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Genetic characterization of members of the genus Contracaecum (Nematoda: Anisakidae) from fish-eating birds from west-central Florida, USA, with evidence of new species

Published online by Cambridge University Press:  13 March 2007

S. D'AMELIO ,
N. B. BARROS ,
S. INGROSSO ,
D. A. FAUQUIER ,
R. RUSSO  and
L. PAGGI
S. D'AMELIO*
Affiliation:
Department of Public Health Science, University of Rome “La Sapienza”, P. le Aldo Moro, 5, 00185 Rome, Italy
N. B. BARROS
Affiliation:
Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL34236, USA
S. INGROSSO
Affiliation:
Department of Public Health Science, University of Rome “La Sapienza”, P. le Aldo Moro, 5, 00185 Rome, Italy
D. A. FAUQUIER
Affiliation:
Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL34236, USA
R. RUSSO
Affiliation:
Department of Pathology, University of California San Diego, La Jolla, CA 92093, USA
L. PAGGI
Affiliation:
Department of Public Health Science, University of Rome “La Sapienza”, P. le Aldo Moro, 5, 00185 Rome, Italy
*
*Corresponding author: Department of Public Health Science, University of Rome “La Sapienza”, P. le Aldo Moro, 5, 00185, Rome, Italy. Tel: +39 0649914671. Fax: +39 0649914644. E-mail: stefano.damelio@uniroma1.it
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Summary

Specimens of Contracaecum spp. from Phalacrocorax auritus and Pelecanus occidentalis from Florida were characterized by sequencing of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) and by PCR-based RFLP analysis of the same gene and of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Analyses of the rrnS sequence data using the MP and UPGMA approaches yielded trees with similar topologies, delineating 3 main clusters. Specimens from Ph. auritus, morphologically assigned to C. rudolphii (s.l.), were part of the cluster comprising also the other 2 species of the C. rudolphii complex (A and B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex, provisionally named as C. rudolphii C. The second cluster comprised 5 individuals from P. occidentalis, which formed a genetically relatively homogeneous group. The rrnS data indicate that these specimens (indicated as Contracaecum sp. 1) are clearly genetically different from the morphologically most closely related species, i.e. C. rudolphii (s.l.) and C. microcephalum, and could represent a new species. The third cluster comprised 7 specimens from P. occidentalis morphologically assigned to C. multipapillatum (s.l.). These were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece, although additional studies are needed to assess their status. PCR-RFLP based markers for the quick identification of these taxa are provided.

Keywords

ContracaecumPhalacrocorax auritusPelecanus occidentalisnuclear and mitochondrial ribosomal DNAFloridaUSA
Type
Research Article
Information
Parasitology , Volume 134 , Issue 7 , July 2007 , pp. 1041 - 1051
Copyright
Copyright © Cambridge University Press 2007

INTRODUCTION

Contracaecum Railliet and Henry, 1912 is one of the largest ascaridoid genera comprising approximately 50 species. Most of these are (at adult-stage) parasites of fish-eating birds, whereas some are parasites of seals and dolphins. According to a recent molecular phylogenetic study (Nadler et al. Reference Nadler, D'Amelio, Fagerholm, Berland and Paggi2000), the latter are more closely related to species of the genus Phocascaris Høst, 1932, from marine mammals than to Contracaecum from birds.

