Hirudinella ventricosa (Pallas, 1774) Baird, 1853 represents a species complex based on ribosomal DNA

Syst Parasitol (2013) 86:197–208 DOI 10.1007/s11230-013-9439-2

Hirudinella ventricosa (Pallas, 1774) Baird, 1853 represents a species complex based on ribosomal DNA Dana M. Calhoun • Stephen S. Curran Eric E. Pulis • Jennifer M. Provaznik • James S. Franks

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Received: 30 October 2012 / Accepted: 14 August 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Digeneans in the genus Hirudinella de Blainville, 1828 (Hirudinellidae) from three species of pelagic fishes, Acanthocybium solandri (Cuvier), Makaira nigricans Lace´pe`de and Thunnus albacares (Bonnaterre), and one benthic fish, Mulloidichthys martinicus (Cuvier), from the Gulf of Mexico are investigated using comparison of ribosomal DNA. Four species are identified based on molecular differences: Hirudinella ventricosa (Pallas, 1774) Baird, 1853 from A. solandri, Hirudinella ahi Yamaguti, 1970 from T. albacares, and two unidentified but distinct species of Hirudinella, herein referred to as Hirudinella sp. A (from both M. nigricans and M. martinicus) and Hirudinella sp. B from M. nigricans. Additionally, H. ahi, based tentatively on morphological identification, is reported from Thunnus thynnus (Linnaeus). This represents the first record of a hirudinellid from M. martinicus and the first record of H. ahi from T. thynnus. A phylogeny of some Hemiurata Skrjabin & Guschanskaja, 1954 using D. M. Calhoun (&)  S. S. Curran  E. E. Pulis  J. S. Franks Department of Coastal Sciences, The University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564, USA e-mail: [email protected] J. M. Provaznik Mississippi Laboratories, National Marine Fisheries Services (NOAA), Southeast Fisheries Science Center (SEFSC), 3209 Frederick Street, Pascagoula, MS 39567, USA

partial fragments of the 28S rDNA sequences is consistent with earlier phylogenies and the position of the Hirudinellidae Dollfus, 1932 is well-supported as a derived group most closely related to the Syncoeliidae Looss, 1899.

Introduction The Hirudinellidae Dollfus, 1932 is a small cosmopolitan family of robust hemiuroid digeneans that inhabit the stomach of pelagic marine fishes. The life history of members of the family is entirely unknown. The family is represented by approximately 50 nominal species, most in the genus Hirudinella de Blainville, 1828. However, the taxonomy of the family has a confusing history (summarised in Nigrelli & Stunkard, 1947; Gibson, 1976; Gibson & Bray, 1977), and remains unresolved because the morphological features traditionally used to separate species in the genus are few in number and are either unreliable or ineffective at the species level (Gibson & Bray, 1977). In the most recent classification of the family, Gibson (2002), following Gibson (1976), and Gibson & Bray (1977, 1979), assigned all nominal species to belong in three monotypic genera. The oldest of these, Hirudinella, is represented by Hirudinella ventricosa (Pallas, 1774) Baird, 1853. The species of Hirudinella are united by having a vitellarium consisting of two lateral fields with their anterior extent at the testicular level and their posterior extent near the middle of the

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hindbody, and a postovarian intercaecal uterus. Hirudinella ventricosa is especially common in the wahoo, Acanthocybium solandri (Cuvier), but it has been reported from a great variety of pelagic fishes, albeit identified as various species in Hirudinella (see Gibson & Bray, 1977). The relationship of the Hirudinellidae in the Hemiurata Skrjabin & Guschanskaja, 1954 has been explored using combined evidence from morphological data and small subunit rDNA including V4 region (Blair et al., 1998) and using small subunit rDNA alone (Pankov et al., 2006). Both studies found a close but unresolved or poorly supported relationship among the Hirudinellidae, Accacoeliidae Odhner, 1911, Derogenidae Nicoll, 1910, Sclerodistomidae Odhner, 1927, Didymozoidae Monticelli, 1888, and Syncoeliidae Looss, 1899. The present study incorporates molecular tools to: (i) assess the identity of several forms of hirudinellids from the Gulf of Mexico belonging to Hirudinella that exhibit some morphological variability, and (ii) investigate the relationship of the Hirudinellidae in the Hemiurata based on partial fragment of the large subunit ribosomal RNA (28S rRNA) gene.

