Introduction
Parasites have long been perceived as having a minor role in ecosystem functioning because of their relatively low biomass compared with other trophic groups (Hudson et al., Reference Hudson, Dobson and Lafferty2006). However, recent evidence has revealed they play a crucial role and have substantial impacts on the ecosystems. Parasites can greatly influence the dynamics of host populations, disrupt interspecific competition, and shape energy flow, among other critical aspects. Thus, parasites emerge as significant drivers of biological diversity by exerting profound effects on ecosystem functions and the structure of food webs (Hudson, Reference Hudson, Thomas, Renaud and Guégan2002; Wood & Johnson, Reference Wood and Johnson2015).
The Chaco-Pampa Plain is a low land at the east of the south part of the Andes formed by sedimentary loessic soils. The area includes Central, North, and Northeast Argentina; Southeast Bolivia; West, Northwest, and Southeast Paraguay; all of Uruguay; and South Brazil. The Pampa, Espinal, Dry Chaco, and Humid Chaco ecoregions are the dominant biomes (Morello et al., Reference Morello, Matteucci, Rodríguez and Silva2012; Bencke et al., Reference Bencke, Chomenko, Sant’Anna, Chomenko and Bencke2016; Mereles et al., Reference Mereles, Céspedes, Egea-Elsam and Spichger2020). Six species of freshwater turtles dominate the basins and related flood-plains in the area: the Chaco Side-necked turtle Acanthochelys pallidipectoris (Freiberg, Reference Freiberg1945); the Black Spine-neck Swamp turtle Acanthochelys spixii (Duméril & Bibron, 1835); the Snake-necked turtle Hydromedusa tectifera Cope, 1870; the Hilaire’s Side-necked turtle Phrynops hilarii (Duméril & Bibron, 1835) (Chelidae); the Scorpion Mud turtle Kinosternon scorpioides (L.) (Kinosternidae); and the Painted turtle Trachemys dorbigni (Duméril & Bibron, 1835) (Emydidae). These species usually arranged in assemblages formed by a single species (usually H. tectifera as in the Cordoba province or south Buenos Aires province: Cabrera et al., Reference Cabrera, Haro and Monguillot1986; Di Pietro et al., Reference Di Pietro, Alcalde, Williams and Cabrera2012) or more commonly by two or three species (e.g., A. pallidipectoris-K. scorpioides in the Argentina Dry Chaco eco-region: Cassano & Alcalde, Reference Cassano and Alcalde2022, H. tectifera-P. hilarii-T. dorbigni, or H. tectifera-A. spixii in Argentina, Uruguay and Brazil Pampa ecoregion: Bujes, Reference Bujes2008; Alcalde et al., Reference Alcalde, Derocco, Rosset and Williams2012). Some of these species are also found in neighbouring ecoregions limiting with the Chaco-Pampa Plain (e.g., Yungas ecoregion in Argentina and Bolivia: K. scorpioides in the Atlantic Rainforest ecoregion in Argentina and Brazil and Cerrado ecoregion in Brazil: H. tectifera: Alcalde et al., Reference Alcalde, Sánchez and Pritchard2021). The Chaco-Pampa plain may also house two turtle species that enter marginally from adjacent Atlantic Rainforest eco-region in Argentina, Brazil and Uruguay: Phrynops williamsi Rhodin & Mittermeier, 1983, and the southernmost lineage (see de Carvalho et al., Reference Carvalho, Vogt, Rojas, Silva Nunes, Fraga, Ávila, Rhodin, Mittermeier, Hrbek and Farias2022) of Phrynops geoffroanus (Schweigger, 1812).
Parasites of the South American freshwater turtles have been understudied, with most records (more than 60) coming from Brazil (see Mascarenhas & Müller, Reference Mascarenhas, Chaviel, Bernardon, Wolter, Coimbra and Müller2022). All turtle species from the Chaco-Pampa plain of Argentina have been studied for internal parasites, with deeper analysis conducted on H. tectifera and P. hilarii: seven species of digeneans and seven nematodes were recorded (Lombardero & Moriena, Reference Lombardero and Moriena1977; Palumbo et al., Reference Palumbo, Capasso, Cassano, Alcalde and Diaz2016, Reference Palumbo, Werneck and Diaz2019, Reference Palumbo, Servián, Sánchez and Diaz2020, Reference Palumbo, Cassano, Alcalde and Diaz2021; Palumbo & Diaz, Reference Palumbo and Diaz2018; Palumbo et al., Reference Palumbo, Servián, Cassano and Diaz2024). Additionally, few ecological studies on some of these turtle species contemplate the analysis of host diet and their parasites (Pereira et al., 2018; Mascarenhas et al., Reference Mascarenhas and Müller2021; Palumbo et al., Reference Palumbo, Cassano, Alcalde and Diaz2021).
The purpose of the present contribution is to increase the knowledge of the internal parasitic assemblages of six species of freshwater turtles from the Argentine Chaco-Pampa plain by providing new parasite-host associations, and additional geographic records for many parasite-host relationships. Some molecular sequences of the studied parasites were supplied as a tool for a better species identification.
Materials and methods
Sampling
Samplings were carried out in different ecoregions of the Argentine Chaco-Pampa plain between 2008 and 2023 (Table 1). Parasite samples proceeded from two sources: (1) necropsied turtles (n=54), most of which were road-kill and a few were from herpetological collections or were field collected individuals that have presumably died from ulcerative shell disease, and (2) field-caught turtles that were stomach flushed (n=433) (Table 1, Fig. 1).
Table 1. Data from the sampled turtles, separated into eviscerated individuals and samples of the stomach contents
Hosts provided by aCentro de Investigaciones Científicas y Transferencia de Tecnología a la Producción (Yanina Prieto); bAlfredo Holley from anthropized environment
Figure 1. Records of helminths parasites studied in South American turtles.
Turtles found dead in the field were refrigerated and transferred to the laboratory for necropsy: the lateral processes, which join the plastron to the carapace, were cut with a grinder and a scalpel was used for the surrounding integument; after the plastron was removed, the musculature was stripped and the cavities were examined. All organs were removed and examined for internal parasites under a stereoscopic microscope (Olympus SZ61, Tokyo, Japan). All collected parasites were preserved in 70% ethanol for their identification (see the following section).
