Introduction
The cestode family Bothriocephalidae Blanchard, 1849 (order Bothriocephalidea) comprises 18 genera and approximately 70 valid species of intestinal parasites infecting marine and freshwater teleost fishes plus three species of Bothriocephalus Rudolphi, 1808, infecting salamanders (Caira & Jensen, Reference Caira and Jensen2017; Kuchta & Scholz, Reference Kuchta, Scholz, Kuchta, Scholz, Bray, Caira and Jensen2017; Caira et al., Reference Caira, Jensen and Barbeau2024). Bothriocephalids exhibit a complex life cycle that involves one or two intermediate hosts. Definitive hosts typically become infected by the ingestion of infected copepod crustaceans that serve as intermediate hosts (Kuchta et al., Reference Kuchta, Scholz and Bray2008b; Kuchta & Scholz, Reference Kuchta, Scholz, Kuchta, Scholz, Bray, Caira and Jensen2017).
Costa Rica represents an important hotspot of aquatic biodiversity with more than 2030 fish species recorded from both freshwater (Angulo, Reference Angulo2021) and marine ecosystems (Froese & Pauly, Reference Froese and Pauly2024). The Synodontidae are small teleost benthic fishes belonging to the aulopiform order which occur in marine and estuarine environments. The genus Synodus comprises 47 recognized species of which only four are found from the coastal waters of Costa Rica (Froese & Pauly, Reference Froese and Pauly2024). Among these, the shorthead lizardfish Synodus scituliceps Jordan & Gilbert is a common demersal predator mainly feeding on small benthic fishes (Rodríguez-Romero et. al., Reference Rodríguez-Romero, López-Martínez, Ochoa Díaz and Herrera-Valdivia2019). This fish is known as a host for few cestode species including Rhinebothrium spp. and tetraphyllideans (Escalante et al., Reference Escalante, Arigaza and Moreno1987; Luque et al., Reference Luque, Iannacone and Farfan1991; Alves et al., Reference Alves, de Chambrier, Scholz and Luque2017), whereas Synodontidae in general have been recorded as host for at least four species of bothriocephalids and two trypanorhinch species (Caira et al., Reference Caira, Jensen and Barbeau2024).
According to previous literature (Rodríguez-Ortíz et al., Reference Rodríguez-Ortíz, García-Prieto and de León2004; Caira & Jensen, Reference Caira and Jensen2017; Solano-Barquero et al., Reference Solano-Barquero, Rojas and Cortés2023; Caira et al., Reference Caira, Jensen and Barbeau2024), approximately 33 cestode species have been reported solely from the Costa Rican marine waters. These include adult stages of the orders Cathetocephalidea, Onchoproteocephalidea, Phyllobothriidea, Rhinebothriidea, Tetrabothriidea, “Tetraphyllidea”, and Trypanorhyncha from elasmobranchs and larval stages of two Phyllobothriidea (Clistobothrium delphini (Bosc, 1802) Caira, Jensen, Pickering, Ruhnke & Gallegher, Reference Cortés and Joyce2020 and Clistobothrium grimaldii (Moniez, 1899) Caira, Jensen, Pickering, Ruhnke & Gallegher, Reference Cortés and Joyce2020 from cetaceans, whereas no records of Bothriocephalidea, and cestodes from teleost fishes in general, exist from Costa Rican waters.
During a parasitological survey of fish from the Pacific coast of Costa Rica, some individuals of a bothriocephalid species were found from the intestine of S. scituliceps. These tapeworms proved to represent a morphologically distinct, previously unknown, species of Anantrum Overstreet, Reference Overstreet1968, which is described here, based on morphological and molecular characters, and phylogenetic analysis.
Materials and methods
Sample collection
On April 7, 2023, seven individuals of the shorthead lizardfish were obtained from off Playa Cuajiniquil (Guanacaste) on the Pacific coast of Costa Rica using nets at benthic depths ranging from 5 to 10 m. They were three females and four males with total length ranging from 15.5 to 21 cm and from 15 to 25 cm, respectively. Fish were obtained under the framework of a project of the Centro de Investigación en Ciencias del Mar y Limnología of the University of Costa Rica “Proyecto BioMar - ACG” (see Cortés & Joyce, Reference Cortés and Joyce2020; Santoro et al., Reference Santoro, López-Verdejo, Angulo, Rojas, Cortés, Pacheco-Chaves and Solano-Barquero2024), aimed at studying the marine biodiversity of the Pacific coast of Costa Rica (permit no. ACG 019-2023). Fish parasites were studied in the frame of a collaborative project between the Stazione Zoologica Anton Dohrn (Italy) and the University of Costa Rica (Costa Rica).