Materials and methods Specimen collection Four different collections of fish stomach were examined for parasites for this study: (i) four yellowfin tuna, Thunnus albacares (Bonnaterre), five Atlantic blue marlin, Makaira nigricans Lace´pe`de, two wahoo, A. solandri, and ten dolphinfish, Coryphaena hippurus Linnaeus, were examined for parasites at a fishing tournament in Biloxi, Mississippi, U.S.A. (6 June 2012); (ii) seven A. solandri, ten T. albacares, six C. hippurus, one swordfish, Xiphias gladius Linnaeus, and one escolar, Lepidocybium flavobrunneum (Smith), were examined for parasites at a fishing tournament in Orange Beach, Alabama, U.S.A. (4 August 2012); (iii) one yellow goat fish, Mulloidichthys martinicus (Cuvier), was collected and examined for parasites during the National Marine Fisheries Service (NMFS) 2007 fall pelagic trawl survey off western Florida, U.S.A. at a depth of 64 m (11 November 2007); and (iv) eleven Atlantic bluefin tuna, Thunnus thynnus (Linnaeus), were collected and examined for parasites by National Oceanic and

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Atmospheric Administration (NOAA) observers participating in a NMFS pelagic observer program on commercial vessels fishing offshore in the Gulf of Mexico at a maximum depth of 320 m (May through June 2012). Worms collected from the first three collections were removed from the stomach and were either killed in nearly boiling agitated tap water and fixed in 10% neutral buffered formalin solution, or directly killed and preserved in 95% ethanol. One formalin-fixed specimen from A. solandri was transferred to 70% ethanol and dissected to observe internal structures. Methods for the fourth collection, conducted by NOAA observers aboard commercial fishing vessels, differed. Observers removed the stomach of each specimen of T. thynnus and placed the stomach in a freezer for an unknown time period while the ship remained at sea. Upon returning to port, the frozen stomachs were transported to the NOAA Laboratory in Pascagoula, Mississippi, U.S.A. In the laboratory stomachs were frozen at -20 °C and stored till thawed for stomach content sorting. During sorting, worms were removed and either re-frozen in separate containers or preserved in 95% ethanol and brought to the Gulf Coast Research Laboratory (Ocean Springs, Mississippi, U.S.A.) for identification. Frozen digeneans were either post-fixed in 10% neutral buffered formalin solution or preserved in 95% ethanol. Worms used in the phylogenetic analysis were fixed with the same methods as the first three collections. Representative formalin-fixed worms were stained with aqueous Meyer’s hematoxylin solution, dehydrated in a graded ethanol series, cleared in clove oil or methyl salicylate, and mounted in Canada balsam on glass slides under cover slips. Some formalin-fixed worms were transferred to 70% ethanol for storage. Representative vouchers, both mounted and in 70% ethanol, are deposited in the Gulf Coast Research Laboratory Museum (GCRLM) (Table 1). DNA extraction, amplification, and sequencing Genomic DNA was extracted from individual worms using Qiagen DNAeasy tissue kit (Qiagen, Inc., Valencia, California) following manufacturer’s instructions. DNA fragments measuring approximately 2,800 base pairs (bp) long comprising the 30 end of the 18S nuclear rRNA gene, internal transcribed spacer (ITS) region (ITS1?5.8S?ITS2), and the 50 end of the 28S gene (including the variable domains D1–D3) were amplified

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Table 1 Hosts, localities, and museum accession numbers for the digeneans used in the present study Species

Host

Locality

Museum no. (GCRL)

Azygia longa Leidy, 1926

Esox niger Lesueur

Pascagoula River, Mississippi (U.S.A.)

GCRL 06511

Hirudinella ahi Yamaguti, 1970

Thunnus albacares (Bonnaterre); Thunnus thynnus (L.) Makaira nigricans Lace´pe`de; Mulloidichthys martinicus (Cuvier) Makaira nigricans Lace´pe`de

Gulf of Mexico

GCRL 06517

Gulf of Mexico

GCRL 06519

Gulf of Mexico

GCRL 06520

Hirudinella ventricosa (Pallas, 1774) Baird, 1853

Acanthocybium solandri (Cuvier)

Gulf of Mexico

GCRL 06518

Lecithochirium microstomum Chandler, 1935

Trichiurus lepturus Linnaeus

Northern Gulf of Mexico

GCRL 06513

Proterometra sp.

Lepomis macrochirus Rafinesque Lepomis microlophus (Gu¨nther)

Hirudinella sp. A Hirudinella sp. B

Thometrema lotzi Curran, Overstreet & Font, 2002

from the extracted DNA by polymerase chain reactions (PCR). Forward primer ITSF (50 -CGC CCG TCG CTA CTA CCG ATT G-30 ) or LSU5 (50 - GGA ATG CAA AGT GGG TGG-30 ) with multiple internal forward primers digl2 (50 -AAG CAT ATC ACT AAG CGG-30 ), 300F (50 -CAA GTA CCG TGA GGG AAA GTT G-30 ), and 900F (50 -CCG TCT TGA AAC ACG GAC CAA G-30 ) and reverse primer 1500R (50 -GCT ATC CTG AGG GAA ACT TCG-30 ) with multiple internal reverse primers, 300R (50 -CAA CTT TCC CTC ACG GTA CTT G-30 ), Digl2r (50 -CCG CTT AGT GAT ATG CTT30 ), and ECD2 (50 -CTT GGT CCG TGT TTC AAG ACG GG-30 ) were used in reactions. The PCR reactions were performed following the protocols described by Tkach et al. (2003). The PCR products were purified with Qiagen QiaquickTM columns, cycle-sequenced using ABI BigDyeTM chemistry (Applied Biosystems, Inc., Carlsbad, California), alcohol-precipitated, and constructed on an ABI 3130 Genetic AnalyzerTM. Contiguous sequences were assembled using SequencherTM (GeneCodes Corp., Ann Arbor, Michigan Version 4.7) and aligned using Clustal W module in BioEdit 7.0.9 (Hall, 1999). The boundaries between the 5.8S gene, ITS2 spacer, and 28S gene fragment were located using the Internal Transcribed Spacer 2 Ribosomal Database (Keller et al., 2009). The boundaries between the 18S and ITS1 gene fragments were

