Introduction
Profilicollis Meyer, 1931 encompasses polymorphid acanthocephalans with long necks, females and males with a fully ovoid proboscis, eggs with concentric membranes and decapods as intermediate hosts (Nickol et al., Reference Nickol, Crompton and Searle1999). The species richness of the genus is still unclear due to the taxonomic status of some forms being unstable as well as the lack of revisionary studies. As currently understood (e.g. Brockerhoff & Smales, Reference Brockerhoff and Smales2002; Tantaleán & Cárdenas, Reference Tantaleán and Cárdenas2004; Amin, Reference Amin2013; Goulding & Cohen, Reference Goulding and Cohen2014), the genus encompasses nine species: P. altmani (Perry, 1942), which includes P. bullocki (Mateo, 1982), P. kenti (Van Cleave, 1947) and P. texensis (Webster, 1948) in its synonymy; P. antarcticus Zdzitowiecki, 1985; P. arcticus (Van Cleave, 1920); P. botulus (Van Cleave, 1916) (type species of the genus); P. chasmagnathi (Holcman-Spector, Mane-Garzon & Dei-Cas, 1978); P. formosus (Schmidt & Kuntz, 1967); P. major (Lundström, 1942); P. novaezelandensis Brockerhoff & Smales, 2002; and P. sphaerocephalus (Bremser in Rudolphi, 1819).
Three species, P. altmani, P. antarcticus and P. bullocki, were traditionally recognized as inhabiting the coasts of the South American Pacific Ocean. Profilicollis antarcticus uses the estuarine crab Hemigrapsus crenulatus as intermediate host. Meanwhile, P. altmani and P. bullocki have the mole crab Emerita analoga as intermediate host and different species of sea birds as definitive hosts (Torres et al., Reference Torres, Schlatter, Montefusco, Gesche, Ruiz and Contreras1993; Riquelme et al., Reference Riquelme, George-Nascimento and Balboa2006). A recent phylogeographic study (Goulding & Cohen, Reference Goulding and Cohen2014) corroborates the morphology-based suggestion made by Tantaleán & Cárdenas (Reference Tantaleán and Cárdenas2004) that P. bullocki is a subjective junior synonym of P. altmani. This latter species has a large bi-oceanic distribution and has been recorded along the Pacific coast of North America (California) and South America (Chile) and along the Atlantic coast of North America (Rhode Island, North Carolina) and the Gulf of Mexico (Florida and Mississippi). Meanwhile, for the South American Atlantic coast the thorny-headed worm P. chasmagnathi has been cited with the crab species Neohelice granulata and Cyrtograpsus angulatus as intermediate hosts (Holcman-Spector et al., Reference Holcman-Spector, Mane-Garzon and Dei-Cas1977; Alda et al., Reference Alda, La Sala, Marcotegui and Martorelli2011; La Sala et al., Reference La Sala, Perez and Martorelli2012) and Larus atlanticus and L. dominicanus as definitive hosts (Diaz et al., Reference Diaz, Cremonte and Navone2011; La Sala et al., Reference La Sala, Perez, Smits and Martorelli2013). There is a mention of cystacanths of P. altmani infecting Emerita brasiliensis and Calidris canutus in Rio, southern Brazil (Buehler et al., Reference Buehler, Bugoni, Dorrestein, González, Pereira, Proença, De Lima Serrano, Baker and Piersma2010). However, this record is ambiguous because the parasite species was mentioned in the publication as either Profilicollis sp. or P. altmani, and no indication of the evidence used to identify the specimens was given.
Here, by means of morphological and molecular evidence, we analyse a sample of Profilicollis retrieved from specimens of the mole crab E. brasiliensis collected on the southern Atlantic coast of Uruguay. We assign these specimens to P. altmani, thereby extending the known geographical range of the acanthocephalan, previously shown to have a Pan-American distribution (Goulding & Cohen, Reference Goulding and Cohen2014).
