Introduction
Members of the family Lecithasteridae (Odhner 1905) occur worldwide and primarily parasitize marine fish (Gibson Reference Gibson, Gibson, Jones and Bray2002). The family has recently undergone taxonomic reorganizations, resulting in the current classification into four accepted subfamilies: Lecithasterinae (Odhner 1905); Macradenininae (Skrjabin and Guschanskaja 1954); Prolecithinae (Yamaguti 1971); and Trifoliovariinae (Yamaguti 1958) (Vainutis and Voronova, Reference Vainutis and Voronova2025a, Reference Vainutis and Voronovab; WoRMS, 2025a). The subfamily Lecithasterinae has also undergone taxonomic revision and now includes only two genera: Lecithaster Lühe, 1901 (23 valid species) and Alloopistholecithum Vainutis and Voronova, 2025 (11 valid species) (Ivashko et al. Reference Ivashko, Atopkin, Van Ha, Van Hien and Besprozvannykh2025; Vainutis and Voronova Reference Vainutis and Voronova2025a, Reference Vainutis and Voronovab).
Six species of lecithasterids digeneans initially classified under the genus Lecithaster have been recorded from South America and Antarctica. However, recent taxonomic revisions have transferred five of these species to the newly established genus Alloopistholecithum. These include: Alloopistholecithum helodes (Overstreet Reference Overstreet1973), which parasitizes fishes of the family Mugilidae in Brazil, Alloopistholecithum intermedium (Szidat Reference Szidat1954), a parasite of brackish-water fishes of the family Clupeidae in Argentina, Alloopistholecithum macrocotyle (Szidat and Graefe Reference Szidat and Graefe1967), a parasite of Nototheniidae in Argentina, Alloopistholecithum micropsi (Zdzitowiecki Reference Zdzitowiecki1992), found in Nototheniidae in Antarctica and subantarctic regions, and Alloopistholecithum pacificum (Figueroa and Puga Reference Figueroa and Puga1991), which parasitizes families Atherinopsidae and Paralichthyidae in Chile (Figueroa and Puga Reference Figueroa and Puga1991; Overstreet Reference Overstreet1973; Szidat Reference Szidat1954; Szidat and Graefe Reference Szidat and Graefe1967; Vainutis and Voronova Reference Vainutis and Voronova2025a, Reference Vainutis and Voronovab; WoRMS 2025b; Zdzitowiecki Reference Zdzitowiecki1992). As noted by Vainutis and Voronova (Reference Vainutis and Voronova2025b), the species Lecithaster falcatus (Amato Reference Amato1983), which parasitizes fishes of the families Serranidae and Sciaenidae in Brazil (Amato Reference Amato1983), remains within its original genus due to insufficient information for reassessment.
Odontesthes bonariensis Valenciennes, the Argentinian silverside, is among the most socio-economically important freshwater fish species in Argentina (Somoza et al. Reference Somoza, Miranda, Berasain, Colautti, Remes Lenicov and Strüssmann2008). Valued for its high-quality meat, it is heavily exploited for both recreational and commercial fishing. Its historical native range includes the La Plata River, the middle and lower Paraná River, and the Salado basin (Ringuelet et al. Reference Ringuelet, Aramburu and Alonso De Aramburu1967). Owing to its economic importance, the species has been translocated across Argentina and to various regions of South America, Europe, Africa, and Asia (Tombari and Volpedo, Reference Tombari, Volpedo, Volpedo and Fernández Reyes2008; Torres-Martínez et al. Reference Torres-Martínez, Mancini, Grosman, Somoza and Strüssmann2025).
In the present study, specimens belonging to the family Lecithasteridae were recovered from O. bonariensis in the brackish waters of Buenos Aires Province. A comprehensive taxonomic analysis revealed that these specimens represent a new species of Alloopistholecithum. This study provides a detailed description of this new species.
Material and methods
Specimen collection
Specimens of O. bonariensis (Argentinian silverside) were collected from Punta Rasa (36°17´S, 56°46´W), Buenos Aires province, Argentina. Fish were captured using cast nets and a landing net, and then transported alive to the laboratory in oxygenated bags. In the laboratory, they were transferred to aerated aquaria containing water from the sampling site. Silversides were euthanized with an overdose of eugenol, and their intestines were examined for parasites. Digeneans were removed, relaxed in hot water, and fixed in 10% formalin for morphological studies. Additional specimens were preserved in 96% ethanol for DNA analysis.