GCRL 06512

Pitt Spring, Florida (U.S.A.)

GCRL 06514

Pascagoula River, Mississippi (U.S.A.)

GCRL 06515 GCRL 06516

estimated using previously annotated sequences from GenBank. The number of bp differences at ITS1, ITS2, 5.8S, 28S genes between sequences of different hirudinellids were counted manually and with the aid of MEGATM (Tamura et al., 2011), and are presented in pairwise matrices (Tables 3, 4). Representative sequences are deposited in GenBank (Table 2). Phylogenetic analysis An alignment was constructed from partial fragments of the 28S rDNA sequences from three studied hirudinellids, plus sequences for 15 species belonging in the Hemiurata and for one species, Olssonium turneri Bray & Gibson, 1980, belonging to the Fellodistomidae Nicoll, 1909 (Suborder Bucephalata La Rue, 1926). Sequences from the species represented in the alignment were either obtained from GenBank or collected during prior studies; the new sequences are deposited in GenBank (Table 2), with vouchers deposited in GCRLM (Table 1). The alignment was trimmed and regions that could not be aligned unambiguously were excluded. Maximum likelihood (ML) analysis was performed using PAUP* (Swofford, 2002 ver. 4.0b10), and Bayesian inference (BI) analysis was performed using MrBayes 3.1.2 software (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). Bivesicula claviformis Yamaguti, 1934 (Suborder Bivesiculata

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Table 2 Hosts, localities, and GenBank accession numbers for the digeneans used in the present study Species

Host

Locality

GenBank Acc. No.

Accacoelium contortum (Rudolphi, 1819)

Mola mola (Linnaeus)

North Sea, United Kingdom

AY222190

Bivesicula claviformis Yamaguti, 1934

Epinephelus quoyanus (Valenciennes)

Lizard Island, Australia

AY222182

Copiatestes filliferus (Leuckart in Sars, 1885) Derogenes varicus (Mu¨ller, 1784)

Trachurus murphyi Nichols

Off New Zealand

AY222188

Hippoglossoides platessoides (Fabricius)

North Sea, United Kingdom

AY222189

Dinurus longisinus Looss, 1907

Coryphaena hippurus (Linnaeus)

Port Royal, Kingston, Jamaica

AY222202

Hemiperina manteri Crowcroft, 1947

Latridopsis forsteri (Castelnau)

Tasmania, Australia

AY222196

Lecithophyllum botryophorum (Olsson, 1868)

Alepocephalus bairdii Goode & Bean

Goban Spur, NE Atlantic

AY222205

Machidatrema chilostoma (Machida, 1980)

Kyphosus vaigiensis (Quoy & Gaimard)

Moorea, French Polynesia

AY222197

Olssonium turneri Bray & Gibson, 1980

Alepocephalus agassizii Goode & Bean Kyphosus cinerascens (Forsska˚l)

Porcupine Seabright, NE Atlantic

AY222283

Off Australia

AY222198

Otodistomum cestoides (van Beneden, 1871)

Raja montagui Fowler

North Sea, United Kingdom

AY222187

Prosogonotrema bilabiatum Viqueras, 1940

Caesio cuning (Bloch)

Off Hawaii, U.S.A

AY222191

Opisthadena dimidia Linton, 1910

Olson et al., 2003; Family Bivesiculidae Yamaguti, 1934), was selected as the outgroup for both analyses based on its phylogenetic position relative to the studied ingroup as estimated by Olson et al. (2003). Analysis by ML was performed using a heuristic search (100 search replicates), 1,000 random addition sequences and tree-bisection-reconnection (TBR) swapping. All included characters were treated as unordered and unweighted. Gaps were treated as missing data. A bootstrap analysis was conducted to establish tree nodal support values using 1,000 replicates with 10 random sequences. Analysis by BI was conducted using the best nucleotide substitution model estimated with jModelTest Version 0.1.1 (Posada & Crandall, 2001; Guindon & Gascuel, 2003; Posada, 2008) as general time reversible with gamma-distributed among site-rate variation (GTR?G) model using the following model parameters: lset nst = 6, rates = gamma, ngen = 1,000,000 and samplefreq = 100. Burn-in value was 2,500 estimated by plotting the log-probabilites against the generations and visualizing plateau in

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parameter values (sumt burnin = 2,500), and nodal support was estimated by posterior probabilities (sumt) (Huelsenbeck & Ronquist, 2001), with all other settings left as default.

