Introduction
The genus Saccocoelioides was established by Szidat (1954) with Saccocoelioides nanii Szidat, 1954 as type species. It is distributed across South, Middle, and North America, infecting a wide range of hosts (Kohn et al. Reference Kohn, Fernandes and Cohen2007; Curran et al. Reference Curran, Pulis, Andres and Overstreet2018; Andrade-Gómez et al. Reference Andrade-Gómez, Sereno-Uribe and Garcia-Varela2019). According to Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018), this genus comprises two groups of species: (i) the diminutive form possesses a smaller body size (<1.7 mm), with few and large eggs that are longer than the pharynx length when fully formed, and the uterus is confined to the hindbody; (ii) the robust group exhibits a larger body size (>1.7 mm), with numerous and small eggs that are shorter than the pharynx length when fully formed, and the uterus entirely fills the hindbody or reaches the forebody in some species. The first group is more specious, while the second one includes species such as Saccocoelioides antonioi Lunaschi, Reference Lunaschi1984, Saccocoelioides elongatus Szidat, 1954, Saccocoelioides guaporense Thatcher & Dossman, 1974, Saccocoelioides magnus Szidat, 1954, Saccocoelioides rotundus Thatcher & Jégu, Reference Thatcher and Jégu1996, and Saccocoelioides szidati Travassos, Freitas & Khon, Reference Travassos, Teixeira de Freitas and Kohn1969.
In Argentina, the following species belonging to the diminutive group have been reported: Saccocoelioides carolae Lunaschi, Reference Lunaschi1984, Saccocoelioides kirchneri Martorelli et al. Reference Martorelli, Montes, Barneche, Reig Cardarella and Curran2022, Saccocoelioides magniovatus Szidat, 1954, Saccocoelioides nanii Szidat, 1954, and Saccocoelioides octavus Szidat, Reference Szidat1970 (Martorelli et al. Reference Martorelli, Montes, Barneche, Reig Cardarella and Curran2022). On the other hand, the species belonging to the robust group found in Argentina are S. antonioi, S. elongatus, S. magnus, and S. szidati (Curran et al. Reference Curran, Pulis, Andres and Overstreet2018; Ostrowski de Nuñez et al. Reference Ostrowski de Nuñez, Arredondo and Gil de Pertierra2017).
During a recent parasitological survey of fishes in Iguazu National Park, Argentina, we collected haploporid digeneans parasitizing several host species. The aim of this work is to describe the new species using an integrative molecular and morphological approach.
Materials and Methods
Collection of samples and morphological study
Specimens of Astyanax dissimilis Garavello & Sampaio, Psalidodon bifasciatus (Garavello & Sampaio), and Bryconamericus ikaa Casciotta, Almirón & Azpelicueta, all members to the Characidae family, were collected with cast nets from the Iguazu River (upstream from the waterfalls) in the Iguazu National Park, Misiones province, Argentina (25°40’34’’S, 54°27’08’’W, Figure 1). They were transported alive to the field laboratory at the Centro de Investigaciones Ecológicas Subtropicales (CIES) in plastic bags containing water from the sampling site, supplied with oxygen. Upon arrival, the fishes were euthanized by cervical dissection, necropsied, and examined for parasites. For the morphological examination, digeneans found in the intestine were heat-killed with hot water and fixed in 10% formalin, while for the molecular study, live specimens were preserved and stored in cold 96% ethanol for later DNA extraction.
Figure 1. Site of collection: Iguazú National Park (*), Misiones province, Argentina.
Morphological analysis
Whole-mount specimens were processed following standard parasitological techniques and stained with hydrochloric carmine (Pritchard and Kruse Reference Pritchard and Kruse1982). Measurements and digital images of the specimens were captured using an Olympus Bx51 microscope equipped with an AmScope MU 1000 10 MP digital camera (United Scope LLC, Irvine, USA). The structures were measured using ImageJ software (Schneider et al. Reference Schneider, Rasband and Eliceiri2012). Drawings were created with the aid of a drawing tube. Measurements are presented as the mean followed by minimum and maximum values in parentheses and are given in micrometers (μm). The type-material was deposited in the Invertebrate Collection of the Museo de La Plata, La Plata, Argentina.