Avian Contracaecum are commonly reported in all regions of the world and occur in a very large number of host species (Mozgovoi, Reference Mozgovoi1953; Hartwich, Reference Hartwich1964; Barus et al. Reference Barus, Sergeeva, Sonin and Ryzhikov1978). Previous multilocus enzyme electrophoretic studies of Contracaecum rudolphii Hartwich, Reference Hartwich1964 (D'Amelio et al. Reference D'Amelio, Nascetti, Mattiucci, Cianchi, Orecchia, Paggi, Berland and Bullini1990; Cianchi et al. Reference Cianchi, Orecchia, Berland, Paggi, D'Amelio, Mattiucci, Nascetti and Bullini1992; Mattiucci et al. Reference Mattiucci, Turchetto, Bragantini and Nascetti2002) have revealed a marked genetic heterogeneity in several genetic loci for specimens collected from Phalacrocorax carbo sinensis (the Eurasian subspecies of the great cormorant) from different geographical localities. These results suggested the co-existence of multiple genetically distinct, reproductively isolated operational taxonomic units (sibling species), namely C. rudolphii A and C. rudolphii B. C. rudolphii A predominantly infects cormorants dwelling in brackish waters of coastal lagoons in Europe, whereas C. rudolphii B is harboured by cormorant colonies living in freshwater lagoons in central Europe (Mattiucci et al. Reference Mattiucci, Turchetto, Bragantini and Nascetti2002). The evaluation of the first (ITS-1) and/or second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) has yielded genetic markers for the specific identification of a range of anisakids from various host groups, including birds (D'Amelio et al. Reference D'Amelio, Mathiopoulos, Santos, Pugachev, Webb, Picanço and Paggi2000; Zhu et al. Reference Zhu, D'Amelio, Paggi and Gasser2000, Reference Zhu, D'Amelio, Hu, Paggi and Gasser2001, Reference Zhu, D'Amelio, Palm, Paggi, George-Nascimento and Gasser2002; Hu et al. Reference Hu, D'Amelio, Zhu, Paggi and Gasser2001; Abollo et al. Reference Abollo, Paggi, Pascual and D'Amelio2003). More recently, a study employing markers in the ITS (Li et al. Reference Li, D'Amelio, Paggi, He, Gasser, Lun, Abollo, Turchetto and Zhu2005) has provided additional support for the hypothesis that C. rudolphii represents a complex of at least 2 sibling species and that Contracaecum septentrionale Kreis, 1955, from Phalacrocorax aristotelis (shag), is a valid, separate species. Extending previous studies, the present investigation focused on the genotypic identification of individual specimens of Contracaecum from the double-crested cormorant (Phalacrocorax auritus) and the brown pelican (Pelecanus occidentalis) from west-central Florida, USA, and compared them with members of the same genus characterized previously using morphological and molecular approaches.

MATERIALS AND METHODS

Parasites

A total of 117 nematodes, comprising 95 from 20 double-crested cormorants (Ph. auritus) and 22 from 4 brown pelicans (P. occidentalis) from Sarasota Bay, on the Gulf coast of Florida (Sarasota and Manatee Counties), were used in the present study (see Table 1). The birds included immatures and adults found dead or which were euthanized in 2004 and 2005 during periods of prolonged and intense ‘red tide’ caused by the dinoflagellate Karenia brevis, which also impacted on marine mammals (cf. Flewelling et al. Reference Flewelling, Naar, Abbott, Baden, Barros, Bossart, Bottein, Hammond, Haubold, Heil, Henry, Jacocks, Leighfield, Pierce, Pitchford, Rommel, Scott, Steindinger, Truby, Van Dolah and Landsberg2005). Nematodes collected from the stomach of these fish-eating birds at necropsy were placed in 70% ethanol. The anterior and posterior ends of individual specimens were removed, cleared in lactophenol for morphological studies, and the remaining part of each worm was used for molecular analysis. For morphological identification, characters considered of diagnostic value for anisakid nematodes (Fagerholm, Reference Fagerholm1991) and those proposed for Contracaecum from birds (Barus et al. Reference Barus, Sergeeva, Sonin and Ryzhikov1978) were considered (e.g., morphology of lips and interlabial tips, length of spicule and morphology of the spicule tip, and patterns of the male caudal papillae). The specimens from double-crested cormorants were identified as Contracaecum rudolphii (s.l.). Fifteen specimens from the brown pelican were identified as Contracaecum multipapillatum (Von Drasche, 1882) Baylis, 1920, whereas 7 specimens did not correspond to any of the described species of Contracaecum, displaying some characters consistent with C. rudolphii or of Contracaecum microcephalum (Rudolphi, 1809).

Table 1. Parasite specimens examined in the present study (C-cor and Pel) and those used for comparative purposes (Pc, A-cor, B-cor, Php), including information regarding their hosts and geographical origin (Cor=cormorant, Pel=pelican, Pc=Pelecanus crispus, Php=Phalacrocorax pygmaeus)

(Representative sequences for each taxon have been submitted to GenBank and have the following Accession numbers: EF014283 (C-cor 52), EF030717 (Pel28), EF030716 (Pel3), EF014280 (Pc1), EF014281 (A-cor31), EF014279 (B-cor41), EF014282 (Php1).)

Twenty-eight specimens used for comparative purposes were identified by microscopy and by using genetic markers defined previously in the internal transcribed spacers of nuclear ribosomal DNA (Li et al. Reference Li, D'Amelio, Paggi, He, Gasser, Lun, Abollo, Turchetto and Zhu2005), and belonged to C. rudolphii complex (A and B) from the great cormorant (Phalacrocorax carbo sinensis) from northeastern Italy, C. multipapillatum from the Dalmatian pelican (Pelecanus crispus) from Greece and C. microcephalum from the small cormorant (Phalacrocorax pygmaeus) from Montenegro (see Table 1).