Identification of the hirudinellids Digeneans were recovered from five of the eight species of pelagic fishes examined for stomach parasites during the four collections from the Gulf of Mexico. Coryphaena hippurus, L. flavobrunneum, and X. gladius were not infected by digeneans. Based on our observations and molecular results, a total of four distinct species belonging in Hirudinella were recovered. Names for these taxa are presented in the results below. Attempts at isolating genomic DNA from our specimens met with varying degrees of success. The entire targeted sequence fragment encompassing the 30 end of the 18S nuclear rDNA gene, ITS1 region, 5.8S gene, ITS2 region, and a fragment of the 50 end of the 28S gene (including variable domains D1–D3) was successfully amplified from three species. Only a

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partial fragment of 28S rDNA gene was successfully sequenced from the single specimen of a fourth species; however, this single fragment differed from the other successfully obtained 28S rDNA fragments (Table 3). A fifth species, Botulus microporus (Monticelli, 1899) Gibson & Bray, 1977 occurred in T. thynnus, but we were unable to extract DNA from the specimens available. Aligned ITS1 regions from three species of Hirudinella were 797 bp long including gaps, the 5.8S gene was identical in these three species, and the aligned ITS2 regions from these three species were 340 bp long including gaps. The aligned fragments of 28S rRNA gene from all four species were trimmed at both ends to match the shortest fragment and measuring 1,117 bp including gaps. Nucleotide differences including gaps with percent difference at ITS1 regions, ITS2 regions, and partial 28S rRNA gene fragments among the species are presented in pairwise matrices (Tables 3, 4). Hirudinella ventricosa (Pallas, 1774) Baird, 1853 Digeneans consistent with the concept of H. ventricosa of Gibson & Bray (1977) occurred in A. solandri (n = 11) at a prevalence of 100% and a mean intensity of 2.81 (1–9). The specimens (based on three heatkilled measured individuals) ranged in body length from 42 to 48 mm, had an oral sucker to ventral sucker width ratio of 1:1.5–1.8, and egg-size 31–37 9 26–28 lm. The body shape of the heat-killed specimens is consistently elongated, with the widest part of the body being in the posterior quarter to third of the body, where it is about twice as wide as the body at the

Table 3 Pairwise sequence comparisons showing variable sites with overall percent difference in parentheses based on the aligned 1,117 bp long partial fragment of the 28S rRNA gene among the four studied species of Hirudinella H. ahi

H. ventricosa

Hirudinella sp. A

Hirudinella sp. B

H. ahi

–

8 (0.7%)

0 (0.0%)

74 (6.6%)

H. ventricosa

–

–

8 (0.7%)

79 (7.1%)

Hirudinella sp. A

–

–

–

74 (6.6%)

Hirudinella sp. B

–

–

–

–

level of the ventral sucker. Maximum body width to maximum body length ratio is 1:3.6–4.0. Nigrelli & Stunkard (1947) reported the following measurements for H. ventricosa (based on approximately twenty mature specimens from A. solandri in various states of fixation): body length ranging from 31 to 58 mm, oral sucker to ventral sucker width ratio of 1:1.9–2.2, and egg-size 31–37 9 20–26 lm. We successfully amplified the entire targeted gene fragment (30 end of the 18S nuclear rRNA gene, ITS1, 5.8S, ITS2, and a fragment of the 50 end of the 28S gene) from seven specimens of H. ventricosa from A. solandri; no intraspecific variation was observed among these replicates.

Hirudinella sp. A A second digenean belonging in Hirudinella occurred in one of five specimens of M. nigricans (one worm) and the single specimen of M. martinicus examined had two adults of these worms. This digenean, though much shorter than H. ventricosa (c.23 mm), had comparable sucker ratio and egg-size, and maximum body width to maximum body length ratio of 1:3.9. Nevertheless, comparison of the successfully amplified targeted gene fragment (30 end of the 18S nuclear rRNA gene, ITS1, 5.8S, ITS2, and a fragment of the 50 end of the 28S gene) from three specimens without intraspecific variation among the three replicates, revealed that this was a species distinct from H. ventricosa (Tables 3, 4). Since we were unable to distinguish this form from H. ventricosa or other nominal species based on the limited number of morphological features available, we subsequently refer to this form as Hirudinella sp. A.

