Syst Parasitol (2014) 89:133–152 DOI 10.1007/s11230-014-9515-2 Four marine digenean parasites of Austrolittorina spp. (Gastropoda: Littorinidae) in New Zealand: morphological and molecular data Katie O’Dwyer • Isabel Blasco-Costa • Robert Poulin • Anna Falty´nkova´ Received: 1 July 2014 / Accepted: 4 August 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Littorinid snails are one particular group obtained. Phylogenetic analyses were carried out at of gastropods identified as important intermediate the superfamily level and along with the morpholog- hosts for a wide range of digenean parasite species, at ical data were used to infer the generic affiliation of least throughout the Northern Hemisphere. However the species. nothing is known of trematode species infecting these snails in the Southern Hemisphere. This study is the first attempt at cataloguing the digenean parasites Introduction infecting littorinids in New Zealand. Examination of over 5,000 individuals of two species of the genus Digenean trematode parasites typically infect a Austrolittorina Rosewater, A. cincta Quoy & Gaim- gastropod as the first intermediate host in their ard and A. antipodum Philippi, from intertidal rocky complex life-cycles. They are common in the marine shores, revealed infections with four digenean species environment, particularly in the intertidal zone representative of a diverse range of families: Philo- (Mouritsen & Poulin, 2002). One abundant group of phthalmidae Looss, 1899, Notocotylidae Lu¨he, 1909, gastropods in the marine intertidal environment is the Renicolidae Dollfus, 1939 and Microphallidae Ward, littorinids (i.e. periwinkles), which are characteristic 1901. This paper provides detailed morphological organisms of the high intertidal or littoral zone and descriptions of the cercariae and intramolluscan have a global distribution (Davies & Williams, 1998). stages of these parasites. Furthermore, partial In the Northern Hemisphere, species rich digenean sequences of the 28S rRNA gene and the mitochon- faunas have been reported for littorinids such as drial gene cytochrome c oxidase subunit 1 (cox1) for Littorina littorea L., L. obtusata L. and L. saxatilis varying numbers of isolates of each species were Olivi (e.g. James, 1968; Granovitch & Mikhailova, 2004; Thieltges et al., 2006, 2009; Blakeslee & Byers, 2008; Granovitch & Maximovich, 2013). K. O’Dwyer (&) Á I. Blasco-Costa Á R. Poulin These include more than 30 species from the families Evolutionary and Ecological Parasitology Group, Echinostomatidae Looss, 1899, Microphallidae Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand Ward, 1901, Notocotylidae Lu¨he, 1909, Heterophy- e-mail: [email protected] idae Leiper, 1909, Renicolidae Dollfus, 1939 and Opecoelidae Ozaki, 1925 (James, 1968; Granovitch A. Falty´nkova´ & Mikhailova, 2004; Blakeslee & Byers, 2008; Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branisˇovska´ 31, Thieltges et al., 2009; Skirnı´sson & Galaktionov, 370 05 Cˇ eske´ Budeˇjovoice, Czech Republic 2002) with up to 12 species per sampling site 123 134 Syst Parasitol (2014) 89:133–152 (Skirnı´sson & Galaktionov, 2002). However, for the using an Olympus CX41 microscope, Olympus DP25 Southern Hemisphere no parasitological investiga- camera and DP2-BSW v. 1.4 software. Measurements tions of periwinkles could be sourced in the literature, were obtained from digital photographs using ImageJ in stark contrast to that available for the Northern (Schneider et al., 2012). Samples for SEM fixed in Hemisphere. glutaraldehyde and formalin were washed in 0.1 M Austrolittorina Rosewater includes five species, all phosphate buffer (three washes, 15 min each), post- distributed in the Southern Hemisphere: Austrolitto- fixed in 1% osmium tetroxide, washed in 0.1 M rina antipodum Philippi and A. cincta Quoy & phosphate buffer (three washes, 15 min each), and Gaimard in New Zealand, A. unifasciata Gray in dehydrated in a graded ethanol series. Samples fixed Australia, and A. fernandezensis Rosewater and A. in 70% ethanol were dehydrated in a graded ethanol araucana d’Orbigny in western South America (Reid series. After dehydration all samples were critical & Williams, 2004). During 2012 and 2013 extensive point-dried and splutter-coated with gold. Samples sampling of A. antipodum and A. cincta was carried were examined using a JEOL JSM 7401-F at an out in the high intertidal zone around the Otago accelerating voltage of 4 kV. The nomenclature of Harbour and Kaikoura, South Island, New Zealand Bayssade-Dufour (1979) was generally accepted for and Paihia, North Island, New Zealand. We found describing the chaetotaxy reconstructed from series frequent infections with larval stages of four digen- of SEM photomicrographs. eans of the families Philophthalmidae Looss, 1899, All measurements are in micrometres and are Notocotylidae, Renicolidae and Microphallidae. Here presented as the range followed by the mean in we provide detailed morphological descriptions of the parentheses. Metrical data in the descriptions are