Introduction
Within the family Haploporidae, Saccocoelioides Szidat, Reference Szidat1954 represents a genus with 26 accepted species (World Register of Marine Species, 2021). In Argentina 10 species were reported according with Ostrowski de Nuñez et al. (Reference Ostrowski de Nuñez, Arredondo and Gil de Pertierra2017). We consider the following eight of those 10 Argentine species as valid: Saccocoelioides nanii Szidat, Reference Szidat1954; Saccocoelioides elongatus Szidat, Reference Szidat1954; Saccocoelioides magniovatus Szidat, Reference Szidat1954; Saccocoelioides magnus Szidat, Reference Szidat1954; Saccocoelioides szidati Travassos, Freitas & Kohn 1969; Saccocoelioides octavus Szidat, Reference Szidat1970; Saccocoelioides antonioni Lunaschi, Reference Lunaschi1984; and Saccocoelioides carolae Lunaschi, Reference Lunaschi1984 (Kohn, Reference Kohn1985; Lunaschi, Reference Lunaschi1996; Kohn et al., Reference Kohn, Fernandes and Cohen2007; Curran et al., Reference Curran, Pulis, Andres and Overstreet2018). We consider two reported species from Argentina to be controversial. Saccocoelioides baciliformis Szidat, Reference Szidat1954 was synonymized with S. octavus (Lunaschi, Reference Lunaschi2002) but recently Gallas & Utz (Reference Gallas and Utz2019) recognize S. baciliformis as a valid species based on their morphological observations from new material recovered from Brazil. In a recent work, Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018) advocated the importance of addressing the condition of existing type-materials, the need for new specimen collections and the need for applying molecular tools for discrimination among Saccocoelioides spp. and other haploporids. Szidat (Reference Szidat1970) speculated that Saccocoelioides spp. might exhibit a high degree of host specificity for their definitive hosts. Considering the high diversity of freshwater fishes in Argentina, which has at least 550 species in 55 families (Mirande & Koerber, Reference Mirande and Koerber2020) the potential for discovery of new species of Saccocoelioides is great. Presently, we are investigating the killifish parasites across Argentina and here present the first result of our work. Cnesterodon decemmaculatus Jenyns is of particular ecological importance in aquatic habitats. It serves as a biological control agent for mosquito larvae (Bonifacio et al., Reference Bonifacio, Usseglio, Hued, Aun and Martori2019); acts as an important consumer of zooplankton and drives changes to the phyloplankton community (Quintans et al., Reference Quintans, Scasso, Loureiro and Yafe2009); and represents an important prey item for top food web-organisms (Ostrowski de Nuñez, Reference Ostrowski de Nuñez1974, Reference Ostrowski de Nuñez1992, Reference Ostrowski de Nuñez1993, Reference Ostrowski de Nuñez1995; Doma & Ostrowski de Núñez, Reference Doma and Ostrowski de Núñez1994; Yafe et al., Reference Yafe, Loureiro, Scasso and Quintans2002). In addition, C. decemmaculatus has been used as a biological indicator of pollution (Bistoni et al., Reference Bistoni, Hued, Videla and Sagretti1999; De la Torre et al., Reference De la Torre, Ferrari and Salibián2005). Therefore, knowledge about the parasite community of C. decemmaculatus increases knowledge about the role played by the fish in the food web of the freshwater community in Lago del Bosque, Argentina.
We collected a haploporid digenean that conformed to the diagnosis for Saccocoelioides by Overstreet & Curran (Reference Overstreet, Curran, Jones, Bray and Gibson2005), among the parasites infecting C. decemmaculatus. It differed from all accepted species and is described as a new species here using morphological and molecular data. In addition, a phylogenetic analysis is conducted using internal transcribed spacer region 2 (ITS2) and 28S rDNA sequences.
Material and methods
Collection of samples and morphological study
Specimens of C. decemmaculatus from Lago del Bosque (La Plata, 34°54′37″S 57°56′16″W) were collected by hand net and transported to the laboratory alive in plastic bags with water from the sample site. Once there, the fish were euthanized by spinal transection, measured in total and standard length, weighed and necropsied. Digeneans were removed from the intestine and heat-killed with hot water and fixed in 10% formalin. Additionally, live specimens were preserved using cold 96% ethanol and stored for later DNA extraction for the molecular study.
