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......

CHAPTER 3 Control of Psoroptesovis in vitro and in vivo using entomopathogenic fungi 22

3.1 Introduction 22 ...... 3.1.1 Sheep 22 scab......

V 24 3.1.2 Life-history of Psoroptesovis ...... 3.1.3 The Psoroptes 25 classification of ovis ......

3.1.4 Prevalence distribution 27 and of scab...... 3.1.5 Control 28 of scab...... 3.1.6 Entomopathogenic fungi 31 to control scab...... 3.2 Methods Results 34 and ......

3.2.1 Analysis 34 ......

3.2.2 The Pathogenicity of Metarhizium anisopliae against Psoroptes ovis

in (Trial 1) 34 vivo ...... in 39 3.2.3 The pathogenicity of two strains of Metarhizium anisopliae vitro ......

3.2.4 Treatment of sheep infected with sheep scab with Metarhizium

(Trial 2) 42 anisopliae ......

3.2.5 The pathogenicity of Metarhizium anisopliae to Psoroptes ovis

in in 45 vitro the presence of sheep skin ......

3.2.6 The pathogenicity of five high temperature tolerant strains of

Psoroptes 48 Metarhiziulm anisopliae against ovis ......

3.2.7 The pathogenicity of Beauveria bassiana against Psoroptes ovis

in 49 vitro ......

3.2.8 Control of Psoroptes ovis using entornopathogenic fungi in vivo

(Trial 3) 55 ...... bassiana 3.2.9 The pathogenicity of Metarhizium anisopliae and Beauveria

in 62 against Psoroptes ovis various formulations in vitro ...... bassiana 3.2.10 The pathogenicity of Metarhizium anisopliae and Beauveria

in formulations in (Trial 4) 67 various vivo ......

3.3 Discussion 73 ......

vi CHAPTER 4 Life-history and population dynamics of Acarus siro 81

4.1 Introduction Acarus 81 to siro ...... 4.1.1 Life-cycle 81 ...... 4.1.2 Effects infestation 83 of ...... 4.2 Materials Methods 84 and ...... 4.2.1 Longevity, development fecundity Acarus 84 and of siro ...... 4.2.2 The dynamics Acarus 86 population of siro ...... 4.2.3 Modelling dynamics 89 the population ofAcarus siro ...... 4.2.4 Analysis 92 ...... 4.3 Results 92 ...... 4.3.1 Longevity, development fecundity Acarus 92 and of siro ...... 4.3.2 The dynamics Acarus 98 population of siro ......

4.3.3Comparison of the modelledand observed population dynamics ofAcarus siro...... 98

4.4 Discussion 110 ......

CHAPTER 5 Effects of entomopathogenic fungi on Acarus siro 117

5.1 Introduction 117 ...... 5.2 Materials Methods 118 and ...... Pathogenicity Metarhizium bassiana 118 5.2.1 of anisopliaeand Beauverla ...... 5.2.2 Horizontal transmission fungal infection 119 of ...... 5.2.3 Effect fungal dynamics Acarus 122 of a pathogenon the population of siro ...... 5.2.4 Analysis 122 ...... 5.3 Results 123 ...... 5.3.1 Pathogenicity Metarhizium basslana 123 of anisopliaeand Beauveria ......

Vil 5.3.2 Horizontal fungal infection 127 transmissionof ...... dynamics Acarus 131 5.3.3 Effect of a fungal pathogenon the population of siro ...... 5.4 Discussion 139 ......

CHAPTER 6 Discussion 143

REFERENCES 157

APPENDIX

Abolins, S., Thind, B., Jackson,V-, Luke, B., Moore, D., Wall, R. and Taylor, M. A. (2007).

Control of the sheepscab mite, Psoroptesovis in vivo and in vitro using fungal pathogens.

VeterinaryParasitology 148: 310 - 317.

Vill ChapterI -Introduction

CHAPTER1

INTRODUCTION

Despite the ongoing health,welfare and economicneed to control parasitesof veterinary and medical importancethere is increasingpressure to reducethe useof many of the insecticides that have formed the primary means of effecting control in the U.K. over the last 50 years

(Kirkwood and Quick, 1981,1982; O'Brien 1999). The reasons for this include the development of resistance, fears over safety of the chemicals to humans and non-targct organismsand concernsover damageto the environment(Stephens et al., 1995; Syngeet al.,

1995; Clark et al., 1996; Cooke et al., 2004). This has lead to calls for increasedresearch into the developmentof alternative treatment methods(Wall, 2007). One such alternative for the control of arthropodpests is the use of entornopathogenicfungi.

1.1 Fungal control using Metarhizium anisoplide and Beauveria bassiana

Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina:Hyphomycetes) and

Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) are two of the most commonly used and researchedfungi (Samishand Rehacek,1999; Inglis et al., 2001; Brooks,

2004; Scholte et al., 2005; Ansari et al., 2007). These species provide good candidatesas control agentsbecause of their wide geographicalspread and host rangeas well as their ability to germinate at relatively low humidity levels (Zimmerman, 1993; Samish and Rehacek, 1999).

Both of these fungal pathogenshave been proved effective against many insect pestsof crops and in certain cases have been made commercially available. One commercially available product containingM. anisopliae is BioGreen", a product marketedas a control agentof the red-

I ChapterI- Introduction

headedcockchafer, Adoryphorus coulon! (Burmeister),in Australia (Inglis et al., 2001). When

BioGreen' was applied to the soil of pastureland there was a 58% reduction in the numberof

pupae, 27 weeks after application (Rath et al., 1995). The two subsequentgenerations after

treatmenthad 68% and 65% fewer larvaethan the controls indicating the long-termeffects of the

fungal pathogen(Rath et al., 1995).

Another commercially available product containing M. anisopliae is Green Muscle"

(Lubilosa, 2004). This product is marketedfor the control of locustsand grasshoppersin regions

of Africa and Australia. A study, conductedbetween November 1998 and March 1999,treated

areasof land infestedwith Locustamigratoria L. nymphsin Australia with an aerial sprayof M

anisopliae (Hunter et al., 1999). At a dose of 3x 1012conidia ha7lthere was less than 10%

survival of treated nymphs (Hunter et al., 1999). Another trial found that approximately50

droplets per CM2of aIX 1012conidia per litre suspensionof Metarhizium flavoviride Gams

Rozsypal were required to achieve 90% mortality of the desert locust Schistocercagregaria

(ForskAl) within 10 days (Bateman et al., 1998). The same study also observedthat locusts

releasedinto an area that had already been treated with the mycoinsecticidebecame infected.

The time taken to achieve 50% mortality was quicker in the groups exposeddirectly to the mycoinsecticidebut there was no significant difference in the averagesurvival time of infected locustsbetween either of the groups(Bateman et al., 1998).

Entornopathogenicfungi have also been tested againstpests of stored-products. When the beetlesRhyzopertha dominica (F.), Sitophilus oryzae (L. ) and Tribolium confusumdu Val were exposed to wheat treated with unformulated, dry conidia of M. anisopliae, mortality increasedwith increasingconcentration (Kavallieratos et al., 2006). After 14 days of exposure at a concentrationof 8x 1010conidia kg" of wheat, mortality levels of 74.6%, 82.3% and 74.4% were achieved for adults of R dominica, S oryzae and T confusum respectively. Control mortality was 6%, 3% and 4.5% respectively (Kavallieratos et al., 2006). The larvae of T confusumare also susceptibleto M anisopliae (Michalaki et aL, 2006). When the larvae were

2 ChapterI- Introduction

exposedto wheat treatedwith dry conidia of M. anisopliaeat a concentrationof 8x 1010conidia

kg"', mortality of over 50% was achieved(Michalaki et al., 2006). Another study using M.

anisopliae mixed with oven ash (burned paper sheets)showed that when applied as a curative

treatment under controlled conditions, mortality levels of 86.7% can be obtained against S.

oryzae(Batta, 2004).

1.2 Fungal control of ectoparasites

As yet there are no commercially available fungal pathogens for the control of

ectoparasites. However, previous studies on a range of parasiteshave shown that there is a

strongpotential for the use of entornopathogenicfungi for the control of ectoparasites.

Metarhizium anisopliae has beenshown to infect and kill many different speciesof tick.

Rhipicephalusappendiculatus Neumann and Amblyommavarlegatwn Fabricius kept on rabbit

earswere shown to be susceptibleto infectionby M. anisopliaebut with relativelylow mortality levels of 34% and 37% respectively(Kaaya et al., 1996). In the samestudy (Kaayaet al., 1996), aIx 10' conidia ml" suspensionof A bassianawas responsiblefor a 34% mortality of adult R. appendiculatus,although it failed to kill any A. variegatum. However, R appendiculatusthat were naturally present on zebu cattle in the field had a much higher level of mortality; 83% death when treated with M. anisopliae and 77% when treated with B. basslana(Kaaya et al.,

1996). Another study recorded90% mortality of adult female Boophilus annulatus Say, 7 days post-inoculation(Gindin et al., 2001). Larval mortality was 100%after 6 days.

A study testing the pathogenicity of 12 different isolates of M anisopliae against

Boophilus microplus (Canestrini), recorded 100% mortality 14 days post-inoculation for 10 of the 12 isolates (Frazzon, 2000). However not all ticks are so susceptible to infection. In

Amblyomma maculawn Koch, mortality of 60% of adult females exposed to M. anisopliae was observed but only 15% mortality in exposed Amblyomma americanum L. females (Kirkland,

3 ChapterI -Introduction

2004). A similar study found that while one strain of M. anisopliae was able to inducemortality

levels of 100% in B. annulatusand RhIpIcephalussanguineus (Latreille), only 5% mortality was

recordedfor Hyalommaexcavatum (Koch) (Gindin, 2002).

The cattle louse,Bovicola bovis (L. ) hasbeen shown to be susceptibleto infection by M.

anisopliae. When maintained in arenas on the skin surface of cattle, 73% of lice became

infected when exposedto a concentrationof Ix 10' conidia ml" of M anisopliae in Tween 80

(Briggs et d, 2006).

Metarhizium anisopliae has also beenshown to be pathogenicto the mosquitovectors of

malaria (Scholte et al., 2005,2007). Field trials using black, cloth targetsimpregnated with M

anisopliae and hung in traditional housesin Tanzaniafound that out of 580 female Anopheles

gambiae Giles sensu stricto collected in the houses, 132 were infected with the pathogen

(Scholte et al., 2005). The Uso values were significantly lower for infected mosquitoes;3.5

days for infected femalescompared to 9.3 daysfor uninfectedfemales (Scholte et al., 2005). In

vitro bioassayshad similar results; 87% of Aedes aegypti (L. ) and 89% of Aedes albopictus

(Skuse)became infected with M anisopliae when exposedto impregnatedfilter papersfor 24 h

(Scholteet al., 2007). This study concludedthat this level of infection could significantly reduce

the transmissionof plasmodium.and thereforewith careful application, fungal pathogenscould

be usedin the control of malaria.

1.3 Fungal control of mite species

The Acari is a large and diversesub-class of the class Arachnida,including many

speciesof parasiteand pest of veterinary, agricultural and economicsignificance. The ability to control these arthropods can be of vital importance to the viability of agriculture, food and farming industries.

4 ChapterI -Introduction

A study testing the pathogenicityof various strains of B, basslana,M. anisopliae and

Paecilomycesfumosoroseus (Wise) against Oligonychusyothersi (McGregor), a pest of tea

crops, found that strainsof B. bassianawere the most pathogenicwith mortalitiesof over 70% at

3 to 4 days post-inoculation (Oliviera et al., 2004). A study testing the pathogenicityof M.

anisopliae on the two-spotted spider mite, Tetranychusutricae Koch found that levels of

infection varied from 3.8% to 29.4% dependingon the isolate used(Chandler, 2005). Mortality

levels were also variable ranging from 6.7% to 43.2% (Chandler,2005). When B. basslanawas

tested against the green mite, Mononychellustanajoa (Bondar) mortality levels of 9% to 91%

were found for B. bassiana,depending on the isolate used (Barreto et al., 2004). The mortality

levels when M. anisopliae was usedranged from 8% to 45%. LT50values rangedfrom 4.2 to 17

days for B. basslanaand 8.6 to 19.8 days for M anisopliae (Barreto et al., 2004). Beauverla

basslanahas also been shown to be pathogenicto the citrus rust mite, Phyllocoptruta oleivora

Ashmead(Alves et al., 2005). A mortality rate of 91% was recordedafter 5 days exposureto 2

ml of a IX108conidia ml" of B. basslanain 0.2% Tween 80 (Alves et al., 2005). The meanLT50

at this concentrationwas 2.74 days (Alves et al., 2005).

Hetarhizium anisopliae has shown great potential as a control agent of Parroa destructorAnderson and Truemanin beehives. In laboratorybioassays, three isolates of M anisopliaewere foundto kill 100%of V destructorwithin 7 daysat a concentrationof lxlO' conidiaml"' andone isolate killed 97%at just IX106conidia ml" (Shawet al., 2002). Fieldtrials recordedinfected mites up to 42 dayspost inoculation even though peak mortality occurred 3 to

4 daysafter inoculation(Kanga et aL, 2003). It was concludedthat M. anisopliaewas just as effectiveas the tau-fluvalinate acaricide, Apistan.