Fish were refrigerated (4 °C) and transferred to the laboratory, where they were studied within 6 h from fishing. During necropsy, the intestine of each individual fish was examined and cestodes were obtained alive under a dissecting microscope (Axio Zoom V16, Zeiss, Switzerland) using the methods described in Santoro et al. (Reference Santoro, Bellisario, Tanduo, Crocetta and Palomba2022, Reference Santoro, Bellisario, Fernández-Álvarez, Crocetta and Palomba2023). Cestodes were washed in physiological saline solution and, when relaxed, they were preserved in 70% ethanol for subsequent morphological and molecular analyses.
Morphological study
For light microscopy, cestodes were stained with Mayer’s acid carmine, dehydrated through a graded ethanol series, cleared in methyl salicylate, and mounted in permanent slides in Canada balsam (Santoro et al., Reference Santoro, López-Verdejo, Angulo, Rojas, Cortés, Pacheco-Chaves and Solano-Barquero2024). In the species description, measurements (in micrometres, except where stated) are reported as range values with mean ± standard deviation in parentheses followed by the total number (n) of observations. Measurements were obtained using a compound microscope (Axio Imager M1, Zeiss) and a dissecting microscope equipped with the ZEN 3.1 imaging system (Zeiss). Drawings were made with the aid of a XP PEN Deco 02 drawing tablet (Deco, Italy) and the software Adobe Illustrator and Adobe Photoshop.
For scanning electron microscopy (SEM) analysis, two specimens were fixed overnight in 2.5% glutaraldehyde, then transferred to 40% ethanol (10 min), rinsed in 0.1 M cacodylate buffer, postfixed in 1% OsO4 for 2 h, and dehydrated in ethanol series, critical point dried, and sputter-coated with platinum. Observations were made using a JEOL JSM 6700F SEM operating at 5.0 kV (JEOL, Japan).
Molecular and phylogenetic analyses
Genomic DNA was extracted from one specimen using the Quick-gDNA Miniprep Kit (Zymo Research, USA), according to the manufacturer protocol. The 28S rRNA segment was amplified using two sets of primer pairs allowing the amplification of two contiguous regions. The primer sets selected were ZX-1 (5’-ACCCGCTGAATTTAAGCAT-3’) - ECD2 (5’-CTTGGTCCGTGTTTCAAGACGGG-3’), and LSU_300F (5’-CAAGTACCGTGAGGGAAAGTTG-3’) - 1500R (5’-GCTATCCTGAGGGAAACTTCG-3’). Polymerase chain reaction (PCR) was performed in 25-μL reactions with 2 μL of DNA sample, 0.6 μL of each primer at 10 mM and 10 μL of MyFi Mix (Bioline Ltd., United Kingdom). The thermocycling amplification program included a preliminary denaturation step at 94 °C (3 min) followed by 40 cycles of 94 °C (30 s), 54 °C (30 s), 72 °C (2 min), and a final extension step at 72 °C (10 min). Amplified products were preserved at 4 °C. Amplicons were visualized in a 1% agarose gel with GelRed (Biotium, UK) stain on a ~35 min, 95 V electrophoresis. Successful PCR products were purified using Agencourt AMPure XP (Beckman Coulter, USA), following the standard manufacturer recommended protocol. Clean PCR products were Sanger sequenced from both strands and from an additional internal primer 1090F (5’-TGAAACACGGACCAAGG-3’) using an Automated Capillary Electrophoresis Sequencer 3730 DNA Analyzer (Applied Biosystems, USA) and the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Life Technologies, USA). The obtained contiguous sequences were assembled and edited using MEGAX v. 11 (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018). Sequence identity was verified using the Nucleotide Basic Local Alignment Search Tool (BLASTn) (Morgulis et al., Reference Morgulis, Coulouris, Raytselis, Madden, Agarwala and Schäffer2008).