Isolation of genomic DNA

DNA was isolated using the Wizard® Genomic DNA purification kit (Promega), according to the manufacturer's protocol. In brief, body portions from individual nematodes were each placed in 600 μl of a mixture containing 0·5 m ethylene diamine tetraacetic acid (EDTA) plus Nuclei Lysis solution and then crushed employing a sterile pestle. An aliquot of 17·5 μl of proteinase K (20 mg/ml; Promega) was added to each tube, which was incubated at 55°C for 3 h. An aliquot of 3 μl of RNase solution (4 mg/ml) was added, and the tubes were incubated at 37°C for 30 min. Subsequently, 200 μl of protein precipitation solution were added, the tubes vortexed and chilled on ice for 5 min, and the DNA precipitated with ethanol. Each DNA pellet was air-dried for 20 min and dissolved in 100 μl of DNA rehydration solution.

PCR amplification

The ITS (plus intervening 5·8S rRNA gene) was amplified by PCR using 4·0 μl of template DNA (20–40 ng), 10 mm Tris-HCl (pH 8·3), 50 mm KCl (Applied Biosystems), 3 mm MgCl2 (Applied Biosystems), 1 mm of dNTPs (Promega), 50 pm of each the forward primer NC5 (5′-GTAGGTGAACCTGCGGAAGGATCATT-3′) and the reverse primer NC2 (5′-TTAGTTTCTTCCTCCGCT-3′) (Zhu et al. Reference Zhu, D'Amelio, Paggi and Gasser2000) and 2·5 U of AmpliTaq Gold™ (Applied Biosystems) in a final volume of 50 μl. The PCR was performed in a GeneAmp PCR System 2400 (Applied Biosystems) under the following conditions: 10 min at 95°C (initial denaturation), 30 cycles of 30 sec at 95°C (denaturation), 40 sec at 52°C (annealing) and 75 sec at 72°C (extension), and a final elongation step of 7 min at 72°C. The amplification of the rrnS was performed using 4·0 μl of template DNA (20–40 ng), 10 mm Tris-HCl (pH 8·3), 50 mm KCl (Applied Biosystems), 3 mm MgCl2 (Applied Biosystems), 1 mm of dNTPs (Promega), 50 pm of the forward primer MH3, 5′-TTGTTCCAGAATAATCGGCTAGACTT, 50 pm of the reverse primer MH4.5, 5′-TCTACTTTACTACAACTTACTCC) and 0·5 μl of AmpliTaq Gold™ (Promega) in a 50 μl final volume of reaction. The conditions of PCR were as follows: 10 min at 95°C (initial denaturation), 35 cycles of 30 sec at 95°C (denaturation), 30 sec at 55°C (annealing) and 30 sec at 72°C (extension), and a final elongation step of 7 min at 72°C.

A negative control (i.e., without genomic DNA) was included in each amplification. Aliquots (5 μl) of individual PCR products were detected on agarose gels (1%), stained with ethidium bromide (10 mg/ml) and detected upon ultraviolet transillumination. Gel images were captured electronically and analysed using the program MULTI-ANALYST (v.1.1, Bio-Rad).

Sequencing and analysis

Twenty-four rrnS amplicons representing the 117 nematode specimens from Florida (indicated as C-cor and Pel in Table 1) were purified by SureClean Product Insert (Bioline), following the manufacturer's instructions. The pellets were re-suspended in 30 μl of H2O and subjected to automated sequencing using BigDye ver. 3.1 Terminator chemistry in a 3730xl DNA sequencer (Applied Biosystem). Nucleotide sequences were aligned using the program ClustalX (Thompson et al. Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997) and adjusted manually after careful checking for misalignments. Analyses were conducted using MEGA (version 3.1). A UPGMA tree was constructed based on Kimura's 2-parameter model (Gamma) (Kimura, Reference Kimura1980). A maximum parsimony (MP) analysis was conducted using an heuristic search (Close-Neighbor-Interchange), and a 70% majority-rule consensus tree was obtained. The robustness of clades of UPGMA and MP trees was assessed using 1000 bootstrap replications. Contracaecum osculatum A, a species reported from pinnipeds, was chosen as an outgroup. The monophyly of Contracaecum species from pinnipeds and their differentiation from avian Contracaecum has been demonstrated previously (Nadler et al. Reference Nadler, D'Amelio, Fagerholm, Berland and Paggi2000).