Table 4 Pairwise sequence comparisons showing variable sites with overall percent difference in parentheses among the three species of Hirudinella studied H. ventricosa

Hirudinella sp. A

H. ahi

H. ventricosa

–

20 (2.5%)

21 (2.6%)

Hirudinella sp. A

11 (3.2%)

–

H. ahi

13 (3.8%)

4 (1.2%)

5 (1.0%) –

Data for ITS1 region of rDNA (797 bp) above diagonal; data for ITS2 region of rDNA (340 bp) below diagonal

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Hirudinella ahi Yamaguti, 1970 A third digenean belonging in Hirudinella but appearing grossly distinct in shape occurred in two of fourteen specimens of T. albacares with a mean intensity of 3.5 (1–6) worms. This form is consistent in overall appearance with Hirudinella ahi Yamaguti, 1970 that was described based on one specimen from T. albacares (as Neothunnus macropterus) off Hawaii. Based on observations from three large mature specimens and two smaller mature individuals, our specimens are roughly the same size as H. ahi (9.5–37.0 mm long vs 28.0 mm) and similar to the original description, the hindbody is elongated and cylindrical, and the body at the level of the ventral sucker is nearly as wide as at the level of the posterior hindbody. Maximum body width to maximum body length ratio is 1:6.6–8.2. The oral sucker width to ventral sucker width ratio of these specimens is 1:2.7–3.0 compared with 1:2.2 in H. ahi, and egg-size is roughly the same (39–45 9 23–28 vs 35–42 9 18–24 lm) (see Yamaguti, 1970). Comparison of the successfully amplified targeted gene fragment (30 end of the 18S nuclear rRNA gene, ITS1, 5.8S, ITS2, and a fragment of the 50 end of the 28S gene) from five specimens without intraspecific variation among the five replicates, confirmed that this form did in fact differ from H. ventricosa and Hirudinella sp. A. Interestingly, the fragments of the 28S rRNA gene from this form and Hirudinella sp. A were identical but these species differed at the ITS1 by 5 bp (1.0%) and ITS2 by 4 bp (1.2%), suggesting they are very closely related but distinct. Since this third digenean resembles H. ahi, was collected from the same host species, and differs genetically from the previously mentioned species, we consider this species tentatively to be H. ahi. Additionally, we collected what we tentatively believe is H. ahi in all of the eleven specimens of T. thynnus examined (mean intensity of 4.9; range 1–11). Unfortunately, we were unable to isolate genomic DNA from any of our specimens of H. ahi from T. thynnus. Specimens from T. thynnus were morphologically indistinguishable from those from T. albacares. Hirudinella sp. B A single digenean representing a fourth species belonging in Hirudinella that strongly resembled H. ahi was collected from M. nigricans. We amplified

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only a fragment of the 28S rDNA gene from this individual. This specimen differs from our specimens of H. ventricosa by being much shorter, measuring 30 mm, with a slender body measuring 4.5 mm in width (maximum body width to maximum body length ratio 1:6.6), and having a relatively small narrow oral sucker (1.0 vs 3.30–4.35 mm). We used tissue from the ventral sucker for DNA extraction, which prevented us from obtaining the oral sucker width to ventral sucker width ratio, but the suckers appear superficially to be similar in shape and size relative to body size as those in H. ahi. The eggs are similar in size to those of H. ahi, but longer than those in H. ventricosa (39–45 vs 31–37 lm). Based on these measurements, the parasitisation of a different host, and the genetic differences at the 28S rRNA gene (see Table 3), we consider this form to be different from H. ventricosa, Hirudinella sp. A, and H. ahi, and subsequently refer to this form as Hirudinella sp. B. Hirudinella sp. B may be further distinguished from Hirudinella sp. A by having longer eggs (39–45 vs c.33 lm).

Phylogenetic analysis The ML and BI analyses produced best trees with slightly differing topology. The best ML tree failed to resolve the clade containing the Hirudinellidae along with Accacoeliidae, Derogenidae, Didymozoidae, Sclerodistomidae, Syncoeliidae. The best tree resulting from the BI is presented as a phylogram with posterior probability values provided over ML bootstrap support values at each node (Fig. 1). The Syncoeliidae represents the most closely related family (included in the analyses) to the Hirudinellidae. Hemipera manteri (Crowcroft, 1947) Yamaguti, 1953 is thought to be a derogenid but forms a branch basal to the rest of the Hemiuroidea. The relationship between the Hemiurata and the Bucephalata was not resolved in the analyses.

Discussion Historically, there has been controversy concerning the validity and number of nominal species in the genus Hirudinella. Nigrelli & Stunkard (1947) provided a historical review of the genus in which they thoroughly summarised the earliest taxonomic works and pointed out some major pitfalls, particularly,

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Fig. 1 The phylogenetic best tree based on analyses of partial sequences of 28S rDNA using Bayesian inference analysis in MrBayes and maximum likelihood algorithm in PAUP*. Numbers above the nodes show posterior probabilities from Bayesian analysis and the numbers below the nodes show bootstrap support values. Bivesicula claviformis was used as an outgroup. Shaded rectangles indicate the placement of the three

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species of Hirudinella studied. Family names are abbreviated in front of species names. Abbreviations: Acc, Accacoeliidae; Azy, Azygiidae; Biv, Bivesiculidae; Der, Derogenidae; Did, Didymozoidae; Fel, Fellodistomidae; Hir, Hirudinellidae; Hem, Hemiuridae; Lec, Lecithasteridae; Scl, Sclerodistomidae; Syn, Syncoeliidae

