International Journal for Parasitology 41 (2011) 861–870 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara The life cycle of Cardicola forsteri (Trematoda: Aporocotylidae), a pathogen of ranched southern bluefin tuna, Thunnus maccoyi q Thomas H. Cribb a,⇑, Robert D. Adlard b, Craig J. Hayward c,d,1, Nathan J. Bott e, David Ellis d,f, Daryl Evans d, Barbara F. Nowak d a School of Biological Sciences, The University of Queensland, Qld 4072, Australia Biodiversity Program, Queensland Museum, South Brisbane, Qld 4101, Australia Aquatic Sciences, South Australian Research and Development Institute, Lincoln Marine Science Centre, P.O. Box 1511, Port Lincoln, SA 5606, Australia d NCMCRS, AMC, University of Tasmania, Locked Bag 1370, Launceston 7250, Tasmania, Australia e Aquatic Sciences, South Australian Research and Development Institute, P.O. Box 120, Henley Beach, SA 5022, Australia f Australian Southern Bluefin Tuna Industry Association, Port Lincoln, SA 5606, Australia b c a r t i c l e i n f o Article history: Received 12 October 2010 Received in revised form 4 February 2011 Accepted 8 March 2011 Available online 27 April 2011 Keywords: Aporocotylidae Life cycle rDNA Terebellidae Cardicola forsteri Southern bluefin tuna Blood fluke a b s t r a c t Aporocotylids (fish blood flukes) are emerging as pathogens of fishes in both marine and freshwater aquaculture. Efforts to control these parasites are hampered by a lack of life cycle information. Here we report on the life cycle of Cardicola forsteri, which is considered a significant pathogen in southern bluefin tuna, Thunnus maccoyi, ranched in South Australia. We surveyed polychaetes, bivalves and gastropods from sites close to tuna pontoons. Infections consistent with the Aporocotylidae were found in terebellid polychaetes, a single Longicarpus modestus and five individuals of Reterebella aloba. All infections were comprised of hundreds of sporocysts in the body cavity of the host, each filled with developing and mature cercariae. Sequences of ITS-2 and lsrDNA from the infection from L. modestus were a perfect match with those of adult C. forsteri from T. maccoyi. This life cycle link is considered confirmed but it is possible that additional terebellid species are infected in South Australia; equally, other species of intermediate host are likely to be involved in other parts of the range of this cosmopolitan trematode. Sequences of the species from R. aloba did not match a known adult but phylogenetic analysis of lsrDNA suggests that it is also a species of Cardicola Short, 1953. These findings show that terebellid polychaetes are a major host group for marine aporocotylids, especially given that Cardicola is the largest marine aporocotylid genus. The two cercarial types are among the smallest known for the family and are unusual, but not unique, in having short, simple tails. We speculate that the form of the tail means that these cercariae are not active swimmers and are thus heavily dependent on currents for dispersal. Control of this parasite might be effected by moving the tuna pontoons appropriate distances to avoid encounter with current-dispersed cercariae, or by increasing the separation of the nets from the sea floor, either by raising the nets or moving to deeper water. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction The family Aporocotylidae comprises trematodes that principally parasitise the vascular system of fishes (Smith, 2002). These blood flukes have a two-host life-cycle involving asexual reproduction in an invertebrate host and sexual reproduction in the fish. As for their close relatives, the schistosomatids of birds and mammals, the cercaria infects the fish by direct penetration q Note: Nucleotide sequence data reported in this paper are available in the GenBank databases under the Accession Nos. JF800668–71 and JF803976–7. ⇑ Corresponding author. Tel.: +61 7 3365 2581; fax: +61 7 3365 4699. E-mail address: T.Cribb@uq.edu.au (T.H. Cribb). 1 Present address: Tohoku University Institute of International Education, 41 Kawauchi Aoba-ku, Sendai, Miyagi 980-8576, Japan. of the skin. The family has emerged as significant pathogens in marine fish aquaculture in Australia and elsewhere (Ogawa et al., 2007; Hayward et al., 2010). Fish blood fluke eggs typically pass from adult trematodes in the heart or elsewhere in the circulatory system to the gills where they lodge, embryonate and hatch, releasing miracidia which seek the intermediate hosts. Eggs may cause significant lesions in the gills and other organs such as the heart itself. The pathogenesis of aporocotylids is typically related more to the reaction to the eggs than to the presence of adult trematodes, a feature shared by the schistosomatids of humans. These flukes can become highly pathogenic in aquaculture when transmission is enhanced. Damaging infections have been reported from carangids, salmonids, scombrids, sparids and tetraodontids (e.g. Rawstron, 1971; Evans, 1974; Ogawa et al., 1989, 2007; Crespo et al., 1992; Ogawa and 0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.03.011 862 T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 Fukudome, 1994; Colquitt et al., 2001; Padros et al., 2001; Aiken et al., 2006). In some cases there is a loss of productivity or minor mortality (Padros et al., 2001) but mass mortalities are also reported (Sommerville and Iqbal, 1991; Ogawa and Fukudome, 1994). Aporocotylid life cycles are relatively simple, requiring just two hosts (a fish and an invertebrate). Invertebrate intermediate hosts include gastropods, bivalves and polychaete worms. The latter group is of special interest because the infections of aporocotylids in polychaetes are the only asexual stages of any digenean that occur in taxa other than molluscs (Cribb et al., 2003). Cercariae develop in sporocysts or rediae within the intermediate host. Known aporocotylid cercariae tend to be relatively distinctive. They are typically small, fork-tailed, lack a ventral sucker, have a well-developed penetrating organ (a specialised oral sucker), and a fin-fold on the cercarial body (Schell, 1974). On the basis of their morphology, several unassociated cercariae have been interpreted as belonging to the Aporocotylidae (e.g. Brant et al., 2006). Knowledge of aporocotylid life cycles is uneven. Freshwater life cycles are known for numerous species of Sanguinicola Plehn, 1905 (e.g. Scheuring, 1920; Meade and Pratt, 1965; Hoffman et al., 1985; Nikitina, 1986; Kirk and Lewis, 1993) and the single species of Paracardicoloides Martin, 1974 (see Nolan and Cribb, 2004b). All of these species infect gastropods. In contrast, marine cycles remain poorly known. The only identified marine species for which the life cycle is known is Aporocotyle simplex Odhner, 1900 which infects flatfishes (Pleuronectiformes) and two terebellid polychaete species in the North Sea (Køie, 1982; Køie and Petersen, 1988). In addition to the cercaria of A. simplex, unidentified aporocotylid infections have been reported from several marine polychaetes and bivalves. Among polychaetes, they have been found in a species of Ampharetidae (Oglesby, 1961), a third terebellid (Martin, 1952), and from two species of Serpulidae (Linton, 1915b; Stunkard, 1929; Martin, 1944b). These polychaetes are filter- or deposit-feeders that share grooved tentacles. Until recently they were grouped as the ‘‘Canalipalpata’’ (Rouse and Fauchald, 1997) but recent phylogenetic analysis (Struck et al., 2007) suggests that the Terebellidae and Ampharetidae belong to the Terebelliformia and the Serpulidae