All the available sequences representatives of the family Bothriocephalidae, according to the latest taxonomic classification (Kuchta et al., Reference Kuchta, Scholz and Bray2008b, Reference Kuchta, Scholz, Brabec and Bray2008a; Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015), were retrieved from GenBank (Table 1) and aligned, together with the sequence generated in this study, using the multiple sequence alignment package T-Coffee (Notredame et al., Reference Notredame, Higgins and Heringa2000). The alignment was then submitted to the transitive consistency score (TCS) to verify the reliability of aligned positions and optimise the phylogenetic topology (Chang et al., Reference Chang, Di Tommaso, Lefort, Gascuel and Notredame2015). In total, 36 sequences were analysed, including the outgroup Grillotia pristiophori Beveridge & Campbell, 2001 (Table 1). Based on recent results indicating a good response of Bayesian inference (BI) in integrative taxonomic studies of parasites of fishes with complicated taxonomy (Kuchta et al., Reference Kuchta, Burianova, Jirků, de Chambrier, Oros, Brabec and Scholz2012; Ailán-Choke et al., Reference Ailán-Choke and Pereira2021; Choudhury et al., Reference Choudhury, Scholz and Beuchel2022), the phylogenetic hypotheses in the present work were inferred using this approach implemented in MrBayes v. 3.2.7 (Ronquist & Huelsenbeck, Reference Ronquist and Huelsenbeck2003). Additionally, maximum likelihood phylogenetic tree was calculated using iQtree v. 1.6.12 (Nguyen et al., Reference Nguyen, Schmidt, Von Haeseler and Minh2015), performing 5000 ultrafast bootstrap approximations to test the phylogenetic reliability. The best fitted evolutionary model was TIM3+I+Γ, as suggested by jModelTest v. 2.1.10 (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012). Posterior probability distributions for the Bayesian analysis were generated using the Markov Chain Monte Carlo (MCMC) method. MCMC searches were run for 10 million generations on two simultaneous runs of four chains and sampled every 1000 generations; the first 25% of samples from the MCMC algorithm were discarded as burn in. The quality of the Bayesian analysis (parameter densities, effective sample size, and burn-in) and the chain convergence were examined in Tracer (Rambaut et al., Reference Rambaut, Drummond, Xie, Baele and Suchard2018), and trees were visualised using Figtree v. 1.4.4 (Rambaut, Reference Rambaut2012). The genetic divergences among taxa for a subset of our dataset, which included the most closely related species to our specimen, were estimated using absolute nucleotide differences and p-distances using MEGAX v. 11 (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018).
Table 1. Information about sequences used in the phylogenetic analysis obtained from GenBank (the sequence generated in this study is shown in bold).
a Reassigned to this genus (see Caira et al., Reference Caira, Jensen and Barbeau2024).
b Outgroup.
Results
Figure 1. Anantrum gallopintoi sp. nov. from Synodus scituliceps. Microphotographs of a ventrally mounted paratype showing the scolex (a) and a section of the spirally twisted strobila with mature proglottids (b).
ZooBank: LSID urn:lsid:zoobank.org:act:C992F0A4-0F83-4909-AD37-0F7BDB799A2B
Orden Bothriocephalidea Kuchta, Scholz, Brabec & Bray, 2008
Family Bothriocephalidae Blanchard, 1849
Anantrum gallopintoi Santoro, López-Verdejo & Occhibove, Reference Santoro, López-Verdejo, Angulo, Rojas, Cortés, Pacheco-Chaves and Solano-Barquero2024 sp. nov.
Based on six specimens as whole mounts and two observed with SEM. Medium-sized worms, flattened dorsoventrally, 119–153 (135.8 ± 14.6; n = 6) mm long, 1603–2522 (1962 ± 383.4; n = 6) maximum wide. Scolex unarmed, elongate, slightly spatulate, without bothria and apical disc (Figures 1a, 3a, 5a), 1445–2655 (2016 ± 432.1; n = 6) long, 550–951 (689 ± 155.2; n = 6) maximum wide; scolex length: width ratio 1:2.2–3.5 (1:2.9 ± 0.5; n = 6). Neck absent (Figures 1a, 3a, 5a). Strobila spirally twisted with rippled margins (Figure 1b), anapolytic, external segmentation absent with indistinguishable number of immature proglottids occupying 10.3%–25.5% (16.7% ± 5.5%; n = 6) of body length (Figure 2). Mature proglottids wider than long (Figures 1b, 3b); gravid proglottids longer than wide (Figure 4a); gravid proglottids (counting reproductive system sets with eggs), 83–154 (104 ± 28.6; n = 6) in number occupying 72.9%–87.8% (82.2% ± 5.5%; n = 6) of body length. Pygidium (posterior end of strobila) longer than wide (Figure 4b) (length: width ratio 1:2.4–4.4 [1:3.1 ± 0.7; n = 6], 1312–2901 [1848 ± 578.2; n = 6] long, 466–649 [584 ± 68.4; n = 6] wide [measured from its posterior extremity to anterior margin of uterus]). Tegument thick. Internal longitudinal muscles well developed. Nerve cord situated lateral on each side of proglottids. Osmoregulatory canals medullary, four pairs on each side of proglottids. Tegumental microtriches not observed.