Specimens were stained with Hill´s haematoxylin and mounted in Canada balsam following standard parasitological techniques (Pritchard and Kruse Reference Pritchard and Kruse1982). Measurements and digital images were obtained using an Olympus BX51 microscope equipped with an AmScope MU 1000 10 MP digital camera. Structural measurements were taken using ImageJ software (Schneider et al. Reference Schneider, Rasband and Eliceiri2012) and are presented as ranges followed by the mean in parentheses, expressed in micrometres (μm). Drawings were prepared using photographs of stained specimens as a reference. Type specimens were deposited in the Helminthological Collection of the Museo de La Plata (MLP-He), La Plata, Buenos Aires, Argentina. Parasitological descriptors follow Bush et al. (Reference Bush, Lafferty, Lutz and Shostak1997). Additionally, type specimens of A. intermedium (MACN-Pa 13/1-2), housed in the Parasitological Collection of the Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, Buenos Aires, Argentina, were examined for comparative purposes.
DNA extraction, amplification, and sequencing
Genomic DNA was extracted from three digeneans recovered from the intestines of silversides using the Wizard® Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol. The 28S rDNA fragment was amplified via polymerase chain reaction (PCR) on an Eppendorf Mastercycler thermal cycler using the forward primer LSU5 (5’-TAG GTC GAC CCG CTG AAY TTA AGC A-3’) (Littlewood et al. Reference Littlewood, Curini-Galletti and Herniou2000) and reverse primer 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3’) (Olson et al. Reference Olson, Cribb, Tkach, Bray and Littlewood2003). PCR reactions were performed using GoTaq Master Mix (Promega), following the protocol and cycling parameters described by Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003). PCR products were sequenced by Macrogen Inc. (South Korea). Sequences were manually edited in Geneious 11 (Biomatters Ltd.) under a free trial licence (http://www.geneious.com). Consensus sequences were assembled, edited, and used to search for homologous sequences in GenBank.
Sequence comparison and phylogenetic analysis
The newly obtained sequences were used to retrieve homologous sequences from GenBank, primarily selecting those belonging to the Lecithasteridae used by Ivashko et al. (Reference Ivashko, Atopkin, Van Ha, Van Hien and Besprozvannykh2025), and including the outgroup Aponurus laguncula Looss, 1907, following the phylogeny of the superfamily Hemiuroidea (Ivashko et al. Reference Ivashko, Atopkin, Van Ha, Van Hien and Besprozvannykh2025). 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 were determined using the Akaike Information Criterion (AIC; Posada and Buckley Reference Posada and Buckley2004) in MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018). The selected model for the 28S rDNA dataset was the general time-reversible model with gamma-distributed rate variation among sites (GTR + G).
Phylogenetic reconstruction was performed using Bayesian inference (BI) in MrBayes v.3.2.3 (Ronquist et al. Reference Ronquist, Teslenkovan, van der Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard and Huelsenbeck2012). Two parallel Metropolis-coupled Markov chain Monte Carlo (MCMC) analyses were run for 20 million generations, sampling every 1,000 generations. The average standard deviation of split frequencies was maintained below 0.01, as recommended by Ronquist et al. (Reference Ronquist, Teslenkovan, van der Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard and Huelsenbeck2012). A majority-rule consensus tree with branch lengths was constructed after discarding the first 25% of sampled trees as burn-in. Node support was assessed using Bayesian posterior probabilities (PP), with PP > 0.95 considered strong support.
Additionally, uncorrected p-distances and nucleotide differences were calculated in MEGA X. The newly generated sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank database.
Results
Family Lecithasteridae Odhner
Lecithasterinae Odhner, 1905
Alloopistholecithum Vainutis and Voronova, 2025
Alloopistholecithum magaliae n. sp.