belongs to the Sabellida and that these groups are phylogenetically distant from each other. Bivalves have also been reported infected by aporocotylids, although none of the parasites has been identified beyond family. Infected bivalve families are Donacidae (Holliman, 1961), Pectenidae (Linton, 1915a), Solecurtidae (Fraser, 1967), Solemyidae (Martin, 1944a) and Veneridae (Holliman, 1961; Wardle, 1979). These five families belong to three of the five main bivalve subclasses, suggesting little phylogenetic specificity of aporocotylid infection among bivalves. The fish blood fluke of southern bluefin tuna (SBT), Thunnus maccoyi, Cardicola forsteri Cribb, Daintith and Munday, 2000, was described only relatively recently (Cribb et al., 2000) but is now considered a significant problem for the ranched Australian SBT industry in Port Lincoln, South Australia; ranching involves the harvesting of wild SBT and their subsequent feeding and growth in captivity prior to sale. Colquitt et al. (2001) characterised the pathogenesis in tuna, finding that large numbers of eggs provoke an inflammatory response in the heart and gills. At that time the parasite was not thought to be associated with mortality. In recent years, however, there has been a steady increase in tuna mortality that is correlated with an increased intensity of infection with C. forsteri (Hayward et al., 2010). Dennis et al. (in press) reported that SBT mortality was associated with severe branchitis which, in turn, was associated with C. forsteri. Losses attributed to this blood fluke are escalating and the SBT industry is anxious to develop control measures. This is presently difficult because, despite previous study (Aiken et al., 2009), the life cycle of the pathogen is unknown. Understanding of the life cycle is critical because the intermediate host might be in the benthos near the sea cages, on net biofouling, or even in animals living on nearby shores. Each possibility has dramatically different implications for potential efforts to control the parasite. In this study, we examined bivalves, polychaetes and gastropods from the environs of SBT pontoons off Port Lincoln, South Australia. Our approach was to seek aporocotylid infections that could then be matched with adult aporocotylids through the comparison of species-specific DNA motifs. 2. Materials and methods 2.1. Collections Five sampling expeditions were undertaken between November 2008 and February 2010, both during and outside the ranching season. The search for the intermediate host or hosts of C. forsteri focussed on bivalves and polychaetes because these taxa have been implicated in the life cycles of marine aporocotylids. A few gastropods (known as hosts of aporocotylids only in fresh water) were examined; these proved to be a relatively minor proportion of fauna at the sites of collection. Among bivalves and polychaetes, we examined all species collected but focussed on some taxa more than others. Thus, scallops (Pectenidae) were examined intensively because this family is known to harbour aporocotylids but mussels (Mytilidae) less intensively because this heavily-studied family has never been shown to harbour aporocotylids and they have been previously examined in some detail at Port Lincoln (Aiken et al., 2009). Among polychaetes we made stronger efforts to examine sedentary families (e.g. Ampharetidae, Sabellidae, Serpulidae and Terebellidae) than errant families. For all invertebrate examinations initial identifications were taken no further than family level except for a few larger, obvious taxa. Invertebrates were collected in several ways. Substantial numbers of benthic grab samples were taken from the immediate vicinity of tuna pontoons. The samples retrieved coarse sandy to muddy substrates and small amounts of hard calcareous structure in the form of dead shells. These samples were immediately sieved with a 1 mm mesh sieve and retained material was transferred into fresh sea water and returned to the laboratory. Divers collected larger benthic invertebrates, notably pectenid scallops, Pinna bicolour (Pinnidae), the large purple fan-worm Myxicola infundibulum (Sabellidae), and hard benthic habitat, mainly in the form of dead encrusted shells. Divers also collected encrusting ‘‘biofouling’’ directly from the nets of tuna pontoons. Finally, various bivalves, polychaetes and gastropods were collected by hand at inter-tidal shore sites as close to tuna pontoons as could be reached. All invertebrates were returned fresh to the laboratory where they were maintained in aerated seawater. Samples were sorted and extracted by a variety of methods. Some sedentary polychaetes were effectively collected by vigorous shaking of hard benthic substrate (dead shells) in sea water followed by sorting of the resulting sediment. Animals were sorted by eye or under a dissecting microscope, identified to family or other taxonomic level and counted. Each invertebrate was dissected individually in sea water. In the case of bivalves and gastropods, most attention was paid to the gonad and digestive gland. In the case of polychaetes, the entire body was pulled apart. For one bivalve species, Hiatella australis, and one polychaete, Galeolaria caespitosa, some were dissected but most were held for 24–48 h in clean sea water and monitored for the presence of emerged cercariae by periodic examination under a dissecting microscope. Aporocotylid asexual stages were preserved in absolute alcohol for subsequent molecular analysis and heat-fixed (as per Cribb and Bray, 2010) and preserved in formalin for morphological study. T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 Specimens for permanent preparations were washed in tap water, stained with Mayer’s haematoxylin, destained in 1% HCl, neutralised in 1% ammonia solution, dehydrated through a graded series of alcohols, cleared in methyl salicylate and mounted in Canada balsam. Measurements were made using a SPOT Insight™ digital camera (Diagnostic Instruments, Inc.) mounted on an Olympus BH-2 compound microscope using SPOT™ imaging software and are in micrometres. Voucher specimens of infected polychaetes were preserved in 70% alcohol for morphological study and small sections in absolute alcohol for potential molecular analysis. 2.2. PCR For parasite molecular analyses, genomic DNA (gDNA) was extracted using a DNeasy Blood and Tissue kit (QIAGEN) according to the manufacturer’s instructions. The quantity of gDNA per sample following extraction was assessed by fluorometry (Wallac, Perkin Elmer, USA) using Quant-iT™ PicoGreenÒ (Invitrogen). The partial D1–D2 domains of the lsrDNA was amplified by PCR using the following oligonucleotide primers: Forward (50 -TAG GTC GAC CCG CTG AAY TTA AGC A-30 ) and Reverse (50 -CTT GGT CCG TGT TTC AAG ACG GG-30 ), while the second internal transcribed spacer of rDNA (ITS-2 rDNA) was amplified using: Forward (50 -AGA ACA TCG ACA TCT TGA AC-30 ) and Reverse (50 -CCT GGT TAG TTT CTT TTC CTC CGC-30 ). All PCR reactions were carried out in 25 ll reactions with Bioline BioMix Red PCR Mastermix (2) (12.5 ll) and PCR primers (final concentration 10 pmol/ll): with the remaining volume made up with RNAase-free H2O, on a MJ Research PTC225 Peltier thermocycler using the following thermocycling conditions: 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s, with a final extension step of 72 °C for 10 min. PCR products were subjected to 1.5% Tris–acetate-EDTA (TAE) agarose gel electrophoresis, stained with GelRed (Biotium) and visualised on a GelDoc (BioRad). Positive PCR products were purified using QIAquick PCR purification kit (QIAGEN), and concentration of products was estimated by fluorometry (Wallac) using Quant-iT™ PicoGreenÒ (Invitrogen). Sequencing of both the forward and reverse strands was carried out using ABI Big Dye Terminator v3.1 chemistry (Applied Biosystems) by the Australian Genome Reference Facility (AGRF), Adelaide, Australia. The contiguous sequences were aligned using Sequencher™ (GeneCodes Corp., Ann Arbor, USA ver. 4.2). 