Zootaxa 2445: 35–52 (2010) ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2010 · Magnolia Press ISSN1175-5334(online edition) Two new Stephanostomum-like cercariae (Digenea: Acanthocolpidae) from Nassarius dorsatus and N. olivaceus (Gastropoda: Nassariidae) in Central Queensland, Australia LEONIE J. BARNETT1, TERRENCE L. MILLER2 & THOMAS H. CRIBB3 1Centre for Environmental Management, CQUniversity Australia, Bruce Highway, North Rockhampton, Queensland 4702, Australia. E-mail: [email protected] 2Biodiversity Program, Queensland Museum, PO Box 3300, South Brisbane, Queensland 4101, Australia. E-mail: [email protected] 3Centre for Marine Studies and Department of Microbiology and Parasitology, The University of Queensland, Brisbane, Queensland 4072, Australia. E-mail: [email protected] Abstract Two new Stephanostomum-like cercariae, Cercaria capricornia VII and Cercaria capricornia VIII (Digenea: Acanthocolpidae), are described from the nassariid gastropods Nassarius dorsatus and Nassarius olivaceus collected from the intertidal zone in the Capricornia region of Central Queensland, Australia. Morphological analysis of these new cercariae was augmented with DNA sequence data from the large subunit (LSU) ribosomal DNA region to aid in identification. Bayesian inference analysis of the LSU rDNA revealed that these putative acanthocolpid cercariae nested well within a clade containing species of Stephanostomum, which along with morphological data, suggests they are species of Stephanostomum. Comparative analysis of LSU rDNA sequences also indicates that these two cercariae are not S. adlardi, S. bicoronatum, S. tantabiddii or S. cf. uku, all species known from Australian fishes. The secondary structure of the internal transcribed spacer 2 (ITS2) rDNA region was inferred for these two cercariae using minimum free energy modelling algorithms. Both cercarial types displayed a four helix ITS2 secondary structure model and differed from each other by two compensatory base changes (CBCs) and nine hemi-CBCs. Key words: Cercaria capricornia; Capricornia; Nassarius dorsatus;Nassarius olivaceus; LSU rDNA; large subunit ribosomal DNA;CBC; compensatory base change; ITS2; internal transcribed spacer 2 Introduction The Acanthocolpidae Lühe, 1906 is a large family of digenean trematodes that infects the gastrointestinal tract of marine fishes. Asexual reproduction occurs in a range of gastropods and metacercariae infect the tissues of fishes which are then eaten by piscivores to complete the life cycle. Although 17 genera were recognised in the family by Bray (2005), Stephanostomum Looss, 1899, with over one hundred species, is by far the largest in the family and one of the largest in marine fishes (Cribb et al. 2002). Despite the size of the genus, the life cycles and cercarial stages have been characterised for only five species (Madhavi & Shameem 1993). A single further Stephanostomum-like cercariae, Cercaria bengalensis VII Gnana Mani, 1994, has been described (Gnana Mani 1994). For such a large and important genus, the range of hosts infected and the morphological consistency of the intra-molluscan stages is of considerable interest. The nassariid gastropods Nassarius dorsatus (Röding) and Nassarius olivaceus (Bruguière) are common scavengers in the intertidal mangroves of the Capricornia region in Central Queensland, Australia. There are ten probable acanthocolpid cercariae reported previously from nassariid gastropods. There are three species of Stephanostomum: those of