cercariae and intramolluscan stages of the four based on live specimens under slight coverslip species and molecular evidence based on phyloge- pressure; measurements of fixed material are pro- netic analyses at the superfamily level using newly- vided separately (where available). Voucher material generated 28S rDNA sequences and, where possible, (in alcohol and permanent mounts in Canada balsam) sequences of the mitochondrial gene cytochrome has been deposited at the Institute of Parasitology, c oxidase subunit 1 (cox1). Academy of Sciences of the Czech Republic under accession numbers HCIP D-700; D-701; D-702 and D-703. Materials and methods DNA was extracted from single ethanol-fixed cercariae/sporocysts/rediae in 150–500 llof5% Austrolittorina antipodum and A. cincta were col- suspension of ChelexÒ in deionised water containing lected in 2012 and 2013 off New Zealand, primarily 0.1 mg/ml proteinase K, followed by incubation around Otago Harbour (-45.80, 170.66) (four sites: overnight at 60°C, boiling at 90°C for 8 min and Aramoana, Portobello Marine Laboratory, Lower centrifugation at 15 000 g for 10 min. Portobello Bay and Weller’s Rock) and off Kaikoura Polymerase chain reaction (PCR) amplifications (-42.43, 173.69) and Paihia (-35.28, 174.09). Snails (total volume 20 ll) were carried out using 1 ll were dissected in the laboratory under a dissecting extraction supernatant, 4 ll MyTaqRed buffer (5 mM microscope and mature cercariae, sporocysts and dNTPs), 10 lM of each primer and 0.5 units MyTaq rediae were photographed live, under natural condi- (Bioline Ltd). Partial (domains D1–D3) 28S rDNA tions and following staining with Neutral Red under sequences were amplified using primers U178 (for- slight coverslip pressure, using a compound micro- ward: 50-GCA CCC GCT GAA YTT AAG-30) and scope. Metacercarial cysts were photographed and L1642 (reverse: 50-CCA GCG CCA TCC ATT TTC measured following encystment on a glass slide. A-30) (Lockyer et al., 2003). The following thermo- Samples of cercariae killed in hot seawater were cycling profile was used: DNA denaturation at 95°C photographed for morphological study and subsam- for 3 min; 40 amplification cycles (94°C for 40 sec, ples were fixed in 100% ethanol for molecular 56°C for 30 sec, 72°C for 1 min 20 sec); and a final analysis and in 2.5% glutaraldehyde, 4% formalin extension step for 4 min at 72°C. Amplification of or 70% ethanol for scanning electron microscopy partial cox1 gene fragments was performed using the (SEM) examination. Photomicrographs were taken primer combination JB3 (forward: 50-TTT TTT GGG 123 Syst Parasitol (2014) 89:133–152 135 CAT CCT GAG GTT TAT-30) (Bowles et al., 1993) 10,000,000 generations. Trees were sampled every and Plag16S-COIdR (reverse: 50-TCG GGG TCT 1,000 generations. ‘‘Burn-in’’ was applied by dis- TTC CGT CT-30) (Blasco-Costa et al., 2012). The carding the first 25% of the sampled trees for each following thermocycling profile was used: DNA dataset. Estimation of consensus tree topology and denaturation at 94°C for 3 min; 40 amplification nodal support was obtained as posterior probability cycles (94°C for 40 sec, 54°C for 35 sec, 72°C for values from the remaining samples. 2 min); and a final extension step for 4 min at 72°C. The newly-generated cox1 sequences were aligned The thermocycling profile for microphallid and using Muscle implemented in MEGA v6 (Tamura renicolid samples was run at a higher annealing et al., 2013) with reference to the amino acid temperature (57°C). translation, using the echinoderm and flatworm The PCR products were purified using exonucle- mitochondrial code (Telford et al., 2000). Genetic ase I, and shrimp alkaline phosphatase, enzymes distances (uncorrected p-distance) were calculated for (Werle et al., 1994). For 20 ll PCR product we each gene/species using MEGA v6. added 1.5 U of Exo1 and 1 U of SAP. PCR amplicons were cycle-sequenced from both strands using the PCR primers [plus 1200R (50-GCA TAG Results TTC ACC ATC TTT CGG-30) for 28S rDNA (Lockyer et al., 2003)] with BigDyeÒ Terminator Examination of over 5,000 individuals of two species Version 3.1 Ready Reaction Cycle Sequencing Kit, of the genus Austrolittorina Rosewater, A. cincta alcohol-precipitated and run on an ABI 3730XL Quoy & Gaimard (1,955 individuals) and A. antip- Analyser (Applied Biosystems, Foster City, Califor- odum Philippi (3,424 individuals), from intertidal nia). Contiguous sequences were assembled and rocky shores at six localities in New Zealand revealed edited using SequencherTM (GeneCodes Corp. v. 5) infections with four digenean species. Periwinkle and submitted to GenBank under accession numbers collections for A. cincta numbered 173–993 individ- KJ868206–KJ868217 (28S rDNA) and KJ868192– ual snails per site (across four sites), and 193–633 per KJ868205 (cox1). site (across five sites) for A. antipodum. Both The newly-generated partial sequences for the littorinid species were found infected with the 28S rRNA gene (931–1,608 bp) were aligned using philophthalmid Parorchis sp.