863 ysis was conducted on the lsrDNA dataset using the TVM+I+G model predicted (proportion of invariable sites = 0.187 and gamma distribution = 1.136) as the best estimator by both the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) in jModelTest. Bayesian inference analysis was run over 10,000,000 generations (ngen = 10,000,000) with two runs each containing four simultaneous Markov Chain Monte Carlo (MCMC) chains (nchains = 4) and every 1,000th tree saved (samplefreq = 1 000). Bayesian analyses used the following parameters: nst = 6, rates = invgamma, ngammacat = 4, and the priors parameters of the combined dataset were set to ratepr = variable. Samples of substitution model parameters, and tree and branch lengths were summarised using the parameters ‘sump burnin = 3,000’ and ‘sumt burnin = 3,000’. These ‘burnin’ parameters were chosen because the log likelihood scores ‘stabilized’ well before 3,000,000 replicates in the Bayesian inference analyses. In addition to the sequences newly obtained here, Dr. M. Nolan provided us with two previously unpublished lsrDNA sequences for Cardicola coeptus Nolan and Cribb, 2006. These and the newly obtained sequences were added to Genbank (http://www.ncbi.nlm.nih.gov/Genbank/) under the Accession No. JF800668 for C. forsteri lsrDNA, JF800669 for Aporocotylid Type A lsrDNA, JF803976– JF803977 for Cardicola coeptus lsrDNA, JF800670 for C. forsteri ITS-2 rDNA and JF800671 for Aporocotylid Type A ITS-2 rDNA. 3. Results 3.1. Field results Table 1 summarises the 9,351 bivalves, polychaetes and gastropods from Port Lincoln waters that were collected and examined for infection with aporocotylid asexual stages. Stages (sporocysts and cercariae) consistent with the Aporocotylidae were found in six individuals of Terebellidae (Polychaeta). Five of the polychaetes could be identified as Reterebella aloba Hutchings and Glasby and the sixth as Longicarpus modestus (Quatrefages). Infections from the five individual R. aloba were indistinguishable from each other and are hereafter referred to as aporocotylid Type A. The infection in L. modestus was immediately distinguishable from those in R. aloba in that the sporocysts were no more than half the length of those from Reterebella sp. This latter species was shown by molecular analysis (below) to be consistent with C. forsteri and is referred to as such hereafter. 2.3. Molecular analysis 3.2. Molecular analyses A dataset incorporating data from the partial lsrDNA region sequenced from larval aporocotylids reported here and data from a range of trematode taxa obtained from GenBank were initially aligned using MUSCLE version 3.7 (Edgar, 2004) with ClustalW sequence weighting and UPGMA (Unweighted Pair Group Method with Arithmetic Mean) clustering for iterations 1 and 2. The resultant alignments were refined by eye using MESQUITE (Maddison, W.P., Maddison, D.R., 2009. Mesquite: a modular system for evolutionary analysis. Version 2.72). After the alignment of the large subunit dataset was edited, the ends of each fragment were trimmed to match the shortest sequence in the alignment. Bayesian inference analysis of the lsrDNA dataset was performed using MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003) run on the CIPRES portal (Miller, M.A., Holder, M.T., Vos, R., Midford, P.E., Liebowitz, T., Chan, L.e.a., 2009. The CIPRES Portals. CIPRES. 2009-08-04. URL: http://www.phylo.org/sub_sections/portal, accessed: 2009-08-04 (archived by WebCite(r) at http:// www.webcitation.org/5imQlJeQa) to explore relationships among these taxa. The software jModelTest version 0.1.1 (Guindon and Gascuel, 2003; Posada, 2008) was used to estimate the best nucleotide substitution model for this dataset. Bayesian inference anal- Sequences of the ITS-2 and partial D1–D2 domains of lsrDNA of the cercariae found in L. modestus were identical to those of adult C. forsteri available from GenBank (DQ059637.1, EF661575.1, EF653387.1 and EF653389.1). Multiple replicates of ITS-2 and partial D1–D2 domains of lsrDNA of Aporocotylidae Type A from R. aloba were identical to each other but did not match any sequence available from GenBank. Initially, the lsrDNA data set was analysed with three bivesiculids (Order Plagiorchiida) as a combined outgroup for a wide range of diplostomidan taxa including all available putative aporocotylids and a wide range of Schistosomatidae, Spirorchiidae, Diplostomoidea, Clinostomidae and Brachylaimoidea. This analysis (not shown) identified that, consistent with the findings of Olson et al. (2003), the Brachylaimoidea was sister to the remainder of the Diplostomida and that the Diplostomoidea was sister to all of the blood fluke taxa. Remaining analyses were therefore performed with a reduced, realigned data set from which the bivesiculids and brachylaimoids were excluded and in which the species of Clinostomidae were defined as the outgroup (Fig. 1). The trimmed lsrDNA alignment comprised 859 bases from the D1/D2 domain of which 864 T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 Table 1 Bivalves, gastropods and polychaetes examined for infection by aporocotylids collected off Port Lincoln, South Australia. Most taxa were identified only to family. Infections were found in six individual terebellid specimens. Group Family Species Total Bivalvia Hiatellidae Limidae Malleidae Mesodesmatidae Mytilidae Hiatella australis 2,413 17 1 268 39 494 24 28 10 306 71 300 10 2 45 38 38 12 151 113 111 64 5 2 13 198 28 117 241 215 267 7 60 25 27 415 22 13 2,261 86 60 734 Ostreidae Pectenidae Gastropoda Polychaeta Pinnidae Pteriidae Tellinidae Veneridae Littorinidae Neritidae Ampharetidae Aphroditidae Capitellidae Cirratulidae Dorvilleidae Eunicidae Glyceridae Goniadidae Hesionidae Lumbrineridae Magelonidae Maldanidae Nephtyidae Nereididae Opheliidae Oweniidae Pectinariidae Phyllodocidae Polynoidae Sabellidae Scalibregmatidae Serpulidae Spionidae Terebellidae Total Paphies cuneata Brachidontes erosus Mytilus galloprovincialis Ostrea angasi Chlamys asperrima Equichlamys bifrons Pecten fumatus Pinna bicolor Electroma georgiana Myxicola infundibulum Galeolaria caespitosa 9,351 467 were parsimony informative. Most major clades were resolved with strong posterior probability support. The 11 schistosomatid genera formed a monophyletic clade with high support. As reported previously by Snyder (2004), the genera of spirorchiids of turtles were paraphyletic relative to the Schistosomatidae (and again did not include the crocodile-infecting genus Griphobilharzia). The Aporocotylidae was not monophyletic. Chimaerohemecus (from a holocephalan) and Sanguinicola (a freshwater genus) formed a clade with low support distinct from all other adult aporocotylids. This clade included the unassociated putative freshwater aporocotylid cercaria reported from Thiara sp. from Australia by Brant et al. (2006); it formed a clade with Sanguinicola with 100% support. All other aporocotylids, all of them marine, formed a clade with 100% support. Within this clade, Paradeontacylix sinensis Liu, 1997 was phylogenetically distant from the five other species of Paradeontacylix for which