5 ChapterI- Introduction

1.4 Mechanisms of infection

The conidia of entornopathogenicfungi such as B. bassianaand M anisopliae,stick to

the surface of a host by random, non-specific, hydrophobic interactions (Charnley, 1989;

Boucias et al., 1991). The conidia are known as dry sporesas they do not have a mucoid layer

surrounding them. Instead they are covered by hydrophobic rodlets that form interwoven

bundles acrossthe surface of the conidium (Charnley, 1989; Boucias et al., 1991). Once the

conidia have attachedto the surfaceof the host, the fungus germinatesand penetratesthe cuticle

(Inglis et al., 2001). This is achievedby the productionof germ tubes or apressoria(Boucias et

al., 1991). The stimulation to produce germ tubes or apressoriavaries with each species. B.

bassianaand M. anisopliae both have wide host rangesand therefore respondto non-specific stimuli. The germination of thesetwo fungi is thought to be stimulatedby non-specificsources of carbon and/or nitrogen as would be found in amino acids, proteins, carbohydratesand some lipids and requiresan exogenousnutrient sourcefor germ tube formation (StIeger et al., 1986;

Charnley, 1989). From the apressoriuma penetrationpeg grows (Clarkson and Charriley, 1996;

Zacharuk, 1970a,b, c) that adheresto the cuticle by secretingmucus similar to that secretedby

Nomuraea r1leyi (Farlow) which has been identified as a mixture of P-1,3 glucans(StIeger et al., 1986;Charnley, 1989). The penetrationof the host cuticle relies upon mechanicalforce and enzymatic degradationbut the relative contribution of either method is not yet known. It is probably dependanton many variablesof the host and pathogeninteraction (Charnley, 1989).

The first barrier that must be penetratedis the epicuticle and wax layer. Penetratingthe wax layer might be achievedthrough the activitiesof lipases,and esterases that are secretedby

M anisopliae and B. basslana(Chamley, 1989; Zacharuk, 1970). However the exact mechanismis still unclearas lipids and estersare not the only componentsof the wax layer

(Charnley,1989). Degradation of the cuticlemay also rely uponthe actionof enzymes(StIeger

1995). The fungalpathogens produce proteases, lipases and chitin deacetylasethat canconvert

6 ChapterI -Introduction

chitin into chitosan,a glucosaminepolymer (Barreto et al., 2004; Nahar et al., 2004). St. Leger

(1986) purified endoproteaseand chitinasefrom culturesof M. anisopliae. Whenthese enzymes

were applied to locust cuticle they were found not only to degradethe cuticle but also provided

possiblesources of nutrient to the growing fungus from the breakdownproducts in the form of

amino sugars. It has been suggestedthat the outer epicuticle is probably breachedthrough

physical force whereasthe inner epicuticle may be broken down by a mixture of endoproteases

producedby the apressorium(Charnley, 1989;Arruda et al., 2005).

Once the epicuticle has been breached,the fungus has to penetratethe procuticle. The

procuticle is the largest part of the cuticle and is constructedfrom chitin fibrils embeddedin a

protein matrix (Charnley, 1989). The degreeof sclerotizationis an important factor influencing

the ability of the fungus to cross the procuticle. More heavily sclerotizedprocuticles are much

more difficult to traverseand may be impassable,as sclerotizedcuticle is comparativelyresistant

to the enzymesproduced during penetrationand also provides a strongerphysical barrier to the

pathogen(StIeger et al., 1986; Chamley, 1989). Heavily sclerotized arthropodsare usually penetratedat the joints of appendages.Hosts with a lower degreeof sclerotizationprovide an easierroute for penetrationthrough the cuticle (Charnley, 1989).

Once the cuticle has been breachedthe morphology of the fungus changesas hyphal bodies are producedwhich then circulate in the haemolymph(Gillespie and Moorhouse,1989) and proliferatejust before the host dies (Ferron, 1981). The hyphaeof M. anisopliae havebeen observed invading the fat body, muscles and gut walls of infected tsetse flies (Glossina morsitansmorsitans Westwood) (Kaaya, 1991). The hyphal bodies of M. anisopliae havebeen found in the haemolymphof S. gregaria 2-3 days after infection (Gillespie et A, 2000). Death of the arthropod usually occurs 3-14 days after infection due to a combination of mechanical damageof tissue,nutrient depletion and toxicosis (Gillespie and Moorhouse,1989).

Some entornopathogenicfungi are able to producetoxins. Thesetoxins may be one of the main causesof host death (Roberts, 1981). Metarhizium anisopliae producesa family of

7 ChapterI- Introduction

cyclic peptide toxins known as destruxins(Clarkson and Chamley 1996; Liu et al., 2004). The

destruxinsare cyclic hexadepsipeptidesconsisting of 5 amino acids and an a-hydroxyl acid (Liu

et al., 2004). The exactmechanisms by which the destruxinskill the hostare not yet known although there is evidence that the inhibition of the hosts immune response within the haernolymphmay be crucial (Huxham et A, 1989;Kershaw et aL, 1999).

Once the host has died, the fungus colonisesthe body and the hyphaegrow through the cuticle and becomeapparent on the surfaceof the host before sporulationtakes place (Gillespie and Moorhouse, 1989). If the outside conditions are not conducive to sporulation the entomogenousfungi can remain inside the host for up to several months (Gillespie and

Moorhouse,1989).

1.5 Aims

The aims of the work describedhere were to examine the potential of two speciesof fungal pathogento act as control agentsfor sheepscab mites (Psoroptesovis (Hering)) (Acari:

Psoroptidae) and also to investigate the effect that fungal pathogens could have on the population dynamicsof the mite speciesAcarus siro L. (Acari: Acaridae). Firstly, following on from previous in vitro studies,the ability of M anisopliae and B. bassianato infect Psoroptes mites on a host was examined(Chapter 3). The effect of skin and fleece and various different formulations were also investigatedboth in vitro and then in vivo (Chapter 3). Subsequently, studieswere undertakento consider transmissionof entornopathogenic,fungi in populationsof mites. However, becauseP. ovis is an obligate ectoparasiteand therefore extremely difficult to study in vivo and in vitro, a model species,A. siro is used. To considerthe effects of a fungal pathogenon population dynamics,populations of A. siro were establishedand maintainedunder constant conditions. Data on the life-history of A. siro, was gatheredand usedto construct a simple model against which populations of A. siro could be compared (Chapter 4). The

8 ChapterI -Introduction pathogenicityof M. anisopliae againstA. s1rowas testedwith direct exposurebetween mites and conidia (Chapter 5). With the pathogenicityestablished, the ability of M. anisopliaeinfection to transmit from infectedcadavers to nawe, live mites was examinedusing small-scale,high densitypopulations and large-scale,low densitypopulations (Chapter 5).

9 Chapter2- GeneralMaterials and Methods

CHAPTER 2

GENERAL MATERIALS AND METHODS

2.1 Culture of Psoroptes ovis

A low level infestation of Psoroptes mites was maintained in the ears of two New

Zealand white rabbits maintained at the University of Bristol, UK under Home Off ice

Licencenumber PPL 30/1636. The infestationof miteswas maintained at low levelsto keep

the pathology and welfare problems to a minimum. Infestation was initiated by placing a

piece of scab, containing mites, inside the ear of a rabbit and then sealing the ear with zinc

oxide adhesive tape (Boots the Chemists Ltd. Nottingham, UK) for 24 h after inoculation.

As many attempts as necessaryat inoculation were made (approximately once a week) until

signs of infestation could be seen. These signs were manifested in scabsthat were visible on

the inner surface of the earswhich resulted from hardening of serousexudates.

2.2 Collection and identification of Psoroptesovis

Scabswere removedfrom the earsof rabbitsusing blunt-nosedforceps. Scabwas

removed as often as required (usually twice per week) to keep the infestation in the ears of the rabbits at a low level. The scab and mites that had been removed were placed into clean,

scalable, plastic containers, 40 mrn in diameter and 60 mm high. Once the mites and scab had been removed from the host, the mites migrated from the scab and crawled up the sides of the container. A fine grade paintbrush was used to pick mites off the sides of the container. These mites were then placed onto a 50 mm diameter petri dish that was floated on 70% ethanol inside a larger 90 mm diameter petri dish. The ethanol collected and killed any mites that crawled out of the inner petri dish.

10 Chapter2- GeneralMaterials and Methods

The life cycle stages of the mites were identified under a binocular microscope according to the descriptions given by Sanderset at (2000). The adults are roughly oval in shape and white in colour, although they can become dark brown if eosinophils are consumed (Fig. 2.1 A, B) (Rafferty and Gray, 1987). The juvenile stagesremain white.

Adult femalesare morphologicallythe largeststage and are approximately0.46 - 0.57 mm in length(Fig 2.1 A) (Sandersel al, 2000). The adultmales are smaller, approximately 0.36

- 0.44 mm, and not as round as the females(Fig. 2.1 B) (Sandersel al, 2000). Sexual dimorphismcan be observedfrom the protonymphstage onwards (Sweatman, 1958; Sanders et al., 2000). Femaleand male nymphs can be distinguishedfrom eachother by the presence of dorsoposteriortubercles on the femalein both nymphalstages (Fig. 2.1 C-F) (Sanderset al., 2000). Adults can be easilysexed by the differencein overall sizebut alsoby a number of otherfeatures. Males have a jointed pretarsiand pulvilli on the first threepairs of legsbut not on the fourth pair, which aremuch shorter (Fig. 2.1 B) (Sanderset al, 2000). Malesalso possessa pair of adanalsuckers and pairedpodosomal lobes. Femaleshave two pairs of metapodosomalsctae on the ventralsurface and a U-shapedvulva on the propodosoma(Fig.

2.1 A) (Sanderset al., 2000;Wall, 2007).

Protonymphscan be distinguishedfrom tritonymphsby the presenceof threepairs of metapodosomalsetae only, ratherthan the five pairsof setaein the tritonymph(Sanders et al., 2007). Tritonymphsare also larger than the protonymphs. Larvae show no sexual dimorphismbut can be identifiedfrom the otherstages due to the presenceof only 3 pairsof legs(Fig. 2.1 G) (Sanderset al., 2000).

11 Chapter2- GeneralMaterials and Methods

Fig. 2.1. The featuresused to identify the different lifecycle stagesof Psoroptesovis. A=

ventralview of an adult female;B= dorsalview of an adult male;C andD= dorsalview of

a femaleprotonymph and tritonymph, respectively; E andF= dorsalview of a male

protonymphand tritonymph respectively; G= ventralview of a larva(Sanders et aL, 2000).

Bar= I OOgm.

A -4 B

log I

vut. 11(ropm

oftumor

D

b

G

Is

12 Chapter2- GeneralMaterials and Methods

2.3 Culture of Acarus siro

Acarus siro mites (strain 9258/2) were obtained, in 1992, from a co-operativc feedmill, Latham, U. K., and reared under laboratory conditions at the Central Science

Laboratory (CSQ, SandHutton, UK. A sub-colony was obtained in 2004 and rearedat the

University of Bristol. For this, the mites were kept in 50 ml conical flasks (Fig. 2.2) (Fisher

Scientific UK Ltd., Loughborough, U. K. ) and fed on a mixture of wholemeal flour

(Sainsbury's SupermarketsLtd., London, U. K. ) and yeast extract (Sigma-Aldrich, Dorset,

U. K. ) in a 3: 1 (w/w) ratio. The flasks were sealed with non-absorbcnt cotton wool. All colonies were maintained at 20 *C and 70% relative humidity, in a controlled climate incubator.

When the density of mites within the flasks was deemedto be too high, i. e. more mites than food was visible when the colony was inspectedunder a binocularmicroscope, the colony was split into severalnew flasks,each containing fresh food. This wasachieved by gently pouringa smallamount of the colonyfrom the flask with the high mite densityinto a new flask with freshfood. Eachnew flask receivedmites from severaldifferent flasks so that no genetic segregationwould occur between colonies. Latex gloves (Semperit

TechnischeProdukte GmbH & Co Vienna, Austria) were worn and sprayedwith 70% ethanolbefore handling mites or food to preventcontamination of the food or mite colonies with fungalpathogens.

13 Chapter2 GeneralMaterials and Methods

Fig. 2.2. Conical flasks containing Acarus siro mites feeding on wholenical flour and yeast extract in a 3: 1 ratio (w/w). Flaskswere sealedwith non absorbentcotton wool.

AWNW&

14 Chapter2- General Materials and Methods

2.4 Collection and identification of Acarus siro mites

Mites were collected from colonies by gently brushing the sides of the conical flask with a fine grade paintbrush to pick up the mites. These mites on the paintbrush were then depositedonto a 50 mm diameter petri dish (Bibby Sterilin, Stone, UK) that was floated on

70% ethanol inside a larger 90 mm diameter petri dish. The ethanol collected and killed any mites that crawled out of the inner petri dish. The life cycle stages of the mites were identified under a binocular microscope and could be picked up with a paint brush. Adult females were identified by their relatively large size and round appearance compared to the other stages. Adult males were identified by their slender body in comparison to adult females and the presence of a spur on the underside of the trochanter of each fore leg.

Tritonymphs and protonymphs were identified by their relative sizes, with tritonymphs being bigger than protonymphs and the larvae were identified by the presence of only 3 pairs of legs rather than the 4 pairs seen in all other stages (Solomon, 1969). Sex was not determined for juvenile stages.

2.5 Incubation of mites

All Psoroptes mites were incubated in their respective treatment groups, in clean incubation chambers constructed from 6x 25 x 75 mm glass blocks (Fig. 2.3). Each block had a 20 mm diameter hole drilled through its centre. However, for Psoroptes mites the bottom of the block was sealed with a fine grade cotton cloth glued to the glass with epoxy resin to which 500 gl of sheepserum had beenadded. Mites were placed in the hole and the chamber was sealed using a glass microscope slide with a5 mm diameter hole drilled through its centre, which was also closed with cotton cloth. Another glass microscopeslide was placed on the underside of the chamber. The slides and glass block were held in place with bulldog clips at eachend of the chamber. The hole in the upper glass slide was to allow for regulation of the humidity of the air within the chamber. The chambersand mites were

15 Chapter2- GeneralMaterials and Methods

incubatedat 30 *C and 90% relativehumidity. A further 200 gI of sheepserum was added

every48 h until all miteshad died.

