Elucidating the Ecology of Bucephalid Parasites Using a Mutation Scanning Approachq
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Molecular and Cellular Probes 18 (2004) 139–146 www.elsevier.com/locate/ymcpr Elucidating the ecology of bucephalid parasites using a mutation scanning approachq Kate S. Hutsona,b,c,*, Craig A. Styana,d, Ian Beveridgeb, Michael J. Keougha, Xingquan Zhub,e, Youssef G. Abs EL-Ostab, Robin B. Gasserb aDepartment of Zoology, The University of Melbourne, Parkville, Vic. 3052, Australia bDepartment of Veterinary Science, The University of Melbourne, Werribee, Vic. 3030, Australia cSchool of Earth and Environmental Sciences, The University of Adelaide, Adelaide, SA 5005, Australia dCentre for Research on Ecological Impacts of Coastal Cities, The University of Sydney, Sydney, NSW 2006, Australia eCollege of Veterinary Medicine, South China Agricultural University, Wushan, Guangzhou 510642, Guangdong Province, China Received 15 September 2003; accepted for publication 11 November 2003 Abstract Nucleotide variation in a portion of the mitochondrial cytochrome c oxidase subunit1 (cox1) gene from asexual stages of bucephalids of southern Australian scallops (Chlamys asperrima, Chlamys bifrons and Pecten fumatus) was investigated using a mutation scanning– sequencing approach. Single-strand conformation polymorphism (SSCP) analysis revealed three main profile types (A, B and C) for parasites isolated from scallops. Sequence analysis revealed that samples represented by profiles B and C had a high degree (97.3%) of sequence similarity, whereas they were ,21% different in sequence from those represented by profile A. These findings suggested that at least two types or species (represented by profile A, or profile B or C) of bucephalid infect scallops, of which both were detected in South Australia, while only one was found in Victoria. The prevalence of bucephalids (and their SSCP haplotypes) appeared to differ among the three species of scallop in South Australia as well as between the two scallop species in Victoria, indicating a degree of host specificity. Adult bucephalids were collected from Eastern Australian Salmon (Arripis trutta), in an attempt to match them with the asexual stages from the scallop hosts. Neither of the two taxa of adult bucephalid (Telorhynchus arripidis and an un-named Telorhynchus species) shared SSCP profiles with the bucephalids from scallops, but were genetically similar, suggesting that the asexual stages from scallops may represent the genus Telorhynchus. This study, which assessed nucleotide sequence variation in a portion of the mitochondrial cox1 gene for bucephalids found in scallops and arripid fish, illustrates the usefulness of the mutation scanning approach to elucidate complex life-cycles of marine parasites. q 2003 Elsevier Ltd. All rights reserved. Keywords: Scallop; Digenea; Bucephalid; Identification; Host specificity; Genetic variation; Single-strand conformation polymorphism analysis; Mitochondrial DNA 1. Introduction host species and dispersive stages [1]. Recently, molecular techniques have proved valuable in enabling the specific Digeneans are important ecological indicators, their identification of parasites where morphological features are transmission pathways providing extensive information on of limited or no value [2]. Consequently, complex parasite host biology, trophic interactions and food-web dynamics life-cycles may be unravelled by genetically matching life [1]. However, digenean life-cycles are complex and difficult stages from different host species. to elucidate, because they usually involve several different Digeneans of the family Bucephalidae parasitise many species of mollusc, including commercially important q Nucleotide sequence data reported in this paper are available in the bivalve molluscs, such as mussels, clams, oysters, cockles EMBL, GenBanke and DDJB databases under accession numbers and scallops [3]. They cause reproductive failure of their AY504854–AY504864. intermediate mollusc host by progressively replacing gonad * Corresponding author. Address: School of Earth and Environmental tissue with asexual life stages, known as sporocysts [3]. Sciences, The University of Adelaide, Adelaide, SA 5005, Australia. Tel.: þ61-8-8303-5282; fax: þ61-8-8303-4364. Millions of two-tailed sexual cercariae are produced within E-mail address: [email protected] (K.S. Hutson). the sporocysts, which escape from the mollusc into water 0890-8508/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2003.11.004 140 K.S. Hutson et al. / Molecular and Cellular Probes 18 (2004) 139–146 and swim until they find their second intermediate (teleost) the unidentified asexual stages of bucephalids from scallops host and penetrate its skin [4]. To complete the cycle, the with known adult stages from a possible