sequences are available. The three species of Cardicola, Cardicola aurata Holzer, Montero, Repulles, Nolan, Sitja-Bobadilla, Alvarez-Pellitero, Zarza and Raga, 2008, C. coeptus Nolan and Cribb, 2006 and C. forsteri, and the infection from R. aloba formed a well-supported clade which in turn formed a strongly supported clade with five species of Paradeontacylix. Unassociated putative freshwater aporocotylid cercariae reported from the gastropod genera Glyptophysa from Australia and Biomphalaria and Segmentorbis from Africa by Brant et al. (2006) did not form clades with any identified taxa. 3.3. Description of life cycle stages 3.3.1. Cardicola forsteri Cribb, Daintith and Munday, 2000 (Fig. 2A and B) Intermediate host: Longicarpus modestus (Quatrefages) (Terebellidae) Australian Museum (AM), Sydney, voucher W37009. Site: off Port Lincoln, South Australia, 34°42.1080 S 135°57.6330 E, 22 m depth. Sporocyst: free in body cavity (coelom). Body a smooth tube tapering towards each end, capable of pronounced extension and contraction in life, 251–426 (328)  65–87 (74) (n = 10). Cercariae in all stages of development from germinal balls to apparently fully developed cercariae; no more than 10 recognisable cercariae present per sporocyst. Birth pore not observed. Cercaria: body elongate, tubular, 66–75 (71)  13–20 (16.4) (n = 5); anterior penetration organ (oral sucker) distinguished by slight constriction, 11–12 (11.6)  13–14 (13.8). Penetration gland cell bodies identifiable in mid-body but numbers not countable. Dorsal fin-fold not detected in living or preserved specimens. Tail simple with no evidence of bifurcation, 40–55 (47) long and 4–6 (4.9) wide. Excretory vesicle small, rounded, at base of tail. Deposited specimens: Queensland Museum, Brisbane (QM), G 232981–9. Remarks: the single infected individual was immediately recognised as being infected when examined under a dissecting microscope. Hundreds of sporocysts were easily visible through the body wall moving freely in the coelom of the worm as it underwent peristaltic movement. No cercariae were found swimming in the water in which the polychaete had been found and none were observed in the body cavity of the polychaete when it was opened to allow sporocysts to be collected. Cercariae could only be observed by rupturing sporocysts. When this was done they wriggled actively and swam feebly. 3.3.2. Aporocotylid Type A. (Fig. 2C and D) Host: Reterebella aloba Hutchings and Glasby (Terebellidae) AM vouchers W37010-1 Site: off Port Lincoln, South Australia, 34°42.1080 S 135°57.6330 E, 22 m depth. Sporocysts: free in body cavity (coelom). Body a smooth tube tapering to point at each end, usually one end noticeably more pointed than other, 866–1530 (1258)  99–206 (171) (n = 10). Cercariae in all stages of development from germinal balls to apparently fully developed cercariae; most well-developed cercariae concentrated at one end of sporocyst; >100 recognisable cercariae present. Birth pore not observed. Cercaria: body elongate, tubular, 86–103 (95)  11–19 (14.8) (n = 7); anterior penetration organ (oral sucker) distinguished by slight constriction, 10–18 (13.8)  11–14 (12.2). Penetration gland cell bodies identifiable in mid-body but numbers not countable. Dorsal fin-fold not detected in living or preserved specimens. Tail simple, with no evidence of bifurcation, 41–51 (45.8) long and 5– 6 (5.6) wide. Excretory vesicle small, rounded, at base of tail. Deposited specimens: QM G 232990–3003. Remarks: four of the five infected individuals of the polychaete were already damaged when found and the infections were immediately obvious because large, active sporocysts were spilling from holes in the body. Sporocysts were found along the length of the body cavity. In one case sporocysts were observed emerging from lateral gonopores of the polychaete. In most of the polychaetes there was no sign of eggs, but in one there were a significant number of eggs which, however, looked malformed. One intact infected R. aloba was kept alive overnight in a Petri dish in clean seawater T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 865 Fig. 1. Bayesian analysis of relationships of aporocotylids and related taxa based on partial lsrDNA sequences. Percentage posterior probability support for clades is indicated at the nodes. but no free-swimming cercariae were found in this water and, as for the infection of C. forsteri, none were found free in the body cavity of infected polychaetes when they were dissected. However, when sporocysts were opened the cercariae wriggled actively and swam feebly. As for the infection in L. modestus, the infection of the intact specimen of R. aloba was easily recognised by large numbers of sporocysts moving freely in the coelom. 4. Discussion Identical lsrDNA and ITS-2 rDNA sequences for the infection from the terebellid polychaete L. modestus and from adult C. forsteri allow us to identify the polychaete infection positively. There is now a substantial literature using rDNA sequences to match trematode life cycles (e.g. Cribb et al., 1998; Jousson and Bartoli, 2000; Brant et al., 2006) and this approach has already been used once previously, for an aporocotylid trematode of Australian freshwater eels (Nolan and Cribb, 2004b). There is also a substantial data set of aporocotylid ITS-2 rDNA sequences that allows variation in this marker to be explored. For Cardicola, 11 described species and four further undescribed species have been sequenced for ITS-2 rDNA (Nolan and Cribb, 2006; Holzer et al., 2008). These sequences differ by 1–61 bases; sequences of C. forsteri differ from all other species at a minimum of 12 bases. Short of experimental transmission 866 T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 Fig. 2. Aporocotylid stages from terebellid polychaetes. (A) Cardicola forsteri sporocyst from Longicarpus modestus. (B) Cardicola forsteri cercariae. (C) Aporocotylid Type A cercariae. (D) Aporocotylid Type A sporocyst from Reterebella aloba. Scale bars (A) 100 lm; (B and C) 50 lm; (D) 200 lm. from polychaete to tuna or vice versa, we can consider that an intermediate host for this life cycle is demonstrated. The identity of the Type A infection remains unknown. However, phylogenetic analysis demonstrates unequivocally that this species falls within a clade of species of Cardicola, suggesting that it will also prove to belong to that genus. Given the similarity of intermediate host and the morphological similarity of the cercaria and asexual stages, this finding is not surprising. The identity of the definitive host of the Type A infection is difficult to predict. The aporocotylid fauna of fishes of southern Australia is imperfectly known. In addition to C. forsteri, species are known from labrids (Nolan and Cribb, 2005), monacanthids (Nolan and Cribb, 2004a) and carangids (Hutson and Whittington, 2006); on the basis that none of these are species of Cardicola, we predict that none of them will match with the present form. We are aware of further undescribed species in the area and it is likely that there are many more undiscovered species in fishes in this area. We conclude that this analysis provides further evidence of the power of a molecular approach for the elucidation of life cycles. Where a direct match between larval and adult stages is found it may be possible to infer a life cycle connection with some confidence. Where there is no match, phylogenetic inference may inform on the likely systematic position of the adult and in some circumstances on the likely host group. In the present case, this analysis