The cloth-basedchambers were usedto incubatePsoroptes mites so that more space

was available when conductingtrials involving sheepskinand fleece. The filter-based

chamberswere alsomore liable to damagewhen the sheepserum was added.

All A. siro mites used in experimentswere incubated in their respective treatment

groups in clean incubation chambersconstructed from 6x 25 x 75 mm glass blocks. Each

block had a 20 mm diameterhole drilled through its centre. A piece of filter paperwas placedover the hole andan indentationwas made. A glassmicroscope slide was placed over the filter paperand the block and slide wereclipped together with a bulldogclip at eachend

(Fig. 2.4). Approximately0.01 g of food was placedin the chamberbefore the additionof the mites. The chambersand miteswere then incubated at 20 'C and 70%r. h. until all mites had died.

16 Chapter 2 Oencral Materials and Mclhods

during hloassý'Ys- Fig. 2.3. F,xim-micntal chamber uscd to inctibatc Psoroples ovis initcs

For details seetext.

Position Of Clips Cotton

6mm

17 Chapter2- GeneralMaterials and Methods

Fig. 2.4. Experimental chamberused to incubateAcarus siro mites during bioassays. For detailssee text.

Position of clips Microscope slide

6 20 mm. mm. --' ftftftft 42.5 mm diameter filter 25 mm 75 mm paperwith indentationin the centre

18 Chapter2- GeneralMaterials and Methods

2.6 Culture and collection of fungal conidia

Both of the fungal pathogensused in this work, M anisopliaeand B. basslana,were

culturedusing the samemethod. The fungi werecultured on platesof PotatoDextrose Agar

(Sigma-Aldrich,Poole, UK) and Yeastextract (Sigma-Aldrich, Poole, UK) (PDAY). The

platesof PDAY were madeby mixing 39 g of PDA with 2g of yeastextract in II of

distilled water. The mixture was then autoclavedat 120'C for a freesteamtime of 15 min.

The liquid agar was then poured into the base of a 90 mm petri dish and left to set. The plates of PDAY were inoculated by pipetting 150 RI of conidia suspendedin silicone oil

(dimethylpolysiloxane hydrolyzate; Sigma-Aldrich, Poole, UK) onto the surface and spreadingthe inoculum over the plates with a sterile glass rod. The PDAY and fungi were then incubated at 25 OC. Conidia were collected from 10 to 14 day-old cultures by gently scraping the conidia off the agar plate using a sterile loop, into a clean, glass universal pot.

Small amounts of conidia of each isolate were stored in eppendorf tubes containing 2 ml of silicone oil and stored in an ultra low temperaturefreezer at - 80 T for future use.

2.7 Formulating conidial suspensions

Oncethe conidia had beencollected, they were readyto be either formulatedin an excipientor usedas unformulated,dry conidia. To makea conidial suspensionusing an oil- basedexcipient (silicone oil, vegetableoil, sunfloweroil or outpue) conidiawere added to 4 ml of the excipient. The concentrationwas calculatedusing an Improved Neubauer

Haernocytometer(Weber Scientific International, Middlesex, UK). The suspensionwas then dilutedto makea concentrationof Ix 109conidia ml-1. The suspensionswere then further dilutedwhen necessary by serialdilution.

Conidiasuspended in waterwith Tween80 (BDH ChemicalsLtd, Poole,UK) were initially suspendedin 4 ml of 0.05%Tween 80 andthen diluted to a concentrationof Ix 10' conidia ml-1 using 0.03% Tween 80. Serial dilutions were carried out using the lower

19 Chapter2- GeneralMaterials and Methods concentration of Tween 80. This lower concentration was used for further dilutions to reduceany toxic effects that Tween 80 may have on the conidia (Butt and Goettel, 1997).

Conidia that were formulated in Codacideo (vegetable oils and polyethoxylated esters;Microcide Ltd, Stanton,UK) werefirst suspendedin 0.4 ml of neatCodacide" before

3.6 ml of water was addedto make4 ml of 10%Codacideo. The conidial suspensionwas thenfurther diluted to the requiredconcentration using 10%Codacidet.

Formulations of conidia in diatomaceousearth were supplied by Dr Belinda Luke at

CABI, Ascot, UK. Concentrationswere calculated by mixing a known volume of conidia with a known volume of diatomaceousearth.

2.8 Inoculation of mites with conidia

When using conidia suspendedin liquid, mites (both A. siro and P. ovis) were inoculated by completely immersing them in 2 ml of suspensionfor I min in a 50 mm diameter Petri-dish lined with a clean filter paper. After I min, the mites were removed and placed onto a clean piece of filter paper for I min to allow excess fluid to run off before being transferredto clean incubation chambers(Fig. 2.3, Fig. 2.4).

When dry conidia were usedto inoculatemites, the appropriatevolume of conidia wasweighed out, suchthat miteswould be exposedto a similar absolutenumber of conidia as if exposedto 2 ml of a1x 108conidia ml-1suspension.

2.9 Determination of the point of death of mites

Death was determinedwhen therewas no movementfrom the mite when touched with a paintbrush. Where severaltreatments were compared,separate paint brusheswere usedfor eachof the treatments.The brusheswere washedin 70% ethanoland wiped dry betweeneach sample.Any dead mites were removedfrom the chambersand surface sterilised in 2% (w/v) sodium hypochlorite solution (Sigma-Aldrich, Dorset, UK) for 30 s and then washed in sterile water for 30 s to remove any conidia that had not penetratedthe

20 Chapter2- GeneralMaterials and Methods mite cuticle beforethe point of death. Eachindividual deadmite was then transferredto a damppiece of sterile filter paperat the baseof a well in a 96 well microtitreplate (Bibby

Sterilin, Stone,UK). The filter paperswere cut into discswith a diameterof 6 mm usinga hole punch and autoclavedbefore being placedinto the wells. The microtitreplates were then sealedwith ParafilmoM (PechineyPlastic Packaging, Chicago, USA) and incubatedat

30 "C. The plates were inspected for mites with fungal infections every day for 2 weeks following the observation of the first infected corpse. Fungal infections were recorded as presentwhen external hyphae could be observedprotruding through the cuticleof a mite.

Proportions of infected mites, as presentedin the results sections of Chapters3 and

5, were calculated by dividing the number of mites showing signs of infection within each group by the number of mites treated in that group.

LT50values were determined by the point at which half of the mites within a group had died.

21 Chapter3- Effects of fungal pathogenson Psoroptesmites

CHAPTER3

CONTROL OF PSOROPTES0 VIS IN VITRO AND IN

VIVO USING ENTOMOPATHOGENIC FUNGI

3.1 Introduction

3.LI Sheepscab

Sheep scab or psoroptic mange, is a diseaseof global importance, caused by the astigmatic mite Psoroptes ovis. The clinical signs of the diseaseinclude wool loss, weight loss, decreasedfeeding, intense pruritus, biting and scratching, epileptiform fitting and can lead to the death of the sheep(Downing, 1936a;Tarry 1974; Kirkwood, 1980). Not only are there serious welfare implications associatedwith the disease,there is also a large economic impact of sheep scab as a result of the reduction in weight gain, damagecaused to the skin and the cost of treatment. In a study in 1986, it was estimatedthat the diseasecould cost the

U. K. L600 million over a 30 year period (Kirkwood, 1986). The samestudy calculatedthat a

30% loss in weight gain would lead to a cost of flOOO for a flock of 100 sheep(Kirkwood,

1986). A more recent study calculated that sheep scab would cost Britain L8.3 million per annum (Nieuwhoff and Bishop, 2005). It was reported that the majority of the expenditure was on prevention (Nicuwhoff and Bishop, 2005). The loss of wool and weight and the damagecaused to the skin, combined with the cost of treatment, meansthat sheepscab is a diseaseof great economic importance, not only in Britain but also in other parts of the world such as Asia, southernAfrica and South America (Kirkwood, 1986).

Although the exact feeding mechanismsarc unknown, it was originally assumedthat mites punctured the skin surface and fed on dermal cells and serous fluid (Shilston, 1915;

Downing, 1936a; Sweatman, 1958). However, it has since been shown that Psoroptesmites

22 Chapter3- Effects of fungal pathogenson Psoroplesmites

feed on the lipids and serousexudates present on the surfaceof the skin and do not bite or burrow (Blake et al., 1978;Sinclair and Kirkwood, 1983;Sinclair and Filan, 1989,1991).

The mouthpartsof P. ovis appearmore suited for abradingthe skin and imbibingthe surface fluids than puncturingthe dermis(Blake et al., 1978;Rafferty and Gray, 1987;Mapstone et aL, 2002). A fluid pumpingaction has beenobserved around the pharyngealregion when

Psoroptesmites are immersed in serum(Rafferty and Gray, 1987).

The presenceof actively feedingmites as well as antigenicmaterial in the faecesis thoughtto trigger a hypersensitivityresponse in the host,leading to inflammationand serous exudateat the skin surface,providing a moresuitable microclimate and more available food for the mite (Sinclair and Kirkwood, 1983;Mathieson and Lehane,1996). Suppressionof the immuneresponse in sheepusing CyclosporinA dramaticallyreduces lesion growth and mite numbers,compared to control sheep,demonstrating the importanceof the immune responsein the developmentof scab(Huntley et al., 2005). It is theseskin secretionsthat dry and hardento form the characteristicyellow scabsfrom which the diseasederives its name.

As the serumon the skin surfacedries to form scabsand wool is lost from the areas of activeinfestation., the conditionsbecome less suitable for the mites,which moveoutwards to the edgeof the lesionswhere wool is still presentand the skin secretionshave yet to dry

(Downing, 1936b). As the populationof mites grows, the lesion size increasesand the diseasespreads across the body of the sheep.The areasof the sheepthat areusually affected are the shouldersand sidesof the body but, as the lesiongrows, up to threequarters of the bodycould be covered(Tarry, 1974).

After the initial infestationthere may be a pre-clinicalphase before the diseaseis detected. This period can be variable in length depending on factors such as sheepbreed, health of the host, initial number of mites and previous exposure (Bates, 1997a,b, 2000).

The population will then undergo a period of rapid growth, usually lasting 40 - 50 days until a peak is reached,after which the numbersof mites plateau(Bates, 1997a,b, 2000; Berriatua,

1999,2001). Transmission of mites betweenhosts is most likely to occur during the plateau

23 Chapter3- Effects of fungal pathogenson Psoroptesmites phasewhen mite numbers are at their highest (Berriatua, 1999,2001). At this stagethe host will either die or the immune responsewill causethe number of mites to decline, leading to the apparent recovery of the sheep. However, some of the sheep that appear to have recoveredmay be harbouring sub-clinical levels of mites which can easily go undetectedand remain as a reservoir for re-infestation within the flock (Babcock and Black, 1933; Downing

1936 ab).

The survival of the mite off-host is highly dependantupon environmentalconditions.

One study which kept mites on scab and wool at room temperature,found that adult females could survive for 16 days (Shilston et al., 1915). Another found that mites could survive for

days 1933). More a mean of 12 (range of I- 38 days) at 21 - 27 'C (Babcock and Black, recent studies have shown that mites may remain alive and infective for 18 - 20 days off- host (0' Brien, 1994; Smith et al., 1999).

3.1.2 Life historyof Psoroptesovis

Psoroptes ovis, has a5 stage life-cycle: egg, larva, protonymph, tritonymph and adult (Sanderset al., 2000). Unlike many other astigmatic mites, P. ovis does not possessa deutonymphstage (Sanders et al., 2000).

Several studies have investigatedthe time spent at the various stagesof development and show that, although there is some variation, on the host each stage in the life-cycle generally requires approximately 2 days; the total time taken to complete the egg to adult life-cycle has been recorded as taking between 10.7 - 16 days (Stockman, 1910; Shilston,

1915; Downing, 1936a,b).

The number of eggs a female can produce in its lifetime has been recorded as ranging from 35 (Downing, 1936 b) to 86 (Shilston et al., 1915) and the number of eggs laid per day per female estimatedto be from 0.5 to 5.6 (Shilston, 1915; Downing, 1936b).

A more recent study created a Leslie matrix model, simulating the population dynamics of P. ovis, used values of 2 days per life-cycle stage, with egg production of 2.9 female eggs per female per day and a female longevity of 16 days (Wall et al., 1999). The

24 Chapter3- Effects of fungal pathogenson Psoroptesmites model gave a good fit with data collected from scab infested sheepand therefore appearsto provide a good estimation for the rate of population growth of P. ovis.

3.1.3 Theclassification of Psoroptesovis

The first descriptionof Psoroptesmites on sheepwas givenby Heringin 1838when he classifiedthem as Sarcoptesovis. It was Gervaishowever in 1841that showedthat the sheepscab mites were differentto thosebelonging in the Sarcoptesgenus and subsequently reclassifiedthem as Psoroptesovis. Then in 1861 the classificationchanged again as

FOrstenburglabelled them as Dermatokoptescommunis before Megnin reassignedthem as

Psoroptesin his 1877review.

Megnin (1877) usedthe speciesname Psoroptes longirostris to describemites that werefound on the bodiesof sheep,cattle, horses and the earsof rabbits. The mitesfound on each host were labelled as different variantsof P. longirostris. Since Megnin's original classificationthere has been various additions and changes to the taxonomyof the Psoroptes genusalthough they havegenerally been referred to as P. communiswith differentvarietal namesappended depending on the host uponwhich the mite is found (Stockmanand Berry,

1913;Shilston, 1915; Hirst, 1922;Downing, 1936; Bates, 1999). However,a reclassification by Sweatman(1958) organisedthe genusinto 5 different speciessplit between2 groups; body mites and ear mites. Psoroptesovis, P. equi and P. natalensiswere groupedas body miteswhile A cunicull andP. cervinuswere grouped as car mites.

