences generated in the present study. Alignment 4 (1119 nt) included 23 28S rDNA sequences of the family Hemiuridae; four sequences generated in the present study. Distance matrices for the alignments were calculated in MEGA ver. X [43].

Results
Morphological and molecular evaluation of collected specimens of digenean trematodes revealed the presence of six species from four families. One species belongs to the family Bucephalidae, one to the family Didymozoidae, one to the family Fellodistomidae and three to the family Hemiuridae. No larval stages of trematodes were recorded in examined fish. A total of 17 novel sequences were generated for five out of six species of trematodes: 28S rDNA (n = 7), ITS2 (n = 5) and cox1 (n = 5). Sequences of Tergestia sp. were not generated.
Remarks: Only one specimen of this species was collected. The specimen corresponds well to the generic diagnosis of the genus Rhipidocotyle Diesing, 1858 provided by Overstreet and Curran [69] in possessing rhynchus consisting of a simple sucker with muscular hood containing five large fleshy lobes, mouth near mid-body, vitellarium in two fields, anterior to ovary, and oblique testes. Morphology of our single specimen, although incomplete and bent, corresponds to the original description of R. angusticolle by Chandler [14], and later updated redescription based on the type and newly collected material by Corkum [17] by rhynchus as a simple sucker with five fleshy lobes and vitelline follicles arranged in symmetrical lateral fields (about 15 follicles each side). However, it differs from the original description by smaller eggs [15-18 Â 9-13 (n = 20) vs 21-22 Â 14 -16].
Rhipidocotyle angusticolle is an intestinal parasite of scombrid fishes from the western Atlantic Ocean. The species was reported in Sarda sarda (Bloch) from Gulf of Mexico [14], and in Euthynnus alletteratus (Rafinesque) and Scomberomorus cavalla (Cuvier) off Grand Isle, Louisiana, USA [17,68]. Shalaby and Hassanine [83] recorded R. angusticolle in the serranid Epinephelus fasciatus (Forsskål) in the Red Sea. However, the specimens were described possessing rhynchus without fleshy lobes and according to Bartoli and Bray [3] the species may not have been correctly identified. In Brazil, R. angusticolle was previously reported from Scomber colias Gmelin by Fabio [22]. Auxis thazard is a new host record for this species.
Molecular results: Comparative sequence analysis of Alignment 1 demonstrated that the sequence of the species found in our study (OP458334) exhibited the lowest divergence with the sequence of R. angusticolle (KT273383) obtained from E. alletteratus collected off Grand Isle, Louisiana, USA, i.e., 0.5% (6 nt). The interspecific divergence between Rhipidocotyle spp. in Alignment 1 ranged from 4.6 to 11.6% (51-127 nt) ( Table 2). Comparative sequence analysis of Alignment 2 showed similar results. The difference between ITS2 sequences of our specimen (OP458341) and R. angusticolle (KT273383) was 1% (5 nt). This is rather low compared to the interspecific divergence in this dataset which ranged from 7 to 28% (35-140 nt) ( Table 2). Intraspecific genetic variation in 28S and ITS2 data has been observed in previous studies of bucephalids. Cutmore et al. [19] reported intraspecific variation of 3 nt and 4 nt in the 28S and ITS2 datasets, respectively for specimens of Dollfustrema durum Nolan, Curran, Miller, Cutmore, Cantacessi & Cribb, 2015 collected from Gymnothorax javanicus (Bleeker) and G. pseudothyrsoideus (Bleeker) in Moreton Bay and from Great Barrier Reef. Corner et al. [18] reported intraspecific variation of 3 nt in the ITS2 dataset for Aenigmatrema undecimtentaculatum Corner, Cribb & Cutmore, 2020 (Bucephalidae) from Sphyraena obtusata Cuvier in Moreton Bay, Australia. Although the genetic divergence between sequences of our specimen and R. angusticolle is slightly higher, there have been similar results obtained by Bray et al. [8] who reported intraspecific variation of 5 nt in the 28S and 5 nt in the ITS2 datasets for a species of lepocreadiid, Preptetos prudhoei Bray, Cutmore & Cribb, 2021 from acanthurid fishes which was associated with geographical distribution (Heron Island vs French Polynesia). Considering the low level of sequence divergence between our specimen and R. angusticolle in relation to other congeners and similarities in morphology, we provisionally consider them as conspecific.