Figure 2. Anantrum gallopintoi sp. nov. from Synodus scituliceps. Line drawings of the strobila sections (paratype, ventral view). The arrowhead indicates where the mature proglottids start.
Figure 3. Anantrum gallopintoi sp. nov. from Synodus scituliceps. Line drawings of the holotype in ventral view. Scolex (a), mature proglottid (b), and genitalia (c). Abbreviations: Gp, genital pore; Mg: Mehlis’ gland; Oo, ootype; Sr, seminal receptacle; Va, vagina; Vs, vaginal sphincter.
Figure 4. Anantrum gallopintoi sp. nov. from Synodus scituliceps. Line drawings of the holotype in ventral view. Gravid proglottid (a), pygidium (b), and egg (c).
Testes medullary, spherical 52–87 (67 ± 8.1; n = 40) in diameter; 50–84 (68 ± 8.4; n = 12) in number per proglottid, forming two longitudinal layers on each side of proglottids (Figures 3b, 4a), 23–50 in number on each side of longitudinal layers. Vas deferens strongly coiled, situated anteromedially. Genital pore dorsal; genital atrium (Figures 5b, 5d, 5e), sub-median, round with irregular tegumental papilliform processes on its margin. Cirrus-sac oval (Figure 3c), large, thick-walled, median, 213–305 (249 ± 29.1; n = 20) long, 160–202 (185 ± 11.9; n = 20) wide. Cirrus elongate, unarmed, 117–201 (159 ± 30.2; n = 9) long, 13–22 (19 ± 3.2; n = 9) wide.
Figure 5. Scanning electron microscopy of Anantrum gallopintoi sp. nov. Scolex (a); dorsal view of mature proglottid showing the genital pore (b, arrowheads); ventral view of mature proglottids showing the irregularly alternating uterine pore (c, arrowheads); genital pore partially obliterated showing contracted tegumental papilliform processes (d); genital pore showing around the tegumental papilliform processed (e); operculate egg coming out of the uterine pore (f).
Ovary bilobed (Figures 3b, 4a), median, transversely elongated near posterior margin of proglottid, 481–847 (651 ± 116.4; n = 20) long, 99–435 (251 ± 84.5; n = 20) wide; isthmus conspicuous. Vagina posterior to cirrus sac in midline of proglottid between ovary lobes, vaginal sphincter 29.4–44.5 (36.5 ± 3.6; n = 20) long, 58.9–75.9 (67.6 ± 5.4; n = 20) wide (Figure 3c). Seminal receptacle round, 37.7–58.87 (50.5 ± 5.1; n = 20) in diameter. Mehlis’ gland round, 37–61 (47 ± 7.1; n = 10) in diameter. Vitelline follicles irregular, densely distributed in two cortical layers around testes (Figures 3b, 4a). Vitelline follicles, 20.9–45.1 (30.1 ± 6.1; n = 20) long, 30.4–52.9 (41.9 ± 5.4; n = 20) wide in mature proglottids, and 43.7–59.7 (50.4 ± 4.4; n = 20) long, 38.5–60.5 (52.5 ± 5.6; n = 20) wide in gravid proglottids. Vitelline reservoir dorsal. Uterus sinuous, coiled, irregularly alternating dextrally or sinistrally to midline, occupying 79%–100% (85% ± 5.6%; n = 20) of proglottid length. Uterine pore ventral (Figures 5c, 5f), anterolateral. Eggs operculate (Figures 4c, 5f), unembryonated, oval 45–61 (53 ± 4.4; n = 25) long, 24–34 (30 ± 2.1; n = 25) wide.
Taxonomic summary
Type host: shorthead lizardfish Synodus scituliceps Jordan & Gilbert, 1882 (Aulopiformes: Synodontidae).
