Syst Parasitol (2010) 76:211–222 DOI 10.1007/s11230-010-9247-x Redescriptions of two species of microcotylid monogeneans from three arripid hosts in southern Australian waters Sarah R. Catalano • Kate S. Hutson • Rodney M. Ratcliff • Ian D. Whittington Received: 17 December 2009 / Accepted: 24 February 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Microcotyle arripis Sandars, 1945 is host, A. truttaceus, from four localities in South redescribed from Arripis georgianus from four Australian waters: Spencer Gulf, Gulf St. Vincent, off localities: Spencer Gulf, Gulf St. Vincent, off Kan- Kangaroo Island and Coffin Bay. Phylogenetic anal- garoo Island and Coffin Bay, South Australia, Aus- ysis of the partial 28S ribosomal RNA gene (28S tralia. Kahawaia truttae (Dillon & Hargis, 1965) rRNA) nucleotide sequences for both microcotylid Lebedev, 1969 is reported from A. trutta off Berma- species and comparison with other available sequence gui, New South Wales and is redescribed from a new data for microcotylid species across four genera contributes to our understanding of relationships in this monogenean family. S. R. Catalano (&) Á K. S. Hutson Á I. D. Whittington Marine Parasitology Laboratory, DX 650 418, School of Earth and Environmental Sciences, The University of Adelaide, North Terrace, Adelaide, SA 5005, Australia Introduction e-mail: [email protected] K. S. Hutson All four species of Arripis Jenyns (Pisces: Arripidae), Discipline of Aquaculture, School of Marine and Tropical A. georgianus (Valenciennes), A. trutta (Forster), Biology, James Cook University, Townsville, QLD 4811, A. truttaceus (Cuvier) and A. xylabion Paulin, are Australia endemic to the waters of southern Australia and New R. M. Ratcliff Zealand (NZ), and are caught by commercial and Infectious Disease Laboratories, Institute of Medical and recreational fishers (see Paulin, 1993, for a review). Veterinary Science, SA Pathology, Adelaide, SA 5000, During a survey of the parasites of A. georgianus, Australia A. trutta and A. truttaceus, two parasite species of the R. M. Ratcliff Microcotylidae Taschenberg, 1879 were recovered. School of Biomedical Science, The University of Microcotyle arripis Sandars, 1945 is redescribed Adelaide, North Terrace, Adelaide, SA 5005, Australia from A. georgianus, as the original description by Sandars (1945) and subsequent redescriptions by I. D. Whittington Parasitology Section, The South Australian Museum, Dillon et al. (1984) and Williams (1991) are based North Terrace, Adelaide, SA 5000, Australia upon limited material. Our study also provides new geographical localities for M. arripis in South I. D. Whittington Australian (SA) waters. Kahawaia truttae (Dillon & Australian Centre for Evolutionary Biology and Biodiversity, The University of Adelaide, North Terrace, Hargis, 1965) Lebedev, 1969, originally described as Adelaide, SA 5005, Australia Gonoplasius truttae Dillon & Hargis, 1965 from 123 212 Syst Parasitol (2010) 76:211–222 A. trutta at Timuru, South Island, NZ (Dillon & Morphological methods Hargis, 1965), is reported from A. trutta off the coast Drawings of preserved, mounted parasites were made of New South Wales (NSW) and from with the aid of a drawing tube and measurements were A. truttaceus from SA waters, providing new geo- made using a computerised digitising system similar to graphical localities for this species in southern that described by Roff & Hopcroft (1986). The termi- Australian waters. This microcotylid is redescribed, nology of Williams (1991) for microcotylid monogen- because the original description by Dillon & Hargis eans is adopted, using buccal organs in preference to (1965) was based on limited material and the buccal suckers, and ovary in preference to germarium. subsequent account by Lebedev (1969) was poorly Unless stated otherwise, all measurements are presented illustrated. To improve our understanding of rela- in micrometres as the mean with the range in parenthe- tionships within the Microcotylidae, newly sequenced ses, followed in brackets by the number of measure- partial 28S ribosomal RNA gene (28S rRNA) nucle- ments made. A dash (-) indicates that measurements otide sequence data for M. arripis from A. georgianus could not be made or were not available. and K. truttae from A. trutta and A. truttaceus are Measurements of lengths refer to the distance along compared with sequence data for other microcotylid the anteroposterior axis, except where otherwise species available in GenBank. noted. Length measurements of structures not orien- tated along the anteroposterior axis in some speci- mens, such as buccal organs, clamps, eggs and genital Materials and methods atrium armature, were measured along the longest axis of these structures. ‘Middle clamps’ are defined