FOLIA PARASITOLOGICA 52: 231–240, 2005 Description of Pseudorhabdosynochus seabassi sp. n. (Monogenea: Diplectanidae) from Lates calcarifer and revision of the phylogenetic position of Diplectanum grouperi (Monogenea: Diplectanidae) based on rDNA sequence data Xiang Y. Wu1, An X. Li1, Xing Q. Zhu2 and Ming Q. Xie1 1Centre for Parasitic Organisms, School of Life Sciences, Sun Yat-sen University, 135 Xingang West Street, Haizhu District, Guangzhou 510275, Guangdong Province, The People’s Republic of China; 2Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou 510642, Guangdong Province, The People’s Republic of China Key words: Pseudorhabdosynochus seabassi, Diplectanum grouperi, PCR, phylogeny, Lates calcarifer Abstract. Pseudorhabdosynochus seabassi sp. n. (Monogenea: Diplectanidae) from the gill filaments of Lates calcarifer Bloch, a marine teleost fish held in floating sea cages in Guangdong Province, China, is described based on morphological observations and molecular data. The shapes of the male copulatory organs (MCO) of Pseudorhabdosynochus spp. were the focus of this study. The typical proximal part of the MCO in most species of Pseudorhabdosynochus is reniform, heavily sclerotized, and divided into four chambers. However, the new species from L. calcarifer has a bulbous proximal region with four concentric layers of apparent muscular origin, instead of a reniform structure with four compartments. This organ is also different in Diplectanum grouperi Bu, Leong, Wong, Woo et Foo, 1999, being sclerotized, cup-shaped, wide proximally with four concentric muscular layers and tubular distally. The 3’ terminal portion of the small subunit ribosomal RNA gene (ssrDNA) and the 5’ terminal region (domains C1-D2) of the large subunit ribosomal RNA gene (lsrDNA) were used to reconstruct the phylogenetic relationships of P. seabassi and D. grouperi with related taxa utilizing maximum-parsimony and neighbour-joining methods. Phylogenetic analyses unequivocally placed D. grouperi amongst Pseudorhabdosynochus using either ssrDNA or lsrDNA data. All species of Pseudorhabdosynochus (including D. grouperi) used in this study clustered together, inferring monophyly. Based on molecular phylogenetic evidence, we propose that D. grouperi from Epinephelus coioides Hamilton be transferred to Pseudorhabdosynochus as P. grouperi comb. n. Diplectanids are a group of monogeneans that mainly americanum Price, 1937, whose inner row of elements in parasitize the gills of serranid fish and have a world-wide the squamodisc forms a complete ring, distinguishing it distribution (Oliver 1993). Many species of these from Diplectanum. The nomenclature and taxonomy of monogeneans occur in cultured fish and are of consid- species in Cycloplectanum and Pseudorhabdosynochus erable economic importance (e.g., Nash et al. 1987, has since become controversial and confusing. Bever- Leong and Wong 1990, Leong 1994). Traditionally, ley-Burton and Suriano (1981) considered that the classification of the Diplectanidae Bychowsky, 1957 has squamodiscs of Cycloplectanum spp. showed inter- been based, to a large extent, on morphology of the specific variation in number and shape of rows, in degree sclerotized components of the haptor (Beverley-Burton of sclerite fusion and overlap pattern, and these varia- and Suriano 1981). Two genera of the family are of par- tions should not be considered of prime importance in ticular interest because of controversies regarding the generic separation. They amended the diagnosis of taxonomic status and phylogenetic positions of some Cycloplectanum and emphasised the importance of the species within them, and have been the focus of a number unique structure of the terminal genitalia (both the male of studies (e.g., Oliver 1968, Kritsky and Bever- copulatory organ [MCO] and vagina). Considering the ley-Burton 1986). One, Diplectanum Diesing, 1858, the confusing nomenclature in these genera, Kritsky and type genus of the family, is characterized in part by Beverley-Burton (1986) presented a historical review presence of a haptor which is constricted from the body and considered Cycloplectanum a junior synonym of proper, with two pairs of hamuli, three transverse bars, Pseudorhabdosynochus Yamaguti, 1958. and dorsal and ventral squamodiscs consisting of con- So far, six diplectanids have been reported from Lates centric rows of sclerites (Yamaguti 1963). The other is calcarifer, two belonging to Pseudorhabdosynochus, Pseudorhabdosynochus Yamaguti, 1958, erected to ac- namely P. latesi Tripathi, 1955 and P. monosquamodis- commodate P. epinepheli Yamaguti, 1958. Subsequently, cusi Balasuriya et Leong, 1995. In this paper, a new Oliver (1968) erected Cycloplectanum for Diplectanum species