Type locality: Gulf of Santa Elena off Playa Cuajiniquil (10°56′04.38″N, 85°42′14.09″W), Guanacaste province, north Pacific coast of Costa Rica (collected on April 7, 2023).
Site of infection: Posterior intestine.
Type material: Holotype (MHNG-PLAT-0159514) and two paratypes (MHNG-PLAT-0159515 and MHNG-PLAT-0159516) in the Parasite Collection of the Natural History Museum of Geneva in Geneve (Switzerland); two paratypes in the Helminthological Collection of Costa Rica (CHCR-215-1 and CHCR-215-2) at the Universidad de Costa Rica, San José (Costa Rica).
Prevalence and intensity: two (males: 15 and 18 cm total length) of seven individuals infected with two and nine worms, respectively.
Etymology: The new species is named after the typical Costa Rican breakfast food.
Remarks
There are only three species of Anantrum, including the new one. These are: A. tortum (Linton, Reference Linton1905) described from Synodus foetens at Beaufort, North Carolina (North Atlantic) (Linton, Reference Linton1905), and later redescribed from S. intermedius at St George’s West, Bermuda (Western Atlantic) (Rees, Reference Rees1969), and S. foetens from South Florida (Everglades National Park, Atlantic) (Overstreet Reference Overstreet1968), and A. histocephalum Jensen & Heckmann, Reference Jensen and Heckmann1977 described from S. lucioceps from coastal waters of Los Angeles County, California (North Pacific) (Jensen & Heckmann, Reference Jensen and Heckmann1977).
Anantrum gallopintoi sp. nov. can be distinguished from both its conspecifics by having a vaginal sphincter, different number of testes per proglottid (50–84 vs 12–16 in A. tortum, and 31–101 in A. histocephalum), and different type host. Anantrum gallopintoi sp. nov. is most closely related to A. tortum; however, it is larger (119–153 vs 36–70 mm), has larger number of sets of reproductive organs along the strobila (mean: 104 vs 45), and has larger cirrus sac (249 × 185 vs 50 × 76 in diameter), and different geographical distribution (Pacific vs Atlantic). Anantrum gallopintoi sp. nov. can be also distinguished from A. histocephalum by body length (119–153 vs 81–552 mm), different scolex shape (elongate vs mushroom shape), presence of tissue-associated scolex in A. histocephalum, and absence vs presence of neck, and different type host. Table 2 lists the main ecological, morphological, and morphometrical differences among the known species of Anantrum.
Table 2. Main comparative data available in literature for the valid species of Anantrum (measurements are expressed as the mean with the range in parentheses).
Molecular and phylogenetic analyses
A sequence of 1438 bp of the 28S rDNA gene was obtained from a specimen of A. gallopintoi sp. nov. which was deposited in GenBank under the accession number PP756387. Results of the query of the BLASTn tool showed that sequences possessing the highest degree of similarity were Anantrum sp. PBI_609 (KR780919), A. tortum isolate PBI_014 (KR780883), and A. tortum (AF286941), with 100% of coverage and about 92% of similarity.
BI and maximum likelihood results were identical, showing the same topology and strong clade and subclade supports, hence only BI tree is shown (Figure 6). The BI analysis from the alignment, of final length 1774 bp solved the tree, clearly separating the genera in the family Bothriocephalidae, and distinguishing between the two clades of freshwater and marine taxa (Figure 6). No sequences were available in GenBank for the remaining genera reported in Caira et al. (Reference Caira, Jensen and Barbeau2024) which included Andycestus Kuchta, Scholz & Bray, 2008, Plicatobothrium Cable & Michaelis, 1967, Plicocestus Kuchta, Scholz & Bray, 2008, and Taphrobothrium Lühe, 1899.
Figure 6. Bayesian inference (BI) tree for the analyses based on the 28S rDNA sequences (1774 bp). Nodal support is given as posterior probabilities. The scale bar indicates the expected number of substitutions per site. The sequence generated in this study is shown in bold. Coloured bars represent fish host habitats (green: coastal; red: freshwater; blue: pelagic). Fish outlines represent, from top to bottom, Trachinocephalus myops, Synodus foetens, and S. scituliceps (the Anantrum spp. hosts, see text). *Reassigned to this genus (see Caira et al., Reference Caira, Jensen and Barbeau2024). #Outgroup.