PCR-RFLP analysis

For each DNA locus, amplicons (95 from specimens from Ph. auritus and 22 from those from P. occidentalis) were digested with the restriction endonuclease Tsp509I (for the ITS), or RsaI or DdeI (for rrnS). Digests were resolved by electrophoresis in 2% agarose gels, stained with ethidium bromide (10 mg/ml), detected upon transillumination and the sizes of fragments determined by comparison with a 100 bp DNA ladder as size marker (Promega).

RESULTS

The rrnS sequences from 24 nematodes from double-crested cormorants and brown pelicans from west-central Florida were aligned with those from the 2 sibling species of the C. rudolphii complex (i.e., C. rudolphii A and C. rudolphii B) from great cormorants (Ph. carbo sinensis) from northeastern Italy, from C. multipapillatum from Dalmatian pelicans (P. crispus) from Greece and from C. microcephalum from small cormorants (P. pygmaeus) from Montenegro. The sequences had an A+T content of 70·0% in average, consistent with percentages obtained previously for mitochondrial genes in ascaridoid nematodes (Hu et al. Reference Hu, D'Amelio, Zhu, Paggi and Gasser2001). Analyses of the mitochondrial rrnS sequence data using the MP (70% majority-rule bootstrap consensus) (Fig. 1) and UPGMA (Fig. 2) approaches yielded trees with similar topologies, delineating 3 main clusters.

Fig. 1. Maximum Parsimony 70% majority-rule bootstrap consensus tree based on rrnS gene sequences. Bootstrap percentages of clades (based on 1000 iterations) are shown at internal nodes.

Fig. 2. UPGMA tree constructed on a genetic distance matrix based on Kimura's 2-parameter model (Gamma), based on rrnS gene sequences. Bootstrap percentages of clades (based on 1000 iterations) are shown at internal nodes.

Cluster 1

The first cluster (cf. Figs 1 and 2), comprises specimens assigned to the morphospecies C. rudolphii (s.l.) and contains 3 subclusters. A bootstrap value of 100 confirmed the robustness of this clustering. Two of these subclusters comprised specimens of C. rudolphii A and C. rudolphii B from cormorants from northeastern Italy, previously characterized by sequencing of the ITS-1 and ITS-2 (cf. Li et al. Reference Li, D'Amelio, Paggi, He, Gasser, Lun, Abollo, Turchetto and Zhu2005). All of the individuals from the double-crested cormorants from Florida formed a single cluster (referred to henceforth as C. rudolphii C), which was distinct from those containing C. rudolphii A and C. rudolphii B.

RFLP analysis of representative rrnS amplicons using RsaI (Fig. 3) produced 4 fragments of 271, 81, 64 and 37 bp for C. rudolphii C, a pattern similar to that of C. rudolphii B (270, 82, 64 and 49 bp) but distinct from C. rudolphii A, which had fragments of 330, 110 and 49 bp. To distinguish C. rudolphii B from C. rudolphii C, amplicons were digested with DdeI. Analysis of amplicons from specimens from Florida displayed fragments of 199, 175 and 84 bp, whereas C. rudolphii A and B revealed fragments of 294 and 183 bp (Fig. 4). Hence, the use of the endonucleases RsaI and DdeI provided a reliable means of delineating the 3 taxa within C. rudolphii (s.l.) by PCR-RFLP. In order to provide independent support for the existence of C. rudolphii C within the complex, PCR-RFLP analysis of ITS amplicons with Tsp509I was conducted, yielding fragments of 480, 220, 170 and 80 bp, compared with fragments of 330, 220, 170 and 80 bp for C. rudolphii A and fragments of 480, 360 and 80 bp for C. rudolphii B (Fig. 5). Using the PCR-RFLP analyses of rrnS and ITS (employing the 3 enzymes), all 95 specimens from Ph. auritus from Florida were identified as C. rudolphii C.

Fig. 3. A representative gel displaying the RFLP profiles following the digestion of rrnS amplicons with restriction endonuclease RsaI. 1–3 C. rudolphii B; 4–6 C. rudolphii A; 7–19 C. rudolphii C; M 100 bp DNA ladder.