Morphological analysis
Whole-mount specimens were made using adult specimens preserved in 10% formalin solution and stained according to standard parasitological techniques using hydrochloric carmine (Pritchard & Kruse, Reference Pritchard and Kruse1982). Digital images of specimens were taken using an Olympus Bx51 microscope equipped with an AmScope MU 1000 10 MP digital camera. Measurements were made using ImageJ software (Schneider et al., Reference Schneider, Rasband and Eliceiri2012). Drawings were made with the aid of a drawing tube. The mean followed by minimum and maximum values in parentheses are given in micrometres (μm). The type-material is 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 two specimens of Saccocoelioides sp. from C. decemmaculatus using Qiagen DNAeasy tissue kit (Qiagen Incorporated, Valencia, California, USA). ITS2 and a fragment of the 5ʹ end of the 28S rDNA gene, including variable domains D1–D3 were targeted for the present study. These regions were obtained from a larger fragment of nuclear rDNA spanning the 3ʹ end of the 18S gene, internal transcribed spacer regions (including ITS1, 5.8S gene, and ITS2), and the 5ʹ end of the 28S rDNA gene. The larger fragment was amplified by polymerase chain reaction (PCR) from both specimens using a PTC-200 Peltier Thermal Cycler (BioRad Incorporated, Hercules, California, USA) using the forward primer LSU5 (5ʹ-TAGGTCGACCCGCTGAAYTTAAGCA-3ʹ) (Littlewood, Reference Littlewood1994) and the reverse primer 1500R (5ʹ-GCTATCCTGAGGGAAACTTCG-3ʹ) (Snyder & Tkach, Reference Snyder and Tkach2001). Additional forward and reverse primers were used in sequencing reactions: DIGL2 (5ʹ-AAGCATATCACTAAGCGG-3ʹ) (Tkach et al., Reference Tkach, Pawlowski and Mariaux2000); 300F (5ʹ-CAAGTACCGTGAGGGAAAGTTG-3ʹ) (Littlewood & Olson, Reference Littlewood, Olson, Littlewood and Bray2001); 900F (5ʹ-CCGTCTTGAAACACGGACCAAG-3ʹ) (Pulis et al., Reference Pulis, Fayton, Curran and Overstreet2013); 300R (5ʹ-CAACTTTCCCTCACGGTACTTG-3ʹ) (Pulis et al., Reference Pulis, Fayton, Curran and Overstreet2013), DIGL2R (5ʹ-CCGCTTAGTGATATGCTT-3ʹ) (Pulis et al., Reference Pulis, Fayton, Curran and Overstreet2013); and ECD2 (5ʹ-CTTGGTCCGTGTTTCAAGACGGG-3ʹ) (Littlewood et al., Reference Littlewood, Rohde and Clough1997). PCR reaction solution volumes used and reaction parameters were conducted following methods in Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003). PCR products were purified using Qiaquick PCR Purification Kit (Qiagen Incorporated) and sequenced using an ABI Prism 3100™ automated capillary sequencer (Applied Biosystems, Foster City, California, USA). We assembled and edited the resulting contiguous sequences of the rDNA fragments using Sequencher™ version 3.1.1 software (GenCodes Corp., Ann Arbor, Michigan, USA). The ITS2 region and the partial 5ʹ end of the 28S rDNA gene were annotated using the ITS2 ribosomal RNA database (Keller et al., Reference Keller, Schleicher, Schultz, Müller, Dandekar and Wolf2009).
Sequence comparison and phylogenetic analysis
The annotated gene regions from both specimens were aligned and compared with comparable sequence fragments from GenBank (table 1) 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 was chosen under the Bayesian information criterion (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 appropriate nucleotide substitution model implemented for the ITS2 rDNA matrix was TVM + I + G and for the 28S rDNA was TIM3 + G.
Collection data and GenBank accession numbers for saccocoelioides spp. analysed in this study. New sequences are in boldface type.
Phylogenetic reconstruction was carried out using Bayesian inference through MrBayes v.3.2.3 (Ronquist et al., Reference Ronquist, Teslenko and van der Mark2012). Phylogenetic trees were constructed 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 1000 generations and the average standard deviation of split frequencies was observed to be less than 0.01, as suggested by Ronquist et al. (Reference Ronquist, Teslenko and van der Mark2012). The robustness of the clades was assessed using Bayesian PP, where PP > 0.90 was considered strongly supported. A majority consensus tree with branch lengths was reconstructed for each run after discarding the first 25% sampled trees.