has also supported the morphology-based suggestions of Ogawa et al. (2007) and Repulles-Albelda et al. (2008) that it is likely that P. sinensis has affinity to Psettarium Goto and Ozaki, 1930 rather than Paradeontacylix. In addition, our analyses cast doubt on whether three cercariae reported from Australian and African snails (species of Glyptophysa from Australia and Biomphalaria and Segmentorbis from Africa) by Brant et al. (2006) belong to the Aporocotylidae, although their final identity is far from clear and they remain taxa of great interest. Here we review the morphology of the two new cercariae relative to those described previously. Table 2 lists the cercariae that were considered. Only the cercariae for A. simplex, C. forsteri (this study), Paracardicoloides yamagutii Martin, 1974 and several species of Sanguinicola have been demonstrated unambiguously to 867 T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 Table 2 Records of known and probable aporocotylid cercariae and their hosts. Aporocotylid species Host group Host species Reference martini Stunkard (1983) (un-named) asymmetrica Holliman (1961) cristulata Holliman (1961) mercenariae Wardle (1979) solemyae Martin (1944) Bivalvia Pectinidae Solecurtidae Donacidae Veneridae Veneridae Solemyidae Pecten irradians Tagelus divisus Donax variabilis Chione cancellata Mercenaria campechiensis Solemya velum Linton (1915a) Fraser (1967) Holliman (1961) Holliman (1961) Wardle (1979) Martin (1944a) Cercaria cristafera Erickson and Wallace (1959) Paracardicoloides yamagutii Martin (1974) Aporocotylid type b Sanguinicola alseae Meade and Pratt (1965) Sanguinicola armata Plehn (1905) Gastropoda Valvatidae Hydrobiidae Hydrobiidae Hydrobiidae Bithyniidae Valvata tricarinata Posticobia brazieri Posticobia brazieri Oxytrema silicula Bithynia leachi Erickson and Wallace (1959) Nolan and Cribb (2004b) Nolan and Cribb (2004b) Meade and Pratt (1965) Sendersky and Dobrovolsky (2004) Rawstron (1971) Hoffman et al. (1985) Schell (1974) Kirk and Lewis (1993) Cercaria Cercaria Cercaria Cercaria Cercaria Cercaria Sanguinicola Sanguinicola Sanguinicola Sanguinicola davisi Wales (1958) fontinalis Hoffman, Fried and Harvey (1985) idahoensis Schell (1974) inermis Plehn (1905) Sanguinicola klamathensis Wales (1958) Sanguinicola lophophora Erickson and Wallace (1959) Sanguinicola rutili Simon-Martin, Rojo-Vazquez and SimonVicente (1987) Hydrobiidae Pleuroceridae Hydrobiidae Lymnaeidae Hydrobiidae Valvatidae Ancylidae Aporocotyle simplex Odhner (1900) Polychaeta Terebellidae Cercaria amphicteis Oglesby (1961) Cercaria hartmanae Martin (1952) Cercaria loossi Stunkard (1929) Ampharetidae Terebellidae Serpulidae belong to the Aporocotylidae although it is provisionally accepted that all of the species listed here do belong to this family. The list of cercariae of species of Sanguinicola, and other unassociated freshwater cercariae likely belonging to that genus, is not comprehensive. However, the reports for this genus that we have considered are relatively uniform in the morphology of the cercariae concerned and we have found this literature somewhat confused. All known putative marine aporocotylid cercariae are listed in Table 2. Unfortunately, given the nature of the present study, a detailed morphological analysis of living samples was not possible; stained and mounted specimens, especially cercariae, revealed little internal morphological detail. Regardless, there are several important observations to be made on the basis of the external form. The most commonly reported aporocotylid cercarial form is that seen for numerous infections from freshwater. In these, the cercarial body has a dorsal fin-fold and the tail is prominent and distinctly forked (e.g. Erickson and Wallace, 1959; Meade and Pratt, 1965; Meade, 1967; Evans and Heckmann, 1973; Kirk and Lewis, 1993; Nolan and Cribb, 2004b), although according to Wales (1958) the body fin-fold is lacking from the cercaria of Sanguinicola davisi Wales, 1958. Four of the six forms known from bivalves are ‘typical’ whereas Cercaria martini Stunkard, 1983 reported by Linton (1915a) from a scallop appears to lack a dorsal fin-fold and the other, Cercaria solemyae Martin, 1944, lacks a dorsal fin-fold and has only a tiny simple tail. Of the forms previously reported from polychaetes, Cercaria loossi Martin, 1944 and Cercaria martini Stunkard, 1983 reported from Hydroides dianthus (Serpulidae) by Linton (1915) have ‘typical’ morphology. The cercariae of A. simplex and Cercaria hartmanae Martin, 1952 both have forked tails (Martin, 1952; Køie, 1982) but lack a fin-fold on the body. Cercaria hartmanae is unique in being reported to have a developing ventral sucker (=acetabulum); this character is of particular interest as no adult aporocotylid is reported to possess any trace of a ventral sucker. Finally, Cercaria amphicteis Oglesby, 1961 lacks a dorsal fin-fold and Oxytrema circumlineata Leptoxis (Mudalia) carinata Lithoglyphus virens Lymnaea auricularia, Lymnaea peregra, Lymnaea stagnalis Fluminicola fusca, Fluminicola seminalis Valvata tricarinata Ancylus fluviatilis Artacama proboscidea Lanassa nordenskioeldi Amphicteis gunneri floridus Lanicides vayssierei Eupomatus dianthus Hydroides hexagonus Meade (1967) Erickson and Wallace (1959) Simon-Martin et al. (1987) Køie (1982) Køie and Petersen (1988) Oglesby (1961) Martin (1952) Linton (1915b) Martin (1944b) has a simple tail. Thus, in lacking forked tails and body fin-folds the forms described here are unusual but not unique for the family. We note that it is possible that absence of a fin-fold might relate to immaturity of the cercariae but, although none were found naturally free-swimming, we think they were essentially mature and that the absence of the fin-fold is real. The two cercariae reported here had maximum body lengths of only about 75 lm (C. forsteri) and 103 lm (Aporocotylid Type A). This is towards the smallest size recorded in the family which is 65–81 lm for the unidentified ‘‘Type b’’ of Nolan and Cribb (2004b) from an Australian freshwater hydrobiid gastropod. Aiken et al. (2009) used a predicted body length of 50 lm as a starting point for body growth in their stochastic simulations of the development of C. forsteri. Although this figure is slightly lower than reported here, it is not at sufficient variance to materially affect their conclusions. There is a continuum of body sizes including lengths exceeding 200 lm (Cercaria cristulata Holliman, 1961, Cercaria cristafera Erickson and Wallace, 1959 and Cercaria mercenariae Wardle, 1979) and even 300 lm (Sanguinicola klamathensis Wales, 1958) (see Erickson and Wallace, 1959; Holliman, 1961; Meade, 1967; Wardle, 1979). The lengths of tails also vary dramatically. The present two species are unusual in having the tail distinctly shorter than the body. By far the smallest tail is that of C. solemyae which is less than 20% of the body length (Martin, 1944a). Cercaria amphicteis has a tail about 80% of the length of the body. In all other reported cercariae the tail is longer than the body. The four species in which the tail is proportionally smallest are also the only four in which the tail is simple rather than forked. It has been established (Cribb et al., 2003) that the forked tail is plesiomorphic for the Digenea. We thus conclude that these simple-tailed cercariae represent a derived morphology within the Aporocotylidae. We speculate that the modification relates to the adoption of a more passive transmission strategy for the cercaria than is seen for larger fork-tailed cercariae. Whether