Site of infection: stomach.
Infection rates: 1 out of 3; 2 specimens per fish. Voucher material: 2 voucher specimens CHIOC-39763 a-b. Remarks: Specimens found in the present study agree well with the generic diagnosis of Tergestia Stossich, 1899 provided by Bray [11] in having oral sucker papillate, muscular flanges on forebody at level of the pharynx, intestinal bifurcation postero-dorsal to the ventral sucker and uterus extending into the post-testicular region. Only two specimens of this species were collected, and their quality restricts observation of the key morphological features designated by Wee et al. [91] used for species differentiation and identification. The DNA sequences of this material were not generated. Therefore, we provide identification of the species to the genus level.
Remarks: Specimens of Didymocystis sp. 6 sensu Louvard et al. [49] were found in capsules in stomach tissue of one fish. Our identification of the species was confirmed based on comparative sequence analysis (see below). The worms were damaged in an attempt to remove them from the capsules, and therefore we do not provide morphological description and identification to the species level using morphological criteria. Didymocystis sp. 6 sensu Louvard et al. [49] is a parasite of the stomach of A. thazard recently reported from Moreton Bay, Queensland, Australia. This is the first record of this species off the Brazilian coast, southwestern Atlantic Ocean.
Digenean trematodes of the family Didymozoidae are common parasites of scombrid fishes [49,54,73]. This family is the most species rich in A. thazard with 24 nominal species reported (Table 1). In Brazil, the family Didymozoidae is represented by 34 species with eight species from the genus Didymocystis; all recorded from scombrids [21,38]. Melanocystis kawakawa is the only didymozoid previously reported in A. thazard in Brazil [38].
Molecular results: Two 28S sequences of Didymocystis sp. 6 sensu Louvard et al. [49] generated in our study were identical. Comparative sequence analysis of Alignment 3 demonstrated that these sequences exhibited the lowest divergence with the sequence of Didymocystis sp. 6 sensu Louvard et al. [49]. The intraspecific divergence between three isolates was 0.2% (3 nt). This is rather low compared to the interspecific divergence in this dataset which ranged from 7.4 to 8.5% (75-88 nt) ( Table 3). Considering the low level of sequence divergence between our specimens and Didymocystis sp. 6 sensu Louvard et al. [49], we consider them conspecific. Comparative sequence analysis between the cox1 sequence of Didymocystis sp. 6 generated in our study and sequences of Didymocystis spp. provided by Louvard et al. [49]   (Based on eight paragenophores and one hologenophore; measurements of paragenophores in Table 4 and hologenophore in description: Body elongate, narrow, rounded anteriorly and posteriorly, dorso-ventrally flattened, 3871 long. Maximum width at level of ventral sucker (n = 6), 971 or posterior body extremity (n = 3). Tegument covered with conspicuous plications. Forebody short, 1213, representing 31% of body length. Ecsoma well-developed, protruded, with irregular tegument, longer than body.
Excretory vesicle and excretory pore not observed. Remarks: Specimens found in the present study correspond well to the generic diagnosis of Dinurus Looss, 1907 provided by Gibson [32] in having large and well developed ecsoma, plicated body surface, long pars prostatica densely invested by gland-cells and linked to the seminal vesicle by distinct aglandular duct, permanent sinus-organ, seminal vesicle constricted into portions and vitellarium seven digitiform tubes (three on one side, four on another).