Trapped turtles for live study (using hookless trot line, netsm or by hand following Semeñiuk et al., Reference Semeñiuk, Alcalde, Sánchez and Cassano2017), were sometimes processed in the field (if feces were not sampled), or carried to the laboratory to collect stomach flushing (according to Legler, Reference Legler1977 with subtle modifications relative to water pumping: see Bonino et al., Reference Bonino, Lescano, Haro and Leynaud2009 and Alcalde et al., Reference Alcalde, Derocco and Rosset2010). Stomach samples were observed under a stereoscopic microscope and all parasites recovered were kept in individual vials with 70% ethanol for further identification (see the following section).
All studied turtles were straight-line carapace length measured (accuracy, 1 mm) and sexed according dimorphic characters and, if necessary, by penis eversion in alive individuals (following Rodrigues et al., Reference Rodrigues, Soares and Silva2014) or trough dissection in necropsied turtles. Alive turtles were also weighted (accuracy, 1 g) and marked (following Cagle, Reference Cagle1939) before release at the site of capture.
Procedures used in this study comply with the current laws for working in Argentina. Permission to work in the study area and for turtle handling was granted by the Dirección de Recursos Naturales of Buenos Aires (No. 102/2014-025 and 69/2016), Corrientes (No. 845), Chaco (No. 375), Entre Rios (No. 001/17), Salta (No. 662), and Santa Fe provinces (No. 198), Argentina.
Morphological identification of parasites
Nematodes were cleared in Amman’s Lactophenol and temporarily mounted, flatworms were stained with hydrochloric carmine, dehydrated in a graded ethanol series, cleared in eugenol, and mounted in natural Canada balsam. Morphological studies were made under a compound microscope (Olympus BX51, Tokyo, Japan).
Adult parasites were determined using keys and specific bibliography (Hedrick, Reference Hedrick1935; Cordero, Reference Cordero1946; Lombardero & Moriena, Reference Lombardero and Moriena1977; Baker, Reference Baker1986; Font & Lotz, Reference Font, Lotz, Bray, Gibson and Jones2009; Mascarenhas & Müller, Reference Mascarenhas and Müller2017; De Sousa et al., Reference De Sousa, Jorge, Carretero, Harris, Roca and Perera2018; Palumbo & Diaz, Reference Palumbo and Diaz2018; Palumbo et al., Reference Palumbo, Servián, Sánchez and Diaz2020, Reference Palumbo, Servián, Cassano and Diaz2024). The prevalence, mean intensity, and abundance of parasites were calculated following Bush et al. (Reference Bush, Lafferty, Font and Shostak1997).
Molecular and polymerase chain reaction methods
A total of nine parasite specimens were isolated in Eppendorf tubes and frozen at –20 °C for subsequent DNA extraction and genotyping (Table 2). DNA was extracted from the isolates using 200 μL of 5% Chelex solution (Bio-Rad Laboratories, CA, USA) containing 0.2 mg/mL Proteinase K (Roche), incubated at 56 °C overnight, followed by 10 min at 95 °C. Nuclear 18S, 28S, and ITS2 rDNA amplification were done using the appropriate primers. Each 50 μL polymerase chain reaction (PCR) contained 25 μL of GoTaq Green Master Mix (Promega, Madison, Wi, USA), 2.5 μL of each primer, 17 μL of water, and 3 μL of extracted DNA.
Table 2. Details of the primers used and method of analysis for each parasite species
PCR amplification for 18S was performed using an Eppendorf Mastercycler ep gradient S, consisting of 94 °C for 15 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 70 s, with a final extension of 72 °C for 240 s, for 28S amplification the program consisting of 94 °C 4 min, followed by 30 cycles of 95 °C for 60 s, 60 °C for 60 s, and 72 °C for 120 s, with a final extension of 72 °C for 4 min, and for ITS2 amplification was one cycle of 95 °C 3 min, 42 °C 2 min, and 72 °C 1 min, followed by six cycles of 95 °C 45 s, 47 °C 45 s, and 72°C 1 min, followed with 35 cycles of 95 °C 20 s, 50 °C 20 s, and 72 °C 1 min, and a final extension of 72 °C for 5 min.
PCR products were further purified and sequenced (Macrogen, South Korea). Sequences were edited and aligned using Chromas v2.6.6 and Gap v4.11.2 (Bonfield et al., Reference Bonfield, Smith and Staden1995) and then compared with the NCBI database using BLAST version 2.2.26 (Altschul et al., Reference Altschul, Madden, Schäffer, Zhang, Zhang, Miller and Lipman1997) to identify sequences with high similarity to DNA sequences obtained.
Phylogenetic analyses
The newly obtained 18S, 28S, and ITS2 sequences were aligned with GenBank available sequences using the CLUSTAL W program (Larkin et al., Reference Larkin, Blackshields and Brown2007). The phylogenetic tree was reconstructed using the Maximum Likelihood method implemented in the Mega 7.0.26 program (Tables 2 and 3). The genetic differences in datasets were also calculated by using uncorrected p distances.
Table 3. List of sequences extracted from GenBank used in the phylogenetic analysis
Results
Parasite diversity
A total of 6230 helminths belonging to 18 taxa (one cestode, 11 digeneans, and six nematodes) were recovered (Table 4). Taxonomical position, host, site of infection, and localities are presented in the following section; additionally, a deep morphological diagnosis and analysis were included in those cases that the finding presents systematic contradictions or drawbacks, if not, only a brief description was provided.
Table 4. List of parasites recorded in this study with details of hosts, localities, number of host (N), prevalence (P), mean intensity (MI), and abundance and range (A)
a Parameters calculated from regurgitated.
Phylum Platyhelminthes
Class Cestoda
Family Proteocephalidae La Rue, 1911
Ophiotaenia La Rue, 1911
Ophiotaenia cohospes Cordero, Reference Cordero1946
Diagnosis: Scolex with four equal, smooth, and elliptical suckers. Acraspedot proglottids, the former is wider than longer, but they become longer changing their ratio when they are mature and gravid. Follicular testes distributed in two lateral fields, ovary bilobed, posterior in the proglottid. Lateral follicular vitellogenic glands. Medullary uterus. Lateral genital pore in median position.