primary host, A. second intermediate host is eaten by a definitive teleost host, trutta. To do this, PCR-based single-strand conformation in which the ingested bucephalid attaches to the intestine of polymorphism analysis (PCR-SSCP) [10] followed by the new host, develops into an adult, reproduces sexually and selective DNA sequencing was employed to characterise releases eggs in the faeces. [4]. Eggs hatch to release motile nucleotide variation in the mitochondrial cytochrome c miracidia, which then infect mollusc intermediate hosts [3]. oxidase subunit 1 gene (cox1) within and among parasite Scallops are well-known hosts of bucephalids. In isolates. Such a mutation scanning approach has been shown Australia, infections have been detected in populations of previously to be effective for studying population genetic Pecten fumatus and Chlamys asperrima from New South structures [11–14]. Wales [4], P. fumatus from Victoria [6], P. fumatus and C. asperrima from Bass Strait (West, personal communi- cation), and P. fumatus, C. asperrima and Chlamys bifrons 2. Materials and methods from South Australia (Styan and Keough, unpublished data). However, the bucephalids which infect these scallops have 2.1. Collection and processing of trematode parasites yet to be identified to genus or species, and their second from scallops and fish intermediate and definitive teleost fish hosts are presently unknown. Sampling locations are shown in Fig. 1. The scallops, Adult bucephalids have been recorded and described for C. asperrima, C. bifrons and P. fumatus, were collected by a number of species of southern Australian fish [7].Of divers in South Australia during October 2000. All three known definitive hosts, Eastern Australian Salmon (Arripis species were collected from under Edithburgh Jetty, Port trutta) is probably the most common and widespread Giles Jetty and Klein Point Jetty in Gulf St Vincent. C. bifrons species, with migratory populations found along the coasts were collected from under Port Hughes Jetty in the Spencer of New South Wales, Tasmania and Victoria [8]. A. trutta is Gulf. P. fumatus was rare in all locations in South Australia. a known host for at least four species of bucephalid parasite, In Port Phillip Bay, P. fumatus was collected off-shore from including Telorhynchoides longicollis, and Telorhynchus Dromana, Mt Martha, Rye, McCrae and Portarlington by arripidis, T. kahawai and T. peacheyii [9]. A closely related divers in October and November 2000; some were also species, the Western Australian Salmon (Arripis truttaceus), collected from a trawler with dredges taken from Clifton could also be a potential definitive host for bucephalids and Springs and Werribee South. C. asperrima was collected occurs in large numbers in South Australia and Western from Rye Pier, Portsea Reef and Point Franklin, Port Phillip Australia. The ranges of the two species of Arripis overlap Bay in November and December 2000. No C. bifrons were in Victorian and Tasmanian waters [8]. Although arripids detected during any collections in Port Phillip Bay. For are common in locations where scallops are also found, it analyses, scallops were treated as coming from either is not known whether bucephalids of scallops represent the Spencer Gulf, Gulf St Vincent (both South Australia) or asexual stages of one or more of those found in A. trutta Port Phillip Bay (Victoria). Only adult scallops (those large or A. truttaceus. enough to have mature gonads in uninfected individuals) It is also not known whether only one species of bucephalid were collected throughout; this corresponded to animals with infects different species of southern Australian scallop. The shell widths greater than ,45 mm (C. asperrima) and prevalence of bucephalid intramolluscan stages has been ,55 mm (P. fumatus and C. bifrons). Parasitised gonads found to differ significantly between different host species were always red in colour and easily distinguished from occurring in sympatry, and can change independently through healthy gonads [6]. For the isolation of DNA, sections time ([5]; Styan and Keough, unpublished data). One (,3mm3) from the end of infected gonads were excised. explanation for these observations might be that separate These were each squashed with a clean slide and then washed species of bucephalid infect different scallop species. A key with physiological saline. A volume of ,200 ml of each problem in resolving this, and addressing questions about the gonad/saline suspension was pipetted into an Eppendorf identity of second intermediate and definitive hosts, is that tube. For the first parasitised samples, the suspensions were species of bucephalids are difficult to distinguish based on the centrifuged and the sediment examined microscopically for morphology of their asexual stages [9]. sporocysts. To obtain