Stephanostomum tenue (Linton, 1898) Linton, 1934 and Stephanostomum dentatum (Linton, 1900) Linton, 1940 (syn. Cercaria dipterocerca Miller and Northup, 1926) from Nassarius obsoletus Say (see Martin 1939; Stunkard 1961) and Stephanostomum cloacum (Srivastava, 1938) Manter & Accepted by N. Dronen: 24 Mar. 2010; published: 5 May 2010 35 Van Cleave, 1951 from Nassarius orissaensis (Preston) (see Madhavi & Shameem 1993). Cercaria bengalensis VII was reported from Nassarius stolatus (Gmelin), and was described as closely resembling the cercariae of S. tenue and Stephanostomum caducum (Looss, 1901) Manter, 1934 (Gnana Mani 1994). Other cercariae reported from nassariids are Cercaria capricornia I–IV Barnett, Smales & Cribb, 2008 from N. olivaceus and Cercaria capricornia V–VI Barnett, Smales & Cribb, 2008 from N. dorsatus. This is a complex of six unusual cercariae which are morphologically very different from cercariae of Stephanostomum in that they have an unusual body shape including a ventral keel (Barnett et al. 2008). Other Stephanostomum cercariae are Stephanostomum baccatum (Nicoll, 1907) Manter, 1934 (syn. Cercaria neptunae Lebour, 1911) from Buccinum undatum Linnaeus, Neptunea decemcostata (Say) and Neptunea antiqua (Linnaeus) (see MacKenzie & Liversidge 1975; Wolfgang 1955) and S. caducum from Euspira pulchella (Risso) (syn. Natica alderi Forbes) (see Køie 1978). Here, we present two new acanthocolpid cercariae from N. dorsatus and N. olivaceus found as part of a study of the parasite fauna of these molluscs in Capricornia. Bayesian inference analysis of partial large subunit (LSU) ribosomal DNA (rDNA) was performed with the aim of exploring the integrity of the putative new cercarial types and their phylogenetic relationships with other acanthocolpid taxa. In addition, we sequenced the internal transcribed spacer 2 (ITS2) rDNA region for these two types and inferred their secondary structures to explore the homology of these structures to each other and to that known for other digeneans. Material and methods Host gastropod and parasite collection. Molluscs were collected by hand from mudflats at Sandy Point, Corio Bay (22°58' S, 150°46' E), Ross Creek, Yeppoon (23°8' S, 150°45' E) and the mouth of Cawarral Creek, Keppel Sands (23°19' S, 150°47' E) in Central Queensland, Australia. A total of 1766 N. dorsatus and 1908 N. olivaceus were collected between August 2004 and May 2006. Molluscs were held in filtered seawater at room temperature (20–28°C) and examined for naturally emerged cercariae every 1–3 days for up to one month. Freshly emerged cercariae were transferred to a cavity block in a small volume of seawater and heat- killed by pouring several volumes of near boiling seawater into the dish. Specimens for morphological analysis were fixed in 5% formalin and specimens for molecular analysis were stored in 70% ethanol or in phosphate buffered saline at -20°C. One or more infected molluscs were dissected to determine the nature of intra-molluscan stages for each cercarial species. Rediae were placed into near boiling seawater and fixed in 5% formalin or 70% ethanol. Representative wholemounts were stained with Mayer's haematoxylin, dehydrated through a graded series of ethanol, cleared in methyl salicylate and mounted in Canada balsam. Wholemounts and wet specimens are deposited in the Queensland Museum, Brisbane, Queensland, Australia. Morphological analysis. Fixed cercariae were examined as temporary wet mounts without flattening. Live specimens and rediae were