Additionally, the proportion (p) of absolute nucleotide sites (p-distance) (Nei & Kumar, Reference Nei and Kumar2000) was obtained (including gaps based on pairwise comparison) 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), with the bootstrap method (1000 replicates) and with nucleotide substitution (transition + transversions) uniform rate. Newly generated sequences of the ITS2 and partial 28S rDNA gene from each sampled specimen were submitted to the National Center for Biotechnology Information GenBank database (http://www.ncbi.nlm).
Result
Morphological analysis
Saccocoelioides kirchneri n. sp.
Saccocoelioides kirchneri n. sp. from Cnesterodon decemmaculatus, holotype, ventral view, scale = 100 μm.
Saccocoelioides kirchneri n. sp. picture of holotype whole specimen, ventral view, scale = 100 μm.
ZooBank: urn:lsid:zoobank.org:pub:2FEB8209-09EF-480D-8D94-B2B5F7A343D8
Description
Measurements based on 10 gravid specimens. Body oval, entirely spinous, 1021 (636–1193) long, 423 (321–542) wide (at ventral sucker level) with pigment present in anterior half of body. Oral sucker subterminal 116 (96–138) long, 139 (109–173) wide. Ventral sucker 138 (126–176) long, 139 (128–186) wide. Oral sucker to ventral sucker width ratio 1:0.97–1.29. Oral sucker to ventral sucker length ratio 1:1.10–1.35. Prepharynx 18 (10–32) long. Pharynx 38 (31–64) long, 51 (42–64) wide. Oesophagus 257 (212–302). Ceca sac-shaped, reaching mid-level between ventral sucker and testis. Forebody 286 (268–296) long, representing 27 (24–30)% of body length. Testis oval, located in the anterior part of the last third of the body, longer than wide, 131 (105–208) long, 109 (97–192) wide. Hermaphroditic sac oval, 124 (80–128) long, 93 (80–128) wide. Oval internal seminal vesicle (n = 3) 71 (62–82 long, 37 (35–40) wide. Elongated external seminal vesicle 99 (93–104) long, 51 (45–54) wide. Genital pore near the middle of the forebody. Ovary slightly elongated 82 (58–112) long, 66 (51–98) wide. Laurer's canal and Mehlis’ gland not observed. Uterus between ventral sucker level and the posterior body end. Vitellarium in two separated clusters of follicles extending from posterior level of ventral sucker to anterior field of testis. Eggs three to 40 in number, 110 (101–118) long, 62 (54–70) wide, generally with poorly developed miracidia, only a few specimens showed miracidia with eyespots. Excretory vesicle Y-shaped, bifurcation at middle testis, crura extended to ovarian level.
Taxonomic summary
Type host. C. decemmaculatus (Jenyns)
Site of infection. Intestine.
Type locality. Lago del Bosque (34°54′37″S 57°56′16″W), La Plata, Buenos Aires, Argentina.
Prevalence of infestation. 47 of 104 fish examined (45%)
Mean intensity. three per infected host
Mean abundance. 1.36 individuals per host examined
Type material. Helminthological Collection of Museo de La Plata, Argentina. Under the Holotype number 7760 MLP-He, and paratypes 7761 MLP-He.
GenBank accession number: MZ504671 and MZ504672, contains small subunit ribosomal RNA, internal, transcribed spacer 1, 5.8S ribosomal RNA, ITS2, and contains D1–D3 domains of large subunit ribosomal RNA
Etymology. The species is named in honour of Néstor Carlos Kirchner, Argentine lawyer and politician, President of Argentina between 2003 and 2007, for his recognition of and support for the development of scientific research in Argentina.