The criteria Sweatman(1958) used to distinguishbetween the specieswere the location on the host's body where the mites were found and the length of the outer opisthosomalseta of the adult males. However,these criteria arenot definitive,as earmites havebeen observed to movefrom the earto the body of the hostand the lengthsof the outer opisthosomalsetae can overlap betweenbody and ear forms (Wright et al., 1984;Bates

1999). A later studyshowed that therewere no morphologicaldifferences between mites of different hosts in sympatricpopulations, only in allopatric populations,and therewere no morphologicaldifferences between ear and body forms(Boyce et al., 1990).

25 Chapter3- Effects of fungal pathogenson Psoroptesmites

Antigenic studies also support the hypothesis that sympatric populations of mites from different hosts may not be ecologically or reproductively isolated (Boyce and Brown

1991). One study has claimed to have shown that it is possible for A ovis and A cuniculi to mate and produce viable offspring, however, this paper is unclear on how this was achieved and this result has never beenvalidated or repeated(Wright et al., 1983). host Psoroples mites have been experimentally transferred from one host to another of a different species. For example, Kirkwood (1985) transferred A ovis from the bodies of sheep to the ears of rabbits, causing a severe infestation, and was then able to transfer the mites back and forth from rabbit to sheep for three generations. The same study also in managed to transfer mites from rabbits to cattle. Sweatman (1958) was successful transferring mites from the ears of rabbits to the earsof sheep.

However, some studies have reported a failure of transferred Psoroples mites to establish(Ochs, 2002). For example,a recentstudy was unable to establishP. cuniculi from the ears of rabbits onto sheepor even onto the backsof rabbits (Ochs,2002). Shilston

(1915) was only able to establishear mites from rabbitsonto sheepfor 17 daysbefore the populationdied out at the secondgeneration. Another studywas unableto producea skin infestationon the backsof rabbitswith P. ovis from sheepbut could inducean infestation within the rabbit's auditory canal; however,an infestationon the backsof sheepor in the auditorycanal could not be establishedusing P. cuniculi (Siegfriedel aL, 2004).

Clearly, establishmentof a new populationis probabilisticand experimentalstudies often suffer from low replicatenumber, so it is difficult to draw conclusionsfrom manyof the smallscale individual studies.

Recentstudies using ELISA testsshow cross-reactivity between various isolatesof

Psoroptes mites taken from different hosts (Boyce and Brown, 1991). Genetic analysis has provided further evidence for the conspecificity of Psoroptes mites (Zahler et al., 1998).

Characterisation of the second internal transcribed spacer of the rRNA in 15 different isolates of Psoroptes mites collected from various hosts from 4 continents, found that the isolates were highly homogenous with no discernable segregation (Zahler et al., 1998).

26 Chapter3- Effects of fungal pathogenson Psoroptesmites

Another study using 9 polymorphic microsatellite markers showed that although there was some genetic segregationbetween mites taken from different speciesthere was not enough difference to suggest separatespecies status (Pegler et al., 2005). Morphological characterisation.of mites from different hostsshowed that there was no clear relationship betweenmorphology of the mites and the different host speciesor geographicregions from wherethey weretaken (Pegler et al., 2005).

In the light of the recent genetic and morphological evidence supporting the grouping of Psoroptesmites into one specieswith phenotypicallyadapted host-derived populations,it hasbeen suggested that the genusshould be synonornizedand that taxonomic priority should be given to the namePsoroptes ovis (Hering) (Wall and Kolbe, 2006).

3.1.4 Prevalenceand distrihutionofscab

Sheepscab is prevalentin manycountries across Europe, Asia, southernAfrica and

SouthAmerica (Kirkwood, 1986). It was introducedbut then eradicatedin Australiaand

New Zealand and it has also largely been eliminated form Norway, Denmark, Sweden,

Canadaand the U. S.A. (Seddon, 1964; Kirkwood, 1986).

Scab has been a problem in Britain for many hundredsof years with the first systematicdata on prevalencedating back to 1807,when 2573 outbreakswere recorded nationally (Kirkwood, 1986). In 1905, the British government made dipping with approved dips compulsory,leading to a reductionin the numberof recordedoutbreaks. By 1914there werejust 226 outbreaks(Kirkwood, 1986). After a slight rise in the prevalenceof scab during the first world war, the numberof casesdecreased until, in 1952,the diseasewas thought to have been eradicated(Kirkwood, 1986). However, in 1971,the diseasewas inadvertentlyreintroduced into Britain, probablyas a result of importing a scab infested sheepfrom Irelandand was initially misdiagnosed(Loxam, 1974;Tarry. 1974). In 1973,27 new outbreakswere recorded.

As the incidenceof scabcontinued to rise,dipping was once again made compulsory in 1976. This led, initially to a decreasein the numberof outbreaks. However,a switch

27 Chapter3- Effects of fungal pathogenson Psoroptesmites from autumnto summerdipping in 1983resulted in an increasein scaboutbreaks (French et al., 1999). Twice-yearly dipping was then madecompulsory in 1984,causing outbreak numbersto declinesubstantially. However, in 1989the summerdip wasremoved (French et al., 1999). In that year,approximately 40 outbreakswere recorded(French el al., 1999). In

1991 the final compulsorydip was abolished;in that year there were approximately120 outbreaks(French et al., 1999). With the deregulationof scab and the abolition of the compulsorydip the incidenceof scabthen increasedand in 1997up to 3000outbreaks were estimatedto haveoccurred nationally (Lewis, 1997).

By 1999,a postalquestionnaire survey estimated that 5000farms were likely to have been infested with scab in a one year period from October 1998 to September1999 (Corke and Broom, 2000). More recently, another postal questionnaire survey estimated that approximately 9% of flocks in Britain had experiencedat least one case of scab between

March 2003 and February 2004 (Bisdroff et al., 2005). However in Scotlandthe prevalence was as high as 17% (Bisdorff et al., 2005). The authors estimated that the number of outbreaksin Britain was close to 7000 (Bisdorff et al., 2005). Clearly the prevalenceof scab in the U. K. is currently growing rapidly.

3.1.5 Control ofscab

The first dip to becomecommercially available was a wettablepowder containing arsenicand sulphur producedby William Cooper in 1843 (Kirkwood, 1986). Previous treatmentmethods included ointments made from tar, butter and lard and washescontaining lime, mercury,nicotine, turpentine or arsenic(Kirkwood, 1986). Followingthe development of the first commercialdip, formulationscontaining coal tar, creosoteand other arsenic preparationswere also usedbut noneof thesedips providedlong term protectionand only persistedin the fleecefor a few daysrequiring a seconddipping 10- 14days later (Kirkwood,

1985). The chemicalswere alsoharmful to the sheepand would damagethe fleece. In 1947,

Downingtested organochlorines including gamma-hexachlorocyclohexane (lindane) and DDT andfound that residualactivity couldlast for up to 3 months.Lindane was approved for usein

28 Chapter3- Effects of fungal pathogenson Psoroptesmites dips in 1948and was not withdrawnfrom the marketuntil 1984(Kirkwood, 1985). The useof lindanewith the combinedeffort andcooperation of farmersand vets is believedto havebeen a majorfactor in theeradication of thedisease in sheepin 1952(Kirkwood, 1986).

The organophosphatesdiazinon and propetamphos,were approvedfor use in dips againstscab in the early 1980s.The organochlorineswere withdrawn from the marketin 1985 afterconcerns about residues in meatand environmental damage (O'Brien, 1999).This meant that the organophosphateswere the only availableformulations for usein dips (Kirkwoodand

Quick, 1981,1982;O'Brien 1999).The pyrethroidflurnethrin was introduced onto the market in 1987and laterhigh cis-cypermethrinwas also registered for use(Bates, 2004). Along with the organophosphates,diazinon and propetamphos,these were the only compoundsavailable for use in dips. In 1999however, all the organophosphatesand pyrethroidswere withdrawn

(O'Brien, 1999). In the caseof pyrethroidsthis wasdue to fearsover environmentaldamage, as they are much more stable and able to persistin the environmentfor longer than the organophosphates(Cooke et aL, 2004). Propetamphoswas withdrawn for economicreasons.

Diazinonwas temporarily withdrawn to allow for the developmentof saferpackaging and then placedback onto the marketin 2001.

An alternativeto dips for the controlof scabare the macrocycliclactones. These were developedin the 1970sas agriculturalpesticides and first usedin veterinarymedicine in the

1980sas anthelmintictreatments against gastrointestinal parasites. Ivermectinis effective againstscab following two subcutaneousinjections of 200 ýig/kg,given seven to ten daysapart

(Soil, 1992;O'Brien et aL, 1993). Two other injectableavcrmectins have been found to be effectivewith just onesubcutaneous injection: moxidectinat 200 gg/kg (O'Brien et aL, 1994,

1996)and dorarnectinat 300 gg/kg (Bateset al., 1995). Howeverthe injectableavermectins, particularlyivermectin, have a relativelyshort period of residualactivity meaningthat great caremust be takento treat every sheepcorrectly and preventreinfestation of the flock from contaminatedhousing (O'Brien, 1999).

NlVtt

Recentlya bolusof ivermectinwas developed to overcomethe problemsof the short

residualactivity seenwith the injectables.A polypropylenecylinder is insertedinto the rumen

of the sheepwhere flexible wings expandfrom the sideto preventthe cylinderfrom leaving the rumen. The bolusreleases ivermectin at a rateof 20 - 40 gg kg7lday"' for 100days and is able to provide 100% therapeuticefficacy (Forbeset al., 1999; O'Brein et al., 1999).

However,while usedin Australia,it is not registeredfor usein the U.K.

Insecticideresistance has become a growingconcern in recentyears. Resistantmites havebeen found in Scotlandand SouthWest England, in 1994,on sheepthat hadbeen dipped threetimes in flumethrinat the recommendeddose (Synge et al., 1995). Organophosphate resistancewas identifiedin 1996with sheepdipped in propetamphosharbouring live mitesup to 70 days(the end of the trial) after exposure(Clark et al., 1996). Resistanceto flumethrin, cypermethrinand diazinon has also been demonstrated in vitro with a largeproportion of mites survivingafter aI minuteimmersion in a varietyof concentrations(Coles and Stafford, 1999).

Thereis growingpressure to banthe useof the remainingdiazinon organophosphate dip. The pressurehas come from groupsand lobbies concerned about the effectsof dipson the environmentas well as the effectsthey may have on humanhealth. A studyof 146 sheep farmers who had experiencedlong-term exposureto organophosphatcdips performed significantly worse than a control group of 143 unexposed quarry workers in neuropsychologicaltests (Stephens et al., 1995).A studyof 612 sheepfarmers, suggested that acute poisoning from organophosphatedips is more likely to be responsiblefor the neurologicalproblems seen in exposedfarmers rather than long-termlow level exposure

(Pilkington el al., 2001). A telephonesurvey of 367 peoplewho believedthat they had becomeill due to organophosphateexposure also concluded that it wasthose individuals that had high levels of exposurethat reportedmore illnessthan thosewith long-terrn,low-level exposure(Fletcher and MacLehose, 2005).

Currentlythe only availableorganophosphate dip is diazinonand the only available alternativesare the injectableavermectins. Resistance, environmental concerns and potential health problemsassociated with the organophophatedips meanthat diazinonmight not be

30 Chapter3- Effects of fungal pathogenson Psoroptesmites available for use much longer. The avcrmectinshave short residualactivity, are expensiveand require long withdrawal periods for meat and milk. When theseproblems are combinedwith the increasing prevalence and incidence of the diseasethere is clearly a pressingneed for alternativetreatments.

One alternative being investigatedis the use of natural plant oils. In vitro bioassays have shown that extracts of Cinnamomumzeylanicum Blume, can induce 100% mortality in mites collected from rabbits earsafter 24 h exposureto 2.5 ml paraffin oil containing 0.3% C zeylanicum oil. (Fichi et al., 2007). The same study also demonstrateda reduction in mite numbers and clinical signs of otoacariasisin infected rabbits treated with the oil. Previous studieshave shown, in vitro, that the compoundscontaining free alcoholic or phenolic groups havethe most potent effect (Perrucciet al., 1995).

Attempts have also been made at producing a vaccine against P. ovis. However, only limited successhas been achieved. Sheep immunized with soluble extracts of P. ovis showeda two-fold decreasein lesion size and a seven-folddecrease in mite numbercompared to the control sheep(Smith et al., 2002). A later experimentthat fractionatedthe extractsto potentially enrich the inoculurn with target antigensfound a three-fold decreasein lesion size with a thirteen-fold decreasein mite numbers (Smith and Pettit, 2004). However, suitable antigenshave yet to be confirmedand tested as targets for usein a vaccine,although there are severalcandidates such as homologues of tropomyosin(Derp 10),paramyosin (Derp 11)and an apolipoprotein-likeIg E reactiveprotein (Der p 14) (Huntley el al., 2004). Although progresshas been made in the identificationof suitabletarget antigens, a potentialvaccine still seemsto be longway in the distance.