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earlier descriptions were grossly inadequate and did not allow for discrimination of the species, vernacular names were often given for hosts, locality data was sometimes absent, and in many cases, original specimens are no longer available for comparison. Hirudinella marina Garcin, 1730, described from the skipjack tuna, Katsuwonus pelamis (Linnaeus) from the Atlantic Ocean, is the oldest named species, and Nigrelli & Stunkard (1947) considered it as the typespecies of the genus. They considered the only other species in the genus to be H. ventricosa on the basis that all of the previously named species could be assigned to either H. marina or H. ventricosa based on morphology. Considering that there is great ambiguity regarding morphological features among specimens belonging in Hirudinella, particularly the body size, shape, state of fixation, size at maturity, and perhaps egg-size, and that he could find no discernible differences in internal anatomy among any forms available for histological comparison, Gibson (1976) advocated that he could not find any evidence to substantiate the presence of more than a single species in the genus. Furthermore, Gibson (1976) pointed out that the opinion that Nigrelli & Stunkard (1947) used to make their decision regarding the type-species of Hirudinella using the International Code of Zoological Nomenclature (version from 1926) was outdated. Consequently, Gibson (1976) considered H. marina to be pre-Linnaean and advocated that the name for the only species in Hirudinella (in 1976) should be Hirudinella ventricosa (Pallas, 1774) Baird, 1853. We tentatively agree with that assessment. However, it is clear based on our molecular results from specimens in the Gulf of Mexico, that more than one species may have been inadvertently included as a single species in the earlier evaluations of the species of Hirudinella. We identified our specimens from A. solandri as H. ventricosa on the basis that the limited number of measurements available were compatible with those reported for that species by Nigrelli & Stunkard (1947), their presence in A. solandri, and the fact that Gibson (1976) and Gibson & Bray (1977) considered H. venticosa to be the only valid species. We believe the slight differences in sucker ratios reported by Nigrelli & Stunkard (1947) can be attributed to the many fixation methods used. Indeed, based on our specimens, body shape in well-fixed specimens appears to be useful for differentiating some species

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in the genus. Specimens collected live, cleaned in saline solution (8.5 ppt), and killed in near boiling water with agitation, fix in such a way that the maximum body width to maximum body length ratio is consistent for a particular species. Specimens that are killed when their fish host is placed in a freezer take a variety of shapes inconsistent with well-fixed specimens. Furthermore, specimens left in their hosts on ice for too long (more than 24–36 h) seem lethargic, unresponsive, or even dead when cleaned in saline solution. When immersed in near boiling water with agitation, these specimens usually fix inconsistently compared with specimens that are moving and responsive prior to being well-fixed. Inconsistent fixation methods have probably led to the vast majority of confusion relating to species in Hirudinella. Chandler (1937) described Hirudinella beebei Chandler, 1937 from Acanthocybium petus (=A. solandri) from off Bermuda, and Yamaguti (1970) reported this species from A. solandri off Hawaii; however Nigrelli & Stunkard (1947), Gibson (1976), and Gibson & Bray (1977) did not agree with Chandler0 s (1937) interpretation of the histological features associated with the digestive system and the configuration of the vitellarium that Chandler (1937) used to differentiate this species. Consequently, they considered H. beebei a junior synonym of H. ventricosa and we tentatively agree with their assessments. Our identification of H. ventricosa is tentative because of the enormous amount of ambiguity in the morphological features traditionally used for identifying hirudinellids (mostly related to quality of fixation), the fact that the type-host for H. ventricosa is unknown, and that no type-material exists for the species. Hirudinella ahi, which we herein report from T. albacares and T. thynnus (for the first time in the latter), has not been reported since its original description, but Gibson & Bray (1977) considered it a junior synonym of H. ventricosa. Consequently, at least some reports published from scombrids in the genus Thunnus South subsequent to Gibson & Bray (1977), may represent this species in our opinion. For example, Eggleston & Bochenek (1990) reported H. ventricosa from T. thynnus off Virginia, and Williams & Bunkley-Williams (1996) reported H. ventricosa from Thunnus alalunga (Bonnaterre), Thunnus atlanticus (Lesson), T. albacares, and T. thynnus off Puerto Rico. Some authors seem to have taken a more cautious approach and did not abide by the