Taxonomic remarks
Saccocoelioides kirchneri n. sp. is the 27th accepted species described and the ninth from Argentina. The new species conforms to a group of congeners that Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018) classified as having relatively ‘diminutive bodies’, which are characterized by having body length ≤1.7 mm long, few, large eggs usually longer than the pharyngeal length, and uterus confined largely or entirely to the hind-body. Prior to this study, the group contained four species reported from Argentina: S. carolae, S. magniovatus, S. nanii and S. octavus; and 14 species reported from outside Argentina: Saccocoelioides agonostomus Dyer, Bunkley-Williams & Williams, 1999; Saccocoelioides beauforti (Hunter & Thomas, 1961) Overstreet, 1971; Saccocoelioides chauhani Lamothe-Argumedo, Reference Lamothe-Argumedo1976; Saccocoelioides cichlidorum (Aguirre-Macedo & Scholz, 2005) García-Varela, Andrade-Gómez & Pinacho-Pinacho, 2017; Saccocoelioides lamothei Aguirre-Macedo & Violante-González, Reference Aguirre-Macedo and Violante-González2008; Saccocoelioides olmecae Andrade-Gómez et al., Reference Andrade-Gómez, Pinacho-Pinacho, Hernández-Orts, Sereno-Uribe and García-Varela2016; Saccocoelioides overstreeti Fernández-Bergiela, Reference Fernández-Bergiela1988; Saccocoelioides ruedasueltensis (Thatcher, Reference Thatcher1978) Curran et al., Reference Curran, Pulis, Andres and Overstreet2018; Saccocoelioides sogandaresi Lumsden, 1963; Saccocoelioides tarpazensis Diaz & Gonzalez, Reference Diaz and Gonzalez1990; Saccocoelioides tilapiae (Nasir & Gómez, Reference Nasir and Gómez1976); Saccocoelioides tkachi Curran et al., Reference Curran, Pulis, Andres and Overstreet2018; Saccocoelioides orosiensis Curran et al., Reference Curran, Pulis, Andres and Overstreet2018; and Saccocoelioides macrospinosum Andrade-Gómez et al., Reference Andrade-Gómez, Sereno-Uribe and Garcia-Varela2019. Key comparative morphological measurements are provided for all 19 species having the ‘diminutive body’ form in table 2.
Compared measurements from adult specimens of Saccocoelioides spp. in the diminutive morphotype according to Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018).
In having ceca that extend posteriorly to the mid-level between the ventral sucker and testis, S. kirchneri n. sp. may be easily distinguished from nine of the diminutive congeners that have ceca extending to the level of the testis. These nine species are S. agonostomus, S. beauforti, S. cichlidorum, S. octavus, S. olmecea, S. orosiensis, S. sogandaresi, S. tilapiae and S. tkachi (Curran et al., Reference Curran, Pulis, Andres and Overstreet2018; Andrade-Gómez et al., Reference Andrade-Gómez, Sereno-Uribe and Garcia-Varela2019). Saccocoelioides lamothei has ceca that may or may not extend posteriorly to the testicular level depending on fixation, but S. lamothei has a larger pharynx than S. kirchneri n. sp. (Aguirre-Macedo & Violante-González, Reference Aguirre-Macedo and Violante-González2008; Andrade-Gómez et al., Reference Andrade-Gómez, Pinacho-Pinacho, Hernández-Orts, Sereno-Uribe and García-Varela2016).
Saccocoelioides kirchneri n. sp. may be distinguished from six of its remaining eight diminutive congeners by having the uterus extending to the posterior body extremity, filling the post-testicular space. In contrast, the six species having the uterus limited to the pre-testicular space are S. carolae, S. chauhani, S. microspinosus, S. nanii, S. overstreeti and S. ruedasueltensis (Szidat, Reference Szidat1954; Lamothe-Argumedo, Reference Lamothe-Argumedo1976; Thatcher, Reference Thatcher1978; Lunaschi, Reference Lunaschi1984; Fernández-Bergiela, Reference Fernández-Bergiela1988; Andrade-Gómez et al., Reference Andrade-Gómez, Sereno-Uribe and Garcia-Varela2019). Only S. kirchneri n. sp., S. magniovatus and S. tarpazensis have a uterus extending into the post-testicular space (Szidat, Reference Szidat1954; Diaz & Gonzalez, Reference Diaz and Gonzalez1990; Curran et al., Reference Curran, Pulis, Andres and Overstreet2018).
Saccocoelioides kirchneri n. sp. differs from S. magniovatus by having a much smaller pharynx (Szidat, Reference Szidat1954). S. kirchneri n. sp. differs from S. tarpazensis by having a much larger body size, larger ventral sucker and smaller eggs (Diaz & Gonzalez, Reference Diaz and Gonzalez1990).