also observed as temporary wet mounts. Measurements were taken using an eyepiece micrometer on an Olympus BX-41 microscope and are given in micrometres as the range followed by the mean in parentheses. Standard deviations were calculated where more than 30 measurements were taken. Drawings were prepared with the aid of a drawing tube. Line drawings of C. capricornia VII and C. capricornia VIII were deposited in MorphBank (http://www.morphbank.net/). Confirmation of prevalence. Of 17 infections, mature cercariae for morphological analysis were not available from one N. olivaceus and this specimen was omitted from the calculation of prevalence. A subset of putatively uninfected molluscs was dissected to confirm the absence of infection. Of 556 N. dorsatus (31.5% of 1766) and 399 N. olivaceus (20.9% of 1908) dissected, there were just two N. dorsatus infected with a trematode similar to those described here, but the cercariae were too immature for morphological comparison. This result suggests that the emergence prevalence reported here closely approximates the actual prevalence. Molecular analysis. Total genomic DNA was extracted from fresh, frozen or alcohol fixed specimens using a QIAGEN® DNeasy® Blood and Tissue Kit following the manufacturer’s protocol, except that the incubation step with Proteinase K was extended to overnight in a rotating incubator at 56°C. Final elution 36 · Zootaxa 2445 © 2010 Magnolia Press BARNETT ET AL. volume was 30–200 μL. Amplification of the partial LSU rDNA region was performed with the primers LSU5 (5’-TAGGTCGACCCGCTGAAYTTAAGCA-3’ Littlewood et al. 2000) and ECD2 (5’- CCTTGGTCCGTGTTTCAAGACGGG-3’ Littlewood et al. 2000) and the ITS2 region with the primers 3S (5’-GGTACCGGTGGATCACGTGGCTAGTG-3’ Morgan & Blair 1995) and ITS2.2 (5’- CCTGGTTAGTTTCTTTTCCTCCGC-3’ Cribb et al. 1998). PCR amplification (total volume 50 μL) was performed using 1X iTaq Buffer (Bio-Rad), 1.25 U iTaq™ DNA polymerase (Bio-Rad), 1.5 mM MgCl , 200 2 μM each dNTP, 0.2 μM each primer and 1–10 μL genomic extract, with the following thermocycling profile: 5 min denaturation at 95°C; 30 cycles of 30 s at 95°C, 30 s at 60°C, 30 s at 72°C; and 4 min extension at 72°C. PCR amplicons were purified using a QIAGEN QIAquick® PCR Purification Kit (with 30 μL elution buffer). Purified amplicons were sequenced (in both directions) using the same primers with ABI BigDye™ v3.1 chemistry at the Australian Genome Research Facility in Brisbane, Queensland. The resulting sequences were edited by eye and contigs constructed using Sequencher™ version 4.5 (GeneCodes Corp.). The sequences for each cercarial type were constructed from multiple replicates (each replicate contig being both a forward and reverse sequence from a single individual) from different host/locality combinations whenever possible. Phylogenetic analyses. The partial LSU rDNA sequences for C. capricornia VII and C. capricornia VIII were aligned with data reported for the species of Acanthocolpidae and Brachycladiidae listed in Table 1. Species of Brachycladiidae were included as this family is considered closely related to the Acanthocolpidae (Bray et al. 2005). Cableia pudica Bray, Cribb & Barker, 1996 was omitted from this analysis as recent molecular phylogenetic analyses have indicated that this species is a member of the Monorchiidae (Bray et al. 2005). Paragonimus westermani (Kerbert, 1878) and Paragonimusiloktsuenensis Chen, 1940 were chosen as outgroup taxa. The partial LSU rDNA dataset was initially aligned using