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Development of Genetic Probes for Rapid Assessment of the Impacts of Marine Invasive Species on Native Biodiversity – Maoricolpus Roseus

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Development of Genetic Probes for Rapid Assessment of the Impacts of Marine Invasive Species on Native Biodiversity – Maoricolpus Roseus Gazameda gunnii Development of genetic probes for rapid assessment of the impacts of marine invasive species on native biodiversity – Maoricolpus roseus Jawahar Patil, Rasanthi Gunasekera, Felicity McEnnulty and Nicholas Bax Final Report for the Department of Environment and Heritage by CSIRO Marine Research November 2004 NHT Genetic Probe Development for Maoricolpus roseus i ” Commonwealth of Australia 2004 ISBN 1 876996 85 4 This work is copyright. Apart from any use permitted under the Copyright Act 1968, no part may be reproduced by any process without prior written permission from the Commonwealth, available from the Department of the Environment and Heritage. Requests and inquiries concerning reproduction and rights should be addressed to the publisher. Publisher: Australian Government Department of the Environment and Heritage John Gorton Building King Edward Terrace Parkes ACT 2600 Australia This publication is also available as a PDF document on the Department of the Environment and Heritage website at: http://www.deh.gov.au/coasts/publications/index.html#pests Authorship: This report is an independent review prepared for the Department of the Environment and Heritage by CSIRO Marine Research. The views and opinions expressed in this publication are those of the authors and do not necessarily reflect those of the Commonwealth Government or the Minister for the Environment and Heritage. The Commonwealth does not accept responsibility for the accuracy or completeness of the contents of this publication, and shall not be liable for any loss or damage that may be occasioned directly or indirectly through the use of, or reliance on, the contents of this publication. Development of genetic probes for rapid assessment of the impacts of marine invasive species on native biodiversity-Maoricolpus roseus. Bibliography. Includes index. ISBN 1 876996 85 4. 1. Maoricolpus roseus - Australia. 2. Aquatic pests - Control - Australia. 3. Pest introduction - Australia. I. Patil, Jawahar G., 1965- . II. Australia. Dept. of Environment and Heritage. III. CSIRO. Marine Research. 594.30994 NHT Genetic Probe Development for Maoricolpus roseus EXECUTIVE SUMMARY The New Zealand Screw shell Maoricolpus roseus unintentionally introduced to south- eastern Tasmania in the 1920s, has now spread out to the 80 metre depth contour off the eastern Victorian and New South Wales coasts and is found as far north as Botany Bay (Bax et al., 2003). This species has colonized more habitat than any other high- impact benthic marine pest in Australia and the risk of its further spread and establishment is very likely owing to its wide temperature and depth tolerance. This project was initiated to develop genetic probes for the rapid detection and assessment of this species as a prelude to determining its impacts on the native flora and fauna as well as to fill our knowledge gaps in its phenology and life history strategies. In particular it is important to know whether this species has a planktonic life history stage that could lead to its dispersal in ships’ ballast water. Obtaining suitable samples of the M. roseus and its closely related species from Australia and its native range in New Zealand, although trivial, were time consuming and samples were often not forthcoming. Specifically, difficulties were encountered in obtaining samples of the native screw shell Gazameda gunnii, which took special attempts involving dedicated divers and a scientific cruise. Maoricolpus roseus samples were obtained from 6 and 2 locations in Australia and New Zealand respectively with the majority of the shells collected being empty. Three sets of gene probes were developed, each of which targeted a unique region in the mitochondrial COI locus. All the three sets of probes were tested against as many closely related species as could be obtained and the reaction conditions optimized for maximum sensitivity and specificity. Further sequence comparison of COI locus of M. roseus and members of Cerithioidea, including the native Australian turritellids, Gazameda iredalei, G. gunnii and Turitella terebra suggest significant differences which should permit the design of specific probes for M. roseus and perhaps for the others such as the G. gunnii. In light of limited sequence variation within and between M. roseus roseus and M. roseus manukauensis, we attempted to amplify the entire mitochondrial genome of M. roseus. In all 13224bp of the expected ~15000 bp of the mtDNA was sequenced and characterized. It is hoped that this information will assist in fine scale genetic discrimination between populations of M. roseus as well as in identifying source population of introduction into Australian waters. A nested polymerase chain reaction (PCR) was used to analyse environmental plankton samples collected in the Derwent Estuary between August 2003 and June 2004. The results suggest that the species does have a planktonic larval existence prior to settlement. Results also support earlier beliefs that the species is predominantly a spring-summer spawner, although M. roseus was detected in plankton samples on two occasions in winter months. The presence of M. roseus in the plankton and water available for ships’ ballast suggests that the risk of this species being spread by shipping may need to be iii managed within the National System for Prevention and Management of Marine Pest Incursions. NHT Genetic Probe Development for Maoricolpus roseus CONTENTS Executive Summary ...............................................................................................................iii 1 Introduction .................................................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2 Objectives ................................................................................................................. 1 2. Methods ......................................................................................................................... 3 2.1 Sample collection ....................................................................................................... 3 2.2 DNA extraction and sequencing ..................................................................................... 3 2.3 “ Maoricolpus roseus –specific” primer design ................................................................... 4 2.4 Detection of larval Maoricolpus roseus ........................................................................... 4 2.5 Detection of Maoricolpus roseus in plankton samples ....................................................... 5 2.6 Amplification and sequencing of partial mitochondrial genome ........................................... 5 3. Results .......................................................................................................................... 7 3.1 Sample collection ....................................................................................................... 7 3.2 DNA Extraction and PCR amplification. ........................................................................ 9 3.3 Sequence Analysis .................................................................................................... 11 3.4 Specificity of the PCR assay ...................................................................................... 16 3.5 Detection of larval Maoricolpus roseus ......................................................................... 18 3.6 Detection of Maoricolpus roseus in environmental samples .............................................. 19 3.7 Sequence of partial mitochondrial genome ..................................................................... 20 4. Discussion .................................................................................................................... 22 4.1 Significance of Maoricolpus roseus in Australia ................................................................ 22 4.2 Probe development, specificity and sensitivity ............................................................... 23 4.2 Mapping the mitochondrial genome ............................................................................. 25 4.3 Testing for a planktonic life history ............................................................................. 26 5. Conclusion ................................................................................................................... 26 6. References .................................................................................................................... 28 Appendix A - Acknowledgements ............................................................................................ 31 Appendix B – Detailed consultancy workplan ............................................................................ 32 Appendix C – Researchers contacted for samples ........................................................................ 33 Appendix D – Turritellid species overlapping Maoricolpus roseus in Australian waters (Wilson 1993) ...................................................................................................................................... 36 35 v Introduction 1 1 INTRODUCTION 1.1 Background The economic cost of aquatic exotic species is significant (Pimental et al., 2000). The extent of the introduced marine species problem in Australia became apparent with the outbreaks of highly visible introduced species
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