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synonymies of Gibson & Bray (1977). Jones (1991) for example, reported Hirudinella sp. from T. alalunga in the Pacific Ocean off New Zealand and de la Serna et al. (2012) reported Hirudinella sp. from T. thynnus in the Mediterranean Sea. Our identification of H. ahi from T. albacares from the Gulf of Mexico is also tentative because of the wide geographic distance between the Gulf of Mexico and the type-locality of the species off Hawaii. Most scombrids, including A. solandri and T. albacares, are widespread oceanic species inhabiting the warm seas worldwide, and it is not unprecedented for a parasite to have a cosmopolitan distribution in a scombrid host. Aiken et al. (2007) investigated the cosmopolitan distribution of tuna parasites using rDNA and found that the blood fluke Cardicola forsteri Cribb, Daintith, & Munday, 2000 occurred in Thynnus maccoyii (Castelnau) off Australia and T. thynnus in the Spanish Mediterranean, and that the polyopisthocotylean Hexostoma thynni (Delaroche, 1811) von Nordmann, 1840 occurred on three species of Thunnus, also from off Australia and in the Spanish Mediterranean. So it is plausible that a hirudinellid species might occupy the entire range of its host. Our identification of Hirudinella sp. A and Hirudinella sp. B is based on differences in the rDNA sequences. The limited number of morphological features and the lack of sufficient molecular data for thorough comparisons with other species prevented us from assigning Hirudinella sp. A, collected from M. nigricans and M. martinicus, to any previously named species. Our two specimens from M. martinicus represent the first report of a hirudinellid in the stomach from a fish of the family Mullidae. Although these two specimens from M. martinicius contain eggs in the uterus, the eggs are smaller and range in size from 20 to 23 lm long by 17–20 lm wide (compared with c.33 9 23–26 lm in the specimen from M. nigricans). Furthermore, members of the Mullidae are nonselective foragers (see Oxenford & Hunte, 1999) suggesting that M. martinicus is probably an abnormal host. The single specimen of Hirudinella sp. B collected from M. nigricans represents yet another unidentifiable species. Williams & Bunkley-Williams (1996) reported that M. nigricans from off Puerto Rico is infected with H. ventricosa. However, these authors seem to have followed the two-species classification of the family presented by Nigrelli & Stunkard (1947).

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We suspect their material could be conspecific with that of Hirudinella sp. A, or Hirudinella sp. B, since all occur in the same host. Given that we collected only one specimen of Hirudinella sp. B, coupled with the fact that we failed to measure the ventral sucker prior to using it for extracting DNA, we are unable to effectively compare the worm with previous descriptions. Our combined morphological observations and molecular data, suggest that the systematic synonymisation of all nominal species of Hirudinella into H. ventricosa is untenable. The genetic differences among the four species of Hirudinella for which we present results here (Tables 3, 4) are comparable to the results of similar studies comparing closely related digenean species. For example, Miller & Cribb (2007) showed that two morphologically similar worms from the red bass Lutjanus bohar (Forsska˚l) from geographically distant sites (Latuterus tkachi Miller & Cribb, 2007 from Lizard Island, Australia and Latuterus maldivensis Miller & Cribb, 2007 from the Maldives), that can be differentiated only by body size, differ by 2 bp (0.4%) at ITS1, 4 bp (1.4%) at ITS2, and 3 bp (0.4%) at 28S. Parker et al. (2010) reported similar genetic differences between two morphologically similar digeneans that parasitise gerreid fishes on either side of the Panamanian Isthmus: Homalometron elongatum Manter, 1947 from the Caribbean Sea differed from Homalometron lesliorum Parker, Curran, Overstreet & Tkach, 2010 from the Pacific Ocean by 12 bp (2.5%) at ITS1, 10 bp (3.2%) at ITS2, and 11 bp (0.8%) at 28S. For comprehensive reviews of studies see Nolan & Cribb (2005) and Olson & Tkach (2005) for reviews of studies demonstrating the effectiveness of ribosomal DNA for discriminating between and among closely related digenean species. We advocate that comparison of ribosomal DNA in combination with a morphological assessment is presently the only effective way to address the identity of species in the Hirudinellidae. We consider H. ventricosa to be a cosmopolitan parasite of A. solandri and H. ahi to be a cosmopolitan parasite of species of Thunnus. The phylogeny presented here includes nineteen species from the Hemiurata in nine families and one species from the Bucephalata (family Fellodistomidae), and agrees largely with previous estimations of the phylogeny of hemiuroid groups that incorporated molecular data (Blair et al., 1998; Cribb et al., 2001;