Fig. 4. A representative gel displaying the RFLP profiles following the digestion of rrnS amplicons with restriction endonuclease DdeI. 1–2 C. rudolphii B; 3–4 C. rudolphii A; 5–7, 9–15, 17 C. rudolphii C; 8, 16, 18 C. multipapillatum (Florida), 19 C. microcephalum; M 100 bp DNA ladder.

Fig. 5. A representative gel displaying the RFLP profiles following the digestion of ITS amplicons with restriction endonuclease Tsp509I. 1–3 C. rudolphii A; 4–6 C. rudolphii B; 7–19 C. rudolphii C; M 100 bp DNA ladder.

Cluster 2

A second cluster, comprising 5 individual specimens from pelicans from Florida (Pel2, Pel3, Pel9, Pel10 and Pel20), was shown to represent a genetically unique entity and thus designated as Contracaecum sp. 1, as distinct from all 3 currently recognized members of the C. rudolphii complex and from C. microcephalum (code Php1) from a small cormorant (Ph. pygmaeus) from the Scutari Lake (Montenegro). There was 70–100% support for Contracaecum sp. 1 being a distinct group (Figs 1 and 2). The position of specimen Pel20 was uncertain, grouping within or external to cluster 2 (Figs 1 and 2).

RFLP analysis of representative rrnS amplicons using DdeI yielded distinct patterns between C. microcephalum and specimens Pel2, Pel3, Pel9 and Pel10. However, a single mutation at nucleotide position 278 for specimen Pel20 revealed the absence of a site for DdeI, such that the profile for the latter specimen was identical to that of C. microcephalum. PCR-RFLP analysis of ITS amplicons with Tsp509I produced fragments of 550 and 380 bp for Contracaecum sp. 1 (Fig. 6, lane 3), as distinct from the patterns produced for each of the 3 recognized members of the C. rudolphii complex (Fig. 6, lanes 4, 5 and 6) and from that of C. microcephalum (Fig. 6, lane 2).

Fig. 6. A representative gel displaying the RFLP profiles following the digestion of the ITS amplicons with restriction endonuclease Tsp509I. 1 C. multipapillatum (Florida); 2 C. microcephalum; 3 Contracaecum sp. 1; 4 C. rudolphii A; 5 C. rudolphii B; 6 C. rudolphii C; M 100 bp DNA ladder.

Cluster 3

A third cluster, well defined by UPGMA tree analysis, comprised the sequence of C. multipapillatum from P. crispus from Greece (specimen's code Pc1) and the remaining individuals from P. occidentalis from Florida (Pel1, Pel4, Pel6, Pel7, Pel12, Pel18 and Pel28). In the MP tree (Fig. 1), specimen Pc1 was related to, but not a sister to, specimens from P. occidentalis. These specimens, morphologically identified as C. multipapillatum, were genetically ‘homogeneous’ but distinct from C. multipapillatum from Greece. Henceforth, they are referred to as C. multipapillatum (Florida). PCR-RFLP analysis of ITS amplicons with Tsp509I produced fragments of 330, 240, 150 and 110 bp for C. multipapillatum (Florida). The same analysis identified 15 specimens from P. occidentalis from Florida as C. multipapillatum (Florida) and 7 specimens as Contracaecum sp. 1.

Mean genetic distance

A matrix of mean genetic distances based on the rrnS sequences (Kimura 2-parameters index), among members grouped according to the topology of the rrnS tree, is given in Table 2. The values of genetic distances between taxa of cluster 1, corresponding to the members of the C. rudolphii complex, range between 0·043 and 0·063, and are the lowest values observed in this study, consistent with their present status as sibling species. Higher values were calculated within and among members of the other clusters, ranging from 0·126 between C. microcephalum and Contracaecum sp. 1, to 0·192 between C. multipapillatum (s.l.) from Greece and Contracaecum sp. 1.