ClustalX version 2.0.9 (Larkin et al. 2007) under the following parameters: pairwise alignment parameters = gap opening 10.00, gap extension 0.10, DNA weight matrix International Union of Biochemistry (IUB); and multiple alignment parameters = gap opening 10.00, gap extension 0.20, delay divergent sequences 30%, DNA weight matrix IUB. The resulting sequence alignment was exported from ClustalX in FASTA and NEXUS formats, and refined by eye using MacClade version 4.08 (Maddison & Maddison 2005). After alignment of the LSU dataset was edited, the ends of each fragment were trimmed to match the shortest sequence in the alignment. Bayesian inference analysis of the LSU dataset was performed using MrBayes version 3.1.2 (Ronquist & Huelsenbeck 2003) to explore relationships between these taxa. Modeltest version 3.7 (Posada & Crandall 1998) was used to estimate the best substitution model for the LSU dataset. Bayesian inference analysis was conducted on the combined dataset using the GTR+I+G model predicted as the best estimator by the Akaike Information Criterion (AIC) in Modeltest. Bayesian inference analysis was run over 1,000,000 generations (ngen=1000000) with two runs each containing four simultaneous Markov Chain Monte Carlo (MCMC) chains (nchains=4) and every 100th tree saved (samplefreq=100). Bayesian analysis used the following parameters: nst=6, rates=invgamma, ngammacat=4, and the MCMC parameters were left at the default settings, 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=3000’ and ‘sumt burnin=3000’. These ‘burnin’ parameters were chosen because the log likelihood scores ‘plateaued’ well before 300,000 replicates in the Bayesian inference analysis. ITS2 rDNA secondary structure prediction. The secondary structure of the ITS2 rDNA region for C. capricornia VII and C. capricornia VIII was predicted using minimum free energy folding algorithms with Mfold software version 3.2 (Zuker 2003). The ITS2 sequence was treated as linear and the folding temperature set at 37ºC for analysis using Mfold. Alignment of the predicted ITS2 secondary structures for these two cercariae was performed using 4SALE (Seibel et al. 2008). Compensatory base change (CBC) analysis was then conducted on the aligned secondary structures using CBCAnalyzer software (Wolf et al. 2005). The predicted folding results from the Mfold analyses were viewed with Pseudoviewer version 3 (Byun & Han 2006). TWO NEW STEPHANOSTOMUM-LIKE CERCARIAE Zootaxa 2445 © 2010 Magnolia Press · 37 TABLE 1. List of taxa and partial LSU rDNA sequences from GenBank used in this study. Species Source: host/locality GenBank accession Acanthocolpidae Cercaria capricornia VII Nassarius dorsatus, Capricornia, Australia FJ809037 Cercaria capricornia VIII Nassarius dorsatus, Capricornia, Australia FJ809036 Monostephanostomum nolani Bray & Cribb, 2007 Carangoides plagiotaenia, Lizard Island, Australia EF506763 Pleorchis polyorchis (Stossich, 1889) Sciaena umbra, Scandola, Corsica DQ248215 Pleorchis uku Yamaguti, 1970 Aprion virescens, Lizard Island, Australia DQ248216 Stephanostomum adlardi Bray, Cribb, Waeschenbach & Plectropomus leopardus,Lizard Island, Australia EF506761 Littlewood, 2007 Stephanostomum baccatum (Nicoll, 1907) Hippoglossus hippoglossus, North Sea, UK DQ248218 Stephanostomum baccatum (Nicoll, 1907) Eutrigla gurnadus, North Sea, UK AY222256 Stephanostomum bicoronatum (Stossich, 1883) Sciaena umbra, Scandola, Corsica DQ248225 Stephanostomum cesticillus (Molin, 1858) Lophius piscatorius, Scandola, Corsica