the modification is associ- 868 T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 ated with the transmission to a pelagic fish as for C. forsteri may become clear as the definitive hosts of other aporocotylid cercariae with small tails are identified. Further variation can be noted in the asexually reproducing generations of aporocotylids. Most studies report development in sporocysts but Ce. hartmanae and Ce. amphicteis are reported from unambiguous rediae (Martin, 1952; Oglesby, 1961). Overall, the morphology of aporocotylid cercariae is exceptionally variable so that almost no character is reliably present in all forms. Almost all of these forms share a prominent anterior penetration organ which is generally interpreted as being a modified oral sucker, however even this is lacking in C. solemyae. In their analysis of the overall phylogeny of the Trematoda using combined ssrDNA and lsrDNA, Olson et al. (2003) reported exceptionally long branch lengths for the Aporocotylidae. These were much longer than, for example, between schistosomatids of tetrapods. Overall we would argue that the morphological variation is consistent with an ancient and highly radiated taxon. The remarkable range of first intermediate hosts of aporocotylids is consistent with the same interpretation. The terebellid identified as intermediate host for C. forsteri, L. modestus, was described from Jervis Bay in New South Wales, Australia, but has since been reported from all Australian states (Hutchings and Glasby, 1988). This species is reported from inter-tidal zones to depths of at least 30 m. The terebellid identified as the host of the aporocotylid Type A is R. aloba, a species reported from New South Wales and South Australia (Hutchings and Glasby, 1988). These identifications bring the number of terebellid species known as hosts of aporocotylids to five. The genera involved in addition to Longicarpus Hutchings and Murray and Reteterebella Hartman are Artacama Malmgren, Lanassa Malmgren and Lanicides Hessle. According to the morphology-based phylogeny of the Terebellidae proposed by Garraffoni and Lana (2008), all belong to the subfamily Terebellinae (syn. Amphitritinae). It appears that the Terebellinae are a major host group for aporocotylids, however due to insufficient sampling it is not possible to conclude that the other terebellid subfamilies (Thelopodinae, Trichobranchinae and Polycirrinae) do not harbour aporocotylids. There are now four aporocotylid species known to infect terebellid polychaetes and another two from other families of polychaetes. There are also six marine species known from bivalves but none from marine gastropods despite their exclusive use in known freshwater cycles. The two known marine life cycles relate to the genera Aporocotyle Odhner, 1900 and Cardicola. These are the two largest marine genera; Aporocotyle has 16 species and Cardicola has approximately 25 species. In general, closely related trematodes tend to infect closely related first intermediate hosts (Cribb et al., 2001). On this basis we propose that all species of these genera are likely to infect terebellids. This prediction extends to the recently described Cardicola orientalis Ogawa, Tanaka, Sugihara and Takami 2009 which infects Pacific bluefin tuna, Thunnus orientalis, in aquaculture in Japan (Ogawa et al., 2010). Unfortunately these results do not allow the prediction of the intermediate hosts of other pathogenic marine aporocotylids. In this context, it is noteworthy that there is a discrepancy in the intermediate hosts of Sanguinicola spp. from freshwater fishes. At least five species have been shown to use pulmonate gastropods as intermediate hosts and a further eight are reported to use orthogastropods (=‘‘prosobranchs’’). Such a deep distinction in the identity of the hosts of a single genus of aporocotylids might cast doubt on the likely consistency of use of other genera of aporocotylids. We acknowledge that this requires considerable further analysis. In respect to the present findings, an interesting aspect of C. forsteri is its cosmopolitan distribution (Bullard et al., 2004; Aiken et al., 2007). In contrast, L. modestus is known only from Australian waters (Hutchings and Glasby, 1988). We predict that C. forsteri infects a mosaic of terebellid species in different parts of the world. In addition, it is entirely possible that more than one terebellid species is infected in the environs of Port Lincoln. In this study, terebellids were not identified to species during initial survey work so as to maximise the numbers of individuals examined; we therefore cannot comment usefully on prevalence of either of the species in the terebellids. The category ‘‘Terebellidae’’ incorporated significant numbers of trichobranchines and polycirrines, none of which were infected. Determination of whether more species are infected would require significant further field-work. These results could contribute to controlling pathogenic infections of this species in Australia and potentially in comparable species elsewhere. The principal implication of the identification of L. modestus as the intermediate host is that it identifies the source of infection to the tuna as the benthic sediment immediately beneath the cages. The free-living stages of aporocotylids, the cercaria and the miracidium, are both small, non-feeding and with life-spans that probably do not often exceed 1 day. Meade and Pratt (1965) reported that cercariae of Sanguinicola alseae (Meade and Pratt, 1965) survived only a few hours at 22 °C but for up to 2 days at 12.8 °C. Holliman (1961) reported survival of about 12 h for both Ce. asymmetrica and Ce. cristulata and Martin and Vazquez (1984) a maximum survival of the cercaria of a species of Sanguinicola of about 15 h. We are unaware of any longevity data for aporocotylid miracidia, but it seems likely that they survive no longer than the cercariae. We predict therefore that any significant physical separation of tuna and polychaete should reduce transmission from polychaete to fish and, perhaps as importantly, from fish to polychaete. The relatively small tail of the cercaria of C. forsteri may be significant in this context. We suspect that the cercaria swims only feebly as it is so small. If this is the case, distribution by currents is likely to be important in transmission ecology. Chambers and Ernst (2005) explored the dispersal capacity of the eggs of the monogenean parasite Benedenia seriolae in waters comparable to those in which SBT are raised. This parasite is also transmitted by a short-lived non-feeding stage, the oncomiracidium. Their key finding was that, in the hydrographic circumstances of their study, sources of infection could affect fish at distances of up to 8 km. Thus, if infection is to be controlled by horizontal movement of the pontoons then the movement may need to be substantial and the direction of prevailing currents will be important. If transmission of C. forsteri is to be reduced by physical separation then such separation might also be vertical, by keeping the nets further from the bottom (changing net depth or moving to deeper water). In this connection, it is noteworthy that Hayward et al. (2010) observed that infections of C. forsteri were heavier and potentially more damaging in SBT in Australia than in any other ranched tuna operation and that the pontoons in Australia are deployed in shallower water than those elsewhere. When considering control measures, it is potentially significant that the single C. forsteri-infected polychaete was collected in