3.1.6 Entomopathogenicfungito controlscab

A potentialalternative to conventionalinsecticides for the controlof scabis the use of entomopathogenicfungi. In vitro trials showedthat M anisopliaeinfected 71% of the adult femalemites which were exposedto 3 ml of a IX107conidia ml-1suspension for 10 s

(Smith et. al., 2000). Hirsutella thompsoniihowever showed no pathogenicityand did not

31 Chapter3- Effects of fungal pathogenson Psoroplesmites

infect any mites(Smith et al., 2000). Anotherstudy had similar successinfecting 77% of P.

ovis which were immersedin a IxIO8 conidiaml' suspension(Brooks and Wall, 2002). It

was also shown in the Brooks and Wall study that cadaversof infectedmites could be

infectiousto live mites that cameinto contactwith the sporulatingcorpse. Chambersthat

had variousratios of live naYvemites and infectedcadavers showed that when 10 infected

cadaverswere housedwith 10 live nalivemites, over 65% of nave mites becameinfected

(Brooks and Wall, 2002). However,it only took I infectedcadaver introduced to 19 live

nalivemites to infect up to 40% of nSfvemites (Brooks and Wall, 2002).

Psoroptesovis completesits life-cycleon the hostand as suchit inhabitsa relatively

stable microclimate. This could be advantageousto the use of entornopathogens,provided that the conditions on the host animal are suitable for the germination of the fungus. The conditions at the skin surface range between 31 'C to 37 'C and in dry conditions the humidity at the skin surface is usually 10% below atmospheric(Wall et. aL 1992). Conidia of M anisopliae have been found to germinateat temperaturesbetween 15 'C and 35 'C with relativehumidities above 92% needed(Walstad et al., 1970). This meansthat temperatures are generally appropriateto allow M. anisopliaeto infect P. ovis on the host animal, although the humidity is often relatively low. Nevertheless,the peak germination temperaturefor M anisopliae is 25 *C (Walstadet al., 1970), hence strain selectionis importantto find an isolatewith a higherpeak germination temperature. Various isolates of

M anisopliaehave been examined for their infectivity againstPsoroples taken from the ears of rabbits,at a range of different temperaturesbetween 28 'C and 40 *C (Brooks et al.,

2004). Comparisonswere madebetween isolates from Brazil, Denmark,France and the

USA suspendedin Tween 80. The numberof fatal infectionswas reducedsignificantly at temperaturesabove 30 T in all excepta Frenchisolate, where there was a slight, although insignificant,increase at 32.5 T (Brooks et al., 2004). No infectionswere seenat 40 T.

Significantly higher levels of infection were seenwhen the conidia were formulatedin siliconeoil ratherthan Tween 80 (Brookset al., 2004).

32 Chapter3- Effects of fungal pathogenson Psoroptesmites

Another fungal pathogenthat could potentiallybe usedfor the treatmentof scabis

Beauveriabassiana. When adult femalemites were exposed, in vitro, to suspensionsof 10'

conidia ml-' in Tween 20,100% of the mites showedsigns of infection and increased

mortality, comparedto the controls (Lekimme et al., 2006). Fungal infection with B.

hassianais also transmissiblebetween infected cadavers and live naYvemites (Lekimme et

al., 2006). When 10 infectedcadavers were housed in chamberswith 10 live naYvemites, all

mites showedsigns of infection at the end of the trial (Lekimmeet al., 2006). Although

several studies have demonstratedthe lethal effects and potential for transmissionof

infection betweenmites, no previousstudies have been able to inducefatal infectionsin

Psoroptesmites in vivo.

The aims of the work describedin this chapterwere to investigatethe effectsof

formulation on the pathogenicity of fungal pathogensand pathogenicity of fungal pathogens

to P. ovis in the presenceof sheepskinand fleece. The potential for M anisopliae and B.

bassiana to be used as a preventative or curative treatments for sheep scab were also

examined in vivo. Becauseof the complexity of the series of experiments describedin this

chapter, they will be presented as methods followed by results for each individual

experiment.

Due to the nature of the collaboration between Central Science Laboratory (CSQ,

Sand Hutton, CAB International (CABI), Ascot and the University of Bristol, there were

several logistical constraints imposed upon the work that could be carried out during the

course of this project. This impacted heavily upon the planning and conduct of the various

trials presentedin this chapter. Previous work (Brooks, 2004) had already screenedmany

isolates of M ansiopliae and identified an appropriate isolate for further testing. Therefore

the project started with in vivo trials rather than with more in vitro work. However, due to a

miscommunication between collaborators, the first in vivo trial used the less virulent Danish

strain of M anisopliae rather than the French strain that was previously identified as the most promising strain. This led to the in vitro bioassay (section 3.2.2) to demonstratethe

33 Chapter3- Effects of fungal pathogenson Psoroptesmites greatervirulence and pathogenicityof the Frenchstrain used throughoutthe rest of this study. Anotherin vivo trial wasthen conducted using the morepathogenic strain.

In vivo and in vitro trials were carriedout alternatelydue to time constraintsand a necessityto demonstratethe effectsof fungalpathogens to A ovis in vivo. The methodsand resultsare presentedin a chronologicalorder to demonstratethe problemsand how they wereaddressed so that a betterunderstanding of the overallproject can be obtained.

3.2 Methods and Results

3.2.1 Analysis

All data were checked for normality using a Kolomogorov-Smirnov test.

Proportional data were arcsine transformed prior to statistical analysis, however, figures show the untransformeddata for clarity.

3.2.2 Thepathogenicity ofMetarhizium anisopliaeagainst Psoroptesovis in vivo (Trial 1)

Materials and methods

An in vivo trial was used to test whether M anisopliae could be used as a pour on treatment for sheep scab. Eighteen pathogen-free,Scottish blackface cross, female sheep, aged between 6 and 9 months were used in this trial. The sheepwere maintained indoors in approvedcontainment facilities at the Central ScienceLaboratory (CSL), SandHutton.

To infest the sheep with P. ovis, a small tuft of fleece (5 mm in diameter) was removedfrom the withers approximately30 cm from the baseof the headand 25 adult femalemites were placedonto the exposedskin. Fleecefrom the surroundingarea was then pulled over the bare skin and mites and held in place with a rubber band. Each sheep receiveda single inoculum.of mites. The sheepwere checkedfor the presenceof mites 14 daysafter the initial seeding.The infestationswere allowedto developfor 28 dayswith no intervention. On day 28 the size and position of the lesionswere recordedand the sheep were divided into 3 groupsof 6 animals. One group of sheepreceived no treatment,one

34 Chapter3- Effectsof fungalpathogens on Psoroplesmites

group of sheephad 100 ml of silicone oil applied per sheepand one group had 100 ml of M.

anisopliae IM1386698 (Danish strain isolated from Agrolls segelum (Denis and

Schiffermuller), Lepidoptera: Noctuidae) conidia in silicone oil at a concentrationof 5x 108

conidia ml-1 applied per sheep. The fungal suspensionand silicone oil were supplied by

CABI Bioscience, Ascot.

The silicone oil and the conidial suspensionwere applied to the dorsal surfaceof the

sheepusing a dial-a-dose drench gun with a spray nozzle (Forlong and Masey, Ripon) in 4

rows of 25 ml from the baseof the head to the baseof the tail. The rows were evenly spaced

across the back of the sheep to ensure an even application of treatment over the dorsal

surface of the sheep. The drench gun was primed with the relevant treatment before

application to the sheep. The sheepthat received a dose of silicone oil were treated first to

prevent any contamination from the conidial suspension. During application, the sheepwere

restrainedby holding the head of the sheepbetween the legs of the handler. After the sheep

had received their treatment they were housed separatelyin their respectivegroups ensuring

that no contact could occur betweenthe groups.

Wash samples from the lesion were taken from each sheep 42 days after the initial

seedingwith mites(14 daysafter the groupshad been treated with siliconeoil or the conidial

suspension).The washsamples were taken along a transectof the lesion,taking two samples

from the edgeof the lesion and one samplefrom the middle of the lesion (Fig. 3.1). This was achievedby flushing aI cm2 areaof the lesionwith phosphatebuffered saline (PBS) usinga syringewith a domeattachment on the nozzle. A securefit wasachieved by pressing the domeof the collectionnozzle against the surfaceof the skin beforeflushing the areawith the PBS. A secondwash samplewas also taken from eachsheep 49 days after the initial seeding(21 days after treatment). The mites were removedfrom the wash samplesby strainingthe PBSthrough a filter paper. The dampenedfilter paperand miteswere placed into a petri dish which was sealedwith parafilm,refrigerated over night and transportedto the University of Bristol usually a maximumof 24 h after collection from the sheep. The following day, up to 20 adult femalemites were removedfrom eachwash sampleusing a

35 Chapter3- Effects of fungal pathogenson Psoroplesmites

fine gradepaint brushthat had beenwashed in 70% ethanol,and incubatedin the standard

incubationchamber (Fig. 2.4) with 500 jil of sheepserum at 30 "C and 90% r.h. If there

were not enoughadult females,female tritonymphs, adult malesand maletritonymphs were

used. Chamberswere checkedfor the presenceof deadmites every24 h. Deadmites were

removedand checked for signsof infectionas described in Chapter2.

At the end of the trial, day 55 (27 daysafter treatment)the sheepwere killed by a

schedule1 methodand the lesionsand surroundingskin were removed. Four samplesof

skin were excisedfrom eachsheep, placed into re-sealableplastic bags and refrigerated over

night at CSL andthen postedto the Universityof Bristol by specialdelivery (sent on day 56

of the trial), arriving the next morning(day 57). The skin sampleswere removedalong a transectof the lesiontaking two samplesfrom the edgeof the lesion,one samplefrom the

middle and one sample from outside of the lesion (Fig. 3.1). The sampleswere

approximately15 x 15 cm squares,stored in sealableplastic bags. Oncethe sampleshad

beenreceived at Bristol up to 30 adult femalemites were removed from eachsample. Mites were removedusing a fine grade paintbrushand forcepsthat had been washedin 70% ethanol. Different forcepsand brushes were used for eachtreatment group. If therewere not enoughadult females,female tritonymphs,adult malesand male tritonymphswere used.

Mites were incubatedin standardincubation chambers and checkedfor signsof infectionas previouslydescribed.

For both of the wash samplesand the skin samples,mites were removedfrom 3 randomly selectedsheep from each group and the samesheep were used for both wash samplesand the skin samples.

Application of the mites,treatment and euthanasia of the sheepwere carriedout by suitablyregistered staff at the CSL.

36 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.1. Diagram depicting the 3 siteswithin a scablesion and I site outside of the lesion that were excised from the sheepskin at the end of the first in vivo trial and sent to the 2 University of Bristol for examination. AI cm areawithin each site was sampledusing PBS washesat days 42 and 49 of the trial. The diagram is not drawn to scale.

Uninfested skin

37 Chapter3- Effects of fungal pathogenson Psoroptesmites

Results

For the wash samplestaken at day 14, a total of 39 mites were removedfrom the control group,47 mites were removedfrom the grouptreated with siliconeoil and 50 were removedfrom the group treatedwith conidia suspendedin silicone oil. For the wash samplestaken at day 21, a total of 56 miteswere removedfrom the controlgroup, 51 mites were removedfrom the grouptreated with siliconeoil and 39 wereremoved from the group treatedwith conidia suspendedin siliconeoil. For the skin samplestaken at the end of the trial (day 27), a total of 187 mites were removedfrom the control group,248 miteswere removedfrom the group treatedwith silicone oil and 202 mites were removedfrom the group treatedwith conidia.suspended in siliconeoil. Hence,for the whole trial, a total of

282 mites removedfrom the control sheep,346 mites removedfrom sheeptreated with siliconeoil and 291 mitesremoved from sheeptreated with conidia,suspended in siliconeoil were examinedfor infection. However, only 2 mites showed any infection with M. anisopliae.Both miteswere removed in the washsamples taken at day 14from two sheepin the grouptreated with conidiasuspended in siliconeoil.

38 Chapter3- Effectsof fungalpathogens on Psoroplesmites

3.2.3 Thepathogenicity oftwo strains of Metarhizium anisopliae in vitro

To try to account for the failure of the fungal inoculation to infect mites in the in vivo trial, an in vitro investigation was undertakento comparethe Danish M anisopliae strainused, against a Frenchstrain.

Materialsand methods

Adult female mites were obtained from the ears of artificially infested rabbits. The mites were then inoculatedwith one of three treatments: silicone oil, M. anisopliae

IM1386697 (French strain isolated from Sitonia discoides Gyllenhal, Coleoptera:

Curculionidae) suspended in silicone oil and M anisopliae IM1386698 (Danish strain) suspendedin silicone oil. The conidial suspensionswere made to a concentrationof Ix 108 conidia ml-1 for both fungal isolates. Once the mites had been inoculated, as described in

Chapter 2, they were transferred in their respective treatment groups to glass incubation chambers (Fig. 2.4) and incubated at 30 'C and 90 % relative humidity. Each chamber housed20 adult female mites. The chamberswere checkedevery 24 h for dead mites which were then removed and checked for signs of infection every 24 h as previously described.

There were 4 replicates for eachtreatment.

Results

The proportion of infected mites was highest for the groups of mites treated with the

Frenchstrain of M. anisopliae(IM1386697) with a meanproportion of 0.91 (± 0.18) (Fig.

3.2.). For the Danish strain,a meanproportion of 0.38 (± 0.32) of mites becameinfected.

Therewere no infectionsin the groupstreated with siliconeoil only. This differencewas highly significant(ANOVA; F2.12ý-- 19.99, P<0.001; Fig. 3.2.). Therewas a significantly greaterproportion of infected mites in the groupsexposed to the Frenchstrain than the groupsof mitesexposed to the Danishstrain (Tukey HSD; P=0.0 13).

39 Chapter3- Effects of fungal pathogenson Psoroptesmites

It seemslikely thereforethat the failure of the first in vivo trial may have been causedby the lack of pathogenicity of the Danish strain used.