Site of infection: Intestine.
Host and localities: Hydromedusa tectifera - Magdalena (35°01′36″S, 57°17′24″W) in Buenos Aires province (Pampa ecoregion) and Tanti (31°22′05″S, 64°34′34″W) and Flor Serrana (31°23′36″S, 64°35′42″W), in Córdoba province (Dry Chaco ecoregion).
Voucher specimens: MLP-He 8083
Remarks: This species was described parasitizing H. tectifera from Uruguay (Cordero, Reference Cordero1946) and subsequently found in anurans from Brazil, Ecuador, and Paraguay (McAllister et al., Reference McAllister, Bursey and Freed2010; Campião et al., Reference Campião, Ribas and Tavares2015). Until now, O. cohospes has not been recorded again in turtles since its description 70 years ago. It is also the first record for this parasite for Argentina.
Class Trematoda
Subclass Digenea
Family Cryptogonimidae Ward, 1917
Caimanicola Freitas & Lent, Reference Freitas and Lent1938
Caimanicola brauni (Mañé-Garzón & Gil, 1961)
Diagnosis: The presence of a crown of 24 spines surrounding the oral sucker, the globose pharynx, the distribution of the reproductive organs (ovary and testis in tandem at the posterior end), and the caecum that end up opening to the exterior at the posterior part of the body, are the main characteristics that identify the specimens as C. brauni (Table 4).
Site of infection: Intestine.
Host and localities: Phrynops hilarii - Magdalena (35°01′36″S, 57°17′24″W) in Buenos Aires province (Pampa ecoregion), and Rafaela (31°15′04″S, 61°30′20″W) in Santa Fe province (Espinal ecoregion).
GenBank acc. number: OR135713 (ITS2), OR137955 (28S)
Voucher specimens: MLP-He 8084
Remarks: This species was described for P. hilarii from Uruguay (Mañé-Garzón & Gil, Reference Mañé-Garzón and Gil1961a) and subsequently recorded for A. spixii and P. hilarii from Brazil (Chaviel et al., Reference Chaviel, Mascarenhas, Bernardon, Coimbra and Müller2020), and experimentally in P. hilarii from Buenos Aires, Argentina (Ostrowski de Núñez, Reference Ostrowski de Núñez1987). The present record represents the first report for a wild turtle from Argentina. Unfortunately, no sequences are available on GenBank for this genus, so comparisons were precluded. This is the first molecular contribution for Caimanicola.
Family Proterodiplostomidae Dubois, Reference Dubois1936
Cheloniodiplostomum Sudarikov, Reference Sudarikov1960
Cheloniodiplostomum argentinense Palumbo & Diaz, Reference Palumbo and Diaz2018
Diagnosis: Elongated body, with a clear difference between forebody and hindbody, elliptical holdfast with proteolytic glands, the vitellaria begins anterior to the bifurcation of the cecum and ends at the posterior border of the ovary. The posterior end of the body is bifurcated: one portion blunt and the other with a funnel-shaped dorsal projection into which the copulatory pouch opens (Table 4).
Site of infection: Intestine.
Host and localities: Phrynops hilarii - La Plata (34°53′26′′S, 57°59′40′′W) in Buenos Aires province (Pampa ecoregion), Aldea Brasilera (31°53′17″S, 60°34′44″W) and Diamante (32°02′52″S, 60°38′10″W) in Entre Ríos province (Espinal ecoregion), and Helvecia (31°06′30″S, 60°05′48″W) in Santa Fe province (Espinal ecoregion).
Historical records in Argentina: Phrynops hilarii - Magdalena (35°01′36″S, 57°17′24″W) in Buenos Aires province (Pampa ecoregion) (Palumbo & Diaz, Reference Palumbo and Diaz2018).
GenBank acc. number: OR135714 (28S)
Cheloniodiplostomum testudinis (Dubois, Reference Dubois1936)
Diagnosis: Short body divided into two parts by a constriction located at the level of the ovary and anterior testis, flattened forebody, and cylindrical hindbody; holdfast rounded; vitellaria extending from near caeca bifurcation to the ovary (Table 4).
Site of infection: Intestine.
Host and localities: Phrynops hilarii - Cañada de Gomez (32°49′45″S, 61°22′57″W) (Pampa eco-region) and Santa Fe City (31°38′21″S, 60°40′54″W) in Santa Fe province (Espinal eco-region).
Historical records in Argentina: Phrynops hilarii and Hydromedusa tectifera - Magdalena (35°01′36″S, 57°17′24″W) Buenos Aires province (Pampa eco-region) (Palumbo & Diaz, Reference Palumbo and Diaz2018). Phrynops hilarii - unknow locality in Corrientes province (Lombardero & Moriena, Reference Lombardero and Moriena1977).
Cheloniodiplostomum sp.
Diagnosis: Elongated body divided into two parts by a constriction located at the level of the ovary, flattened forebody and cylindrical hindbody; oral sucker rounded, small, subterminal, pre pharynx absent, pharynx rounded and short, esophagus short, caeca bifurcating above acetabulum and reaching the posterior edge of the posterior testis; acetabulum rounded, similar in size to oral sucker; large and rounded holdfast located in forebody, with proteolytic glands in its posterior edge; testes in tandem, without cirrus and cirrus sac; ovary rounded pretesticular, ootype intertesticular; vitellaria extending from near pharynx to anterior testis; uterus completely in hindbody, few elliptical eggs observed; posterior end of the body with muscle pouch shape (Table 4, Fig. 2).
Figure 2. Stained whole-mount micrographs of Cheloniodiplostomum sp. Scale bar 400 μm.
Site of infection: Intestine.
Hosts and localities: Hydromedusa tectifera and Phrynops hilarii - La Plata (34°53′26′′S, 57°59′40′′W) in Buenos Aires province (Pampa eco-region).
GenBank acc. number: OR135719 (28S), OR135721 (ITS2).
