Final report Area-wide control of buffalo fly and prevention of southward spread using Wolbachia Project code: B.AHE.0242 Prepared by: Peter James Queensland Alliance for Agriculture and Food Innovation, University of Queensland Date published: 30 November 2020 PUBLISHED BY Meat and Livestock Australia Limited PO Box 1961 NORTH SYDNEY NSW 2059 Meat & Livestock Australia acknowledges the matching funds provided by the Australian Government to support the research and development detailed in this publication. This publication is published by Meat & Livestock Australia Limited ABN 39 081 678 364 (MLA). Care is taken to ensure the accuracy of the information contained in this publication. However MLA cannot accept responsibility for the accuracy or completeness of the information or opinions contained in the publication. You should make your own enquiries before making decisions concerning your interests. Reproduction in whole or in part of this publication is prohibited without prior written consent of MLA. B.AHE.0242 – Wolbachia control of buffalo fly Contents Abstract ...................................................................................................... 4 Executive summary ..................................................................................... 5 1. Background ......................................................................................... 7 1.1 Buffalo flies .................................................................................. 7 1.1.1 Description and biology .................................................................................... 7 1.1.2 Economic and welfare effects ........................................................................... 7 1.1.3. BF lesions.......................................................................................................... 8 1.1.4 Southerly spread of BF ...................................................................................... 9 1.2 Control of buffalo flies ................................................................ 11 1.2.1 Current controls (and problems) ..................................................................... 11 1.2.2 Past control of southerly spread (and problems) ............................................ 11 1.3 Potential for the use of area-wide approaches against buffalo flies in Australia ................................................................................. 12 1.3.1 Chemical-based area wide programmes ............................................................ 12 1.3.2 Autocidal and biologically based approaches ..................................................... 12 1.3.3 Wolbachia and area wide control of buffalo flies ............................................... 14 2. Methodology ..................................................................................... 18 2.1 BF colony ................................................................................... 18 2.2 Adaptation of Wolbachia in Haematobia cell lines ....................... 18 2.2.1 Establishment of cell lines............................................................................... 18 2.2.2 Infection of cell lines with wAlbB, w Mel and wMelPop Wolbachia................. 19 2.3 Wolbachia assays ....................................................................... 19 2.3.1 Detection and quantification .......................................................................... 19 2.3.2 Assay to visualise Wolbachia infection dynamics??......................................... 20 2.3.3 Wolbachia isolation from HIE-18 cells for microinjection ................................ 20 2.4 Transinfection of BF with Wolbachia ........................................... 20 2.4.1 Microinjection of eggs .................................................................................... 20 2.4.2 Microinjection of pupae ................................................................................. 21 2.4.3 Microinjection of adults .................................................................................... 21 Page 2 of 45 B.AHE.0242 – Wolbachia control of buffalo fly 2.5 Assessment of fitness effects ...................................................... 21 2.5.1 Survival ............................................................................................................. 21 2.5.2 Adult emergence rate ..................................................................................... 22 2.5.3 Egg production ................................................................................................. 22 3. Results and Discussion ....................................................................... 22 3.1.1 Haematobia cell line establishment ........................................... 22 3.1.2 Species confirmation using PCR and chromosomal analysis ........ 22 3.1.3 Transinfection of cell line establishment and maintenance ......... 23 3.1.3.1 Wolbachia replication in HIE‐18 cell lines........................................................ 23 3.2 Microinjection ............................................................................ 24 3.2.1 Microinjection of eggs .................................................................................... 24 3.2.2 Microinjection of adults .................................................................................. 25 3.2.2.1 Growth of Wolbachia in injected females ....................................................... 26 3.2.2.2 Distribution of Wolbachia infection in injected female BF ............................. 26 3.2.2.3 Survival of Wolbachia infected females:........................................................ 28 3.3 Microinjection of pupae ................................................................................. 29 3.3.1 Wolbachia growth and tissue distribution in pupal –infected buffalo flies ...... 29 3.3.1 Effect of pupal injection with Wolbachia on survival of emergent buffalo flies 31 3.3.2 Effect of pupal injection with Wolbachia on adult emergence ........................ 32 3.3.4 Effect of pupal injection with Wolbachia on egg production by buffalo flies ... 32 4 Conclusion ......................................................................................... 34 4.1 Key findings ................................................................................ 36 4.2 Benefits to industry .................................................................... 36 5 Future research and recommendations ............................................... 37 6 References ......................................................................................... 37 7 Appendix ................................................... Error! Bookmark not defined. 7.1 Heading.............................................. Error! Bookmark not defined. 7.1.1 Sub heading ........................................................ Error! Bookmark not defined. Page 3 of 45 B.AHE.0242 – Wolbachia control of buffalo fly Abstract Buffalo flies (BF) are the second most costly disease issue in northern Australian cattle causing production loss and welfare impacts and are steadily expanding their range into southern production areas. Modelling indicates the potential for the establishment of sustaining populations of BF as far south as South Australia and south east Western Australia by 2030 under the effects of climate change. Wolbachia are maternally transmitted intracellular bacteria of widespread interest in the development of novel controls for insect pests and disease vectors. Wolbachia could be used in area wide control of BF to collapse overwintering populations, to prevent the spread of Stephanofilaria nematodes associated with the development of buffalo fly lesions or potentially for eradication of regional populations or incursion foci. We have shown that BF is a competent host for Wolbachia and that the three Wolbachia strains tested, originally isolated from mosquitoes and Drosophila, successfully infected and grew in transinfected BF. Furthermore we have developed pupal and adult microinjection techniques that can produce ovarian infections in female BF, essential for spread of the bacteria through BF populations, and shown that Wolbachia caused a reduction in lifespan, reduced fecundity and egg hatch and reduced and delayed pupal emergence in infected flies. We are now well placed to produce Wolbachia-transinfected BF lines suitable for use in future biocontrol programs for BF population suppression or local eradication. Page 4 of 45 B.AHE.0242 – Wolbachia control of buffalo fly Executive summary Buffalo flies (BF), Haematobia irritans exigua, are the second most costly disease issue in northern Australian cattle, causing production loss and welfare impacts, and are rapidly spreading southwards. Modelling indicates the potential for sustaining populations of BF as far south as areas of South Australia and south east Western Australia by 2030 under the effects of climate change. Currently, control methods for BF are almost exclusively applied on a herd-by-herd basis. In the past legislatively supported regulatory programmes based on compulsory chemical treatments and regulated cattle movements were implemented to prevent southerly incursion but these were not effective and were eventually abandoned. Modelling studies indicate the relative inefficiency of herd-by-herd approaches and suggest that application of control techniques on an area-wide basis, targeting the entire population, can be more efficient. This would appear