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Olson et al., 2003; Pankov et al., 2006). This latest phylogeny uses lsr DNA to estimate the position of the Hirudinellidae for the first time. The resulting tree (Fig. 1) indicates with strong support (also for the first time) that the Hirudinellidae is closely related to the Syncoeliidae. The Syncoeliidae is a marine family of robust digeneans comprising 11 species in four genera whose adults are usually found associated with the gills, stomach, or mouth of elasmobranchs or teleosts (Yamaguti, 1971; Gibson & Bray, 1977; Gibson, 2002; Curran & Overstreet, 2000). Pelagic, benthopelagic, and benthic fishes serve as definitive hosts, and the metacercaria for a syncoeliid species, Copiatestes filiferus (Leuckart in Sars, 1885) Gibson & Bray, 1977, is known to occur in the haemocoel of euphausiid crustaceans (Nematoscelis megalops Sars and Thysanoessa gregaria Sars) in the South Atlantic Ocean (Gibson, 1976; Gibson & Bray, 1977). Dollfus (1966) reported the presence of a metacercaria from the external surface of the euphausiid Nyctiphanes couchii (Bell) from the surface of the Atlantic Ocean (off Cape Verde, equatorial West Africa) and Overstreet (1970) which was later identified as belonging to the syncoeliid genus Paronatrema Dollfus, 1937. Additionally, Overstreet (1970) reported the presence of a metacercaria belonging to either Syncoelium Looss, 1899 or Copiatestes Crowcroft, 1948 from the external surface of the copepod Candacia pachydactyla (Dana) from the Atlantic Ocean (off the mouth of the Amazon River). Since many of the 11 species of syncoelliids use definitive hosts that occur in benthic or bentho-pelagic habitats, it is likely that vertical migration of crustaceans or the use of paratenic hosts may play a role in the life history of the Syncoeliidae. Considering the close phylogenetic relationship between the Syncoeliidae and the Hirudinellidae demonstrated here, and that many of the Hemiuroidea utilise an arthropod in their life-cycle, the presence of a crustacean in the life history of Hirudinella spp. is plausible. And since the fish hosts of Hirudinella spp. are largely pelagic, a pelagic crustacean likely plays a role in the hirudinellid life-cycle. Furthermore, our discovery of an adult hirudinellid (Hirudinella sp. A) in a bentho-pelagic mullid host (M. martinicus), suggests the possible presence of vertical migration of the intermediate host, involvement of a paratenic host, or an accidental host. Regardless, until the metacercariae of Hirudinella spp. are identified

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(preferably using rRNA gene sequences), it will not be possible to determine where, when, or at what depth a host becomes infected, nor how long a host is infected for. The present study establishes a benchmark for identifying the metacercariae of hirudinellids in the Gulf of Mexico. Various scombrid fishes comprise important commercial and recreational fisheries worldwide (DeVries et al., 2002; Franks et al., 2008; ICCAT, 2012). Discriminating between scombrid fish stocks and populations is an important component of fisheries management and generally, certain parasites can serve as biological tags or markers for discriminating between stocks or populations of marine fish (see Kabata et al., 1988; Williams et al., 1992; MacKenzie & Abaunza, 1998; Blaylock et al., 2003; Mosquera et al., 2003; Smith et al., 2004). Specific criteria for assessing the usefulness of a particular parasite for such a task have been established by numerous authorities (see Kabata, 1963; Williams et al., 1992; Mosquera et al., 2003). Accepted fundamental criteria based on these authorities and are not fulfilled by Hirudinella spp. are: (i) There should be significantly different levels of infection in the studied host group in different parts of a study area; (ii) Parasites with a simple life-cycle using a single host make the best markers, while those with multiple life history stages and hosts make more complicated markers; and (iii) Parasites should be easily detected and identified. Species of the genus Hirudinella have been used as biological tags in some studies involving scombrids (e.g. Nakamura & Yuen, 1961; Watertor, 1973; Manooch & Hogarth, 1983; Eggleston & Bochenek, 1990; Smith et al., 2004) despite not fulfilling some of these basic criteria. We contend that caution should be employed when using hirudinellids as biological markers. This study underscores the importance of establishing the species identity of the hirudinellid being used as a biological marker. Though the worms are huge and easily detected in the stomachs of scombrid hosts, they are not easy to identify without employing molecular techniques. As this study shows, identification based solely on morphological identification is inadequate for species level taxonomy in this family. Comparison of rDNA sequences, particularly of the internal transcribed spacer regions, shows that Hirudinella is not a monotypic genus.

Syst Parasitol (2013) 86:197–208 Acknowledgements We would like to thank Robin M. Overstreet for advice, and Lynnae C. Manuel, Jean Jovonvich Alvillar, and Janet Wright, for their assistance with DNA extractions (all from The University of Southern Mississippi, USM). We are also grateful to: Vasyl V. Tkach (University of North Dakota) for his great efforts in attempting to extract DNA from problematic worms; Kenneth Keene from the Southeast Fisheries Science Center Pelagic Observer Program in Miami, Florida, U.S.A. for facilitating collections; Bobby Carter, director of the Mississippi Gulf Coast Billfish Classic, and participating anglers; Bill Haffner, director of the Mobile Big Game Fishing Club, and participating anglers; Paul Grammer (USM), Sarah Ashworth (USM) and Michael Buchanan (Mississippi Department of Marine Resources) for facilitating collections. Two anonymous reviewers provided helpful suggestions for improving the manuscript. This material is based on work supported by the National Science Foundation under Grant No. 0529684, RAPID 1055071, as well as USDC, NOAA award no. NA08NOS4730322. The work was also supported by Mississippi Department of Marine Resources SubGrant S-11-USM-GCRL and USDI/MS DMR MSCIAP MS.R.798 Award M10AF20151.

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