EFFECT OF PHYSIOLOGICAL AND BEHAVIOURAL CHARACTERISTICS OF PARASITOIDS ON HOST SPECIFICITY TESTING OUTCOMES AND THE BIOLOGICAL CONTROL OF PAROPSIS CHARYBDIS A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University By Tara J. Murray Lincoln University 2010 To Barbara Barratt and Kath Dickinson, outstanding scientists, inspiring mentors and patient friends - without your enthusiastic and supportive introduction to scientific research I wouldn’t have had the determination or desire to keep going when the road got rough. Things have rarely gone as planned, but thanks to you I know there are many exciting research opportunities awaiting me, and some amazing scientists that I can look forward to working with in the future. Abstract of thesis submitted in partial fulfilment of the requirements for the Degree of Ph.D. Effect of physiological and behavioural characteristics of parasitoids on host specificity testing outcomes and the biological control of Paropsis charybdis By Tara J. Murray An established host-parasitoid-hyperparasitoid system was used to investigate how the physiological and behavioural characteristics of parasitoids influence the outcomes of laboratory- based host specificity tests. The characteristics of the two pteromalid egg parasitoids, Enoggera nassaui (Girault) and Neopolycystus insectifurax Girault, were assessed and interpreted in regard to the particular host specificity testing methods used and the control of the eucalypt defoliating beetle Paropsis charybdis Stål (Chrysomelidae) in New Zealand. The physiology of N. insectifurax was examined to determine how to increase production of female parasitoids that were physiologically capable and motivated to parasitise P. charybdis eggs in laboratory trials. Neopolycystus insectifurax were found to be more synovigenic than E. nassaui . Provisioning them with honey and host stimuli for three days, and allowing females to parasitise hosts in isolation (i.e. in the absence of competition) was an effective means of achieving these goals. No-choice tests were conducted in Petri dish arenas with the four paropsine beetles established in New Zealand. All four were found to be within the physiological host ranges of E. nassaui and N. insectifurax , but their quality as hosts, as indicated by the percent parasitised and offspring sex ratios, varied. The results of paired choice tests between three of the four species agreed with those of no-choice tests in most instances. However, the host Trachymela catenata (Chapuis), which was parasitised at very low levels by E. nassaui in no-choice tests, was not accepted by that species in paired choice tests. A much stronger preference by N. insectifurax for P. charybdis over T. catenata was recorded in the paired choice test than expected considering the latter was parasitised at a high level in the no-choice test. The presence of the target host in paired choice tests reduced acceptance of lower ranked hosts. Both no-choice and choice tests failed to predict that eggs of the i acacia feeding beetle Dicranosterna semipunctata (Chapuis) would not be within the ecological host range of E. nassaui and N. insectifurax . Behavioural observations were made of interspecific competition between E. nassaui and N. insectifurax for access to P. charybdis eggs. Two very different oviposition strategies were identified. Neopolycystus insectifurax were characterised by taking possession of, and aggressively guarding host eggs during and after oviposition. They also appeared to selectively oviposit into host eggs already parasitised by E. nassaui, but did not emerge from significantly more multi- parasitised hosts than E. nassaui . Enoggera nassaui did not engage in contests and fled when approached by N. insectifurax . Although often prohibited from ovipositing by N. insectifurax, E. nassaui were able to locate and begin ovipositing more quickly, and did not remain to guard eggs after oviposition. It is hypothesised that although N. insectifurax have a competitive advantage in a Petri dish arena, E. nassaui may be able to locate and parasitise more host eggs in the field in New Zealand, where competition for hosts in is relatively low. The biology of the newly established encyrtid Baeoanusia albifunicle Girault was assessed. It was confirmed to be a direct obligate hyperparasitoid able to exploit E. nassaui but not N. insectifurax . Field and database surveys found that all three parasitoids have become established in many climatically different parts of New Zealand. Physiological characteristics were identified that may allow B. albifunicle to reduced effective parasitism of P. charybdis by E. nassaui to below 10%. However, the fact that hyperparasitism still prevents P. charybdis larvae from emerging, and that B. albifunicle does not attack N. insectifurax, may preclude any significant impact on the biological control of P. charybdis . Overall, parasitoid ovigeny and behavioural interactions with other parasitoids were recognised as key characteristics having the potential to influence host acceptance in the laboratory and the successful biological control of P. charybdis in the field. It is recommended that such characteristics be considered in the design and implementation of host specificity tests and might best be assessed by conducting behavioural observations during parasitoid colony maintenance and the earliest stages of host specificity testing. Keywords: biological control, host specificity testing, no-choice test, choice test, parasitoid, hyperparasitoid, parasitoid behaviour, Eucalyptus , Acacia , Paropsis charybdis , Enoggera nassaui , Neopolycystus insectifurax , Baeoanusia albifunicle ii ACKNOWLEDGEMENTS This research was funded by the Foundation for Research Science and Technology through contracts to Scion (No. CO4X0302 and No. CO4X0807), and through contract CO2X0501, the Better Border Biosecurity (B3) Programme (www.b3nz.org) and was supported by the Forest Biosecurity Research Council to Scion. Additional funding was gratefully received through the following scholarships: Gordon Williams Postgraduate Scholarship in Ecological Sciences; MacMillan Brown Agricultural Research Scholarship; New Zealand Plant Protection Society Inc. Research Scholarship; Robert C. Bruce Trust Research Grant; Royal Society of New Zealand Travel Grant; Todd Foundation Awards for Excellence. I would like to thank the members of the New Zealand Farm Forestry Association and the Acacia Melanoxylon Interest Group Organisation, in particular Dean Satchell and Ian Brown, for their assistance locating and accessing field sites for insect collection. Individuals who have contributed their time and experience to help me conduct this research include Richard Parker (COHFE), Yaffa Gould and Richard Lainson (SDR Clinical Technology) for assistance with The Observer technology; Mark Kimberley (Scion) and James Ross (Lincoln) for statistical support; Geoff Tribe (Agricultural Research Council of South Africa) and Jo Berry (MAF) for providing and identifying parasitoid voucher specimens; Dave Hayes, Pam Taylor, Andrea Sharpe, Caro Gous (Scion) Diane Jones (MAF) and Kathleen Camp (University of California, Riverside) for advice and technical assistance in maintaining insect colonies. I would like to acknowledge my supervisors Bruce Chapman and Sue Worner (Lincoln University) and Sarah Mansfield (University of Sydney) for guiding me through the PhD process despite the distance, as well as Toni Withers (Scion) and Barbara Barratt (AgResearch) for their continued assistance and advice throughout this research. Finally, I would like to thank everyone at FBP, a great bunch to be ‘vital’ with, going forward. Special thanks to Damien, Michelle, Phil, fine Islay single malts, and my running shoes, for suffering most of my whinging and making for some good times between chapters. iii TABLE OF CONTENTS Abstract..................................................................................................................................i Acknowledgements ............................................................................................................ iii Table of Contents................................................................................................................iv List of Figures.....................................................................................................................vii List of Tables ........................................................................................................................x Chapter 1: Introduction ......................................................................................................1 1.1 Classical Biological Control..................................................................................1 1.2 Evaluating the Risks of Biological Control Agents.................................................2 1.3 Host Specificity Testing of Parasitoids....................................................................4 1.4 Improving Host Specificity Testing.........................................................................5 1.5 Study System in Which to Examine Host-Specificity Testing................................8 1.6 Thesis Goals...........................................................................................................12 Chapter 2: Insect Cultures & Preparing Parasitoids for Experimental Trials ...........14 2.1 Introduction............................................................................................................14