Table 2. Matrix of genetic distances, using Kimura 2-parameters index, between taxa based on groups identified by UPGMA and Maximum Parsimony analysis

Preliminary morphological examination of C. rudolphii C and Contracaecum sp. 1

Morphological examination of the 2 taxa supports their genetic identification. Specifically, C. rudolphii C differs significantly from both C. rudolphii A and C. rudolphii B in the length of the spicules, which shows non-overlapping values (A: 6·50–7·90, B: 8·11–10·00, C: 5·20–5·96, in mm). Contracaecum sp. 1 differs from all congeners other than C. microcephalum and C. rudolphii (s.l.). Contracaecum sp. 1 can be distinguished from C. microcephalum by (i) the length of spicules (in mm) being longer (4·82–5·83) than the latter (1·43–3·65) according to the descriptions by Hartwich (Reference Hartwich1964) and Barus et al. (Reference Barus, Sergeeva, Sonin and Ryzhikov1978) and by (ii) the shape of the distal tip of the spicule having a longer free distal tip (distance of most distal insertion of alae to rounded distal point of spicules). This new species can be distinguished from C. rudolphii sensu Hartwich, Reference Hartwich1964 by (i) interlabial tips rounded and not bifurcated, (ii) lips slightly longer than wide, and (iii) dorsal lip with a slight deepening on upper margin rather than a deep depression. A formal description of the 2 species and a detailed analysis of the morphological differentiation of C. rudolphii C from the other members of the C. rudolphii complex and of Contracaecum sp. 1 from congeners will be the subject of separate articles.

DISCUSSION

There are few records of Contracaecum species from the double-crested cormorant from the southeastern USA. The following species have been recorded: C. multipapillatum from Louisiana (Deardorff and Overstreet, Reference Deardorff and Overstreet1980), Contracaecum specimens identified as C. microcephalum from Mississippi, Louisiana and Florida (Deardorff and Overstreet, Reference Deardorff and Overstreet1980) but with a spicule length and a tip morphology in accordance with C. rudolphii.

Several studies of members of the genus Contraceacum from the brown pelican from the Gulf of Mexico and Caribbean Sea have been carried out, and the following records exist: C. mexicanum, a species described from Acapulco, Mexico (Flores–Barroeta, Reference Flores-Barroeta1957), from Venezuela (Diaz-Ungria, Reference Diaz-Ungria1978, Reference Diaz-Ungria1979) and Puerto Rico (Dyer et al. Reference Dyer, Williams, Mignucci-Giannoni, Jiménez-Marrero, Bunkley-Williams, Moore and Pence2002); C. multipapillatum from the Gulf of Mexico (Courtney and Forrester, Reference Courtney and Forrester1974; Courtney et al. Reference Courtney, Forrester and White1977; Deardoff and Overstreet, Reference Deardorff and Overstreet1980; Grimes et al. Reference Grimes, Suto, Greve and Albers1989), and Puerto Rico (Dyer et al. Reference Dyer, Williams, Mignucci-Giannoni, Jiménez-Marrero, Bunkley-Williams, Moore and Pence2002); C. rudolphii (sometimes reported as C. spiculigerum) from the Gulf of Mexico (Hutton, Reference Hutton1964; Huizinga, Reference Huizinga1966, Reference Huizinga1971; Courtney and Forrester, Reference Courtney and Forrester1974; Deardoff and Overstreet, Reference Deardorff and Overstreet1980) and Puerto Rico (Bunkley-Williams and Williams, Reference Bunkley-Williams and Williams1994); Contracaecum sp. or spp. from the Gulf of Mexico (Courtney et al. Reference Courtney, Forrester and White1977; Humphrey et al. Reference Humphrey, Courtney and Forrester1978; Deardorff and Overstreet, Reference Deardorff and Overstreet1980; Greve et al. Reference Greve, Albers, Suto and Grimes1986; Dronen et al. Reference Dronen, Blend and Anderson2003).