Molecular analyses
Genetic variation at the ITS2 and 28S rDNA of S. kirchneri n. sp. with 13 congeners are shown in tables 3 and 4. The range of variation were 0.00–0.09 of p-distance and 0–23 the number of nucleotides of the ITS2 rDNA. The genetic distance of the ITS2 rDNA (table 3) shows a close similarity among S. kirchneri n. sp., S. beauforti, S. chauhani, S. cihlidorum, S. macrospinosus, S. nanii, S. olmecae, S. sogandaresi, S. tkachi, and (P-value = 0.03) with the smallest genetic distance exhibited among S. kirchneri n. sp., S. chauhani, S. macrospinosus and S. tkachi (nucleotide base difference = 7). Genetic distance shows similarity at the partial 28S rDNA gene among all the species having a diminutive body (table 4). The range of genetic distance were 0.00–0.05 of p-distance and 0–40 the number of nucleotide of 28S rDNA. Genetic distance is highest among S. beauforti, S. macrospinosus, S. olmecae and S. sogandaresi. (P-value = 0.01 and nucleotide base difference = 4). The ITS2 tree (fig. 3) and 28S rDNA tree (fig. 4) were constructed with an alignment measuring 380 base pairs (bp) from 51 taxa (ITS2) and 1334 bp from 57 taxa (28S rDNA). Well-supported clades separating both the ‘diminutive’ and ‘large’ morphotypes of Saccocoelioides spp. were recovered in both trees (figs 3 and 4) as in the analysis of Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018). S. kirchneri n. sp. branches within the diminutive group of taxa in both trees. In fig. 3 S. kirchneri n. sp. is clustered with S. tkachi, S. cichlidorum and S. lamothei, but with low posterior probabilities. In the diminutive body branch of fig. 4 several nodes are well supported, but the one containing S. lamothei and S. kirchneri n. sp. is not one of them.
Phylogram resulting from Bayesian inference of internal transcribed spacer region 2 rDNA gene sequences of Saccocoelioides kirchneri n. sp. and Saccocoelioides spp. rooted with Intromugil alachuaensis. Black bar marking the diminutive morphotype species.
Phylogram resulting from Bayesian inference of partial 28S rDNA gene sequences of Saccocoelioides kirchneri n. sp. and Saccocoelioides spp. rooted with Intromugil alachuaensis. Black bar marking the diminutive morphotype species.
Genetic divergence among species of Saccocoelioides spp., estimated through of uncorrected proportion (p) of absolute nucleotide sites (p-distance) of the internal transcribed spacer region 2 rDNA under, and the number of the nucleotide difference above, the black diagonal. Variable sites including gaps based on pairwise comparison of 380 sites. The IG column have the values of the p-distance inside the group. Abbreviation: n/c, not calculated.
Genetic divergence among species of Saccocoelioides spp., estimated by uncorrected proportion (p) of absolute nucleotide sites (p-distance) of the 28S rDNA under, and the number of the nucleotide difference above, the black diagonal. Variable sites including gaps based on pairwise comparison of 1334 sites. The IG column have the values of the p-distance inside the group. Abbreviation: n/c, not calculated.
Discussion
The new species infects C. decemmaculatus, possesses the typical characteristics of Saccocoelioides, and falls within the diminutive morphotype of Curran et al. (Reference Curran, Pulis, Andres and Overstreet2018).
The elucidation of the species within Saccocoelioides is very difficult using only morphological features, and it is necessary to complement this with molecular studies. Despite that, Curran et al. et al. (Reference Curran, Pulis, Andres and Overstreet2018) made a division between the species of this genus in two main groups based mainly on overall body size and egg size in relation to the pharynx. The novelty of S. kirchneri n. sp. also is supported by the molecular analysis. According to the 28S rDNA tree (fig. 4), the species clustered with S. lamothei, S. cichlidorum and S. tkachi, but the morphology, geographical distribution and the host infected for each of those species could separate them from the new species. The new species was collected from C. decenmaculatus a Poeciliidae from the Rio de la Plata basin in contrast with the nearest species S. carolae (in Cichlasoma facetum (Jenyns) a Cichlidae from Rio de la Plata basin, Argentina, South America), S. cichlidorum (in Vieja maculicauda (Regan) a Cichlidae from Torsuani River, Nicaragua, Middle America), S. lamothei (in Dormitator latifrons (Richardson) an Elotridae from Tres Palos and Coyuca, Mexico, Middle America) and S. tkachi (parasite in Astyanax aeneus (Günther) a Characidae from Animas and Tempisque (and tributaries) Rivers, Costa Rica, Middle America).
The descriptions of the new species show an interesting question about the specificity of this genus as was stated by Szidat (Reference Szidat1970), showing a high coevolution with the host.
In addition, new studies using DNA are necessary to elucidate the real affiliation of several species. We are sure that new species of Saccocoelioides are waiting to be described in Argentina.
Acknowledgements
We thank the Actividades Pesqueras, Acuicultura y Control Pesquero of Buenos Aires Province for the sample authorization, Centro de Estudios Parasitológicos y Vectores for providing the place and equipment where these studies were made and thank M. Marcia Montes for the drawings. This research was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0015- CONICET-CCT La Plata to SRM).
Financial support
This research was financially supported by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0015- CONICET-CCT La Plata to SRM).
Conflicts of interest
None.
Ethical standards
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