DQ248226 Stephanostomum gaidropsari Bartoli & Cribb, 2001 Gaidropsarus mediterraneus, Marseille, France DQ248221 Stephanostomum interruptum Sparks & Thatcher, 1958 Menticirrhus americanus, Gulf of Mexico, USA DQ248223 Stephanostomum minutum (Looss, 1901) Uranoscopus scaber, Scandola, Corsica DQ248224 Stephanostomum pristis (Deslongchamps, 1824) Phycis phycis, Scandola, Corsica DQ248222 Stephanostomum tantabiddii Bray & Cribb, 2004 Carangoides fulvoguttatus, Ningaloo, Australia DQ248220 Stephanostomum cf. uku Yamaguti, 1970 Aprion virescens, Lizard Island, Australia DQ248219 Tormopsolus orientalis (Yamaguti, 1934) Seriola dumerili, Scandola, Corsica DQ248217 Brachycladiidae Zalophotrema hepaticum Stunkard & Alvey, 1929 Zalophus californianus, California, USA AY222255 Paragonimidae Paragonimus iloktsuenensis Chen, 1940 Rattus norvegicus, Amami Island, Japan AY116875 Paragonimus westermani (Kerbert, 1878) Canis familiaris, Hyogo, Japan AY116874 Results Two different types of acanthocolpid cercariae with morphology similar to described Stephanostomum cercariae emerged naturally from N. dorsatus and N. olivaceus. Morphological data – intramolluscan stages Diagnosis: Distomate cercariae. Dorsoventrally flattened body, longer than wide. Tegument spinose. Eye- spots present. Oral sucker ventrally subterminal. Excretory vesicle Y-shaped. Tail simple, lacking spines, chaetae or fins. 38 · Zootaxa 2445 © 2010 Magnolia Press BARNETT ET AL. Cercaria capricornia VII (Fig. 1) Host: Nassarius dorsatus (Röding), (Gastropoda, Nassariidae). Other host: Nassarius olivaceus (Bruguière), (Gastropoda, Nassariidae). Locality: Cawarral Creek, Keppel Sands, Queensland (23°19' S; 150°47' E). Habitat: Intertidal mudflats. FIGURE 1. a–d. Cercaria capricornia VII, naturally emerged from Nassarius dorsatus and N. olivaceus.a. Body, heat- killed, in ventral view; b. Entire cercaria, heat-killed, from side view; c. Live cercaria, body folded for swimming, from ventral view; d. Redia, fixed. Scale bars a, b, d = 50 μm; c not to scale. TWO NEW STEPHANOSTOMUM-LIKE CERCARIAE Zootaxa 2445 © 2010 Magnolia Press · 39 TABLE 2. Morphometric comparison between Cercariae capricornia VII and VIII and published Stephanostomum and Stephanostomum-like cercariae (Gnana Mani 1994; Køie 1978; Madhavi & Shameem 1993; Martin 1939; Stunkard 1961; Wolfgang 1955). C. capricornia S. dentatum S. tenue S. caducum S. cloacum S. baccatum C. bengalensis VII VII VIII Total body 155–250 182–240 135–145 145–380 220–260 480–500 320–500 160–195 length (195.2) (205.3) (240) (240) (181) Maximum body 50–85 62–83 46–53 45–86 115–140 154–166 90–140 59–74 width (69.2) (70.5) (64) (125) (62) Forebody 75–125 95–138 length (97.2) (112.4) Tail length 142–210 215–270 220–240 183 250–300 300–320 540–640 210–244 (175.6) (253.6) (194) Tail width 17–28 20–30 16–20 31 25–35 28–32 20–23 (20.6) (22.1) (20) Oral sucker 30–40 35–43 24–25 31 42–50 72–78 60–80 31–35 length (34.4) (38.2) (48) Oral sucker 27–38 32–38 24–25 30 72–78 60–80 31–35 width (33.4) (35.4) Ventral sucker 17–33 27–35 23–24 33 35–38 62 50–70 31–35 length (29.1) (30.6) (37) Ventral sucker 20–35 27–35 23–24 30 35–40 66 50–70 31–35 width (30.4) (31.3) (38) Pharynx length 10 25–48 25–52 15 40–44 31–34 Excretory 37–75 42–55 88–100 vesicle length (52.1) (49.7) Excretory 32–58 32–48 58–70 vesicle width (45.1) (39.3) Eyes 7–10 13 x 10– 9–12 8 (diam.) 13 (diam.) (diam.) Glands (central 2 + 3 2 + 3 2 + 3 2 + 2 2 + 3 2 + 2 2 + 0 5 + 5 + lateral) Oral sucker– 32–55 37–68 eye distance (46.6) (53.6) Eye–eye 