as Collection of hosts and parasites being located halfway along the total haptor length and Monogeneans were collected from the gills of Arripis ‘posterior clamps’ are defined as being located at the georgianus and A. truttaceus at four localities in SA posterior end of the haptor. Occasionally bodies and waters (Spencer Gulf, Gulf St. Vincent, off Kangaroo haptors were curved, so multiple linear measurements Island and Coffin Bay; see ‘Results’ for geographical were made and summed to give the total length. Body coordinates), from A. trutta at one location off NSW width was measured across the ovary, and haptor (Bermagui; see ‘Results’ for geographical coordi- width was measured across the anterior margin. nates) and from Sillaginodes punctatus (Cuvier) in SA waters (specific locality unknown). Most fish Molecular methods were examined fresh; others were frozen immediately Monogenean parasites for molecular analyses were upon collection and processed later. Gills were preserved in 95% undenatured ethanol and collected removed, placed in individual Petri dishes of seawater from the gills of four fish species, A. georgianus, A. and studied for parasites. Monogeneans were trutta, A. truttaceus and S. punctatus (Table 1). removed and preserved in either 10% formalin or Additional monogenean sequences were obtained 95% undenatured ethanol. Representative specimens from GenBank (Table 1). were slivered along the body margin with the slivered Total genomic DNA (gDNA) was extracted using section preserved in 95% ethanol and the remaining the Gentra Kit (Gentra Systems) to digest tissue and section preserved in formalin. Parasites preserved in release DNA, followed by the RNeasy Mini Kit formalin were washed twice in MiliQ water, stained (animal tissue protocol incorporating QIAshredder in Mayer’s haematoxylin, destained in 3% HCl, pretreatment, QIAGEN GmbH, Hilden, Germany) to dehydrated in an ethanol series (70%, 90%, 95% and extract the DNA. PCR amplification of partial 28S 100%), cleared in cedar wood oil and mounted on a rRNA sequence was carried out using two universal slide beneath a coverslip in Canada balsam. primers (Jovelin & Justine, 2001), modified with a 5’ Tissue samples were collected from each fish, M13 tag, C1’M13 (5’GTAAAACGACGGCCAGAC stored in undenatured ethanol and lodged with the CCGCTGAATTTAAGCAT-3’) and reverse D2M13 Australian Biological Tissue Collection (ABTC) at (5’-CAGGAAACAGCTATGACTCCGTGTTTCAA- the South Australian Museum Australia (SAMA), GACGG-3’), corresponding to positions 25 and 1126 North Terrace, Adelaide, South Australia 5000, of the complete Mus musculus 28S rRNA nucleotide Australia (ABTC 108509–777). sequence (Hassouna et al., 1984). Two additional 123 Syst Parasitol (2010) 76:211–222 213 Table 1 Polyopisthocotylean monogeneans: specimens amplified, sequenced and analysed in this study (– = not applicable) Family Monogenean species Host species Number of GenBank Source individuals number analysed Microcotylidae Kahawaia truttae Arripis truttaceus 52 GU263831 Present study (Dillon & Hargis, 1965) Microcotylidae Kahawaia truttae Arripis trutta 13 GU263832 Present study Microcotylidae Microcotyle arripis Sandars, 1945 Arripis georgianus 1 GU263830 Present study Microcotylidae Polylabris sillaginaea Sillaginodes punctatus 10 GU289509 Present study (Woolcock, 1936) Microcotylidae Microcotyle erythrinii Pagellus erythrinus – AM157221 Badets et al. van Beneden & Hesse, 1863 (unpublished) Microcotylidae Microcotyle sebastis Goto, 1984 Sebastes sp. – AF382051 Olson & Littlewood (2002) Microcotylidae Microcotylidae sp. M10 Sebastes sp. – EF653385 Aiken et al. (2007) Microcotylidae Diplostamenides sciaenae Not specified – FJ432589 Su (unpublished) (Goto, 1894) Discocotylidae Discocotyle sagittata Goto, 1984 Salmo trutta – AF382036 Olson & Littlewood (2002) a Species identified from Hayward (1996) primers were designed for improved amplification of analysis (1,000 replicates). Percentages over 50% were fish fin clips, SC-FM13 (5’-GTAAAACGACGGC- included on the neighbour-joining (NJ) tree. Represen- CAGACCTCAGATCAGACGAGACAAC-3’) and tative sequences were deposited in GenBank (Table 1). reverse SC-RM13 (5’-CAGGAAACAGCTATGACC GTGCGTTAGACTCCTTGGTC-3’). Specimens requested and deposited Amplification reactions were conducted in a final Five voucher specimens of Kahawaia truttae (as volume of 25 ll containing: 2.5 ll of GeneAmp 10x Gonoplasius truttae, W. 14952–56), lodged by Dillon PCR Buffer II (Applied Biosystems, Inc. [ABI], & Hargis (1965) collected from A. trutta in NZ, were Foster City, CA, USA), 1.5 mM MgCl2 (ABI), borrowed from the Australian Museum (6 College 200 lM of each dNTP, 10 pmol of each primer, Street, Sydney, NSW, Australia) for comparison with 1.25 U of AmpliTaq Gold (ABI) and