February 2010, only a few weeks after the first tuna had been transferred to pontoons in the region for the year in late December 2009. We think it unlikely that the polychaete was infected to the point of the heavy infection in this short time-frame. More likely the infection had survived since the previous season. Almost all tuna had been harvested by September 2009, suggesting that infections in polychaetes have a longevity of at least 4–5 months. Nothing is known about the longevity of L. modestus but other species such as Eupolymnia nebulosa in the Mediterranean (Bhaud and Gremare, 1991) and Neoleprea streptochaeta in the sub-Antarctic (Duchene, 1980) are reported to have life-spans of about four and at least 5 years, respectively. Thus, it seems likely that infected L. modestus would easily be able to survive the period for which there are no tuna in the Port Lincoln area rather than establishing T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 new infections every season. Thus, on the basis of these limited data, we predict that if sites are to be left fallow then this would need to be for at least an entire season and possibly longer. Previous efforts to control fish blood flukes have been very limited. Combs (1968) proposed the use of an electrical grid to control trematode cercariae, including those of aporocotylids, in hatchery water supplies; the method does not appear to have been adopted. Sapozhnikov et al. (1986) described prophylactic measures that excluded aporocotylids from Russian freshwater fish farms. Sapozhnikov (1988) also reported local eradication of the freshwater species Sanguinicola inermis Plehn, 1905 by applying a combination of chemical, biological and ecological measures against its lymnaeid gastropod intermediate hosts. Such methods appear to have little application in systems such as are affected by species like C. forsteri for which eradication appears an unlikely ultimate goal. Acknowledgements We thank Brenton Ebert (Skipper of the Breakwater Bay), Jason Nichols and Leo Mantilla from the South Australian Research and Development Institute for their help on the boat. We thank Melanie Andrews, Nicole Gunter, Elizabeth Nosworthy, Kirsten Rough and Lexie Walker for help both on the water and in the laboratory. We thank the many divers for the provision of large quantities of benthic samples. We thank Dr. Matt Nolan for generously making available unpublished sequences. We thank Dr. Terry Miller of the Queensland Museum, Australia for help with the phylogenetic analysis and Dr. Pat Hutchings of the Australian Museum for the identification of terebellid polychaetes. This project was financially supported by the Fisheries Research and Development Corporation, Australia. References Aiken, H., Hayward, C.J., Cameron, A., Nowak, B., 2009. Simulating blood fluke, Cardicola forsteri, infection in farmed southern bluefin tuna, Thunnus maccoyii, using stochastic models. Aquaculture 293, 204–210. Aiken, H.M., Bott, N.J., Mladineo, I., Montero, F.E., Nowak, B.F., Hayward, C.J., 2007. Molecular evidence for cosmopolitan distribution of platyhelminth parasites of tunas (Thunnus spp.). Fish Fish 8, 167–180. Aiken, H.M., Hayward, C.J., Nowak, B.F., 2006. An epizootic and its decline of a blood fluke, Cardicola forsteri, in farmed southern bluefin tuna, Thunnus maccoyii. Aquaculture 254, 40–45. Bhaud, M., Gremare, A., 1991. Reproductive cycle of Eupolymnia nebulosa (Polychaeta, Terebellidae) in the Western Mediterranean Sea. Ophelia 295, 304. Brant, S.V., Morgan, J.A.T., Mkoji, G.M., Snyder, S.D., Rajapakse, R., Loker, E.S., 2006. An approach to revealing blood fluke life cycles, taxonomy, and diversity: provision of key reference data including DNA sequence from single life cycle stages. J. Parasitol. 92, 77–88. Bullard, S.A., Goldstein, R.J., Goodwin, R.H., Overstreet, R.M., 2004. Cardicola forsteri (Digenea: Sanguinicolidae) from the heart of a northern bluefin tuna, Thunnus thynnus (Scombridae), in the northwest Atlantic Ocean. Comp. Parasitol. 71, 245–246. Chambers, C.B., Ernst, I., 2005. Dispersal of the skin fluke Benedenia seriolae (Monogenea: Capsalidae) by tidal currents and implications for sea-cage fanning of Seriola spp. Aquaculture 250, 60–69. Colquitt, S.E., Munday, B.L., Daintith, M., 2001. Pathological findings in southern bluefin tuna, Thunnus maccoyii (Castelnau), infected with Cardicola forsteri (Cribb, Daintith & Munday, 2000) (Digenea: Sanguinicolidae), a blood fluke. J. Fish Dis. 24, 225–229. Combs, B.D., 1968. An electrical grid for controlling trematode cercariae in hatchery water supplies. Prog. Fish-Cult. 30, 67–75. Crespo, S., Grau, A., Padros, F., 1992. Sanguinicoliasis in the cultured amberjack Seriola dumerili Risso, from the Spanish Mediterranean area. B. Eur. Assoc. Fish Pat. 12, 157–159. Cribb, T.H., Anderson, G.R., Adlard, R.D., Bray, R.A., 1998. A DNA-based demonstration of a three-host life-cycle for the Bivesiculidae (Platyhelminthes: Digenea). Int. J. Parasitol. 28, 1791–1795. Cribb, T.H., Bray, R.A., 2010. Gut wash, body soak, blender and heat-fixation: approaches to the effective collection, fixation and preservation of trematodes of fishes. Syst. Parasitol. 76, 1–7. Cribb, T.H., Bray, R.A., Littlewood, D.T.J., 2001. The nature and evolution of the association between digeneans, molluscs and fishes. Int. J. Parasitol. 31, 997– 1011. Cribb, T.H., Bray, R.A., Olson, P.D., Littlewood, D.T.J., 2003. Life cycle evolution in the Digenea: a new perspective from phylogeny. Adv. Parasitol. 54, 197–254. 869 Cribb, T.H., Daintith, M., Munday, B.L., 2000. A new blood-fluke, Cardicola forsteri, (Digenea: Sanguinicolidae) of southern blue-fin tuna (Thunnus maccoyii) in aquaculture. Trans. R. Soc. S. Aust. 124, 117–120. Dennis, M.M., Landos, M., D’D’Antignana, T., in press. Case–control study of epidemic mortality and Cardicola forsteri-associated disease in farmed southern bluefin tuna (Thunnus maccoyii) of South Australia. Vet. Pathol. doi:10.1177/0300985810388524. Duchene, J.C., 1980. 1st data on reproduction and growth of Neoleprea streptochaeta, polychaetous annelid, in sub-Antarctic province. Ann. Inst. Oceanogr. Paris (Nouv. Ser.) 56, 109–115. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Erickson, D.G., Wallace, F.G., 1959. Studies on blood flukes of the genus Sanguinicola. J. Parasitol. 45, 310–322. Evans, W.A., 1974. Growth, mortality, and hematology of cutthroat trout experimentally infected with the blood fluke Sanguinicola klamathensis. J. Wildl. Dis. 10, 341–346. Evans, W.A., Heckmann, R.A., 1973. The life history of Sanguinicola klamathensis. Life Sci. 13, 1285–1291. Fraser, T.H., 1967. Contributions to the biology of Tagelus divisus (Tellinacea: Pelecypoda) in Biscayne Bay. Florida. Bull. Mar. Sci. 17, 111–132. Garraffoni, A.R.S., Lana, P.C., 2008. Phylogenetic relationships within the Terebellidae (Polychaeta: Terebellida) based on morphological characters. Invertebr. Syst. 22, 605–626. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Hayward, C.J., Ellis, D., Foote, D., Wilkinson, R.J., Crosbie, P.B.B., Bott, N.J., Nowak, B.F., 2010. Concurrent epizootic hyperinfections of sea lice (predominantly Caligus chiastos) and blood flukes (Cardicola forsteri) in ranched Southern Bluefin tuna. Vet. Parasitol. 