DNA extraction, amplification, and sequencing
Total genomic DNA was extracted from Saccocoelioides sp. on P. bifasciatus, A. dissimilis, and B. ikaa (one specimen per host) using a PURO-Genomic DNA kit (PB-L Productos Bio-Logicos® S.A., Argentina) following the manufacturer’s protocol. Amplification of each gene was conducted using polymerase chain reaction (PCR) on an Eppendorf Mastercycler thermal cycler (Hamburg, Germany). The fragment of the 28S rDNA gene (28S) was amplified with the forward primer LSU-5 (5’-TAG GTC GAC CCG CTG AAY TTA AGC A-3’) and the reverse primer 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3’) as described by Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003). PCR thermocycling conditions followed Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003). The fragment of the mtDNA COI gene (COI) was amplified using the primer pair DICE 1F (5’-ATT AAC CCT CAC TAA ATT WCN TTR GAT CAT AAG-3’) and DICE 14R (5’-TAA TAC GAC TCA CTA TAC CHA CMR TAA ACA TAT GAT G-3’) as described by Van Steenkiste et al. (Reference Van Steenkiste, Locke, Castelin, Marcogliese and Abbott2015). PCR conditions were as follows: 94 °C for 2 min; 5 cycles of 95 °C for 30 s, 48 °C for 40 s, 72 °C for 1 min; followed by 40 cycles of 94°C for 30 s, 56°C for 40 s, 72°C for 1 min; and a final extension at 72°C for 10 min. Both strands of each PCR product were sequenced using an ABI 3730XLs sequencer (Macrogen Inc., Seoul, South Korea). The sequences were assembled using Geneious v.6.0.5 (http://www.geneious.com) and utilized to search for homologous sequences in GenBank (Table 1).
Table 1. Collection data and GenBank accession numbers for Saccocoelioides spp. analyzed in this study; new species in bold
Sequence comparison and phylogenetic analysis
Sequences were aligned using the online version of MAFFT v.7 (Katoh et al. Reference Katoh, Rozewicki and Yamada2019). The best partitioning scheme and substitution model for the DNA partition were chosen based on the Bayesian information criterion (BIC; Schwarz Reference Schwarz1978) using the ‘greedy’ search strategy in Partition Finder v.1.1.1 (Lanfear et al. Reference Lanfear, Calcott, Kainer, Mayer and Stamatakis2014). The optimal nucleotide substitution models were HKY+G for the 28S sequences, and F81+I, GTR+G, and TrN+G for the first, second, and third codon positions of the COI sequences, respectively.
Phylogenetic reconstruction was conducted using Bayesian inference (BI) in MrBayes v.3.2.3 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). Phylogenetic trees were generated using two parallel analyses of Metropolis-coupled Markov chain Monte Carlo (MCMC) for 20 million generations each to estimate the posterior probability (PP) distribution. Topologies were sampled every 1,000 generations, and the average standard deviation of split frequencies was maintained below 0.01, as recommended by Ronquist et al. (Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). The robustness of the clades was evaluated using Bayesian PP, with PP > 0.95 considered strongly supported. A majority consensus tree with branch lengths was constructed for each run after discarding the initial 25% of sampled trees.
The proportion (p) of absolute nucleotide sites (p-distance) (Nei and Kumar Reference Nei and Kumar2000) was calculated to compare the genetic distance between lineages. The p-value matrix was obtained using MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018), employing the bootstrap method (1,000 replicates) and uniform rate nucleotide substitution (transition + transversions). Newly generated sequences (Table 1) were submitted to the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov).
Results
Morphological analysis
Saccocoelioides miguelmontesi n. sp. (Figure 2)
Figure 2. Saccocoelioides miguelmontesi n. sp. Ventral view: photograph and drawing.
Scale bar = 100 μm.
Zoobank: urn:lsid:zoobank.org:act:38F64EEA-C89F-4251-8323-32B45287F28B
Description
Body elongate, tegument entirely spinous, with dispersed eye spot pigment in anterior half, 888 (858–969) long, 287 (268–315) wide at ventral sucker level. Oral sucker subterminal, 96 (91–102) long, 112 (108–117) wide. Ventral sucker 106 (96–123) long, 94 (89–103) wide. Oral/ventral sucker ratio 1:1.05–1.22 long, 1:0.82–0.88 wide. Prepharynx 35 (23–43) long. Pharynx 57 (50–63) long, 57 (51–66) wide. Esophagus 179 (165–209) long. Ceca sac-shaped, not reaching pretesticular region. Forebody 355 (328–389) long, representing 38 (36–40)% of body length. Testis oval, near mid hindbody, longer than wide, 246 (238–263) long, 121 (108–129) wide. Hermaphroditic sac oval, 125 (111–141) long, 78 (73–83) wide. External seminal vesicle globose, 79 (61–112) long, 59 (46–70) wide. Genital pore opening medially between ventral sucker and pharynx. Ovary slightly elongated longitudinally, 81 (70–89) long, 53 (50–60) wide. Laurer’s canal and Mehlis’ gland not observed. Uterus extending between hermaphroditic sac and posterior body end. Vitellarium arranged in 2 clusters of follicles extending from posterior level of ventral sucker to anterior margin of testis. Eggs 60 (59–61) long, 29 (28–29) wide; fully developed eggs containing miracidia with eye spots. Pharynx/egg ratio length 1:0.91–1.03 long. Excretory vesicle Y-shaped.