12–28 7–23 distance (20.3) (14.4) Stylet (length) no no no yes (12) yes (6) yes no yes (5) Other locality: Sandy Point, Corio Bay (22°58' S, 150°46' E), Ross Creek, Yeppoon, Queensland (23°8' S, 150°45' E). Location in host (redia): Digestive gland, gonads. Prevalence of emergence: 0.57% (10 of 1766 N. dorsatus), 0.21% (4 of 1908 N. olivaceus). Material deposited: Queensland Museum; G231698–G231720. Molecular sequence data: ITS2, 6 replicates; LSU, 6 replicates. GenBank accession numbers: ITS2 (FJ809039); LSU (FJ809037). MorphBank accession number: 515656. Dates of collection: August/November 2004, February/May–June/November 2005 and March/May 2006. Description: Redia. (Fig. 1d, based on 10 unflattened specimens): Body elongate, cylindrical with posterior extremity tapering, 530–1210 (703.0) long by 60–120 (75.5) wide. Mouth opens terminally. Pharynx 25–32.5 (29.3) long by 20–25 (23.8) wide. In reproductive and digestive glands. 40 · Zootaxa 2445 © 2010 Magnolia Press BARNETT ET AL. Cercaria. (Figs 1a–c, based on 140 naturally emerged specimens): Body elliptical, longer than wide, widest point posterior to ventral sucker, 155–250 (195.2 ± 16.1) long by 50–85 (69.2 ± 5.9) wide; length/ width 2.27–4.17 (2.84 ± 0.3). Tegument spinose; spines arranged in regular rows. Tail simple, long, cylindrical, gradually tapering terminally, lacking spines, chaetae, fins, 142–210 (175.6 ± 15.2) long by 17–28 (20.6 ± 2.1) wide. Oral sucker 30–40 (34.4 ± 2.2) long by 27–38 (33.4 ± 2.0) wide. Ventral sucker 17–33 (29.1 ± 2.2) long by 20–35 (30.4 ± 1.9) wide. Mouth subterminal, opening anteroventrally, surrounded by spines; spines around mouth aperture slightly larger than on surrounding tegument. Forebody 75–125 (97.2 ± 8.9) long; forebody/hindbody 0.80–1.34 (0.99 ± 0.07). Pharynx generally obscured by central glands, pyriform, 10 long by 12 wide (n=1). Penetration glands in 5 pairs; 2 pairs median and immediately anterior to ventral sucker and 3 pairs lateral or anterolateral to ventral sucker. Excretory vesicle 37–75 (52.1 ± 6.1) long by 32– 58 (45.1 ± 4.7) wide; V-shaped when full, Y-shaped when empty; excretory duct appears to continue length of tail. Eye-spots dense, spherical, 12–28 (20.3 ± 2.9) apart, 32–55 (46.6 ± 5.0) from anterior end; 7–10 diameter (n=24). Remarks: Large numbers of cercariae emerge, sometimes on consecutive days, with pauses between emergences (few days to weeks). Cercariae generally emerge before 8 am. Naturally emergent cercariae are free-swimming and strongly positively phototactic. When swimming, the hindbody is folded ventrally (Fig. 1c) and the tail lashes from side to side for movement. When resting, the hindbody remains folded ventrally. When first emerged, cercariae swim actively towards light, frequently forming swarms near the surface. After about 24 hours, cercariae become less active. Cercariae were never observed to encyst. Cercaria capricornia VII can be distinguished from the cercariae of S. tenue,S. cloacum, S. caducum and C. bengalensis VII by the absence of a stylet (Gnana Mani 1994; Køie 1978; Madhavi & Shameem 1993; Martin 1939). Stephanostomum baccatum is larger and has no lateral penetration glands (Wolfgang 1955). Stephanostomum dentatum has a tail with lateral cuticular fins and is photonegative (Stunkard 1961). Morphometric comparison between C. capricornia VII and C. capricornia VIII, S. tenue, S. cloacum, S. caducum,C. bengalensis VII, S. baccatum and S. dentatum is given in Table 2. Cercaria capricornia VIII (Fig. 2) Host: Nassarius dorsatus (Röding) (Gastropoda, Nassariidae). Other host: None. Locality:Cawarral Creek, Keppel Sands, Queensland (23°19' S; 150°47' E). Habitat: Intertidal mudflats. Location in host (redia): Digestive gland, gonads. Prevalence of emergence: 0.11% (2 of 1766 N. dorsatus). Material deposited: Queensland Museum; G231721–G231724. Molecular sequence data: ITS2, 1 replicate; LSU, 1 replicate. GenBank accession numbers: ITS2 (FJ809038); LSU (FJ809036). MorphBank accession number: 515658. Dates of collection: August and November 2004. Description: Redia. (Fig. 2c, based on 10 unflattened specimens): Body elongate, cylindrical with posterior extremity tapering, 240–580 (416.0) long by 35–50 (41.5) wide. Mouth opens terminally. Pharynx 27–30 (28.5) long by 20–25 (23.3) wide. In reproductive and digestive glands. Cercaria. (Figs 2a–b, based on 30 naturally emerged specimens): Body elliptical, longer than wide, widest point anterior to ventral sucker, 182–240 (205.3 ± 13.1) long by 62–83 (70.5 ± 4.7) wide; length/width ratio 2.48–3.31 (2.92 ± 1.2). Tegument spinose; spines arranged in regular rows. Tail simple, long, cylindrical, gradually tapering terminally, lacking spines, chaetae, fins, 215–270 (253.6 ± 13.4) long by 20–30 (22.1 ± 2.2) wide. Oral sucker 35–43 (38.2 ± 1.8) long by 32–38 (35.4 ± 1.9) wide. Ventral sucker 27–35 (30.6 ± 1.7) long by 27–35 (31.3 ± 1.8) wide. Mouth subterminal, opening anteroventrally, surrounded by spines; spines around mouth aperture slightly larger than on surrounding tegument. Forebody 95–138 (112.4 ± 9.8) long; forebody/ TWO NEW STEPHANOSTOMUM-LIKE CERCARIAE Zootaxa 2445 © 2010 Magnolia Press · 41 hindbody ratio 1.08–1.34 (1.21 ± 0.07). Pharynx obscured by central glands. Penetration glands in 5 pairs; 2 pairs median and immediately anterior to ventral sucker and 3 pairs lateral or anterolateral to ventral sucker. Excretory vesicle 42–55 (49.7 ± 2.8) long by 32–48 (39.3 ± 3.5) wide; V-shaped when full, Y-shaped when empty; excretory duct appears to continue length of tail. Eye-spots dense, oblong, 7–23 (14.4 ± 2.9) apart, 37– 68 (53.6 ± 6.1) from anterior end; 13 long by 10–13 wide (n=7). FIGURE 2. a–c. Cercaria capricornia VIII, naturally emerged from Nassarius dorsatus.a. Body, heat-killed, in ventral view; b. Entire cercaria, heat-killed, from side view; c. Redia, fixed. Scale bars a, b, c = 50 μm. 42 · Zootaxa 2445 © 2010 Magnolia Press BARNETT ET AL. FIGURE 3. Relationships between Cercaria capricornia VII and C. capricornia VIII and the remainder of the taxa examined based on Bayesian inference analysis of partial LSU rDNA. Posterior probabilities are shown at the nodes with values < 50 not shown. TWO NEW STEPHANOSTOMUM-LIKE CERCARIAE Zootaxa 2445 © 2010 Magnolia Press · 43 FIGURE 4. Inferred secondary structure of the ITS2 rDNA region for based on minimum free-energy modelling for a. Cercaria capricornia VII and b. C. capricornia VIII. Helices one through four are indicated with Roman numerals. Complete compensatory base changes (CBCs) and hemi-CBCs are indicated in grey. Remarks: Large numbers of cercariae emerge, sometimes on consecutive days, with pauses between emergences (few days to weeks). Cercariae generally emerge before 8 am. Naturally emergent cercariae are free-swimming and strongly positively phototactic. When swimming, the hindbody is folded ventrally and the tail lashes from side to side for movement, similar to C. capricornia VII (see Fig. 1c). When resting, the 44 · Zootaxa 2445 © 2010 Magnolia Press BARNETT ET AL.