of Pseudorhabdosynochus collected from gills of Address for correspondence: A.X. Li, Centre for Parasitic Organisms, School of Life Sciences, Sun Yat-sen University, 135 Xingang West Street, Haizhu District, Guangzhou 510275, Guangdong Province, The People’s Republic of China. Phone: ++86 208 411 5113; Fax: +86 208 411 5113; E-mail: [email protected] 231 L. calcarifer is described based on morphological char- were fixed in 70% ethanol under a cover-slip, stained with acters. However, there are limitations using morpho- carmine, differentiated in acid-alcohol, dehydrated in serial logical approaches as some studies on Gyrodactylidae concentrations of ethanol, cleared in xylene, and mounted in (Mo 1991) have demonstrated variation in the mor- Canada balsam. The descriptive terminology and numbering of phology of haptoral sclerites on which Diplectanidae and the haptoral parts follow Bu et al. (1999). Drawings were made from specimens originated from the same population used for most other monogeneans are mainly based (Oliver 1987). molecular analyses. Measurements are presented in microme- Molecular approaches using genetic markers in small tres. and large subunit ribosomal DNA (ssrDNA and lsrDNA) DNA extraction and PCR amplification. Prior to DNA offer alternatives and complement morphology for iden- extraction, individual parasites were removed from Bleasure’s tification and systematics of monogeneans (e.g., Cun- glue, placed in 0.5 ml Eppendorf tubes and dipped in 500 µl ningham et al. 1995, Mollaret et al. 1997, Littlewood et al. TE9 (500 mM Tris-HCl, 200 mM EDTA, and 10 mM NaCl, pH 1998, Olson and Littlewood 2002). A few studies have 9.0) for 2–3 h. They were then placed in 0.5 ml tubes containing examined phylogenetic relationships of monogeneans at 20 µl lysis buffer (0.45% NP-40, 0.45% Tween-20, 1 mM the subclass and family levels using molecular ap- EDTA, 10 mM Tris-HCl and 20 µg/ml proteinase K) and in- proaches (Mollaret et al. 2000, Jovelin and Justine 2001, cubated at 65°C for 1 h, followed by incubation at 95°C for 15 Olson and Littlewood 2002). However, little is known min to inactivate the proteinase K. about phylogenetic relationships within Monogenea at A region comprising partial ssrDNA and the entire internal the generic level, although this could be important to transcribed spacer 1 (ITS1) region was amplified using primers understand host-parasite relationships (Desdevises et al. S1 (5’-ATTCCGATAACGAACGAGACT-3’) and IR8 (5’-GCTAGCTGCGTTCTTCATCGA-3’) as previously de- 2000, 2002, Huyse et al. 2003, Šimková et al. 2003). scribed (Šimková et al. 2003). Each amplification reaction was Therefore, another objective of the present study is to performed in a final volume of 50 µl containing 9 µl of lysate, investigate the phylogenetic status of Diplectanum 1.5 mM of MgCl2, 1 × buffer (TakaRa), 200 µM of each dNTP, grouperi Bu, Leong, Wong, Woo et Foo, 1999 which 0.8 µM of each PCR primer and 2.5 U of Ex Taq polymerase parasitizes the gills of Epinephelus coioides Hamilton (TakaRa) in a thermocycler (MJ Research) under the following and is very similar to Pseudorhabdosynochus spp. in conditions: 4 min at 95°C (initial denaturation), followed by 35 terms of host species and morphological features, such as cycles of 1 min at 92°C (denaturation), 1 min at 53°C (an- the four muscular layers in the proximal part of MCO, nealing) and 1.5 min at 72°C (extension) and a final extension which is similar to that of P. latesi and P. at 72°C for 10 min. monosquamodiscusi. It was described, however, as a The C1-D2 lsrDNA region was amplified using the uni- species of Diplectanum considering the cupshaped MCO versal primer C1 (5’-ACCCGCTGAATTTAAGCAT-3’) and as a whole. We used the 3’ terminal portion of ssrDNA the reverse primer D2 (5’-TGGTCCGTGTTTCAAGAC-3’). and the 5’ terminal region of lsrDNA (domains C1-D2), Amplification was performed as described above, except that the lysate was 6 µl in each reaction. The cycling conditions as both have been widely used to estimate phylogenetic were as following: 5 min at 94°C (initial denaturation), fol- relationships among monogeneans (e.g., Cunningham et lowed by 30 cycles of 1 min at 94°C (denaturation), 1 min at al. 1995, Mollaret et al. 1997, Littlewood et al. 1998, 56°C (annealing) and 1 min at 72°C (extension) and a final Desdevises et al. 2000, Mollaret et al. 2000, Chisholm et extension at 72°C for 10 min. Samples with host DNA or al. 2001, Jovelin and Justine 2001, Olson and Littlewood without genomic DNA were included in each amplification run 2002). The validity and phylogenetic position of the new as controls. PCR products were examined on 1% agarose gels, species described here was also confirmed by molecular stained