Taxonomic summary
Type host: Psalidodon bifasciatus (Garavello & Sampaio).
Other hosts: Astyanax dissimilis Garavello & Sampaio, Bryconamericus ikaa Casciotta, Almirón & Azpelicueta.
Site of infection: Pyloric ceca.
Type locality: Iguazu River (upstream from the waterfalls), Iguazu National Park, Misiones province, Argentina.
Prevalence of infection: 30% (9/30) in P. bifasciatus, 17% (5/30) in A. dissimilis, and 27% (8/30) in B. ikaa.
Type material: Holotype MLP-He 8081and paratype MLP-He 8082.
Etymology: The species epithet “miguelmontesi” was selected to honor Martin Montes’ father, Miguel Angel Montes, for his unconditional love and support.
Remarks
The new species cannot be assigned to either of the two morphological types within Saccocoelioides classified by Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018). Certainly, it may not be included in the robust group because its body size is smaller than 1.7 mm, and it may not belong to the diminutive group because the mature eggs are smaller or equal in length to the pharynx length and the uterus is not confined in the hindbody.
Although the new species displays an intermediate configuration between the two forms, it is important to compare the new species primarily to the robust species, focusing on the ratio of mature egg to pharynx length, which is either smaller or equal in the robust species.
A comparison between the measurements of the 6 species within the robust morphotype and S. miguelmontesi n. sp. is shown in Table 2.
Table 2. Comparison between measurements of adult specimens of Saccocoelioides spp. belonging to the robust morphotype (Curran et al. Reference Curran, Pulis, Andres and Overstreet2018) and S. miguelmontesi n. sp.
Abbreviations: B., Body; E., Egg; Ex., External; H., Hermaphroditic; L, Length; O., Oral; Oes., Oesophagus; Ov., Ovary; P., Pharynx; Ppx., Prepharynx; S., Sucker; Sem., Seminal; T., Testicle; V., Ventral; Ves., Vesicle; W., Width; *calculated on the drawings or the measures given by the authors; † not assigned by Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018) in any group
Saccocoelioides miguelmontesi n. sp. is distinguished from the robust species by having a smaller body size (except for S. guaporense and S. rotundus, which show overlapping values) and smaller eggs (except for S. magnus). Most of the measurements of the robust species are smaller than those of the new species.
Saccocoelioides miguelmontesi n. sp. has smaller oral and ventral suckers, pharynx width, egg length, oral/ventral sucker width ratio, oral sucker/pharynx width ratio, and post-cecal/body length percentage than those of S. guaporense and S. rotundus. In comparison with S. guaporense, S. miguelmontesi n. sp. has smaller ventral sucker and pharynx length, hermaphroditic sac width, sucker length ratio, and post-testicular/body length percentage. In comparison with the latter species, the new species has smaller body width, testes, ovary and egg width, and body width/body length percentage, while it has a larger forebody/body length percentage and post-testicular/body length percentage. Saccocoelioides miguelmontesi n. sp differs from S. magnus in possessing smaller values for all measurements (Table 2).
Molecular analyses
The partial sequences of 28S from Saccocoelioides miguelmontesi n.sp. were obtained from one specimen from P. bifasciatus (1296 bp) and one from A. dissimilis (1359 bp). Once trimmed with the Gblocks program http://www.phylogeny.fr/one_task.cgi?task_type=gblocks, these new sequences exhibited no differences in nucleotide composition between them. They were subsequently combined with sequences obtained from GenBank (Table 1) to construct a matrix with 57 taxa and 1334 bp including gaps. In the 28S tree reconstructed through Bayesian analysis (Figure 3), S. miguelmontesi n. sp. appeared sister to S. elongatus, forming a cluster with S. magnus; both S. elongatus and S. magnus belonged to the robust group (sensu Curran et al. Reference Curran, Pulis, Andres and Overstreet2018). The genetic divergence and number of nucleotide differences among the 28S partial sequences of Saccocoelioides spp. ranged from 0 to 5% and from 0 to 40 bp, respectively. Saccocoelioides miguelmontesi n. sp., S. elongatus, and S. magnus were separated by a p-distance of 1% further supporting their close relationship (Table 3).