In the present study, the genetic data obtained support the existence of 2 new species, and possibly a third. The first new taxon comprises nematodes collected from the stomach of double-crested cormorants from Florida, morphologically assigned to C. rudolphii (s.l.). These specimens were found to be part of the cluster comprising the other 2 species of the complex (C. rudolphii A and C. rudolphii B), but representing a genetically distinct group, potentially corresponding to a distinct lineage within the complex. Although allopatric with respect to the existing species of the C. rudolphii complex, described from central and southern Europe and, recently, from Qinghai Lake, China (Zhu et al. Reference Zhu, D'Amelio, Gasser, Yang, Paggi, He, Lin, Song, Ai and Li2007), this third clade seems to represent a new species within the C. rudolphii complex (here provisionally named as C. rudolphii C). In anisakid nematodes, speciation processes, and associated levels of genetic variation, are not always followed by morphological differentiation. Speciation processes have led to a number of sibling species within this group of nematodes, including 3 species within Anisakis simplex (s.l.) (Mattiucci et al. Reference Mattiucci, Nascetti, Cianchi, Paggi, Arduino, Margolis, Brattey, Webb, D'Amelio, Orecchia and Bullini1997), 5 species within Pseudoterranova decipiens (s.l.) (Paggi et al. Reference Paggi, Nascetti, Cianchi, Orecchia, Mattiucci, D'Amelio, Berland, Brattey, Smith and Bullini1991, Reference Paggi, Mattiucci, Gibson, Berland, Nascetti, Cianchi and Bullini2000; Mattiucci et al. Reference Mattiucci, Paggi, Nascetti, Ishikura, Kikuchi, Sato, Cianchi and Bullini1998; Zhu et al. Reference Zhu, D'Amelio, Palm, Paggi, George-Nascimento and Gasser2002), 5 species within C. osculatum (s.l.) (Nascetti et al. Reference Nascetti, Cianchi, Mattiucci, D'Amelio, Orecchia, Paggi, Brattey, Berland and Smith1993; Orecchia et al. Reference Orecchia, Mattiucci, D'Amelio, Paggi, Plotz, Cianchi, Nascetti, Arduino and Bullini1994; Zhu et al. Reference Zhu, D'Amelio, Paggi and Gasser2000; Hu et al. Reference Hu, D'Amelio, Zhu, Paggi and Gasser2001). Regarding anisakids from fish-eating birds, sibling species (C. rudolphii A and C. rudolphii B) were proposed within the C. rudolphii complex using allozyme markers (D'Amelio et al. Reference D'Amelio, Nascetti, Mattiucci, Cianchi, Orecchia, Paggi, Berland and Bullini1990; Mattiucci et al. Reference Mattiucci, Turchetto, Bragantini and Nascetti2002) and later confirmed by rDNA sequence data (Li et al. Reference Li, D'Amelio, Paggi, He, Gasser, Lun, Abollo, Turchetto and Zhu2005). The assessment at species level classification based on haploid data has been discussed recently by Padgett et al. (Reference Padgett, Nadler, Munson, Sacks and Boyce2005), who stated that “genetically defined clades, corresponding to distinct evolutionary lineages are consistent with their recognition as separate species”. Also, the genetic differentiation of the C. rudolphii C clade from both C. rudolphii A and C. rudolphii B based on nuclear (diploid) ITS data clearly supports its recognition as a distinct species within the complex.

C. rudolphii C is a sister taxon to C. rudolphii A. From a geographical perspective, one could expect that species A and B should be more related to each other, as both species are found in sympatry in Italy. However, species A and B have a broad geographical distribution. For instance, C. rudolphii A has been detected in the Atlantic coast of Spain (Abollo et al. Reference Abollo, Gestal and Pascual2001), whereas C. rudolphii B has been recorded in inland waters in central Europe and China (Zhu et al. Reference Zhu, D'Amelio, Gasser, Yang, Paggi, He, Lin, Song, Ai and Li2007). From an ecological viewpoint, Mattiucci et al. (Reference Mattiucci, Turchetto, Bragantini and Nascetti2002) considered C. rudolphii A as a species occurring in brackish waters, as is C. rudolphii C, in contrast to C. rudolphii B which occurs mostly in freshwater habitats. Therefore, it is possible that the tree topology representing the 3 sibling species reported in this study may reflect ecological niches rather than their geographical distributions.

Five individuals from P. occidentalis from Florida formed a genetically relatively homogeneous group (Contracaecum sp. 1) compared with members of the C. rudolphii complex as well as C. microcephalum, indicating the existence of a distinct cluster. The rrnS sequence data indicate that these specimens are clearly genetically different both from all current members of the C. rudolphii complex and from C. microcephalum, and may represent an additional species. Morphologically, such specimens have no bifurcated interlabia as is typical for C. microcephalum, but their spicule length and spicule tip shape is consistent with these features for C. rudolphii. Specimens showing the same morphological characters were described by Deardorff and Overstreet (Reference Deardorff and Overstreet1980) from Ph. auritus and P. occidentalis from the northern Gulf of Mexico, suggesting that they could represent a new species. The possibility of a new species of Contracaecum from white and brown pelicans from Galveston Bay, Texas, has also been proposed by Dronen et al. (Reference Dronen, Blend and Anderson2003).

