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Research-Information.Bristol.Ac.Uk This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Abolins, Stephen Ralph Title: Biological control using entomopathogenic fungi : an evaluation against the mites Psoroptes ovis (Hering) and Acarus siro L. General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Biological control using entomopathogenic fungi: an evaluation against the mites Psoroptes ovis (Hering) and Acartis siro L. by Stephen Ralph Abolins A disseilation submilted to the University ol'Bristol in accordance Nvith the rcquircnicnts of the degree of Doctor ofilhilosophy in the Faculty of Scicilce. April, 2008 Word count: 36,138 Biological control using entomopathogenic fungi: an evaluation against the mites Psoroptesovis (Hering) and Acarus siro L. by Stephen Ralph Abolins A dissertationsubmitted to the Universityof Bristol in accordancewith the requirements of the degreeof Doctor of Philosophy in the Faculty of Science. April, 2008 Word count: 36,138 ABSTRACT The reductionin availableinsecticides for the treatmentof sheepscab, has prompted interest in the development of non-insecticidal alternatives such as the use of entornopathogenicfungi for the control of Psoroptesovis. The work presentedhere demonstratesthat undercontrolled conditions it is possibleto inducelethal infectionsin A ovis, in vivo, usingthe fungalpathogens Metarhizium anisopliae and Beauveria bassiana. Initial in vivo trials, in which conidiaof M anisopliaewere applieddirectly to the lesionsof sheepexperimentally infested with scab,resulted in few or no infectedmites being recovered. This may have beendue to the inability of the conidia to penetratethe fleece. Undermore controlled conditions, batches of 20 adult femalePsoroptes mites were confined in 25 mm diameterarenas glued to the skin of 6 sheep,with 6 arenasper sheep.Some mites wereexposed to the fungi for 48 h in vitro prior to beingplaced on the hostwhile othermites had no prior exposureand were placeddirectly onto the skin of a treatedhost. For both speciesof fungi, the highestlevels of infectionwere achieved when the miteswere exposed for 48 h in vitro prior to 48 h exposurein vivo; infectionrates of 100%for B. bassianaand 92.9% (±3.34) for M. anisoplide were observed. The lowest levels of infection were observedwith the conidia in diatomaceousearth; even with prior exposurein vitro only 20.8%(±6.74) became infected with M. anisopliaeand 88.6% (±4.58) with A bassiana. Further arena-basedin vivo trials showedthat formulation had a significant but highly variable effect on pathogenicitymaking it difficult to discerngeneral patterns. It seemslikely that formulationwill be an importantfactor in the developmentfor a treatment of sheepscab so that viable fungal conidiacan be deliveredto the skin surface,where the mitesare present. Due to the difficulty of studyingP. ovis in vivo and in vitro, a modelsystem using Acarussiro, was designed.The populationdynamics of the mite in the absenceof a fungal pathogenwere first studied over an 80 day period, under conditionsof restrictedfood availability (0.3g; wholemealflour: yeast, 3: 1, w/w) at 20 OCand 70% r.h.. The populations initially consistedof 50 adult femaleand 50 adult malemites and repeatedsampling showed that it grew to reach a peak of 10080mites, 54 days post-establishment,after which it declinedas a result of food shortage. A Leslie matrix model constructedfrom the life- history data gatheredin the presentstudy, proved to be too simpleto accuratelymodel the dynamicsof a populationthat hada high degreeof intrinsicvariability. It was also shownthat A. siro is susceptibleto infection by M. anisoplideand A bassiana,with 92% (-± 7) and 99% (± 2) of mites infectedwhen exposedto unformulated conidia for 1 min. Infected mites had lower LT50values of 3-5 days comparedto the controls,9.5 days(±2.8). Horizontaltransmission was possiblebetween cadavers infected with M anisopliaeand live, na1vemites; 30% (± 10) of live, naYvemites becameinfected. Transmissionalso occurred between live, exposedmites and live, naYvemites; 43% (± 12)of live, na*fvemites becameinfected. Transmissionwas shown to be possiblein small but densepopulations of naYvemites when 20 infectedcadavers were introducedto populations of 100adults (1: 1 sexratio). However,there was no evidenceof transmissionor persistence of the fungal pathogenin largerbut initially lessdense populations. It may be that a density thresholdexists, below which the fungal pathogencannot persist and transmit within a populationof mites. This study showsthat fungal pathogenshave clear potentialto be valuableagents contributing to the control of sheepscab mites but many problemsmust be overcome. Further work needs to be conductedto identify suitable formulations and application methodsand to assessthe transmissionof the fungal pathogensthrough populationsof Psoroplesmites in vivo. Il ACKNOWLEDGEMENTS, First and foremost I would like to thank ProfessorRichard Wall for his help, patience,encouragement and guidance throughout my Ph.D. I am grateful to staff at the CSL including Prof. Mike Taylor, Vicky Jackson, BhushyThind andLouise Ford for their helpwith the animaltrials andsupply of mites. I am also grateful to Dr Dave Moore, Dr Belinda Luke and Steve Edgington at CABI for the supply of fungal conidia and helpful comments. I am also very thankful for the help of Patricia Wells for spending a summer in the laboratory,counting a ridiculouslylarge number of mites. Thanks also go to the staff in the biological sciences animal facility for the maintenanceof the rabbits. I am also very grateful for the support from the members of the Veterinary Parasitology and Ecology group: Jenny Broughan, Kat Pegler, Betty Bisdorff, Laura Briggs, Jan Van Dijk, Luke Dickson, Ryan Jefferies, JacquelineLusat but especially Hannah Rose for providing accommodationand lots of tea. I would also like to thank RishaPatel and Jon Flandersfor letting me a room and cooking somegreat curries(Risha, not Jon!), as well as my many other friends in Bristol who mademy time herevery enjoyable. I am gratefulfor the fundingreceived from DEFRA. Specialthanks go to my parents,Linda and GunarAbolins for their constanthelp and support. III AUTHOR'S DECLARATION I declarethat the work in this dissertationwas carried out in accordancewith the Regulationsof the University of Bristol. The work is original exceptwhere indicatedby specialreference in the text and no part of the dissertationhas been submitted for any other degree. Any views expressed in the dissertation are those of the author and in no way representthose of the University of Bristol. The dissertationhas not been presentedto any other University for examination eitherin the UnitedKingdom or overseas. Signed: Date: 1 J/7/0 jr IV CONTENTS CHAPTER I Introduction 1 1.1Fungal control usingMelarhizium anisoplide and Beauveria bassiana ........................................................................................................... 1 1.2 Fungal control of ectoparasites ...................................................................... 1.3 Fungal 4 control of mite species........................................................................ 1.4 Mechanisms infection 6 of .............................................................................. 1.5 Aims ...................................................................................................... 10 CHAPTER 2 General Materials and Methods 2.1 Culture Psoroptes 10 of ovis.............................................................................. 2.2 Collection identification Psoroptes 10 and of ovis................................................... 2.3 Culture Acarus 13 of siro .................................................................................. 2.4 Collection identification 15 and of Acarus siro mites................................................ 2.5 Incubation 15 of mites..................................................................................... 2.6 Culture fungal 19 and collection of conidia ............................................................ 2.7 Formulating 19 conidial suspensions.......... 0............ 0.................. *. *......... *............ 2.8 Inoculation 20 of mites with conidia ................................................................... 2.9 Determination the death 20 of point of of mites.....................................................
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