Description (based on nine specimens, measurements in Table 1). Body fusiform, smooth (Figure 1). Tegument without spines. Pre-oral lobe present. Oral sucker subterminal. Prepharynx absent. Pharynx spherical. Oesophagus short. Intestinal bifurcation immediately posterior to the pharynx. Caeca short, not extending beyond anterior limit of vitellarium. Ventral sucker large, in anterior third of body, larger than oral sucker. Testes round, smooth, symmetrical, between ventral sucker and ovary. Seminal vesicle large, saccular, dorsal to the ventral sucker, reaching upper margin of testes. Pars prostatic elongated, lined with vesicular cells, and surrounded by numerous prostatic cells. Sinus sac oval, between anterior edge of ventral sucker and intestinal bifurcation, penetrated by hermaphroditic duct. Genital pore median, at level of intestinal bifurcation. Pit connected to thin-walled sac on midline posterior to genital pore. Ovary with four rounded lobes, mid-hindbody, between posterior edge of testes and anterior margin of vitellarium. Seminal receptacle round to elongated, anterodorsal to ovary. Vitellarium with seven drop-shaped lobes, immediately posteroventral to ovary. Uterine loops located between posterior margin of ventral sucker and posterior end of body, filled with eggs. Eggs numerous, small, oval. Excretory vesicle Y-shaped, pore terminal.
Measurements (in micrometres) of Alloopistholecithum magaliae n. sp. and other Alloopistholecithum spp. from South America and Antarctic water. New specimen measurements are in bold.
Alloopistholecithum magaliae n. sp. from the intestine of Odontesthes bonariensis. a. Holotype, entire specimen, ventral view; b. Holotype, light micrograph of whole mature specimen. Scale bar = 100 μm.
Taxonomy summary
Type host: Odontesthes bonariensis (Valenciennes)
Site of infection: Intestine
Type locality: Punta Rasa (36°17´S 56°46´W) Buenos Aires, Argentina
Other locality: Collector Channel of Salado River (35° 50´S, 57°25´W) Buenos Aires, Argentina
Prevalence, mean intensity, and abundance: 85% (22/26), 16.18, and 13.69 (Punta Rasa)
Type material: Holotypes MLP-He 8331, Paratypes MLP-He 8332, Voucher MLP 8333
Etymology: The specific epithet ‘magaliae’ honours Magalí Montes, Martin’s sister, as a way of recognizing the deep emotional influence she has had on his life
Remarks
This study describes Alloopistholecithum magaliae n. sp. as a new species from Argentina. The new species is compared with all congeners reported from South America, including one from the Pacific Ocean (A. pacificum), two from the Antarctic Ocean (A. macrocotyle and A. micropsi), and two from the Atlantic Ocean (A. helodes and A. intermedium). Additionally, the type material of A. intermedium, comprising three specimens (holotype and two paratypes), was reexamined; only the holotype was sufficiently relaxed to allow measurement of diagnostic structures (see Table 1 and Supplementary Material 1).
Alloopistholecithum magaliae n. sp. is distinguished by the following combination of features: a markedly smaller oral sucker relative to the acetabulum; short caeca that do not extend beyond the anterior margin of the vitellarium; smooth, spherical to oval testes; a quadrilobed ovary with lobes slightly smaller than the testes; and a uterus occupying the entire posterior extremity of the body, among other traits.
The new species differs from all South American congeners by its short caeca, which do not extend beyond the vitellarium. It can be distinguished from A. intermedium by the size and shape of the testes, which are larger and have a small lobe or notch in Szidat’s species (A. intermedium; Szidat, Reference Szidat1954). Alloopistholecithum magaliae n. sp. also differs from A. macrocotyle, A. micropsi, and A. pacificum in the relative size of the suckers: in the new species, the acetabulum is approximately three times larger than the oral sucker, whereas in the latter three species, it is less than twice as large. In A. micropsi and A. pacificum, the uterus does not extend beyond the vitellarium, whereas in the new species it reaches the posterior end of the body (Figueroa and Puga Reference Figueroa and Puga1991; Zdzitowiecki Reference Zdzitowiecki1992). Alloopistholecithum macrocotyle and A. micropsi are further characterized by larger body size and larger internal structures, including the eggs (Zdzitowiecki Reference Zdzitowiecki1992). The new species is also distinguished from A. helodes by its larger body size and smaller eggs (Overstreet Reference Overstreet1973).