Figure 3. Phylogram resulting from Bayesian inference (20,000,000 generations) of partial 28S rDNA gene sequences of Saccocoelioides miguelmontesi n. sp. rooted by Forticulcita platina. Branch support values indicate posterior probabilities ≥95%. Gray bar = robust group; black bar = diminutive group.
Table 3. Uncorrected p-distances of the 28S rDNA gene calculated in MEGA X with variance estimation, bootstrap method (500 replicates), and uniform nucleotide substitution (transition + transversion) rates below the diagonal line expressed in percentage; number of nucleotide differences above the diagonal line
The partial sequences of COI from Saccocoelioides miguelmontesi n. sp. were obtained from the hosts P. bifasciatus (693 bp), A. dissimilis (726 bp), and B. ikaa (618 bp), one specimen each. Once trimmed to the shorter sequences, these new sequences exhibited no differences in nucleotide composition among them. They were subsequently combined with sequences obtained from GenBank (Table 1), resulting in a matrix with 45 taxa and 777 bp.
In the COI tree (Figure 4), S. miguelmontesi n. sp. occupies a single branch that represents the “robust group”. The p-distances and number of nucleotide differences among Saccocoelioides spp. varied from 10 to 23% and from 32 to 70 bp, respectively. The p-distance between S. miguelmontesi n. sp., S. olmecae, and S. orosiensis was 18% (Table 4). The intragroup p-distance among the sequences of the new species was 0%.
Figure 4. Phylogram resulting from Bayesian inference (20,000,000 generations) of COI mtDNA gene sequences of Saccocoelioides miguelmontesi n. sp. rooted by Forticulcita sp. Branch support values indicate posterior probabilities ≥85%.
Table 4. Uncorrected p-distances of the COI mtDNA gene calculated in MEGA X applying variance estimation, bootstrap method (500 replicates), and uniform nucleotide substitution (transition + transversion) rates below the diagonal line; number of nucleotide differences above the diagonal line
Discussion
According to the COI tree and p-distance analysis, the sequenced specimens obtained from the three fish hosts belong to the same species. The sequences deposited in GenBank only represent the diminutive group, making the ones reported here the first of this group to be documented. The phylogenetic analysis of 28S sequences and the p-distance analysis showed that S. miguelmontesi n. sp. was more closely related to S. elongatus and S. magnus, which were the only robust species sequenced.
Although the genus Saccocoelioides has been proposed to be highly host-specific (Szidat Reference Szidat1970; Ostrowski de Nuñez pers. com.), S. miguelmontesi n. sp. was found parasitising three different fish hosts of different genera. According to Mirande (Reference Mirande2010), those genera belong to Characidae, but Astyanax sp. and Psalidodon sp. are inside the Stethaprioninae, and Bryconamericus sp. belongs to Stevardiinae. These subfamilies are not directly related. Recently, Terán et al. (Reference Terán, Benitez and Mirande2020) clarified the systematics of “Astyanax sp.”, transferring A. bifasciatus to the resurrected Psalidodon genus. Interestingly, during dissection we noted that the hosts shared food items (e.g., algae and aquatic plants) suitable for cercarial encystment. It is possible that these fishes acquired the infection by ingesting metacercariae either attached to the substrate or floating freely in the water column.
Saccocoelioides miguelmontesi n. sp. was found upstream from the Iguazu waterfalls. This region harbours a highly diverse endemic ichthyofauna (Casciotta et al., Reference Casciotta, Almirón, Ciotek, Giorgis, Říčan, Piálek, Dragová, Croci, Montes, Iwaszkiw and Puentes2016) that has been poorly investigated for digeneans, thus representing a potential source of new parasite species. This assumption may be confirmed by studies of other digenean species infecting fishes upstream from the Iguazu waterfalls, such as Prosthenhystera gatii Montes, Barneche, Croci, Rodriguez Gil, Curran, Ferrari, Casciotta & Martorelli, Reference Montes, Barneche, Croci, Rodriguez Gil, Curran, Ferrari, Casciotta and Martorelli2020, and Creptotrema guacurarii (Montes, Barneche, Croci, Balcazar, Almirón, Martorelli & Pérez-Ponce de León, Reference Montes, Barneche, Croci, Balcazar, Almirón, Martorelli and Pérez-Ponce de León2021) Franceschini, Aguiar, Zago, de Oliveira Fadel Yamada, Bertholdi Ebert & da Silva, 2021 (Montes et al., Reference Montes, Barneche, Croci, Rodriguez Gil, Curran, Ferrari, Casciotta and Martorelli2020; Reference Montes, Barneche, Croci, Balcazar, Almirón, Martorelli and Pérez-Ponce de León2021).