Additionally, our specimens resemble specimens of D. scombri Yamaguti, 1934 more closely than any other congeners in possessing body of similar length (3526-5337 vs 4780 in holotype), similar oral/ventral sucker ratio (1:1.14-1.31 vs 1:1.06 in holotype), preoral lobe, tegument covered with conspicuous plications and vitellarium composed of seven elongate digitiform lobes. However, they differ from D. scombri in possessing ecsoma longer than body (1:1.49-2.48 vs 1:0.40 in holotype), longer and wider pharynx (226-364 Â 181-286 vs 160-140 in holotype), longer and wider seminal vesicle Table 3. Nucleotide comparison of the partial 28S rDNA sequences of the Didymozoinae based on 978 nt long alignment. P-distance (%) is given below diagonal and the number of variable nucleotides above diagonal.   (346-368 Â 144-155 vs 190-74 in holotype) and longer extension of uterus within ecsoma (up to one-fifth length of ecsoma vs up to one-tenth in holotype). Dinurus euthynni is a parasite of the stomach of scombrid fishes from the western Pacific Ocean. Since the original description, Mamaev [56] reported this species in A. thazard and Euthynnus affinis (Cantor) from the South China Sea, and Lester et al. [44] in the type host, K. pelamis from Helen Reef, Ponape, Papua New Guinea, Solomon Islands, Coral Sea, Fiji and Norfolk Island. This is the first record of D. euthynni in A. thazard off the Brazilian coast, southwestern Atlantic Ocean. Newly collected material of D. euthynni in the present study represents the fourth record of this species and provides the first detailed morphological description supplemented with DNA sequence data.  Table 4 and hologenophore in description): Body elongate, dorso-ventrally flattened, 1493 long. Maximum width close to posterior body extremity, 356. Tegument slightly rugose up to level of vitellarium. Forebody short 333, representing 22% of body length. Ecsoma well developed, withdrawn, protruded or partially extruded.
Excretory vesicle not observed; excretory pore terminal. Remarks: Specimens found in the present study correspond well to the generic diagnosis of Lecithochirium Lühe, 1901 provided by Gibson and Bray [31] and Gibson [32] in having well developed ecsoma, pre-oral lobe, tubular pars prostatica, vitellarium of two lateral masses divided into 3 and 4 short lobes, and eggs without polar filament.
Due to the morphological complexity of Lecithochirium, Bray [10] proposed a provisional key to species-groups. The species are classified into 24 groups based on modifications of the oral sucker, ratio of suckers, the presence or absence of presomatic pit, type of terminal genitalia, and shape of vitellarium. According to this classification, L. floridense belong to the "Musculus-group" based on the absence of the presomatic pit, the presence of the vitellarium of compact masses with distinct and short digitiform lobes, the terminal genitalia of the "musculus" type, the non-muscular seminal vesicle, and the absence of the internal elevations in ventral sucker.
Among the species belonging to the "Musculus-group", our material most resembles L. floridense described by Manter [57] from the stomach of Paralichthys sp. (type host) and many other fish species in Tortugas, Florida, USA and later reported by Bullard et al. [12] from the stomach of the lionfish, Pterois cf. volitans in the Atlantic Ocean, off Beaufort, North Carolina, USA, particularly in possessing a preoral lobe, well developed ecsoma, genital pore at the level of the pharynx, bipartite seminal vesicle, suckers of similar ratio (1:2.34-2.76 vs 1:2.0-3.0 in Manter [58] vs 1:2.0-2.40 in Bullard et al. [12]), and eggs of similar size (12-15 Â 9-12 vs 12-16 Â 9-10 in Manter [58] vs 14-17 Â 7-10 in Bullard et al. [12]). Our material differs from material of Manter [58] and Bullard et al. [12] in exhibiting higher minima for body size and size of most internal organs, and in lower maxima for body size, ecsoma and forebody length from specimens in material of Manter [58] ( Table 4). Identification of the species based on morphological data was supported by the phylogenetic analyses (see below).