Our new species clustered with other sequences of the genus Anantrum, forming the most basal lineage of the genus. The most closely related taxa are members of the genus Clestobothrium and bothriocephalids from other synodontid fishes, such as Penetrocephalus ganapattii and Oncodiscus sauridae. The tree resolved well the phylogeny of Bothriocephalidae (Figure 6), in agreement with Brabec et al. (Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015), who reconstructed the molecular phylogeny of Bothriocephalidea. Freshwater Bothriocephalidae consisted of two groups, the first comprising Nearctic species of Bothriocephalus sensu lato with Holarctic distribution (Clade J in Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015). The second included Bothriocephalus travassosi Tubangui, 1938 on a separate branch, and subsequently Senga spp. Dollfus, 1934, Schyzocotyle spp. Akhmerov, 1960, and a group of bothriocephalids designated as Clade K in Brabec et al. (Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015). As expected, this group also included Regobothrium microhamulinum Scholz, Takemoto & Kuchta, Reference Scholz, Takemoto and Kuchta2017 which grouped with African taxa (Scholz et al., Reference Scholz, Takemoto and Kuchta2017). Marine taxa comprised almost all parasites of coastal demersal marine teleosts with the exception of Bothriocephalus manubriformis (Linton, 1889) Ariola, 1900. The most basal taxon was represented by Ptychobothrium belones (Dujardin, 1845) Lönnberg, 1889, whereas on the subsequent branch separated Clade G (Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015), then Anantrum species, and finally Bothriocephalus spp. (Clade H according to Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015), which was sister to the three Clestobothrium Lühe, 1899 species available (Fig. 6).
The close relationship between Anantrum species and Clade H + Clestobothrium species was also confirmed by the results of pairwise distances shown in Table 3. The present analysis clearly showed that our sequence was phylogenetically different from other species of Anantrum. In addition to be located on a well-supported separate branch of Anantrum genus, demonstrating its distinctiveness, the described species presented genetic distances between congeners ranging from 0.060 to 0.069 (Table 3), well above commonly used thresholds for species discrimination. In Clestobothrium or Bothriocephalus, smaller differences could be observed among congeners, similar to those between other Anantrum species; thus, the A. gallopintoi sp. nov. higher degree of dissimilarity confirmed its status as new species.
Table 3. Differences among representatives of the genera Anantrum, Bothriocephalus, and Clestobothrium for 28S rDNA sequences, p-distances (below the diagonal), and pairwise nucleotide differences (above the diagonal) (alignment 1774 bp); the sequence generated in this study is shown in bold.
Discussion
The family Bothriocephalidae was established by Bray et al. (Reference Bray, Jones, Andersen, Khalil, Jones and Bray1994) and later slightly modified in the identification key by Kuchta et al. (Reference Kuchta, Scholz and Bray2008b) to accommodate the cestode genera of aquatic environment by having a median genital pore. The present specimens well agree with the diagnostic morphological characters of the genus Anantrum, as previously described in Kuchta et al. (Reference Kuchta, Scholz and Bray2008b). In particular, the main characters used for their identification as belonging to Anantrum were: the spirally twisted strobila with rippled margins, the absence of strobila segmentation, and the elongated scolex without bothria (Bray et al., Reference Bray, Jones, Andersen, Khalil, Jones and Bray1994; Kuchta et al., Reference Kuchta, Scholz and Bray2008b).
Species of the genus Anantrum are parasites with strong host species-specificity for marine teleost of Synodus spp. Members of this genus have been previously identified in S. foetens, S. intermedius, and S. lucioceps along its range of distribution (Linton, Reference Linton1905; Overstreet, Reference Overstreet1968; Rees, Reference Rees1969; Jensen & Heckmann, Reference Jensen and Heckmann1977). An additional undescribed putative new species found in Trachinocephalus myops (previously assigned to the genus Synodus) from the Gulf of Mexico and referred to in GenBank as Anantrum sp. n. PBI_609 (KR780919) has been deposited by Brabec et al. (Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015). The latter is confirmed by our phylogenetic analysis to be a distinct entity from known species of Anantrum waiting to be described as new species.
Morphological, molecular, and phylogenetic analyses of the newly described species supported its status as a new taxon. Anantrum gallopintoi sp. nov. parasitizes S. scituliceps, an endemic species of the eastern Pacific with a distributional range limited to the coasts from Mexico to Chile and the Galapagos Islands. The geographical distribution of the present host does not overlap the distributional ranges of other Synodus and Trachinocephalus spp. in which the other Anantrum spp. have been found (Froese & Pauly, Reference Froese and Pauly2024). According to the present finding, S. scituliceps represents a new host record and Costa Rica a new geographical record for Anantrum species, and, in general, for a bothriocephalid.