Other species – Alloopistholecithum gibbosum (Rudolphi 1802), Alloopistholecithum leiostomi (Overstreet 1970), Alloopistholecithum maeoticum (Naidenova 1974), Alloopistholecithum minimum (Martin and Multani 1970), Alloopistholecithum salmonis (Yamaguti 1934), and Alloopistholecithum sandugaense (Vainutis and Voronova, 2025) – have been reported from the Holarctic region. Despite their geographic distribution, these species can be morphologically distinguished from the new species, with the exception of A. maeoticum, which is considered ‘uncertain’ species according to WoRMS (2025c). Eggs are larger in A. gibbosum, A. leiostomi, A. minimum, and A. salmonis compared with those of Alloopistholecithum magaliae n. sp. and A. sandugaense. A. gibbosum shares a similar body size but has a smaller sucker ratio and larger ovary and vitellarium. A. leiostomi exhibits a narrower body, smaller ventral sucker, and reduced seminal vesicle; A. minimum has a smaller body, ventral sucker, and hermaphroditic sac; and A. salmonis possesses a larger body and wider pharynx. Although A. sandugaense has eggs similar in size to those of the new species, it displays a slightly larger body, a broader oral sucker, a large hermaphroditic sac and ovary, and a distinctly larger vitellarium. However, A. sandugaense differs from Alloopistholecithum magaliae n. sp. by its smaller sucker ratio and shorter forebody length (as percentage of total body length).
Molecular analyses
Three 28S rDNA sequences of the new species were obtained, ranging from 1065 to 1118 bp in length. The final alignment comprised 1068 bp and included 37 terminals. Alloopistholecithum magaliae n. sp. is recovered as the sister species of Alloopistholecithum sp. (ON524172), previously reported by Rodríguez-Romeu et al. (Reference Rodríguez-Romeu, Soler-Membrives, Padrós, Dallarés, Carreras-Colom, Carrassón and Constenla2022) from Engraulis encrasicolus (Linnaeus 1758) in Spain (Figure 2). The p-distance (Table 2) between the new species and Alloopistholecithum sp. (ON524172) is 2% (13 bp), 4% (27 bp) with A. micropsi, and 5% (28 bp) from the remaining species of Alloopistholecithum.
Phylogram resulting from Bayesian Inference (20,000,000 generations) of partial 28S rDNA gene sequences of Alloopistholecithum magaliae n. sp. rooted by Aponurus laguncula. Branch support values indicate posterior probabilities ≥0.95.
Uncorrected p-distances (below diagonal, in percentage) and nucleotide differences (above diagonal) in the 28S rDNA gene among Lecithasteridae species, calculated in MEGA X. New sequences generated in this study are shown in bold.
Discussion
The digeneans found in the intestine of Argentinian silversides from brackish waters belong to the genus Alloopistholecithum and represent a new species, A. magaliae n. sp. This species can be distinguished from the five other South American congeners based on both morphological and molecular evidence. The most distinctive features include smooth testes lacking notches, a uterus extending to the posterior extremity of the body, and short caeca that do not reach beyond the anterior margin of the vitellarium.
Montes (Reference Montes2012) reported the presence of Lecithaster confusus Odhner, 1905 in specimens of Jenynsia lineata (Jenyns) from the Río de la Plata estuary and Micropogonias furnieri (Linnaeus) from the Bahía Blanca estuary, both in Buenos Aires Province. However, Ostrowski de Núñez et al. (Reference Ostrowski de Núñez, Arredondo and Gil de Pertierra2017) suggested that this identification was erroneous. Based on the present findings, it is likely that the specimens from J. lineata correspond to A. magaliae n. sp., whereas those from M. furnieri may represent another, as yet undescribed, lecithasterid species. Unfortunately, the poor preservation of the material described by Montes (Reference Montes2012) and the absence of molecular data prevent a definitive identification.
Determining whether members of Lecithasterinae should be assigned to Lecithaster or Alloopistholecithum based solely on morphology remains challenging due to several factors: a scarcity of distinctive morphological features, high intraspecific variation, wide host ranges, and incomplete descriptions based on poorly preserved material (Vainutis and Voronova, Reference Vainutis and Voronova2025a). Comprehensive taxonomic studies integrating morphology and molecular data are therefore essential to clarify the systematics of the genus as additional species are discovered. For South American representatives, sequencing A. intermedium and specimens identified as Lecithaster sp. parasitizing Odontesthes spp., J. lineata, and M. furnieri is particularly important (Carballo et al. Reference Carballo, Cremonte, Navone and Timi2012; Drago Reference Drago2012; Flores et al. Reference Flores, Semenas, Rauque, Vega, Fernandez and Lattuca2016; Montes Reference Montes2012).