173, 107–115. Hoffman, G.L., Fried, B., Harvey, J.E., 1985. Sanguinicola fontinalis sp. nov. (Digenea: Sanguinicolidae): a blood parasite of brook trout, Salvelinus fontinalis (Mitchill), and longnose dace, Rhinichthys cataractae (Valenciennes). J. Fish Dis. 8, 529– 538. Holliman, R.B., 1961. Larval trematodes from the Apalachee Bay area, Florida, with a checklist of known marine cercariae arranged in a key to their superfamilies. Tulane stud. zool. 9, 2–74. Holzer, A.S., Montero, F.E., Repulles, A., Nolan, M.J., Sitja-Bobadilla, A., AlvarezPellitero, P., Zarza, C., Raga, J.A., 2008. Cardicola aurata sp. n. (Digenea: Sanguinicolidae) from Mediterranean Sparus aurata L. (Teleostei: Sparidae), its unexpected phylogenetic relationship with Paradeontacylix McIntosh. Parasitol. Int. 57, 472–482. Hutchings, P.A., Glasby, C.J., 1988. The Amphitritinae (Polychaeta: Terebellidae) from Australia. Rec. Aust. Mus. 40, 1–60. Hutson, K.S., Whittington, I.D., 2006. Paradeontacylix godfreyi n. sp. (Digenea: Sanguinicolidae) from the heart of wild Seriola lalandi (Perciformes: Carangidae) in southern Australia. Zootaxa 1151, 55–68. Jousson, O., Bartoli, P., 2000. The life cycle of Opecoeloides columbellae (Pagenstecher, 1863) n. Comb. (Digenea, Opecoelidae): evidence from molecules and morphology. Int. J. Parasitol. 30, 747–760. Kirk, R.S., Lewis, J.W., 1993. The life-cycle and morphology of Sanguinicola inermis Plehn, 1905 (Digenea: Sanguinicolidae). Syst. Parasitol. 25, 125–133. Køie, M., 1982. The redia, cercaria and early stages of Aporocotyle simplex Odhner, 1900 (Sanguinicolidae) – a digenetic trematode which has a polychaete annelid as the only intermediate host. Ophelia 21, 115–145. Køie, M., Petersen, M.E., 1988. A new annelid intermediate host (Lanassa nordenskioeldi Malmgren, 1866) (Polychaeta: Terebellidae) for Aporocotyle sp. and a new final host family (Pisces: Bothidae) for Aporocotyle simplex Odhner, 1900 (Digenea: Sanguinicolidae). J. Parasitol. 74, 499–502. Linton, E., 1915a. Note on trematode sporocysts and cercariae in marine mollusks of the Woods Hole region. Biol. Bull. Mar. Biol. Lab. Woods Hole 28, 198–209. Linton, E., 1915b. Sporocysts in an annelid. Biol. Bull. Mar. Biol. Lab. Woods Hole 28, 115–118. Martin, S., Vazquez, R., 1984. Biology and behavior of the cercariae of a Sanguinicola sp. in the River Cilloruelo (Salamanca, Spain). Ann. Parasitol. Hum. Comp. 59, 231–236. Martin, W.E., 1944a. Cercaria solemyae n. sp., probably a blood fluke, from the marine pelecypod, Solemya velum. J. Parasitol. 30, 191–195. Martin, W.E., 1944b. Studies on trematodes of Woods Hole IV. Additional observations upon Cercaria loossi Stunkard developing in an annelid. Trans. Am. Microsc. Soc. 63, 237–243. Martin, W.E., 1952. Another annelid first intermediate host of a digenetic trematode. J. Parasitol. 38, 1–4. Meade, T.G., 1967. Life history studies on Cardicola klamathensis (Wales, 1958) Meade and Pratt, 1965 (Trematoda: Sanguinicolidae). Proc. Helm. Soc. Wash. 34, 210–212. Meade, T.G., Pratt, I., 1965. Description and life history of Cardicola alseae sp. n. (Trematoda: Sanguinicolidae). J. Parasitol. 51, 575–578. Nikitina, E.N., 1986. Trematode larvae in snails of Lake Glubokoe. Hydrobiologia 141, 139–141. Nolan, M.J., Cribb, T.H., 2004a. Ankistromeces mariae n. g., n. sp. (Digenea: Sanguinicolidae) from Meuschenia freycineti (Monacanthidae) off Tasmania. Syst. Parasitol. 57, 151–157. Nolan, M.J., Cribb, T.H., 2004b. The life cycle of Paracardicoloides yamagutii Martin, 1974 (Digenea: Sanguinicolidae). Folia Parasitol. 51, 320–326. 870 T.H. Cribb et al. / International Journal for Parasitology 41 (2011) 861–870 Nolan, M.J., Cribb, T.H., 2005. Sanguinicola maritimus n. sp. (Digenea: Sanguinicolidae) from Labridae (Teleostei: Perciformes) of southern Australian waters. Syst. Parasitol. 61, 99–106. Nolan, M.J., Cribb, T.H., 2006. Cardicola Short, 1953 and Braya n. Gen. (Digenea: Sanguinicolidae) from five families of tropical Indo-Pacific fishes. Zootaxa 1265, 1–80. Ogawa, K., Fukudome, M., 1994. Mass mortality caused by blood fluke (Paradeontacylix) among amberjack (Seriola dumerili) imported to Japan. Fish Pathol. 29, 265–269. Ogawa, K., Hattori, K., Hatai, K., Kubota, S., 1989. Histopathology of cultured marine fish, Seriola purpurascens (Carangidae) infected with Paradeontacylix spp. (Trematoda: Sanguinicolidae) in its vascular system. Fish Pathol. 24, 75–81. Ogawa, K., Nagano, T., Akai, N., Sugita, A., Hall, K.A., 2007. Blood fluke infection of cultured tiger puffer Takifugu rubripes imported from China to Japan. Fish Pathol. 42, 91–99. Ogawa, K., Tanaka, S., Sugihara, Y., Takami, I., 2010. A new blood fluke of the genus Cardicola (Trematoda: Sanguinicolidae) from Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan. Parasitol. Int. 59, 44–48. Oglesby, L.C., 1961. A new cercaria from an annelid. J. Parasitol. 47, 233–236. Olson, P.D., Cribb, T.H., Tkach, V.V., Bray, R.A., Littlewood, D.T.J., 2003. Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda). Int. J. Parasitol. 33, 733–755. Padros, F., Zarza, C., Crespo, S., 2001. Histopathology of cultured sea bream Sparus aurata infected with sanguinicolid trematodes. Dis. Aquat. Org. 44, 47–52. Posada, D., 2008. JModelTest: Phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Rawstron, R.R., 1971. Harvest and survival of rainbow trout infected with Sanguinicola davisi Wales. Calif. Fish Game 57, 253–256. Repulles-Albelda, A., Montero, F.E., Holzer, A.S., Ogawa, K., Hutson, K.S., Raga, J.A., 2008. Speciation of the Paradeontacylix spp. (Sanguinicolidae) of Seriola dumerili. Two new species of the genus Paradeontacylix from the Mediterranean. Parasitol. Int. 57, 405–414. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rouse, G.W., Fauchald, K., 1997. Cladistics and polychaetes. Zool. Scr. 26, 139–204. Sapozhnikov, G.I., 1988. Sanguinicola inermis infestation in carp and its control. Vet. Moscow 8, 36–38. Sapozhnikov, G.I., Vasil’kov, G.V., Osetrov, V.S., 1986. Prophylaxis of trematode infections in fish. Vet. Moscow 5, 51–53. Schell, S.C., 1974. The life history of Sanguinicola idahoensis sp. n. (Trematoda: Sanguinicolidae), a blood parasite of steelhead trout, Salmo gairdneri Richardson. J. Parasitol. 60, 561–566. Scheuring, L., 1920. Die Lebensgeschichte eines Karpfenparasiten (Sanguinicola inermis Plehn). Allg. FischZeitg. 45, 225–230. Sendersky, I.V., Dobrovolsky, A.A., 2004. Morphology and chaetotaxy of Sanguinicola armata cercariae (Trematoda: Sanguinicolidae). Parazitologiya 38, 310–321. Simon-Martin, F., Rojo-Vazquez, F.A., Simon-Vicente, F., 1987. Sanguinicola rutili n.sp. (Digenea: Sanguinicolidae) parasito del sistema circulatorio de Rutilus arcasi (Cyprinidae) en la provincia de Salamanca. Rev. Iber. Parasitol. 47, 253– 261. Smith, J.W., 2002. Family Sanguinicolidae Graff, 1907. In: Gibson, D.I., Jones, A., Bray, R.A. (Eds.), Keys to the digenean parasites of vertebrates. CAB International, Cambridge, pp. 433–452. Snyder, S.D., 2004. Phylogeny and paraphyly among tetrapod blood flukes (Digenea: Schistosomatidae and Spirorchiidae). Int. J. Parasitol. 34, 1385–1392. Sommerville, C., Iqbal, N.A.M., 1991. The process of infection, migration, growth and development of Sanguinicola inermis Plehn, 1905 (Digenea: Sanguinicolidae) in carp, Cyprinus carpio L. J. Fish Dis. 14, 211–219. Struck, T.H., Schult, N., Kusen, T., Hickman, E., Bleidorn, C., McHugh, D., Halanych, K.M., 2007. Annelid phylogeny and the status of Sipuncula and Echiura. BMC Evol. Biol. 7, 1–11. Stunkard, H.W., 1929. Further observations on the cercaria which occurs in the marine annelid, Hydroides dianthus. J. Parasitol. 16, 106. Wales, J.H., 1958. Two new blood fluke parasites of trout. Calif. Fish Game 44, 125– 136. Wardle, W.J., 1979. A new marine cercaria (Digenea: Aporocotylidae) from the southern quahog Mercenaria campechiensis. Contrib. Mar. Sci. 22, 53–56.