40 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.2. The proportion of adult female mites that becameinfected with M. anisopliae after exposureto silicone oil only, conidia of a French strain (IM1386697) suspendedin silicone oil and conidia of a Danish strain (IM1386698)suspended in silicone oil at 30 IC and 90% r.h.. Conidia of both strains were suspendedat a concentrationof Ix 10' conidia ml". Each datapoint representsI replicateof 20 adult femalemites. Therewere 4 replicatesfor each group.

1.00 pI

0.80 0 V

JOR C 0 0.60 E %I-0 C 0 E 0 CL0 0.40 0 a- CL 0

0.20

0.00 Siliconeoil Siliconeoil Siliconeoil + conidia + conidia IM1386697 IM1386698 (French) (Danish)

Treatment

41 Chapter3- Effects of fungal pathogenson Psoroplesmites

3.2.4 Treatmentofsheep infected with sheepscab with Metarhizium anisopliae (Trial 2)

A secondin vivo trial was usedto investigatethe potential use of M anisopliae

against sheepscab using a more carefully controlled application procedure.

Materialsand methods

Nine pathogen-free,poll Dorsetfemale sheep, aged between 6 and 12months were

usedin this trial. As for the previoussheep trial the animalswere maintained indoors in

approvedcontainment facilities at theCentral Science Laboratory (CSQ, Sand Hutton.

On day I of the trial, eachsheep had an areaof fleecesheared down to the skinto

createan areaof unshearedfleece 10 x 10 cm square,surrounded by a margin of sheared

fleece 2 cm,wide. This margin of shearedfleece was designedto act as a deterrentto

migration for the mites that were placedwithin the enclosed10 x 10 cm squarearea. The

sheepwere divided into groups of 3 and housedin separateunits dependingupon the

treatmenteach group would receive. The first group,to act as a control,was inoculatedwith

miteson day I of the trial andreceived no treatment.The secondgroup was inoculated with

mites on day I of the trial and then exposedto conidiaof M anisopliaeIM1386697 on day

21 to act as a curativetreatment. The third group was exposedto conidiaof M. anisopliae

IM1386697on day 20 of the trial and then inoculatedwith mites on day 21. The same

methodof inoculationwith 25 adult femalemites was usedin this trial as for the previous

sheeptrial, exceptthat the mites were placedinto the centreof the areaof skin surrounded

by the shearedmargin. For the groupstreated with M anisopliae,sheep were exposedto

0.4gof the unformulatedconidia (I x 1011conidia g"'). The conidiawere appliedby parting the fleecein 3 evenlyspaced lines horizontallyand then 3 lines vertically andfinally oneline

diagonallyacross the 10 x 10 cm squarewith the conidia sprinkledevenly into the parted fleece.

By the end of the trial the control sheephad beenexposed to mitesfor 42 dayswith no treatment. The curativegroup had beenexposed to mites for 42 daysbut were treated with M anisopliaeconidia on day 21. The preventativegroup had beentreated with M.

42 Chapter3- Effectsof fungalpathogens on Psoroplesmites anisopliae conidia on day 20, before inoculation with mites on day 21, and therefore had only been exposedto mites for 21 days.

At the end of the trial, day 42, the sheepwere killed by a scheduleI methodand the skin containingand surroundingthe lesionswas excisedand placedinto re-sealable plastic bagsand refrigeratedover night. The following day (day 44), 3 groupsof 30 adult femalemites were removedfrom eachskin samplein the sameway as describedfor the previoussheep trial. Thesemites were then incubatedwith 30 mitesper chamber(Fig. 2.4) at 30 *C and 90% r.h. with 500 pI of sheepserum. Eachchamber was inspectedevery 24 h for dead mites which were removedand checkedfor signs of infection as describedin Chapter2.

The skin samples and any remaining mites were then frozen at -13 T (± 2 T).

After 28 days in deep freeze(day 72), the sampleswere thawedat room temperatureand then washedwith distilled water in an attemptto removeas many mites as possible. The mites removedby the washingwere placedinto 90 mm petri disheswith dampfilter paper and incubatedat 30 'C and 90 % R.H. and inspectedevery 24 h for 14 daysto checkfor signsof infection.

Results

Inspectionsmade during the trial showedthat not all of the sheepinoculated with P. ovis developedlesions. The sheepthat showedno obvioussigns of infestationduring the trial werenot found to haveany mitespresent on the skin samplesinspected at the endof the trial. Therewere 4 sheepin total whereno mitescould be found, including I sheepfrom the control group, 2 sheepfrom the curativegroup and I sheepfrom the preventativegroup.

This meantthat on day 44 of the trial, 180mites were examined from the controlgroup, 180 mites were examinedfrom the preventativegroup and 90 mites were examinedfrom the curativegroup. However,none of thesemites showed any signsof infection. On day 72 of the trial, 203 mites were removedfrom the control group,63 mites were removedfrom the

43 Chaptcr 3- Effects of fungal pathogcnson Psoroplesmites preventative group and 104 mites were removed form the curative group. Surprisingly, only

4 mites,removed from the controlsheep B909, showed signs of infection(Table 3.1).

Table 3.1. The number of mites removed from the skin samplesof each sheepafter euthanasia(day 44) andfrom the washof the skin on day 72 andthe numberof infected mitesfound from eachsheep.

Sheep Treatment Mites removed Mites removed Numberof day44 day 72 (wash) infectedmites B909 Control 90 106 4 B919 Control 90 97 0 B900 Preventative 90 21 0 B904 Preventative 90 42 0 B91 I Curative 90 104 0

44 Chapter3- Effects of fungal pathogenson Psoroplesmites

3.Z5 Thepathogenicity ofMetarhizium anisopliaeto Psoroptesovis in vitro in the presence ofsheep skin

To try to accountfor the secondfailure to infect mitesin vivo, it wasconsidered that the skin or fleeceitself may havesome inhibitory compounds.An in vitro bioassaywas conductedto testthis.

Materialsand methods

The skin of a freshly-killed lamb with no history of treatmentfor P. ovis was obtainedfrom an abattoir. A seriesof 20 mm diametercircles were cut from the skin andthe fleece attachedto these circles was cut to a length of 20 mm. Once a disc of skin and fleece had received its appropriatetreatment, it was placed into an incubation chamber(Fig. 2.4).

Four treatmentgroups were used:untreated fleece only, fleeceto which conidiaof

M anisopliaesuspended in siliconeoil hadbeen added, fleece to which only siliconeoil had beenadded and, as a positivecontrol, cotton cloth to which conidiasuspended in siliconeoil hadbeen added.

Treatments containing conidia used a suspension of Ix 108 conidia ml-1 of M. anisopliae IM1386697(French strain) in silicone oil (dimethylpolysiloxanehydrolyzate,

Sigma-Aldrich,Dorset, UK). The silicone oil, or silicone oil with its suspendedconidia, were applied using a household plant sprayer (Hozelock Group Ltd. Aylesbury, UK) fixed

20 cm directly abovethe skin or cloth usinga clampstand. This distancebetween the nozzle of the sprayerand the discs of skin or cloth was chosento give an evenapplication of the treatmentacross the skin or cloth and to simulatea possibleapplication method for useon sheep.The sprayerdelivered a standard0.08 ml of conidiasuspended in siliconeoil to each surface resulting in the application of 8x 106conidia per disc.

After application, the skin and cloth were placed into the chambersand left for Ih before the addition of 20 adult, female Psoroptesmites, collected from the ears of experimentallyinfested rabbits on the sameday that they wereused in the experiment.

45 Chapter3- Effectsof fungalpathogens on Psoroplesmites

In the cloth treatment, 500 gl of ovine serumwas also added(Sigma-Aldrich, poole,

UK) before the addition of the mites. Five replicates of each treatment were set up. The miteswere then left in their treatmentchambers for 24 h at 30 T and90% relative humidity.

After 24 h, all mites were removedfrom the chambersand transferred,in their respective treatment groups, to clean incubation chamberswhere they were incubatedat 30 IC and 90% r.h. with 500 gI of sheepserum. The chamberswere inspectedevery 24 h for the presenceof dead mites which were removed with a fine grade paint brush and checked for signs of infection as previously described. Different paint brushes were used for each of the treatments. The brusheswere washedin 70% ethanol and wiped dry betweeneach sample.

Results

In the groupsthat receivedno treatmentor siliconeoil only, therewere no infected mites (Fig. 3.3). In the group that was exposedto silicone oil plus conidia on the skin, a meanof 0.52 (± 0.34) mites becameinfected and for the group exposedto siliconeoil plus conidia on the cloth, a meanof 0.34 (± 0.31) mites becameinfected. This differencewas highly significant (ANOVA; F3,19= 8.4, P=0.001; Fig. 3.3). There was no significant differencein the proportionof infectedmites in the two groupsexposed to siliconeoil and conidia whetheron fleeceor cloth (Tukey HSD post-hoctest, P>0.05; Fig. 3.3). Clearly the sheepfleece itself did not inhibit germinationof conidiaand infectionof mites.

46 Chapter3- Effectsof fungalpathogens on Psoroplesmites

Fig. 3.3. Mean proportion of mites (± S.D. ) showing signs of fungal infection after 24 h exposureto untreatedfleece, fleece treatedwith silicone oil, fleece treated with silicone oil plus Metarhizium anisopliae (IM1386697) conidia (I x 108conidia ml") and cloth treated with silicone oil plus M anisophae (IM1386697) conidia (I x IOconidia ml").

1.0

0.9

0.8

0.7

0.6 E 4- 0 0.5 r_ 0 E 0 0.4 0 CLA- 0.3

0.2

0.1

0.0

Fleeceonly Fleece+ Fleece+ Cloth + silicone siliconeoil siliconeoil oil and conidia and conidia (IM1386697) (IM1386697)

Treatment

UNNER 47 OF BRt=' LIBRARY Chapter3- Effects of fungal pathogenson Psoroptesmites

3-Z6 Thepathogenicity offive high temperaturetolerant strains of Metarhizium ani sopI iae against Psoroptesovis in vitro

One possible reasonfor the failure of the first two in vivo trials was that the temperatureat the sheepskin wastoo high to allow germinationof the coniclia. An in vitro trial was conductedto test 5 new strainsof M. anisopliaethat hadexhibited tolerance to high temperaturesfor their pathogenicity againstP. ovis

Materialsand methods

The standardinoculation method as describedin Chapter2, was usedto inoculate adult female mites with the 5 new strains of M anisopliae at 37 "C and 90% r. h. Each strain was suspendedin silicone oil to give a concentrationof Ix 10' conidia ml"'. Each treatment was tested against 4 replicates of 20 mites. Another 4 replicates of 20 adult female mites was set up that were treated with silicone oil only. Table 3.2 gives information on the strains used.

Table 3.2. The strainsof Metarhiziumanisopliae, reference numbers and the original host from which the fungi were isolated.

Species Referencenumber Origin (ARSEF)

M. anisopliae 1545 Scotinopharacoarclata (Hemiptera:Pentatomidae) M. anisopliae var. acridum 2575 Curculio caryac (Coleoptera: Curculionidae)

M anisopliae var. acridum 3609 Patanga succincta (Orthoptera: Acrididae)

M. anisopliae var. acridum 3619 Oxya multidentata (Orthoptera: Acrididae)

M. anisopliae var. acridum 324 A ustracris gultulosa (Orthoptera: Acrididae)

48 Chapter3- Effectsof fungalpathogens on Psoroptesmites

Results

There were no infected mites found in any of the treatment groups. None of the strainstested could infect mitesat 37 'C.

3.2.7 Thepathogenicity of Beauveriabassiana against Psoroptesovis in vitro

It was decidedalso to considerthe pathogenicityof A bassianaas an alternativeto

M anslopliae. Using the standard inoculation method described in Chapter 2, the pathogenicityof B. bassianawas testedagainst adult female A ovis. The strain of B. bassianawas obtainedfrom a naturallyinfected mite found on a sheepat the CSL andwill be describedas CSL strain 1.

Materialsand methods

The conidia of B. hassianawere suspendedin siliconeoil at a concentrationof Ix

108conidia ml-1. Therewere 5 replicatesof 20 mitesused. A further 5 replicatesof 20 mites were setup andtreated with siliconeoil only ascontrols.

In a further experiment,the pathogenicityof B. bassianaformulated in 0.05%

Tween 80 at various concentrationswas testedagainst P. ovis. For eachconcentration, 3 replicatesof 20 adult female mites were inoculatedusing the standardmethod. The

105,1 X 106,1 X 107,1 X log, IX 10 ' concentrationstested were: Tween80 only, IX conidia ml-1. A further3 replicatesof 20 miteswere left untreatedto act as a control.

For both experiments,mites were collectedfrom the earsof experimentallyinfested rabbitskept at CSL the day beforethe experimentaltests and sent to the Universityof Bristol ovemightby specialdelivery.

Results

A mean proportion of 0.58 (± 0.08) of mites inoculated with B. bassiana suspended in silicone oil at I X108conidia ml"' becameinfected, whereas none of the mites exposedto siliconeoil becameinfected (Fig. 3.4). The mites exposedto B. hasslanahad a meanUso

49 Chapter3- Effectsof fungalpathogens on Psoroplesmites of 6 (± 0.55) days and the mites exposedto silicone oil had an LT5oof 6.4 (4-0.71) days (Fig.

3.5). This was not a significant differcncc (ANOVA; Fl, g=1.0, P>0.05).

When B. bassiana was formulated in Tween 80, a high proportion of infected mites were found at all the concentrationstested. No infected mites were found in the groups exposedto Tween 80 only or groupsthat receivedno treatment(Fig. 3.6). This was a significant difference (ANOVA; F6.14= 974.06, P<0.001; Fig. 3.6). However, there was no significant difference in the proportion infected between the groups treated with B. bassianasuspended in Tween 80, regardlessof concentration(Tukey HSD; P>0.05). All of the groups treated with B. bassiana had significantly more infected mites than the control or

Tween 80 only groups (Tukey HSD; P<0.001).