Voucher specimens: MLP-He 8085
Remarks: Recently Achatz et al., (Reference Achatz, Brito, Fecchio and Tkach2021) suggested that the genus Cheloniodiplostomum Sudarikov, Reference Sudarikov1960 should be considered as a junior synonym of Herpetodiplostomum Dubois, Reference Dubois1936, basing their proposal on the fact that the host specificity (turtles vs. alligators), the limits of the vitellarium (anterior to the acetabulum vs. posterior to the acetabulum), and the presence/absence of papillae on the holdfast would not be sufficient characteristics to differentiate both genera. Although these authors demonstrate that there are species of both genera that present papillae on the holdfast, they failed in found any Cheloniodiplostomum species parasitizing alligators. Molecularly, they have sequenced a single species precluding any comparison between Cheloniodiplostomum and Herpetodiplostomum. We follow the classification first proposed by Sudarikov (Reference Sudarikov1960) then followed by subsequent authors (Dubois, Reference Dubois1968, Reference Dubois1982; Niewiadomska, Reference Niewiadomska, Gibson, Jones and Bray2002; Mascarenhas & Müller, Reference Mascarenhas and Müller2021).
Four species of Cheloniodiplostomum are recognized parasitizing neotropical turtles: Ch. brevis (Dubois, Reference Dubois1979) parasitizing P. geoffroanus (Colombia) and K. scorpioides (United States) (MacCallum, Reference MacCallum1921; Dubois, Reference Dubois1979); Ch. delillei (Zerecero, Reference Zerecero1947) recorded for Chelydra serpentina (L.) and Claudius angustatus Cope, Reference Cope1875, both from Mexico (Thatcher, Reference Thatcher1963, Reference Thatcher1964); Ch. testudinis described for a turtle (genus and species unknown), P. geoffroanus (Brazil), and P. hilarii and H. tectifera (Argentina) (Dubois, Reference Dubois1936; Lombardero & Moriena, Reference Lombardero and Moriena1977; Fernandes & Kohn, Reference Fernandes and Kohn2014; Silva, Reference Silva2014; Mascarenhas et al., Reference Mascarenhas, Bernardon and Müller2016; Palumbo & Diaz, Reference Palumbo and Diaz2018); and Ch. argentinense parasitizing P. hilarii from Argentina (Palumbo & Diaz, Reference Palumbo and Diaz2018).
Phylogenetic analysis of partial 28S rRNA gene sequences shows that Cheloniodiplostomum and Herpetodiplostomum species form a strongly supported clade (Fig. 3A). This group composed only by species that parasitize turtles, while the other genera form a clade which species parasitize crocodiles, lizards and amphibians but not turtles.
Figure 3. Phylogenetic tree based on Cheloniodiplostomum rDNA sequences newly obtained in this study compare with Diplostomidae sequences available in GenBank using the Maximum Likelihood method with a distance matrix calculation with K2P. The numbers at the nodes represent the percentages of 1000 bootstrap replicates (A). Phylogenetic tree based on Thelandros rDNA sequence newly obtained in this study compare with Pharyngodonidae sequences available in GenBank using the Maximum Likelihood method with a distance matrix calculation with T92. The numbers at the nodes represent the percentages of 1000 bootstrap replicates (B). Sequences are identified by GenBank accession numbers, taxa names, and hosts.
Herpetodiplostomum Dubois, Reference Dubois1936
Herpetodiplostomum duboisi Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1969
Diagnosis: Elongated body, with a clear difference between forebody and hindbody; rounded holdfast with proteolytic glands; the vitellaria start posteriorly to the bifurcation of the caecum and ends at posterior border of the anterior testis. The posterior end of the body is bifurcated: one portion blunt and the other with a funnel-shaped dorsal projection into which the copulatory pouch opens (Table 4).
Site of infection: Intestine.
Host and localities: Phrynops williamsi - Comandante Andresito (25°40′22′′S, 54°02′22′′W) in Misiones province (Atlantic Rainforest eco-region).
Voucher specimens: MLP-He 8086
Remarks: There are six species recorded within the genus Herpetodiplostomum: H. caimanicola (Dollfus, Reference Dollfus1935), H. duboisi, H. gavialis (Narain, Reference Narain1930), H. ovalis Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1969, H. vogti Achatz et al., Reference Achatz, Brito, Fecchio and Tkach2021, and H. wolffhugeli Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1969. Two of them are parasites of crocodiles, H. caimanicola (Brazil) and H. gavialis (India) and share the following characteristics: acetabulum twice as big as the oral sucker, vitellaria extend close to acetabulum than to the ovary; with proteolytic glands on the holdfast, but without proteolytic glands below it. On the other hand, the rest of the species have the acetabulum similar in size than the oral sucker; the vitellarium extends from anterior to the acetabulum to anterior testis; with proteolytic glands below the holdfast and, all have turtles as definitive host (Narain, Reference Narain1930; Dubois, Reference Dubois1936; Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1969; Achatz et al., Reference Achatz, Brito, Fecchio and Tkach2021), so they should probably be transferred to Cheloniodiplostomum depending on future phylogenetic analyses with sequences confirming what is presumed from morphology and hosts specificity.
The species H. duboisi has not been recorded since its original description parasitizing P. hilarii in Artigas, Uruguay (Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1969). Moreover, this is the first record of internal parasites for P. williamsi.
Family Cladorchiidae Fischoeder, 1901
Nematophila Travassos, 1934
Nematophila grandis (Diesing, 1839)
Diagnosis: Large, leaf-shaped body with a cup-shaped terminal oral sucker and a large muscular acetabulum at the posterior end of the body. Testis intracecal, anterior to the ovary. Vitellaria extracecal (Table 4).
Site of infection: Intestine.
Host and localities: Phrynops hilarii: Ciudad Autónoma de Buenos Aires (34°36′28′′S, 58°21′28′′W), Buenos Aires province (Pampa ecoregion).
Historical records in Argentina: Phrynops hilarii - unknow locality in Corrientes province (Lombardero & Moriena, Reference Lombardero and Moriena1977).
Remarks: This widely distributed species was previously recorded for several turtle species from Argentina, Brazil, Ecuador, French Guyana, Paraguay, Peru, and Venezuela (Mascarenhas & Müller, Reference Mascarenhas and Müller2021). This finding represents the second record of the species in Argentina and the first for Buenos Aires.