Lecithochirium floridense is a parasite of the stomach of a variety of marine fish species. After the original description, Manter [58] re-evaluated the material collected in Manter [58] and observed that there was more than one species among what he identified as L. floridense. Thereafter, he provided an updated list of hosts, which consisted of 21 species from 13 families. To date, L. floridense has been reported from fishes belonging to at least 16 families with the majority of records coming from the western Atlantic Ocean [12,15,48,59,65,76,81,85,88]. Parukhin [70] reported this species from Haplobrotula gnathopus (Regan) and Scorpaena scrofa Regan (=Scorpaena natalensis) collected off the South African coastline, i.e., Atlantic and Indian oceans. The only records of L. floridense from Rhomboplites aurorubens (Cuvier) (Lutjanidae) and Pterois volitans (Linnaeus) (Scorpaenidae) by Claxton et al. [15] and from Syacium papillosum (Linnaeus) (Cyclopsettidae) by Vidal-Martinez et al. [88] were supplemented with DNA sequence data, ITS1-5.8S-ITS2 and 28S rDNA, respectively. Our record of A. thazard infected with L. floridense off the Brazilian coast represents a new host and a new geographical record for this species.
Remarks: Specimens found in the present study correspond well to the generic diagnosis of Lecithochirium in characters as mentioned above. Following the key to species-groups of Lecithochirium proposed by Bray [10], our specimens belong to the "Synodi-group" based on the presence of the glandular presomatic pit, vitellarium of compact masses with distinct and short digitiform lobes, the terminal genitalia of the "musculus" type, the non-muscular seminal vesicle, and the absence of the internal elevations in ventral sucker.
In comparison with species from the "Synodi-group", our specimens can be distinguished from L. exodium McFarlane, (tripartite vs bipartite) and in shape of sinus-sac (pyriform vs transversely oval).
Although limited with sequence data, we followed a model of trematode species recognition proposed by Bray et al. [8] for the identification of our specimens as L. synodi based on their morphology (see above) and geographical distribution of their hosts. Lecithochirium bothi and L. kawakawa have thus far been reported to have their geographical distributions in the Indian and Pacific oceans [7,10,90,94], whereas L. synodi has been reported in the north-western Atlantic Ocean [37,57,59]. Although L. synodi has been reported from the unicorn leatherjacket filefish Al. monoceros in the South China Sea, north-western Pacific Ocean by Wang [90], this record should be interpreted with caution. The specimens of Wang [90] are morphologically similar to other two species, L. bothi and L. kawakawa reported in the Pacific Ocean. Previous reports on fish host spectrum of the three species suggest that all have low host specificity. Lecithochirium bothi was reported in bothiid and mullid fishes, L. kawakawa in platycephalid, scombrid and sparid fishes, and L. synodi in monacanthid, paralichthyid and synodontid fishes. In our study, L. synodi was for the first time reported from scombrid fish A. thazard and this is the first record of L. synodi off the Brazilian coast, south-western Atlantic Ocean. Our newly collected material of L. synodi has allowed us to provide detailed morphological description of the species and generate DNA sequence data.

Discussion
Despite its wide distribution in the Atlantic Ocean and its economic value for regional commercial fisheries, A. thazard has not been a frequent target of fish parasitological investigations in Brazil. The present study is the first to apply both morphological and molecular techniques to explore the diversity of digenean trematodes of the frigate tuna A. thazard collected off the coast of Rio de Janeiro, Brazil. Despite a small sample size of A. thazard in our study, we recorded six species of digenean trematodes, namely: the bucephalid Rhipidocotyle cf. angusticolle, the didymozoid Didymocystis sp. 6 sensu Louvard et al. [49], the fellodistomid Tergestia sp., and three hemiurids D. euthynni, L. floridense and L. synodi. For three of these species -R. cf. angusticolle, L. floridense and L. synodithe frigate tuna was reported as a new host and four -Didymocystis sp. 6 sensu Louvard et al. [49], D. euthynni, L. floridense and L. synodiwere reported in Brazil for the first time. With our new records, the number of digenean trematodes of A. thazard in Brazil increased from two to eight species, with hemiuroid trematodes being most diverse in this fish.
Specimens putatively identified in our study as R. angusticolle were found for the first time in the frigate tuna. Rhipidocotyle angusticolle is a stenoxenous parasite and has previously been reported only in scombrid fishes in the western Atlantic Ocean [14,17,68]. With 64 species parasitizing freshwater and marine fishes worldwide [68] the genus Rhipidocotyle is represented by only four species in marine fishes in Brazil, namely: R. angusticolle, R. fluminensis Vicente & Santos, 1973, R. pentagonum (Ozaki, 1924), and R. quadriculatum Kohn, 1961. They are all parasites of scombrid fishes in the region [21].