There was no significant difference in LT30 between any of the different concentration groups exposed to B. bassiana. The mites in the control groups had significantly longer LT50 values than any of the groups of mites exposed to conidia

(ANOVA; F6,14 = 20.9,P<0.00 1; Fig. 3.7).

The resultsshow that this strain of B. bassianais highly pathogenicto P. ovis evenat relativelylow concentrations.

50 Chapter3- Effcctsof fungalpathogens on Psoroptesmites

Fig. 3.4. Proportion of adult female mites infected with Beauveria basslanawhen exposed to silicone oil only or A bassianasuspended in silicone oil at a concentrationof Ix 108 conidiaml" at 30 T and90% r. h.. Eachdata point representsI replicateof 20 adult females.

0.7

00

0.6

0.5 00

4- 0.4

E 4- 0

0 0.3 u 0 0. 0 0.2

0.1

0 000 Silicone Siliconeoil oil + conidia (B. bassiana)

Treatment

51 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.5. LT50(± S.D. ) of mites surviving after exposureto silicone oil or Beauveria bassianaconidia suspended in siliconeoil at a concentrationof Ix 108conidia ml" at 30 T and 90% r.h.. There were 4 replicatesused for each group.

7.0

6.5

"a 6.0 E i-

5.5

5.0 Silicone Siliconeoil oil + conidia (B. bassiana)

Treatment

52 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.6. Meanproportion of mitesinfected with Beauveriabassiana when exposed to no treatment,Tween 80 only or A basslanaconidia suspended in Tween80 at various concentrationsat 30 "C and90% r. h.. Therewere 3 replicatesused for eachgroup.

1.0 m 00 mmm

0.8 10

C U) Is 0.6 E %I-.0 c 0 %E CL0 0.4 2 IL

0.2

0.0 06 1X107 JX108 JX109 Control Tween 1x1O5 1X, 80

Concentration (conidia mi")

53 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.7. LT50 of adult female mites after exposure to no treatment (control), Twcen 80 only or Beauveria basslana conidia suspended in Tween 80 at various concentrations; Ix 105,1 x

101 107,1 108 Ix 109 30 "C 90% h.. There were 3 replicates ,IX X and conidia ml" at and r. of 20 mites used for each treatment. Letters show groups between which there was no significant difference (P > 0.05).

b

7

6

5 U, >1 Co a, b 7 E F- 3

b, ca 2 00 w so

1

0 Control Tween 1X105 1X1 0" 1xio, 1xio, 1xio, 80

Concentration (conidia ml*')

54 Chapter3- Effectsof fungalpathogens on Psoroptesmites

3.2.8 Control of Psoroptesovis using entomopathogenkfungi in vivo (Trial 3)

A yet more controlled approachwas usedfor the third in vivo trial to investigate whether or not A ovis can be infected by fungal pathogenson a host animal.

Materialsand methods

Six, pathogen-free,poll Dorsetsheep aged between 6 to 12 monthswere used in this trial. The sheep were maintained indoors as a single group, on straw in approved containmentfacilities, at the CSL.

Eachsheep had six circular rubberarenas glued to the skin on its back (threeeither side of the midline) giving a total of 36 arenas(Fig. 3.8). Prior to attachment,the fleece was shorn close to the skin and the arenaswere attachedwith surgical glue. Each arena had an internal diameter of 25 mm and was 10 mm high. Each arena was sealed with fine mesh cotton held in place with a tight fitting rubber washer placed on the inside of the arena(Fig.

3.8).

Treatments were allocated to specific sheep/arenasat random, with the constraint that no onesheep had more than onetreatment of any particulartype andthat eachsheep had one arenato which no treatmentwas applied. Thus,the skin in a total of six arenasacted as controlsand had no treatmentapplied. The skin of six of the arenasreceived 0.01 g of dry sporesof M anisophae(isolate IM1386697) at a concentrationof 1.8x 1010conidia g"', three arenaswere treated with 2 ml of the sameisolate of M. anisopliaeconidia suspended at 1.5x

109conidia ml" in sheepdip excipient(70% Octyl oil plus stabilizers,diluted to 15%W/V in water)and three arenas were treatedwith 0.01g of the sameisolate of M anisopliaeconidia mixed in 0.001gof diatomaceousearth giving a concentrationof 1.6 x 1010conidia g". A further six arenaswere treatedwith 0.01 g of a mixture of two different isolatesof M anisopliae (ARSEF3297 isolated from Boophilus spp., Acari: Ixodidae, Mexico) and

(ARSEF4556isolated from Boophilusspp., Acari: Ixodidae,U. S. A. ) at a concentrationof 8 x 109conidia g".

55 Chapter3- Effects of fungal pathogenson Psoroplesmites

Beauveriabassiana (CSL strain 1) was also examined.For this, six arenaswere treatedwith 0.01 g of dry sporesof B. bassiana(1.2 x 1011conidia g"), threearenas were treatedwith 2 ml of B. hassianaconidia suspendedat Ix 1010conidia ml-1in sheepdip excipientand three arenas were treated with 0.01g of B. bassianaconidia mixed in 0.001g of diatomaceousearth (1.1 x 10"conidia g"). The skin in eachof the arenaswas treatedIh beforemites were applied.

For the trial, 25-30adult femalePsoroptes mites were placed inside each arena. The miteshad beenremoved from infestedsheep 72 h prior to beingplaced on the experimental sheep. Prior to applicationto the experimentalsheep, half of the groupsof mites were maintainedin cleanchambers at 90%r. h. and30 I'C andoffered 500 gl of sheepserum.

The other half of the mites wereexposed to formulationsof eitherM anisoplideor

A bassiana. Among thesegroups, the mitesto be exposedto dry conidiaon the hostwere first dippedin suspensionsof Ix 108conidia ml-' of eitherM. anisopliaeor A bassianain

Tween80 for I min. The miteswere then transferred to a dry pieceof filter paperfor I min to allow any excessfluid to run off beforebeing placedinto a clean incubationchamber.

The mitesto be exposedon the sheepto conidia in sheepdip excipicntor conidiamixed in diatomaceousearth were placedonto filter paperin a 45 min diameterPetri-dish which had beentreated with the samevolume and concentration of the treatmentto which theywere to be exposedon the sheepfor 48 h.

Hence,on the experimentalsheep, half the arenascontaining dry spores,contained mites that had been pre-exposed,while half containedmites that had not. All the arenas treatedwith conidia.in dip excipientand all the arenastreated with conidia in diatomaceous earth containedmites that had been pre-exposed. Table 3.3 summarizesthe treatment regimesused.

At the end of a 48 h period on host, the sheepwere euthanizedby a scheduleI methodand the skin within and surroundingthe chamberswas excised. The skin andarenas were then packedinto individual sealableplastic bagsand refrigeratedat 5 IC over night.

The following day the mites were removedfrom the arenas,surface sterilised with 2%

56 Chapter3 Elffectsof fungal pathogenson Psoroplesmites sodium hypochlorite solution, placed into clean incubation chambers (Fig. 2.4) with 500 gl of sheep serum and incubated at 30 'C and 90% r. h. The chambers were inspected every 24 h for the presence of dead mites. A further 200 ld ofsheep serum was added every 48 h until all the mites had died. Dead mites were treated as describedpreviously and fungal infections were recordedwhen external hyphaecould be observedprotruding through the cuticle.

Fig. 3.8 Rubber arena attached to the skin on the dorsurn of a sheep. The arenas had an internal diameter of 25 mm and a height of 10 mm.

57 Chapter3- Effectsof fungalpathogens on Psoroptesmites

Table3.3. Summaryof the fungalpathogens, excipients, concentrations and amounts applied to the arenason the experimentalsheep and the exposuremethod usedto inoculate adult, female mites for the third in vivo trial.

Fungal species Excipient Concentration Amount Exposuremethod applied M anisopliae Dry conidia 1.8 x 10'0 O.Olg 48 h on host (IMI386697) conidia g"

M anisoplide Dry conidia 1.8 x 1010 O.Olg 48 h in lab and QM1386697) conidia g" 48 h on host

9 h in lab M anisopliae Sheepdip 1.5 x 10 2ml 48 and (IMI386697) excipient conidia ml-l 48 h on host h in lab M anisoplide Diatomaceous 1.6 x 1010 O.Olg 48 and (IMI386697) earth conicliag-l + O.Oolg 48 h on host

A bassiana Dry coniclia 1.2 x 1011 O.olg 48 h on host (CSLI) conidia g"'

B. bassiana Dry coniclia 1.2 x 1011 O.olg 48 h in lab and (CSLI) conicliag-l 48 h on host

B. bassiana Sheepdip Ix loll 2ml 48 h in lab and (CSLI) excipient conicliaml-l 48 h on host

A bassiana Diatomaceous 1.1 x1oll O.olg 48 h in lab and (CSLI) earth conidia g7l + O.oolg 48 h on host

h host M. anisopliae Dry conidia 8x 10' O.olg 48 on (ARSEF3297 + coniclia g-1 ARSEF4556)

lab M, anisopliae Dry coniclia 8x 109 O.olg 48 h in and (ARSEF3297 + conicliag"' 48 h on host ARSEF4556)

Control (no fungal pathogen)

58 Chapter3- Effectsof fungalpathogens on Psoroptesmites

Results

There were no infected mites in any of the control groups. Infected mites were found in the treatment groups but there was no significant difference in the proportion infectedbetween the groupof mitesthat wereexposed to the fungi for 48 h in vitro followed by 48 h exposure in vivo and the group of mites that only had the 48 h exposure in vivo

(ANOVA; F2,12-'ý 11.15, P>0.05; Fig. 3.9). There was however, a significant interaction, as those mites exposed to the mixed strains of M. anisopliae for 48 h in vitro prior to exposure for 48 h in vivo showed fewer infections than those mites exposed to the fungus for only 48 h in vivo (F2,12ý-- 11.15, P=0.002; Fig. 3.9).

Overall, B. bassiana infected a significantly greater number of mites than M anisopliae (IM1386697) when compared in different formulations (F2,12= 9.93, P=0.003;

infection (F, 12 Fig. 3.10). But formulationalso had a significanteffect on the level of ,

24.42, P<0.001; Fig. 3.10). Unformulated, dry conidia were significantly more infectious than conidiaformulated in sheepdip excipientor diatomaceousearth for bothM anisopliae andA bassiana(Tukey HSD posthoc test,P<0.05). However,for B. bassiana,the level of infectiousnessremained high when in both sheepdip excipientand in diatomaceousearth, whereasfor M anisopliaethe level of infection observedwith thesetwo formulationswas significantly lower (Fig. 3.10).

In this trial, the ability of M anisopliae and B. hassiana to infect mites on a sheep wasdemonstrated but undervery tightly constrainedconditions.

59 Chapter3- Effects of fungal pathogenson Psoroptesmites

Fig. 3.9. Mean proportion of adult female mites (± S.D. ) showing fungal infection when treatedwith Metarhizium anisopliae (IM1386697),a mixture of M. anisopliae isolates

(ARSEF3297 and ARSEF4556) or an isolate of Beauveria bassiana(CSLI) either for 48 h in vivo only (open squares)or 48 h in vitro followed by 48 h exposurein vivo (solid circles).

1.0

0.9 0 0.8 0) C., 0.7 U) C, E 0.6 0 0 0.5 t 0 0. 0.4 0.2

ccC, 0.3 2 0.2

0.1

0.0 II M. anisopliae M. anisopliae B. bassiana (IM1386697) (mixed strains)

Fungal pathogen

60 Chapter3 -Effects of fungal pathogenson Psoroplesmites

Fig. 3.10. Mean proportion (± S.D. ) of adult female mites showing fungal infection when treatedwith either Metarhizium anisopliae (solid circles) or Beauveria basslana(open squares)in vivo when formulated as dry spores,in sheepdip excipient or with diatomaceous earth.

1.0

0.9

,a0.8 a)

Ju 0.7 c 0.6

0.5 t 0 CL 0.4 0 I- CL 0.3 C

0.2

0.1

0.0

Unformulated Sheepdip Diatomaceous dry conidia excipient earth

Formulation

61 Chapter3- Effects of fungal pathogenson Psoroplesmites

3.2.9 Thepathogenicity of Metarhiziurn anisopliaeand Beauveria bassianaagainst

Psoroptesovis in variousformulations in vitro

It was consideredthat one of the problems in in vivo trials I and 2 may have been associatedwith the formulation of the conidia, which might have preventedpenetration to the skin surface. Therefore an in vitro bioassaywas designedto examinethe effects of formulation on the pathogenicity of fungal pathogens.

Materialsand methods

Conidia of Metarhiziumanisopliae IM1386697 (French strain) were suspendedin various excipientsat a concentrationof Ix 10' conidia ml*'. The cxcipientsused were: vegetable oil (rapeseedoil, Sainsburys,UK), Codacideo,Outputý and sunflower oil

(Sainsburys,UK). Codacideois a commerciallyavailable oil-based adjuvant, containing rapeseedand emulsifiers (Microcide Ltd. U.K.; Jenkinsand Thomas, 1996). It is designedto encapsulatethe particlesof the active ingredient,improving the solubility of hydrophobic compoundsin waterand also improvingthe spreadof the active ingredient. Outpue is also a commerciallyavailable oil-based adjuvant consisting of 60% mineral oils and 40% non- ionic surfactants(Syngenta Crop ProtectionLtd. Switzerland). Each excipient,with and without fungal conidia, as well as a control group of no treatment,was tested for its pathogeniceffects by inoculating4 replicatesof 20 adult femalemites using the standard methodas describedpreviously. The miteswere then incubatedin the standardincubation chambers(Fig. 2.4) at 30 'C and 90% r.h. with 500 gi of sheepserum. The chamberswere inspectedevery 24 h for the presenceof deadmites which were removedwith a fine grade paint brushand checked for signsof infectionas previouslydescribed.