Materials and methods
Thirty-seven specimens of E. brasilensis were collected by hand on two sandy beaches of Uruguay: Cabo Polonio, Rocha (34°24.200′S, 53°47.700′W; December 2014) and Arachania, Rocha (34°37.179′S, 54°8.800′W; January 2016). Mole crabs were stored in 95% ethanol, transported to the laboratory and dissected for the presence of cystacanths. These were placed on distilled water to force osmotic eversion of the proboscis, examined under a stereomicroscope and identified using Nickol et al. (Reference Nickol, Heard and Smith2002).
Genetic comparisons and phylogenetic analyses were based on DNA sequences of the mitochondrial cytochrome oxidase I gene (hereafter COI; a fragment of 578 base pairs (bp)) gathered from four specimens. These sequences were integrated to a matrix composed of all COI sequences of Profilicollis available in GenBank (see fig. 1), with most sequences being recorded by Goulding & Cohen (Reference Goulding and Cohen2014). To enlarge the taxonomic coverage of Profilicollis, we also sequenced two specimens of P. antarcticus retrieved from specimens of the estuarine crab H. crenulatus collected at Niebla, Los Ríos, Chile (39°52.446′S, 73°24.041′W). As such, 206 sequences of Profilicollis were analysed. The outgroup was formed with sequences of two species of Polymorphus, the sister genus of Profilicollis (García-Varela et al., Reference García-Varela, Pérez-Ponce de León, Aznar and Nadler2013). All sequences were obtained using universal primers presented by Folmer et al. (Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994) and the protocol outlined by García-Varela & Nadler (Reference García-Varela and Nadler2006). Amplicons were sequenced using an external sequencing service (Macrogen, Inc. Seoul, South Korea). New DNA sequences were edited using CodonCode (Codon-Code, Dedham, Massachusetts, USA) and deposited in GenBank (KU928251–KU928256).
Genealogical relationships of haplotypes of the COI gene of the genus Profilicollis using Bayesian analysis; support values correspond to posterior probability (only those for species and relationships among species are shown) with GenBank accession numbers given at the terminal labels. NP, northern Pacific Ocean; SP, southern Pacific Ocean; NA, northern Atlantic Ocean; UY, Uruguayan Atlantic Ocean.
Sequences were aligned in MEGA 6 (Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013) using default parameter values. Observed genetic distances (p) were calculated in MEGA 6. Phylogenetic relationships were inferred via Bayesian analyses (BA) using MrBayes 3.1 (Ronquist & Huelsenbeck, Reference Ronquist and Huelsenbeck2003). To reduce computational time we analysed a matrix consisting of only one sequence per haplotypic class (e.g. both specimens of P. antarcticus analysed share the same haplotype), as such the matrix included 77 sequences of Profilicollis. Analysis of each matrix consisted of two independent runs, each with five heated and one cold Markov chain. The substitution model HKY + G was selected using jModelTest (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012). All model parameters were estimated in MrBayes. Uniform-interval priors were assumed for all parameters except base composition and substitution model parameters, which assumed a Dirichlet prior. Runs were allowed to proceed for 20 million generations, with trees sampled every 1000 generations per chain. To check for convergence on a stable log-likelihood value, we plotted the log-likelihood values against generation time for each. The first 25% of the trees were discarded as burn-in and the remaining trees were used to compute a 50% majority rule consensus tree and to obtain posterior probability (PP) estimates for each clade.
Results and discussion
Of the 37 specimens of E. brasiliensis examined, three were infected with Profilicollis (prevalence = 8.1%), with one, one and two parasites each (total = 4; mean intensity = 1.3). These four Profilicollis were sequenced and three distinct haplotypes were found. Observed average divergence of the Uruguayan sequences is 1.0%. In the genealogical analysis the haplotypes do not form a monophyletic group (fig. 1) and fall within the large clade of P. altmani, which is strongly supported (PP = 1). As such, the Bayesian analysis of DNA sequences suggests that cystacanths recovered from E. brasiliensis on the southern Atlantic coast belong to the same species, P. altmani, that has already been recorded on the Californian and Chilean Pacific coasts, as well as on the Atlantic coast of North America. Observed p distances among species of Profilicollis ranged from 22.2 to 25.1%.