The scarcity of genetic data for Alloopistholecithum – only about half of the known species have been sequenced – limits the formulation of hypotheses regarding their regional or global distribution, as illustrated in the present phylogeny (Figure 2). Incorporating additional sequences from the remaining species will facilitate biogeographical and evolutionary interpretations.
The discovery of A. magaliae n. sp. in O. bonariensis is particularly noteworthy. Although parasites of Argentinian silversides have been extensively studied in various regions of Argentina (Arredondo Reference Arredondo2013; Drago Reference Drago2012; Flores et al. Reference Flores, Semenas, Rauque, Vega, Fernandez and Lattuca2016), none of these works detected this digenean. Its presence likely reflects an affinity for brackish environments, as the specimens examined were collected in the Río de la Plata estuary. In contrast, previous surveys of silversides were conducted in freshwater habitats, including Pampas lagoons (Drago Reference Drago2012), Patagonian lakes and reservoirs (Flores et al. Reference Flores, Semenas, Rauque, Vega, Fernandez and Lattuca2016), and the lower Paraná River basin (Arredondo Reference Arredondo2013). This observation is consistent with Gibson (Reference Gibson, Gibson, Jones and Bray2002), who noted that most lecithasterids are predominantly marine. Alloopistholecithum magaliae n. sp. has thus far been recorded only in brackish waters, whereas A. intermedium was found in the Río de la Plata near Buenos Aires, where freshwater predominates (Szidat Reference Szidat1954). The hosts of both species, R. melanostoma and O. bonariensis, are euryhaline fishes capable of migrating between marine and freshwater environments (Almirón et al. Reference Almirón, Casciotta, Ciotek and Giorgis2015; Somoza et al. Reference Somoza, Miranda, Berasain, Colautti, Remes Lenicov and Strüssmann2008). Further work is needed to elucidate the timing and conditions of host infection by these digeneans.
Finally, O. bonariensis is a commercially important species, valued for its high-quality flesh and popularity as a game fish. Due to its ability to thrive in both freshwater and brackish habitats, it has been widely translocated within Argentina and introduced to other countries (Dyer Reference Dyer2006; Somoza et al. Reference Somoza, Miranda, Berasain, Colautti, Remes Lenicov and Strüssmann2008; Tombari and Volpedo, Reference Tombari, Volpedo, Volpedo and Fernández Reyes2008; Torres-Martínez et al. Reference Torres-Martínez, Mancini, Grosman, Somoza and Strüssmann2025). This raises concerns about the potential spread of this parasite in populations inhabiting brackish waters. More comprehensive surveys are required to assess the ecological and economic impact of this digenean on its host.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25101090.
Data availability statement
All the material will be deposited in Museums and the sequences deposited on GenBank.
Acknowledgements
We are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires and to CEPAVE for providing facilities and equipment, to Ministerio de Medio Ambiente de la Provincia de Buenos Aires for providing collection permits (DI-2019-49-GDEBA-DANPOPDS), and to M. Marcia Montes for the drawings.
Author contribution
Martin M. Montes: conceptualization, writing original draft
Nathalia J. Arredondo: writing original draft
Fabricio Gómez: data curation
Paula S. Marcotegui: review and editing
Marina Ibañez Shimabukuro: methodology
Jorge A. Barneche: methodology
Sergio R. Martorelli: supervision, review, and editing
Funding
This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CCT-CONICET-La Plata; N.J.A., grant number 11220150100705CO; M.M.M., grant number 11220200101713CO) and Fondo para la Investigación Científica y Tecnológica (FONCyT) (M.M.M., grants number PICT-2020-SERIEA-01531; N.J.A. grant number PICT-2020-SERIEA-00660).
Competing interests
On behalf of all authors, the corresponding author states that there is no conflict of interest.
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 laboratory animals.
Consent to participate
All the authors give their consent to participate in this work.
Consent for publication
All the authors give their consent to the publication of this work.
Code availability
Not applicable.
Use of artificial intelligence
We used AI tools (ChatGPT) during the preparation of this manuscript exclusively for English language correction. This use has been transparently declared in accordance with the journal’s policy and ethical requirements.