Family Echinostomatidae Looss, 1899
Prionosomoides Freitas & Dobbin, Reference Freitas and Dobbin1967
Prionosomoides phrynopsis (Mañé-Garzón & Gil, 1961)
Diagnosis: Peristomal disc with 47 teeth arranged in a single row, oral sucker smaller than the ventral sucker, ovary pretesticular, testis in tandem near the middle of the body (Table 4).
Site of infection: Intestine.
Host and localities: Phrynops hilarii - Magdalena (35°01′36″S, 57°17′24″W), Buenos Aires province (Pampa ecoregion), Rafaela (31°15′04″S, 61°30′20″W) and Santa Fe Capital (31°38′21″S, 60°40′54″W) in Santa Fe province (both at Espinal ecoregion), Gualeguaychú (Pampa ecoregion, 32°57′30″S, 58°34′59″W) and Diamante (Espinal ecoregion, 32°02′52″S, 60°38′10″W) both in Entre Ríos province; San Miguel de Tucumán (26°49′11″S, 65°12′52″W), in Tucumán province (ecoregion not assigned because the studied specimen came from unknown origin and proceed from an anthropized artificial pond).
GenBank acc. number: OR135717 (28S)
Voucher specimens: MLP-He 8087
Remarks: Prionosomoides includes three species that parasitize freshwater turtles: Pr. scalaris Freitas & Dobbin, Reference Freitas and Dobbin1967 parasitizes P. geoffroanus from Brazil (Freitas & Dobbin Jr., Reference Freitas and Dobbin1967) and P. hilarii from Argentina (Lombardero & Moriena, Reference Lombardero and Moriena1977); Pr. taiwanensis Fischathal & Kuntz, Reference Fischathal and Kuntz1975 parasitizes Mauremys reevesii (Gray, Reference Gray, Griffith and Pidgeon1831) from Taiwan (Fischthal & Kuntz, Reference Fischathal and Kuntz1975); and Pr. phrynopsis was reported for P. hilarii from Uruguay (Mañé-Garzón & Gil, Reference Mañé-Garzón and Gil1961a). This finding is a new country record for Pr. phrynopsis from Argentina.
Family Telorchiidae Loos, 1899
Telorchis Luhe, 1899
Telorchis dubius Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1968
Diagnosis: Lanceolate body shape, small oral sucker similar in size to the acetabulum, pre-pharynx absent, small pharynx, juxtaovarian large cirrus pouch, oval-shaped ovary, small testis arranged in tandem and vitellaria extending beyond the ovary (Table 4, Fig. 4A).
Figure 4. Line drawings of Telorchis species. T. dubius (A), T. devincenzii (B), T. birabeni (C), and T. diaphanus (D). Scale bars 200 μm.
Site of infection: Intestine.
Host and localities: Trachemys dorbigni - Concordia (31°21′22′′S, 58°05′42′′W) and Federal (30°40′54′′S, 58°47′20′′W) in Entre Ríos province (Espinal eco-region).
GenBank acc. number: OR135716 (ITS2)
Voucher specimens: MLP-He 8088
Remarks: This species was recorded only for T. dorbigni from Uruguay (Mañé-Garzón & Holcman-Spector, Reference Mañé-Garzón and Holcman-Spector1968). This is the second register of this host for this parasite and the first record for Argentina. In addition, we present for the first time the sequence of the ITS2 gene of T. dubius. The p-distances analysis shows it is close to T. diaphanus.
Telorchis devincenzii Mañé-Garzón & Gil, 1961
Diagnosis: Oral sucker larger than acetabulum, presence of prepharynx, globose pharynx, juxtaovarian cirrus pouch and vitellogenic glands of cecal and extracecal position (Table 4, Fig. 4B).
Site of infection: Intestine.
Host and localities: Hydromedusa tectifera - La Plata (34°51′38′′S, 58°03′54′′W) in Buenos Aires province (Pampa eco-region).
Voucher specimens: MLP-He 8089
Remarks: After its finding and description parasitizing H. tectifera from Canelones, Uruguay (Mañé-Garzón & Gil, Reference Mañé-Garzón and Gil1961b) the species was not recorded again. Catto & Amato, (Reference Catto and Amato1993) transferred some Telorchis species (but not T. devincenzii) to Pseudotelorchis Yamaguti, 1971 but late authors (Fernandes & Kohn, Reference Fernandes and Kohn2014; Mascarenhas & Müller, Reference Mascarenhas and Müller2021) have erroneously interpreted that Catto & Amato, (Reference Catto and Amato1993) transferred T. devincenzii to Pseudotelorchis, and consequently they have mistakenly included their findings of T. devincenzii as P. devincenzii. It should be clearly stated that the morphological characteristics of T. devincenzii are proper to Telorchis and not fill those of Pseudotelorchis.
Telorchis birabeni Mañé-Garzón & Gil, 1961
Diagnosis: Oral sucker bigger than acetabulum, presence of a pre-pharynx and a globose pharynx, ovary and acetabulum widely separated, small testis in tandem, and vitellaria extending from the ovary to the anterior testis (Table 4, Fig. 4C).
Site of infection: Intestine.
Host and localities: Phrynops hilarii - La Plata (34°53′26′′S, 57°59′40′′W) in Buenos Aires province (Pampa eco-region); Gualeguaychú (32°57′30″S, 58°34′59″W) in Entre Ríos province (Pampa eco-region); Cañada de Gomez (32°49′45″S, 61°22′57″W), Helvecia (31°06′30″S, 60°05′48″W), Rafaela (31°15′04″S, 61°30′20″W) and Santa Fe Capital (31°38′21″S, 60°40′54″W) in Santa Fe province (Espinal eco-region). Phrynops williamsi - Comandante Andresito (25°40′22′′S, 54°02′22′′W) in Misiones province (Atlantic Rainforest eco-region).
Historical records in Argentina: Phrynops hilarii - unknow locality in Corrientes province (Lombardero & Moriena, Reference Lombardero and Moriena1977).