A morphological assessment of the cystacanths collected in Uruguay points in the same direction, as it clearly differentiates them from P. chasmagnathi and P. bullocki, the other southern South American thorny-headed worms, in the number of hooks per head row as well as in the shape of the proboscis (table 1; Holcman-Spector et al., Reference Holcman-Spector, Mane-Garzon and Dei-Cas1977; Balboa et al., Reference Balboa, Hinojosa, Riquelme, Rodríguez, Bustos and George-Nascimento2009). Cystacanths collected in Uruguay have an elongated body and an ovoid proboscis, with 26–29 longitudinal rows of 14–15 hooks each. Similarly, cystacanths from the Pacific coast of Chile have between 14 and 16 hooks per row (Balboa et al., Reference Balboa, Hinojosa, Riquelme, Rodríguez, Bustos and George-Nascimento2009; reported as P. bullocki). Interestingly, specimens of P. altmani from the Atlantic and Pacific coasts of the USA have 12 hooks in each longitudinal row (Nickol et al., Reference Nickol, Heard and Smith2002).
Morphometrics (measurements in μm and mean values given in brackets) of cystacanths of Profilicollis altmani from the USA, Chile and Uruguay.
Taken as a whole, the evidence suggests that cystacanths recovered from E. brasiliensis along the southern Atlantic coast belong to the same species, P. altmani, already recorded on the Californian and Chilean Pacific coast as well as on the Atlantic coast of North America. These results confirm that the acanthocephalan P. altmani has a Pan-American distribution with much variation (table 1), although the biological significance of this at the intermediate host stage remains unclear.
We also enlarge the list of intermediate hosts of P. altmani. Goulding & Cohen (Reference Goulding and Cohen2014) reported cystacanths of P. altmani parasitizing the mole crab species E. analoga, E. talpoidea, E. benedicti and E. rathbunae. Here we show that P. altmani also parasitizes E. brasiliensis (see also Buehler et al., Reference Buehler, Bugoni, Dorrestein, González, Pereira, Proença, De Lima Serrano, Baker and Piersma2010).
As on the southern Pacific coast of South America, on the South American Atlantic coast two species of Profilicollis are also found. Profilicollis chasmagnathi uses crabs from estuarine environments as intermediate hosts (Holcman-Spector et al., Reference Holcman-Spector, Mane-Garzon and Dei-Cas1977; Martorelli, Reference Martorelli1989; La Sala et al., Reference La Sala, Perez and Martorelli2012) and the gulls L. atlanticus and L. dominicanus as definitive hosts (e.g. Díaz et al., Reference Diaz, Cremonte and Navone2011; La Sala et al., Reference La Sala, Perez, Smits and Martorelli2013). Profilicollis altmani has the mole crab E. brasiliensis, which inhabits sandy beaches, as intermediate host. Future studies should clarify the definitive host of P. altmani in this environment (but see Buehler et al., Reference Buehler, Bugoni, Dorrestein, González, Pereira, Proença, De Lima Serrano, Baker and Piersma2010, who reported a mortality event of the sandpiper Calidris canutus that may be linked to the presence of P. altmani). Finally, the recording of P. altmani on the South American Atlantic coast is of relevance, given that infection with this parasite has caused the mortality of marine mammals in North America (Kreuder et al., Reference Kreuder, Miller, Jessup, Lowenstine, Harris, Ames, Carpenter, Conrad and Mazet2003; Mayer et al., Reference Mayer, Murray and Miller2003). Similarly, there are suggestions that P. altmani can infect humans and domestic animals that accidently ingest cystacanths when eating mole crabs (Tantaleán et al., Reference Tantaleán, Cárdenas and Güere2002; Rojas & Sebastián, Reference Rojas and Sebastián2011).
Acknowledgements
Danilo Calliari (Uruguay) kindly provided the sample of Emerita brasiliensis from Cabo Polonio. Kennia Morales assisted us in the laboratory work. Two anonymous reviewers made valuable suggestions on an earlier version of this paper.
Financial support
This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (grant number FONDECYT 1141055).
Conflict of interest
None.