Seven specimens from P. occidentalis from Florida were shown to be genetically homogeneous and related to but quite distinct from C. multipapillatum from Greece. According to their morphology, these specimens have shown to be related to C. multipapillatum (s.l.), a species that may represent a complex of species, according to preliminary data presented by Nascetti et al. (Reference Nascetti, Mattiucci, Cianchi, Berland, Bullini and Paggi2000). A species similar morphologically to C. multipapillatum and named C. mexicanum was described by Flores–Barroeta (Reference Flores-Barroeta1957). However, the original description of C. mexicanum reports spicule lengths ranging from 2·6 to 2·8 mm, whereas the individuals studied herein possessed spicule lengths ranging from 1·62 to 2·10 mm, consistent with C. multipapillatum (s.l.). The 7 specimens examined in the present study may be members of a distinct taxon within a C. multipapillatum complex, although further investigation is needed to test this hypothesis.

The 2 host species (the double-crested cormorant and the brown pelican) investigated herein were shown to harbour multiple genetically distinct groups/species of Contracaecum, i.e., C. rudolphii C in the double-crested cormorants and Contracaecum sp. 1 and C. multipapillatum (s.l.) in the brown pelicans. These bird species are sympatric in Sarasota Bay and other areas of the Gulf of Mexico and have largely overlapping dietary habits (Hatch and Weseloh, Reference Hatch, Weseloh, Poole and Gill1999; Shield, Reference Shields, Poole and Gill2002), suggesting that these differences could be the product of adaptation of adult forms to their definitive hosts. Therefore, host specificity seems to play a more important role than trophic ecology.

The present study provides evidence for at least 2 new species in the genus Contracaecum. A detailed analysis of the morphological differentiation of C. rudolphii C from the other members of the C. rudolphii complex and of Contracaecum sp. 1 from congeners will permit their formal description and nomenclatural designation.

We wish to thank very much the editor (Professor Robin B. Gasser) and three anonymous referees, whose comments and suggestions have significantly improved the manuscript. This research was partially supported by grants from the Ministero delle Politiche Agricole, Alimentari e Forestali – Sesto Piano Triennale della Pesca e dell'Acquacoltura.

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Figure 0

Table 1. Parasite specimens examined in the present study (C-cor and Pel) and those used for comparative purposes (Pc, A-cor, B-cor, Php), including information regarding their hosts and geographical origin (Cor=cormorant, Pel=pelican, Pc=Pelecanus crispus, Php=Phalacrocorax pygmaeus)(Representative sequences for each taxon have been submitted to GenBank and have the following Accession numbers: EF014283 (C-cor 52), EF030717 (Pel28), EF030716 (Pel3), EF014280 (Pc1), EF014281 (A-cor31), EF014279 (B-cor41), EF014282 (Php1).)

Figure 1

Fig. 1. Maximum Parsimony 70% majority-rule bootstrap consensus tree based on rrnS gene sequences. Bootstrap percentages of clades (based on 1000 iterations) are shown at internal nodes.

Figure 2

Fig. 2. UPGMA tree constructed on a genetic distance matrix based on Kimura's 2-parameter model (Gamma), based on rrnS gene sequences. Bootstrap percentages of clades (based on 1000 iterations) are shown at internal nodes.

Figure 3

Fig. 3. A representative gel displaying the RFLP profiles following the digestion of rrnS amplicons with restriction endonuclease RsaI. 1–3 C. rudolphii B; 4–6 C. rudolphii A; 7–19 C. rudolphii C; M 100 bp DNA ladder.

Figure 4

Fig. 4. A representative gel displaying the RFLP profiles following the digestion of rrnS amplicons with restriction endonuclease DdeI. 1–2 C. rudolphii B; 3–4 C. rudolphii A; 5–7, 9–15, 17 C. rudolphii C; 8, 16, 18 C. multipapillatum (Florida), 19 C. microcephalum; M 100 bp DNA ladder.

Figure 5

Fig. 5. A representative gel displaying the RFLP profiles following the digestion of ITS amplicons with restriction endonuclease Tsp509I. 1–3 C. rudolphii A; 4–6 C. rudolphii B; 7–19 C. rudolphii C; M 100 bp DNA ladder.

Figure 6

Fig. 6. A representative gel displaying the RFLP profiles following the digestion of the ITS amplicons with restriction endonuclease Tsp509I. 1 C. multipapillatum (Florida); 2 C. microcephalum; 3 Contracaecum sp. 1; 4 C. rudolphii A; 5 C. rudolphii B; 6 C. rudolphii C; M 100 bp DNA ladder.

Figure 7

Table 2. Matrix of genetic distances, using Kimura 2-parameters index, between taxa based on groups identified by UPGMA and Maximum Parsimony analysis