GenBank acc. number: OR135715 (ITS2, PW), OR135718 (ITS2, PH)
Voucher specimens: MLP-He 8090
Remarks: The species was previously recorded for P. hilarii from Argentina, Brazil and Uruguay, and for P. geoffroanus from Brazil (Mañé-Garzón & Gil, 1961; Lombardero & Moriena, Reference Lombardero and Moriena1977; Silva, Reference Silva2014; Mascarenhas et al., Reference Mascarenhas, Bernardon and Müller2016). This is the first record for P. hilarii for Buenos Aires, Entre Ríos and Santa Fe Argentine provinces, and the first record for P. williamsi. In addition, the two new ITS2 gene sequences provided a good complement to morphology to better differentiate species of the genus (Table 5).
Table 5. Number of per site base differences based on pairwise comparisons of the sequenced fragment at 5′ end of ITS2 rDNA gene among the sequences newly obtained in this study and one sequence of Telorchis species available in GenBank. Positions containing gaps and missing data were eliminated
Telorchis diaphanus Freitas & Dobbin Jr., Reference Freitas and Dobbin1959
Diagnosis: Oral sucker slightly larger than the acetabulum, prepharynx absent, small pharynx, cirrus sac reaches the upper edge of the ovary, oval-shaped ovary, large testis in tandem, extra cecal vitellaria with asymmetrical distribution (Table 4, Fig. 4D).
Site of infection: Intestine.
Hosts and localities: Phrynops hilarii - Bella Vista (28°30′46′′S, 59°01′49′′W) in Corrientes province (Esteros del Ibera ecoregion). Trachemys dorbigni - Federal (30°40′54′′S, 58°47′20′′W) in Entre Ríos province (Espinal ecoregion).
GenBank acc. number: OR135722 (28S, TD), OR135720 (ITS2, TD)
Voucher specimens: MLP-He 8091
Remarks: This species was described for K. scorpioides from Pernambuco, Brazil (Freitas & Dobbin Jr., Reference Freitas and Dobbin1959). This is the first record of T. diaphanus for P. hilarii and T. dorbigni from Argentina. Table 5 shows that T. diaphanus is more closely related to T. dubius than to the rest of the species of the genus. The 28S gene sequences for the individuals found in both turtle species studied here constitute the first of this gene for T. diaphanus.
Phylum Nematoda
Class Chromadorea
Family Kathlaniidae Lane, 1914
Falcaustra Lane, 1915
Falcaustra affinis (Leidy, 1856)
Diagnosis: Three long vesicular lips; cephalic papillae present; small deirids; esophagus divided into pharynx, body, isthmus, and bulb. Males with a muscular ventral pseudoventosa; the ventral region between the cloaca and pseudoventosa contains 35 to 43 pairs of muscles. Ten pairs of caudal papillae present, three precloacal and seven poscloacal. Sclerotized spicules nearly equal in size. Well-developed gubernaculum. Females with vagina anteriorly directed, conical tail, with a pair of phasmidia near the tip. Eggs elliptical, thin-walled (Table 4).
Site of infection: Intestine.
Host and localities: Trachemys dorbigni - La Plata (35°01′18″S, 57°51′25″W) in Buenos Aires province (Pampa ecoregion), Concordia (31°21′22′′S, 58°05′42′′W) and Federal (30°40′54′′S, 58°47′20′′W) in Entre Ríos province (Espinal ecoregion).
Voucher specimens: MLP-He 8092
Remarks: This species was originally recorded from North American turtles and amphibians (Baker, Reference Baker1986). Recently, Mascarenhas & Müller (Reference Mascarenhas and Müller2015) found this species parasitizing T. dorbigni from Rio Grande do Sul, Brazil. Our finding represents the southernmost record for the species and the first geographic record for Argentina.
Family Pharyngodonidae Travassos, 1919
Thelandros Wedl, 1862
Thelandros sp.
General description (based on four gravid females): Robust body 3.44 (2.7-4.25) mm long and 412 (298-554) μm wide, with cuticle annulations. Mouth opening triangular, surrounded by three bilobed lips, with a pedunculate amphid in each ventrolateral lobe. Total length of esophagus 382 (350-402) μm, corpus 250 (245-255) μm, isthmus 20 (16-22) μm, and bulb 94 (91-98) μm. Nerve ring and excretory pore at 120 (112-135) μm and 473 (418-544) μm from anterior end, respectively. Vulva post equatorial, 1.42 (1.10-1.84) mm from the posterior end. The anus opens at the end of the body, which ends in a caudal filament 366 (327-388) μm long. Eggs oval, 84 (81-88) μm x 41 (36-49) μm, with terminal operculum (Table 4, Fig. 5).
Figure 5. Line drawings of Thelandros sp. detailing anterior end, lateral view (A), posterior end, ventral view (B), vulva (C), and egg (50 μm scale) (D). Scale bars 100 μm.
Site of infection: Stomach.
Hosts and localities: Acanthochelys pallidipectoris - Vera (29°26′14″S, 60°08′03″W) in Santa Fe province (Humid Chaco ecoregion); Las Lajitas (24°43′30″S, 64°09′32″W) in Salta province (Dry Chaco ecoregion). Phrynops hilarii - General José de San Martin (26°24′49′′S, 59°22′02′′W) in Chaco province (Dry Chaco ecoregion).
GenBank acc. number: OR135723 (18S)
Voucher specimens: MLP-He 8093
Remarks: The cephalic structures, the morphology of the esophagus, the position of the excretory pore and vulva, the pedunculate tail, and the shape of the eggs indicate that these specimens belong to Thelandros (De Sousa et al., Reference De Sousa, Jorge, Carretero, Harris, Roca and Perera2018). Unfortunately, only females were found, so it was not possible to reach their specific identity. This is the first record of the genus for freshwater turtles. The phylogenetic analysis made with 18S gene sequences of the family Pharyngodonidae shows a stronger association between the species of Thelandros recorded in lizards (KY541834 and KJ778073) and the species described here than with the rest of the species of the family (Fig. 3B).
Pharyngodonidae gen. et sp. indet.