The other two known species of the genus Anantrum had historically been described or redescribed based solely on their traditional morphological characters. Unfortunately, they are still poorly known. For instance, in the redescription of A. tortum, Overstreet (Reference Overstreet1968) mentioned that the genital atrium has approximately 30 papillae which are placed in four circular rows. Rees (Reference Rees1969) redescribing the same species mentioned that the genital atrium is lined by tegument raised into prominent closely packed papilliform processes. The present study of the tegumental surface of the genital atrium of A. gallopintoi sp. nov., as revealed by SEM, allowed to detect the presence of some tegumental papilliform processes on its external margin, which could obliterate the pore when contracted, as suggested previously by Rees (Reference Rees1969). Moreover, Linton (Reference Linton1905) did not mention whether the egg of A. tortum is operculate or not, whereas Overstreet (Reference Overstreet1968) and Rees (Reference Rees1969) redescribed the same cestode species with operculate and unoperculate eggs, respectively. All these incongruences suggest that morphological characters of this cestode family should be also investigated using SEM because they may be difficult to assess using traditional microscopy alone, as confirmed in the present study.
Members of the most closely related genera to Anantrum (i.e. Bothriocephalus species in Clade H + Clestobothrium spp.) present remarkable morphological differences and host associations, including hosts with dissimilar biological and ecological features, as well as a wide geographic distribution (Bray et al., Reference Bray, Jones, Andersen, Khalil, Jones and Bray1994; Kuchta et al., Reference Kuchta, Scholz and Bray2008b). In addition, Anantrum species are associated with members of the Synodontinae sub-family presenting significant differences in their distribution and ecology. However, this is not uncommon for Bothriocephalidea, and for Bothriocephalidae in particular (Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015). For example, similar morphological features appear in not closely related lineages across the phylogenetic tree of the Bothriocephalidea, being likely the result of convergent evolution (Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015). Furthermore, the same species might occur in various orders of teleosts, differing in their biology, ecology, habitat, and other characters potentially affecting parasite transmission, whereas, at the same time, be phylogenetically most related to species recovered from very distant geographic locations (e.g. R. microhamulinum) (Scholz et al., Reference Scholz, Takemoto and Kuchta2017). Most Bothriocaphalidae lack morphological synapomorphies and can only be defined unequivocally by a combination of biological and ecological characteristics (Brabec et al., Reference Brabec, Waeschenbach, Scholz, Littlewood and Kuchta2015; Kuchta et al., Reference Kuchta, Scholz and Bray2008b). Therefore, results of the present analysis, in agreement with the latest taxonomic classification of the Bothriocephalidae family, highlights how the integration of molecular and morphological characterisation is essential to clarify taxonomic relationships of these cestodes. Future efforts should be focused on finding novel morphological, biological, and ecological traits, in addition to increasing efforts to expand resolution of molecular markers, to improve taxonomic reliability.
Acknowledgements
We are thankful to Naidely Vidaurre, Gabryele Malcher, Pablo Rojas, and Gilbert Ampie for their assistance during fish sampling, as well as to Alexa Rodriguez and Marialetizia Palomba for their technical support during the fish dissection.
Financial support
Fish sampling was partially supported by the “Proyecto BioMar-ACG (808-B9-508)” of the Centro de Investigación en Ciencias del Mar y Limnología (CIMAR) supported by the Guanacaste Dry Forest Conservation Fund and the University of Costa Rica. The parasitological study was supported by the Stazione Zoologica A. Dohrn (SZ) in the frame of the project between the SZ and the University of Costa Rica aimed at studying the parasite biodiversity of fishes in the coastal waters of Costa Rica.
Competing interest
No potential conflict of interest was reported by the authors.
Ethical standard
The fish collection was authorized by the Ministerio de Ambiente y Energía and Sistema Nacional de Áreas de Conservacion of Costa Rica under the framework of a project of the Centro de Investigación en Ciencias del Mar y Limnología of the University of Costa Rica “Proyecto BioMar-ACG” (permit n. ACG 019-2023). Animal procedures were performed in full compliance with the ethical standards of the relevant national and institutional guides on the care and use for animal experimentation.
