Developing a rapid molecular identification technique to improve egg production based fish biomass assessments Richard J. Saunders, Shannon Kjeldsen, Roger Huerlimann, Thor Saunders, Shane Penny, Andrew Tobin and Dean Jerry 2019 FRDC Project No 2014/022 © Year Fisheries Research and Development Corporation. All rights reserved. ISBN 978-0-6485037-0-5 Developing a rapid molecular identification technique to improve egg production based fish biomass assessments FRDC Project No 2014/022 2019 Ownership of Intellectual property rights Unless otherwise noted, copyright (and any other intellectual property rights, if any) in this publication is owned by the Fisheries Research and Development Corporation [and if applicable insert research provider organisation/s e.g. CSIRO Marine Research] This publication (and any information sourced from it) should be attributed to Saunders, R.J., Kjeldsen, S., Huerlimann, R., Saunders, T., Penny, S., Tobin, A. and Jerry, D. (2019). Developing a rapid molecular identification technique to improve egg production based fish biomass assessments. FRDC Final Report 2014/022. Creative Commons licence All material in this publication is licensed under a Creative Commons Attribution 3.0 Australia Licence, save for content supplied by third parties, logos and the Commonwealth Coat of Arms. Creative Commons Attribution 3.0 Australia Licence is a standard form licence agreement that allows you to copy, distribute, transmit and adapt this publication provided you attribute the work. A summary of the licence terms is available from creativecommons.org/licenses/by/3.0/au/deed.en. The full licence terms are available from creativecommons.org/licenses/by/3.0/au/legalcode. Inquiries regarding the licence and any use of this document should be sent to: [email protected] Disclaimer The authors do not warrant that the information in this document is free from errors or omissions. The authors do not accept any form of liability, be it contractual, tortious, or otherwise, for the contents of this document or for any consequences arising from its use or any reliance placed upon it. The information, opinions and advice contained in this document may not relate, or be relevant, to a readers particular circumstances. Opinions expressed by the authors are the individual opinions expressed by those persons and are not necessarily those of the publisher, research provider or the FRDC. The Fisheries Research and Development Corporation plans, invests in and manages fisheries research and development throughout Australia. It is a statutory authority within the portfolio of the federal Minister for Agriculture, Fisheries and Forestry, jointly funded by the Australian Government and the fishing industry. I Researcher Contact Details FRDC Contact Details Name: Richard Saunders Address: 25 Geils Court Address: 1 James Cook Dr Deakin ACT 2600 Douglas QLD 4814 Phone: 02 6285 0400 Phone: +64 2 6423965 Fax: 02 6285 0499 Fax: n/a Email: [email protected] Email: [email protected] Web: www.frdc.com.au In submitting this report, the researcher has agreed to FRDC publishing this material in its edited form. II Contents CONTENTS ....................................................................................................................................... III ACKNOWLEDGMENTS ..................................................................................................................... VI ABBREVIATIONS ............................................................................................................................. VII EXECUTIVE SUMMARY .................................................................................................................. VIII 1 INTRODUCTION ............................................................................................................................. 1 OBJECTIVES....................................................................................................................................... 5 2 METHODS ...................................................................................................................................... 6 3 RESULTS ....................................................................................................................................... 17 4 DISCUSSION ................................................................................................................................. 31 5 CONCLUSION ............................................................................................................................... 36 7 IMPLICATIONS ............................................................................................................................. 40 8 RECOMMENDATIONS .................................................................................................................. 40 9 FURTHER DEVELOPMENT ............................................................................................................ 40 10 EXTENSION AND ADOPTION...................................................................................................... 41 11 PROJECT COVERAGE .................................................................................................................. 41 APPENDICES ................................................................................................................................... 42 FRDC FINAL REPORT CHECKLIST ..................................................................................................... 58 III Tables Table 1 List of species for which species-specific probes were developed in the project and the rationale for their inclusion. ......................................................................................................................................... 6 Table 2. Probe sequences for each species. Note: Only a single gene could be targeted for some species due to the highly conserved nature of these genes across species. .......................................................... 12 Table 3. Universal primer sets used in the study. ....................................................................................... 13 Table 4. General PCR recipe ........................................................................................................................ 14 Table 5. PCR conditions for COI and 16S gene ............................................................................................ 14 Table 6. Sequencing results for identification of 10 unknown eggs collected during the October 2015 egg survey. ......................................................................................................................................................... 20 Table 7. 2016 egg collections. *Helix Experiment eggs were preserved in a range of solutions, numbers in brackets are for those eggs preserved in 100% EtOH................................................................................. 21 Table 8. Comparison of identification success between sequencing and the bead array (on Magpix platform) for 2016 samples. ........................................................................................................................ 28 Table 9. The impact of preservation on the opacity and shape of barramundi eggs. 100% EtOH = 100% ethanol, 70% EtOH = 70% ethanol, RNAL8R = RNA L8R, F+100% = eggs washed in 5% formalin and preserved in 100% ethanol, Formalin 5% = Formalin 5%. .......................................................................... 29 Table 10. The impact of different preservatives on the subjective ability to stage barramundi eggs. 100% EtOH = 100% ethanol, 70% EtOH = 70% ethanol, RNAL8R = RNA L8R, F+100% = eggs washed in 5% formalin and preserved in 100% ethanol, Formalin 5% = Formalin 5%. .................................................... 29 Table 11. Rate of PCR success using a variety of preservation methods on wild collected plankton samples. 100% EtOH = 100% ethanol, 70% EtOH = 70% ethanol, RNAL8R = RNA L8R, F+100% = eggs washed in 5% formalin and preserved in 100% ethanol, Formalin 5% = Formalin 5% .............................. 30 Table 12. Range of published methods for fish egg identification identifying DNA amplification failure rates. ............................................................................................................................................................ 32 Table 13. Issues and solutions for the bead array method (on Magpix platform). ................................... 34 IV Figures Figure 1. Flow chart of molecular egg identification approaches. ............................................................... 3 Figure 2. (A) Map of Qld illustrating study region highlighted by a black square. (B) Zoomed study area showing sites of plankton samples collected in October 2015 research survey. Black dots are twin-ring vertical tows, x is 24 hour neuston sampling site. ........................................................................................ 8 Figure 3. Map illustrating study region highlighted by a black square with zoomed study area showing sites of plankton samples collected in Spring 2016 plankton surveys. Black dots are the sites of the twin- ring vertical tows. .......................................................................................................................................... 9 Figure 4. Map illustrating study region in the NT highlighted by a black square with zoomed study area showing sites of plankton samples collected