General description (based on three gravid females): Small and cylindrical body, truncated anteriorly and narrow at the posterior end, 2.35-2.75 mm long and 113-125 mm wide. Mouth surrounded by three triangular lips. Without buccal capsule, esophagus 289-310 μm long, ending in a bulb 100-115 μm × 120-127 μm. Excretory pore opens at 355-389 μm from anterior end. Vulva opens 730-764 μm from caudal end, at mid-posterior part of body. One to five elliptical eggs 59-61 μm × 45-49 μm (Fig. 6).
Figure 6. Line drawings of Pharyngodonidae gen. sp. indet. Complete individual (A) (scale 200 μm), detail anterior end (B), and vulva (C) in lateral view. Scale bars 100 μm.
Site of infection: Stomach.
Hosts and localities: Acanthochelys pallidipectoris - Joaquín V. González (29°26′14″S, 60°08′03″W) in Salta province (Dry Chaco ecoregion). Kinosternon scorpioides - Miraflores (25°38′16″S, 60°56′03″W) in Chaco province and Las Lajitas (24°43′30″S, 64°09′32″W) in Salta province (Dry Chaco ecoregion).
Voucher specimens: MLP-He 8094
Remarks: One female was found in feces of A. pallidipectoris and two females were recovered from regurgitates of K. scorpioides. Because of the condition of the specimens and the fact that males were not found, it was not possible to reach genus and species level taxonomic identifications. This finding represents the first record of the family for K. scorpioides.
Family Camallanidae Railliet & Henry, 1915
Camallanus Railliet & Henry, 1915
Camallanus emydidius Mascarenhas & Müller, Reference Mascarenhas and Müller2017
Diagnosis: The arrangement of ridges on the cephalic capsule, the number and distribution of caudal papillae (seven precloacal, two adcloacal, and four poscloacal), the morphology of the spicules in males, and the morphology of the vulva and number of mucrons in females, place these individuals within C. emydidius (Table 4).
Site of infection: Stomach and intestine.
Host and localities: Trachemys dorbigni - Federal (30°40′54′′S, 58°47′20′′W) in Entre Ríos province (Espinal ecoregion).
Historical records in Argentina: Trachemys dorbigni - Concordia (31°21′22′′S, 58°05′42′′W) in Entre Ríos province (Espinal ecoregion) (Palumbo et al., Reference Palumbo, Servián, Cassano and Diaz2024).
Voucher specimens: MLP He-8060
Remarks: Camallanus emydidius was originally described parasitizing T. dorbigni from Rio Grande do Sul, Brazil (Mascarenhas & Müller, Reference Mascarenhas and Müller2017) and was later recorded on H. tectifera from the same locality (Chaviel et al., Reference Chaviel, Mascarenhas, Bernardon, Coimbra and Müller2020). This record represents a new locality for C. emydidius. Additionally, there are records of other species of Camallanidae in Argentina: C. pallidipectoris Palumbo, Servián, Cassano & Diaz, Reference Palumbo, Servián, Cassano and Diaz2024 in A. pallidipectoris and P. hilarii in Santa Fe and Entre Ríos, C. spiculatus Palumbo, Servián, Cassano & Diaz, Reference Palumbo, Servián, Cassano and Diaz2024 in A. spixii and P. hilarii in Corrientes and Serpinema sp. in K. scorpioides in Formosa (Palumbo et al., Reference Palumbo, Servián, Cassano and Diaz2024).
Family Gnathostomatidae Railliet, 1895
Spiroxys Schneider, 1866
Spiroxys contortus (Rudolphi, 1819)
Diagnosis: Oral opening surrounded by two trilobed lips, the middle lobe has a truncated “tooth” at its apex; deirids located posterior to the excretory pore. Males with four pairs of precloacal papillae and seven pairs postcloacal. Spicules of equal size, supported by a cuticular gubernaculum. Vulva cuticular, located posterior to midbody. Vagina directed anteriorly, oviparous.
Site of infection: Stomach and intestine.
Host and localities: Hydromedusa tectifera - Tanti (31°22′05″S, 64°34′34″W) and Flor Serrana (31°23′36″S, 64°35′42″W) streams in Córdoba province (Dry Chaco ecoregion). Phrynops hilarii - Diamante (32°02′52″S, 60°38′10″W) in Entre Ríos province (Espinal ecoregion), Bella Vista (28°30′46′′S, 59°01′49′′W) in Corrientes province (Esteros del Ibera ecoregion) and Vera (29°26′14″S, 60°08′03″W) in Santa Fe province (Humid Chaco ecoregion). Trachemys dorbigni - La Plata (34°53′26′′S, 57°59′40′′W) in Buenos Aires province (Pampa ecoregion) and Concordia (31°21′22′′S, 58°05′42′′W) in Entre Ríos province (Espinal ecoregion).
Historical records in Argentina: Hydromedusa tectifera and Phrynops hilarii - Magdalena (35°01′36″S, 57°17′24″W) in Buenos Aires province (Pampa ecoregion) (Palumbo et al., Reference Palumbo, Capasso, Cassano, Alcalde and Diaz2016).
Remarks: This species was previously recorded from A. spixii, H. tectifera, P. hilarii, and T. dorbigni from Brazil (Mascarenhas & Müller Reference Mascarenhas and Müller2021), and from H. tectifera and P. hilarii from Argentina (Palumbo et al., Reference Palumbo, Capasso, Cassano, Alcalde and Diaz2016). The 18S ribosomal gene of S. contortus found parasitizing a H. tectifera from Buenos Aires could be partially sequenced. Comparison with sequences obtained from specimens found parasitizing Emys orbicularis (L.) from Poland confirm the wide geographic range of S. contortus and the ability to adapt to different environments and hosts (Demkowska-Kutrzepa et al., Reference Demkowska-Kutrzepa, Szczepaniak, Rocze-Karczmarz, Palumbo, Studzinska, Rózanski and Tomczuk2021). This is the first record of S. contortus for T. dorbigni from Argentina.
Family Hedruridae Railliet, 1916
Hedruris Nitzch, 1821
Hedruris dratini Palumbo et al., Reference Palumbo, Servián, Sánchez and Diaz2020
Diagnosis: The same position of the excretory pore and deirids with respect to the anterior end, the number and arrangement of the caudal papillae in males, and the shape of eggs, place these specimens within the species H. dratini.