The first records of two hemiuroids D. euthynni and Didymocystis sp. 6 sensu Louvard et al. [49] in the Atlantic Ocean demonstrate that the geographical distribution of these species is wider than formally known. Previous records were restricted to the Pacific Ocean [44,49,56,92]. The wide distribution of these species is most likely associated with the distribution of the frigate tuna. Mamaev [56] reported D. euthynni from A. thazard in the South China Sea and Louvard et al. [49] reported Didymocystis sp. 6 from A. thazard in Moreton Bay, Australia. The identification of Didymocystis sp. 6 sensu Louvard et al. [49] was based on DNA sequence comparison, while DNA sequence data for D. euthynni were not available for comparison. Thus, based on DNA sequence data, Didymocystis sp. 6 sensu Louvard et al. [49] was confirmed to be an oioxenous parasite infecting A. thazard, whereas D. euthynni is known as a stenoxenous parasite infecting fishes from the family Scombridae.
Lecithochirium is one of the most species-rich genera within the Hemiuridae, with over 100 species parasitizing marine fishes from several orders [54]. The taxonomy of the genus remains in a controversial state due to the presence of morphologically similar species, poor morphological descriptions for the majority of species, and the lack of DNA sequences which hinders elucidation of its composition based on phylogenetic analysis [10,31,54,60]. There is an obvious need for a thorough revision of the genus based on additional datasets that combine information on species morphology, DNA sequences and host distribution as proposed by Bray et al. [8]. It is worth noting that the findings of the current study do not support the previous study of Sokolov et al. [84] that published the DNA sequence of He. luehei. The results of our phylogenetic and comparative sequence analyses demonstrated that the sequence of the isolate identified as He. luehei clustered among the isolates of L. floridense. Thus, our results indicate incorrect identification of the He. luehei isolate. Sequences of L. floridense were not included in the analysis of Sokolov et al. [84] and, consequently, the phylogenetic position of the genus Hemiurus being closely related to the genus Lecithochirium within the Hemiuridae was erroneous.
Digenean trematodes represent the most diverse group of parasites in A. thazard. Previous studies performed by Mamaev [56] and Yamaguti [94] showed high species richness of digeneans in A. thazard in the Pacific Ocean, with 12 and 8 species being found, respectively (see Table 1). Recently, however, Louvard et al. [49] found eight didymozoids in the frigate tuna from Moreton Bay, Australia. The fauna of digeneans of A. thazard in the Atlantic Ocean, including the results of our study, currently accounts for ten species. Prior to our study, R. pentagonum was the only species recorded in the frigate tuna in both the Atlantic and Indo-Pacific regions. In our study, D. euthynni and Didymocystis sp. 6 sensu Louvard et al. [49] known from the frigate tuna in the South China Sea [56] and in Moreton Bay, Australia were discovered in the frigate tuna in the Atlantic Ocean for the first time.
To date, 63 nominal species of digenean trematodes from 10 families, including our data, have been reported in scombrid fishes in Brazil [21,38]. The skipjack tuna K. pelamis and blackfin tuna Th. atlanticus (Lesson) are the scombrid hosts with the highest diversity of digeneans in the region, each known as the host for at least 15 digenean species [21,38]. Of the 63 species of digeneans, there are only three speciestwo bucephalids, R. angusticolle and R. pentagonum, and one didymozoid M. kawakawathat A. thazard shares with the other species of scombrid fishes in the region, namely: E. alletteratus, K. pelamis, Th. atlanticus, and Scomber colias. We believe that this information is likely to change when the diversity of digeneans from A. thazard becomes better known in Brazil.
The present study brings to light new information on the digenean diversity of the frigate tuna in Brazil and presents novel sequence data and data on host association and geographical distribution of six digenean species. Further large-scale investigations including seasonal monitoring and the application of an integrative taxonomic approach will uncover the true species diversity of digenean trematodes in A. thazard from the Atlantic Ocean and beyond.