From a morphological viewpoint, the new species shows an intermediate configuration between the diminutive and robust forms described for Saccocoelioides by Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018). Certainly, it possesses a small body size, eggs and pharynx of equal length, and a uterus mainly concentrated in the hindbody, with some loops extending to the forebody. Moreover, the phylogenetic analysis of 28S sequences placed the new species in a well-supported clade with two robust species. Considering the aforementioned facts, we propose to use egg length relative to pharynx length rather than body size as the main criterion to discriminate between Saccocoelioides species in both groups. As a result, the “diminutive group” could be characterized as having mature eggs longer than the pharynx, and the “robust group” as having eggs shorter or equal in length to the pharynx. Under this classification, the diminutive group would comprise the following 19 species: Saccocoelioides agonostomus Dyer, Bunkley-Williams & Williams, 1999; Saccocoelioides beauforti (Hunter & Thomas, 1961) Overstreet, 1971; Saccocoelioides carolae Lunaschi, Reference Lunaschi1984; Saccocoelioides chauhani Lamothe-Argumedo, 1976; Saccocoelioides cichlidorum (Aguirre-Macedo & Scholz, 2005) García-Varela, Andrade-Gómez & Pinacho-Pinacho, 2017; Saccocoelioides kirchneri (Martorelli, Montes, Barneche, Reig Cardarella & Curran, Reference Martorelli, Montes, Barneche, Reig Cardarella and Curran2022); Saccocoelioides lamothei Aguirre-Macedo & Violante-González, 2008; Saccocoelioides macrospinosus Andrade-Gómez, Sereno-Uribe & García-Varela 2019; Saccocoelioides magniovatus Szidat, 1954; Saccocoelioides nanii Szidat, 1954; Saccocoelioides octavus Szidat, Reference Szidat1970; Saccocoelioides olmecae Andrade-Gómez, Pinacho-Pinacho, Hernández-Orts, Sereno-Uribe & García-Varela, 2016; Saccocoelioides overstreeti Fernandez-Bargiela, 1987; Saccocoelioides ruedasueltensis (Thatcher, 1978) Curran, Pulis, Andres & Overstreet, Reference Curran, Pulis, Andres and Overstreet2018; Saccocoelioides sogandaresi Lumsden, 1963; Saccocoelioides tarpazensis Díaz & González, 1990; Saccocoelioides tilapiae (Nasir & Gomez, 1976); Saccocoelioides tkachi Curran, Pulis, Andres & Overstreet, Reference Curran, Pulis, Andres and Overstreet2018; and Saccocoelioides orosiensis Curran, Pulis, Andres & Overstreet, Reference Curran, Pulis, Andres and Overstreet2018). On the other hand, the robust group would comprise the following seven species: S. antonioi, S. elongatus, S. guaporense, S. magnus, S. rotundus, S. szidati, and S. miguelmontesi n. sp.
Results from morphological and molecular studies, new and more complete information on life cycles, and the description of larval forms will help elucidate the phylogeny of the Saccocoelioides species. Argentina harbors about 550 freshwater fish species in 56 families (Mirande and Koerber Reference Mirande and Koerber2020) serving as potential hosts for Saccocoelioides species. Among them, only Saccocoelioides species in seven families were reported: Anostomidae (S. magniovatus, S. szidati), Characidae (S. octavus), Cichlidae (S. carolae), Curimatidae (S. antonioi, S. magnus), Loricariidae (S. nanii), Poecillidae (S. kirchneri), and Prochilodontidae (S. elongatus, S. nanii). Considering the significant number of families without registered Saccocoelioides species, the likelihood of there being more than one species for these families suggests a potential abundance of undiscovered Saccocoelioides species.
Acknowledgements
We are grateful to CONICET and CEPAVE for providing facilities and equipment, and the Buenos Aires Environment Ministry for providing permits. We thank M. Marcia Montes for the line drawings and Silvia Pietrokovsky for English language revisions.
Financial support
This work was funded by research grants PIP 1713 from CONICET and PICT-2020-SERIEA-01531 from Fondo Para La Investigación Científica Y Tecnológica (FONCyT), both to M.M.M.
Competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of animals.