Site of infection: Stomach and intestine.
Host and localities: Hydromedusa tectifera - La Plata (34°51′38′′S, 58°03′54′′W) in Buenos Aires province (Pampa ecoregion). Trachemys dorbigni - La Plata (34°53′26′′S, 57°59′40′′W) in Buenos Aires province (Pampa ecoregion).
Historical records in Argentina: Hydromedusa tectifera and Phrynops hilarii - La Plata (34°53′02′′S, 58°02′30′′W) in Buenos Aires province (Pampa ecoregion) (Palumbo et al., Reference Palumbo, Servián, Sánchez and Diaz2020)
Remarks: Hedruris dratini was previously recorded for H. tectifera and P. hilarii from Argentina (Palumbo et al., Reference Palumbo, Servián, Sánchez and Diaz2020). This is the first record of the species for T. dorbigni.
Comments on parasite ecology
A total of 487 turtles were analysed, of which 433 were examined only from stomach contents, whereas the rest (54) were analysed entirely from viscera. Of the total number of turtles, 180 were parasitized (P = 37.03) by at least one of the 18 taxa found and 6230 helminths specimens were counted. Most of the eviscerated turtles were parasitized (P = 92.59, MI = 88.16, A = 2733). In contrast, parasites were found in 129 of the hosts analysed from stomach contents (P = 26.59, MI = 27.32, A = 3497).
The Pampa ecoregion had the highest species richness (S = 14), followed by Espinal ecoregion (S = 10), the Humid (S = 5) and Dry (S = 4) Chaco, and finally Rainforest and Esteros del Ibera. According to the host the highest specific richness (Fig. 7) in this study was found in P. hilarii (S = 11), followed by T. dorbigni (S = 8), while the lowest richness was found in K. scorpioides (S = 1). Spiroxys contortus was the species with the largest distribution (found in five ecoregions and three host species).
Figure 7. Number of species of endoparasites recorded per turtle species.
Discussion
A total of 21 new geographic records were documented, increasing to 36 the total parasite records for freshwater turtles in different Argentine Chaco-Pampa plain ecoregions (Pampa, Espinal, Dry Chaco, Humid Chaco ecoregions) and neighbouring ones (Atlantic Rainforest and Esteros del Ibera ecoregions) (Mascarenhas & Müller, Reference Mascarenhas and Müller2021). Fourteen new parasite-host associations were reported, with A. pallidipectoris having the highest number of records (4), followed by T. dorbigni (3), P. hilarii, P. williamsi and H. tectifera (2), and K. scorpioides (1). Notably, the findings in A. pallidipectoris and P. williamsi are significant as only two parasite species were reported previously for the former turtle species (Palumbo et al., Reference Palumbo, Servián, Cassano and Diaz2024), whereas no parasites were known for the second species throughout their distribution. Figure 8 illustrates the contribution of this study to each host species, highlighting the records for Argentina and the species documented in other countries but not yet in Argentina.
Figure 8. Number of endoparasite species recorded only in Argentina (Only Arg), species recorded both in Argentina and elsewhere (Shared species) and species recorded outside Argentina (ROW), per turtle species.
The turtle P. hilarii shares habitat and parasites with other turtle species along different Argentine provinces and ecoregions. Thus, P. hilarii shares four parasitic species with H. tectifera (i.e., C. testudinis, H. orestiae, H. dratini, and S. contortus), two with T. dorbigni (i.e., T. diaphanus and S. contortus), and two with A. pallidipectoris (i.e., Thelandros sp. and Camallanus pallidipectoris). It is also observed that A. pallidipectoris and K. scorpioides were unique in this study harbouring the nematode Pharyngodonidae gen. et sp. indet. A recent study compared the parasitic fauna of three species of freshwater turtles from southern Brazil and mentioned 11 helminth species for A. spixii and eight species for both H. tectifera and P. hilarii (Mascarenhas et al. Reference Mascarenhas, Chaviel, Bernardon, Wolter, Coimbra and Müller2022). Authors stated that the parasite community of A. spixii is more similar to that of H. tectifera than that of P. hilarii. In Argentina, A. spixii does not share environments with H. tectifera but does with P. hilarii, enlarging the chances to acquiring the same nematode species (Palumbo et al., Reference Palumbo, Servián, Cassano and Diaz2024). The greater number of shared helminth species was recorded between H. tectifera and P. hilarii (4).
The most commonly used technique for searching endohelminth parasites is the capture and evisceration of hosts followed by processing of their internal organs. However, when working with larger vertebrates and considering that some species of turtles are endangered (e.g., A. pallidipectoris), euthanasia is not possible. The complete hosts analyzed in this study consisted of specimens found dead in the wild or housed in herpetological collections. Thus, to increase the collection of internal parasite samples, noninvasive technique such as stomach flushing was employed. Most of the parasitic species found in stomach contents were also found in eviscerated hosts of the same species and locations. Moreover, this procedure allowed us the detection of Pharyngodonidae gen. sp. in A. pallidipectoris and K. scorpioides, Thelandros sp. in A. pallidipectoris and P. hilarii, and H. dratini in P. hilarii, which could not be recorded through evisceration. These results indicate that flushing is a valuable technique for describing parasite fauna in this type of host.
Finally, it is important to emphasize how the integrative taxonomy (i.e., different morphological and molecular tools) helped us to solve and elucidate taxonomical hypothesis and systematic problems. For this reason, the contribution of sequences of different genes, even if not used in the work itself (such as the sequences of Caimanicola brauni or Prionosomoides phrynopsis) can be very useful to other researchers in future studies. Therefore, the most commonly used sequences (28S, 18S, ITS, etc.) should be an additional character provided in each species description.
Acknowledgments
We thank Dr. Marina Ibáñez Shimabukuro for her collaboration in DNA extraction and replication and Dr. Adriana Manzano and Dr. Alfredo Holley for providing the hosts. This work is the ILPLA’s scientific contribution N°1260.
Funding
This study was partially funding by UNLP N828 (Dir. JID).
Competing interest
Authors declare they have no financial interests.
Ethical approval
Not applicable.