The experimentwas repeatedreplacing M. anisopliaewith A bassiana(CSL strain

1). The concentrationused for the conidialsuspensions was the same,Ix 10' conidiami".

Again, 20 adult femalemites were inoculatedusing the samemethod as beforebut this time therewere 5 replicatesfor eachgroup.

62 Chapter3- Effects of fungal pathogenson Psoroplesmites

Results

The highest proportion of infected mites was found in the group treated with M anisopliae conidia suspendedin vegetable oil where all of the mites became infected (Fig.

3.10). A high proportion of infection (0.99 d: 0.03) was also achieved with M anisopliae suspendedin Codacide'D(Fig. 3.11). The groups treated with M. anisopliac suspendedin

Outpue or sunflower oil had a lower proportion of infected mites at only 0.29 (± 0.125) and

0.25 (± 0.21) respectively (Fig. 3.11). There were no infected mites in the control group that received no treatment or any of the groups that were exposedto excipient only. The effect of treatment on the number of mites infected was highly significant overall (ANOVA; F8,2s=

109.03, P<0.001) but there was no significant difference in the number of infected mites when exposedto M anisopliae in vegetableoil and those mites exposedto M. anisopliae in

Codacidel) (Tukey HSD; P>0.05; Fig. 3.11). There was also no significant difference between mites exposed to M anisopliae suspendedin Outpue and those mites exposedto

M anisopliae suspendedin sunflower oil (P > 0.05; Fig. 3.11). Each group of mites that were exposed to M anisoplide showed significantly higher levels of infection than the groups of mites exposedto excipient only or no treatment (P < 0.05).

When the experiment was repeated with R bassiana suspended in the various formulations, surprisingly a much lower number of infections was observed compared to when M anisopliae was used (Fig. 3.12). Again, there were no infected mites for those exposed to excipient only. The highest proportion of infected mites (0.06 ± 0.065) was achievedwith B. bassiana suspendedin sunflower oil. All of the other treatmentscontaining

B. bassiana conidia had a mean proportion of 0.01 (-± 0.02), infected mites. There was a significant difference in the number of infected mites between the different treatmentswhen the controlswere included (ANOVA; F8,36= 2.94, P=0.012). However,it was only the mites exposedto A bassianasuspended in sunflower oil that had significantly greater numbersof infectedmites than the control group and the excipient control group (Tukey HSD; P=0.0 16;Fig. 3.12).

63 Chapter3- Effcctsof fungalpathogcns on Psoroptesmites

There was no significantdifference in the numberof infectedmites betweenthe groups that were exposedto B. basslanain any of the formulations (P = 0.07; Fig. 3.12).

64 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.11. Proportion of adult female mites showing infection after exposureto Melarhizium

109 anisopliae conidia suspendedin various formulations at a concentrationof Ix conidia females. There ml-1at 30 'C and 90% r. h.. Each data point representsI replicate of 20 adult were 4 replicates usedfor eachtreatment. Untreatedand excipient only controls are not shown.

1.0 .

0.8

0- 0.6 E 4- 0 S c 0 %E S 0CL 0.4 0 I- S IL S S 0.2 S S S

0.0 III Vegetable oil Output Coclacide Sunflower oil + coniclia + coniclia + coniclia + coniclia

Formulation

65 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.12. Proportion of adult female mites showing infection after exposureto Beauveria

bassianaconidia suspendedin various formulations at a concentrationof Ix 109conidia mi" females. There at 30 T and 90% r.h.. Each data point representsI replicate of 20 adult

were 5 replicates used for eachtreatment. Untreated and excipient only controls are not

shown.

0.16 I

0.14

0.12

*a

JQRI 0.10 .

0.08 0 c 0 E 0 0. 0.06 0 I- CL IIII

0.04

0.02

0.00 Vegetable oil Output Coclacide Sunflower oil + coniclia + coniclia + coniclia + coniclia

Formulation

66 Chapter3- Effectsof fungalpathogens on Psoroplesmites

3.2.10 Thepathogenicity of Metarhiziumanisopliae and Beauveriabassiana in various formulationsin vivo (Trial 4)

The fourth in vivo trial was designedto examinethe effects of formulationand fleecelength on the pathogenicityof fungalpathogens on the hostunder the sameconditions asused in in vivo trial 3.

Materialsand methods

Ten pathogenand insecticidefree, poll Dorset sheepwere used. The sheepwere maintainedindoors as a single group, on straw in approvedcontainment facilities, at the

CSL. Six circular rubber arenas,as used in the previous in vivo trial, with an intemal diameterof 25 mm and height of 10 mm were attachedto the dorsal surfaceof the sheep using surgical glue. Twenty four hours after the arenaswere attached,approximately 25 adult,female P. ovis miteswere placedinto 58 of the arenas.The remainingtwo arenashad no mitesplaced in them. Therewas no pre-exposurein this trial.

The Psoroptesmites were obtainedfrom experimentallyinfested sheep the day beforethe trial and maintainedin standardincubation chambers with 500 ýdof sheepserum and incubatedat 30 'C and 90% rh. Seventytwo hoursafter the mites had beenplaced into the arenas,the sheepskin enclosedwithin eacharena received a doseof oneof the following treatments. Six arenaswere treated with 0.1 ml of M. anisopliae in CodacideID at a concentrationof Ix 108conidia ml-', anothersix were treatedwith 0.1 ml of M anisopliae in sunfloweroil at a concentrationof Ix 109conidia ml", anothersix weretreated with 0.06g of M anisopliaewith 0.001gof diatomaceousearth and anothersix were treatedwith 0.06g of unformulatedM. anisopliae(Table 3.4). Thesetreatment groups were repeatedfor B. bassiana,so that, six arenaswere treatedwith 0.1 ml of B. bassianain Codacideoat a concentrationof Ix 108conidia ml-', anothersix weretreated with 0.1 ml of B. bassianain sunfloweroil at a concentrationof Ix 109conidia mi", anothersix weretreated with 0.009g of B. bassianawith 0.001gof diatomaceousearth and anothersix were treatedwith 0.009g of unformulatedB. bassiana(Table 3.4). In 10 of the arenas,no treatmentwas appliedto

67 Chapter3- Effects of fungal pathogenson Psoroplesmites provide controls. For eachtreatment, as well as the controls,half of the arenashad fleece that had beensheared down to the skin beforethe additionof the mitesand half of the arenas had fleecethat had beensheared to approximately10 mm in length. This was doneto test for any effectsthat fleece length may have on the pathogenicityof the fungi. Therefore, there were 3 replicates of each treatmentat each fleece length. All treatmentsare summarisedin Table3.4.

Once the treatmentshad been applied,the arenaswere sealedwith a fine grade cottonmesh held in placewith a rubberwasher. The day that the treatmentswere applied to the sheepwas recordedas day 0. The arenaswere left untouchedfor 4 daysafter which tile sheepwere euthanizedand the skin containingthe arenaswas excised. The skin, with the arenasattached, were placedinto sealableplastic bags and refrigeratedover night. The next day (day 5), all visible mites were removedfrom the arenawith a paintbrushand forceps beforeremoving the arenaitself washingthe skin with distilled waterto try and removeany moremites that werenot visible upon first inspection.The washwas filtered througha fine gradenylon cloth to catchall stagesof mites. The fleecewas inspectedone more time after being washed. Different paint brushesand forcepswere usedfor the controlsand the also for the different speciesof fungi. The brushesand forcepswere washedin 70% ethanol betweentreatments.

All mites were surface sterilised and live mites were placed into the standard incubationchamber (Fig. 2.4) while dead mites were treated as previously describedin

Chapter2. Chamberswere given 500 RI of sheepserum and incubatedat 30 'C and90% r. h. and were checkedevery 24 h for the presenceof dead mites which were removedand checkedfor signsof infectionas describedpreviously,

To confirm the pathogenicityof the fungal formulations,each of the treatments appliedto the arenason the sheepwere also usedto inoculatemites in vitro. The standard method(as previouslydescribed) was usedto inoculate20 adult femalemites with the same concentrationsand amountsof eachof the treatmentsapplied to the sheep.Tile miteswere then movedto cleanchambers with 500 [LIof sheepserum and incubatedat 30 OCand 90%

68 Chapter3- Effects of fungal pathogenson Psoroplesmites r.h. The chamberswere checked5 daysafter inoculationand everyday afterwardsto check for the presenceof deadmites which were removedand checkedfor infectionas previously described.There were three replicates of eachtreatment.

Table 3.4. Summary of the fungal pathogens,excipient, concentration,amounts and the lengthof fleecewithin the arenaused in the secondarena based in vivo trial.

Fungal Species Fleece Excipient Amount Concentration length applied M anisopliae Short Codacide' 0.1 ml Ix 10' conidia ml" M anisoplide Short Diatomaceous 0.06g + Ix 109conidia 9" earth 0.001g M anisopliae Short Sunflower oil 0.1 ml I xIO' conidia ml*' M anisopliae Short Dry conidia 0.06g I xIO' conidia g" M. anisopliae Long Codacide" 0.1 ml Ix 10' conidia ml*1 M anisopliae Long Diatomaceous 0.06g + I xl 09conidia gý' earth 0.001g M anisopliae Long Sunflower oil 0.1 ml I x109 conidia ml"' M anisopliae Long Dry conidia 0.06g I x109 conidia g7l B. bassiana Short Codacide'r 0.1 ml Ix 108conidia ml-1 B. bassiana Short Diatomaceous 0.009g + I xl 09conidia g*1 earth 0.001g B. bassiana Short Sunflower oil 0.1 ml I x109 conidia ml" B. bassiana Short Dry conidia 0.009g I xI 09conidia gý' B. bassiana Long Codacides 0.1 ml Ix 108conid ia ml" B. bassiana Long Diatomaceous 0.0099+ 1 x109conidia g7l earth 0.00 1g B. bassiana Long Sunflower oil 0.1 ml I xl 09conidia mi" B. bassiana Long Dry conidia 0,009g I xl 09conidia g" None Short None Long

69 Chapter3- Effectsof fungalpathogens on Psoroplesmites

Results

The proportion of adult female mites infected and the proportion of adult female

mites surviving the 5 day in vivo period, were each analysed individually using 3-way

ANOVAs with fungal species,formulation and fleece length as the independentfactors each

time.

When the controls are excluded from the analysis, formulation had a significant

effect on the proportion of adults infected (F3,32= 7.46, P=0.001) as those mites exposedto

conidia formulated in Codacide" had a lower proportion of infection than all other

formulations (Fig. 3.13). There was no significant effect of fungal speciesor fleece length

on the proportion of infected mites and there were no significant interactions.

The species of fungus had a significant effect on the proportion of adult females that were alive when removed from the arenas (Fl = 5.56, P=0.025) with a lower proportion , 32"

of mites surviving the 5 day period in vivo when exposedto M. anisopliae (Fig. 3.14). There was also a significant effect of formulation on the proportion of adult femalessurviving the 5

day in vivo period (F3 = 9.76, P<0.001; Fig. 3.14). A f'I lower proportionof , 32 signi icantly mitessurvived the 5 day in vivo periodwhen exposed to diatomaceousearth than any of the othertreatments (P < 0.002),which did not differ significantlyfrom eachother (P > 0.05).

70 Chapter3- Effectsof fungalpathogens on Psoroplesmites

Fig. 3.13. Meanproportion (± S.D. ) of adult femalemites showing infection after 5 days exposure,in vivo, to Metarhiziumanisopliae (solid circles),Beauveria bassiana (open squares)in various formulations and no treatment (solid triangle).

1.0

0.8

42

E 0.6 te-0 m 0 %E 00. 0h. CL 0.4

2

0.2

0.0

Control Coclacide Diatomaceous Sunflower oil Unformulated + coniclia earth + coniclia dry coniclia + coniclia Formulation

71 Chapter3- Effects of fungal pathogenson Psoroplesmites

Fig. 3.14. Meanproportion of adultfemale mites that wererecovered alive from the arenas after exposureto Metarhiziumanisopliae (solid circles),Beauveria bassiana (open squares) and no treatment (black triangle) in vivo for 5 days.

1.0

0.8

0 0

0.6

0 0.4 c 0 0 CL 0 CLI- 0.2 c

2 0.0

Control Coclacide Diatornaceous Sunflower oil Unformulated + coniclia earth + coniclia dry coniclia + coniclia

Formulation

72 Chapter3- EfTectsof fungalpathogens on Psoroplesmitcs

3.3 Discussion

The key result presentedin this chapteris the demonstrationthat undercontrolled conditionsit is possiblefor the fungal pathogensM anisopliaeand B. bassianato infcct A

ovis in vivo, This and previousstudies have demonstrated the efficacyof fungal pathogens to P. ovis in vitro, but wereunable to achieveinfection of the miteson a live host. By using

rubberarenas attached to the dorsalsurface of the sheep,the presentstudy was able to induce

lethalinfections in P. ovis at a level comparableto previousin vitro work.

Severalstudies have investigatedthe use of M anisopliaeand A bassianaagainst induced veterinary ectoparasitesin vivo. AIx 108conidia ml*' suspensionof M. anisopliae

mortalities of 30 and 37% in the adult ticks R appendiculatusNeumann and A. variegatun?

Fabricius, respectively, as they fed on the ears of rabbits (Kaaya et al., 1996). In the same

study (Kaaya et al., 1996), aIx 108conidia ml-1 suspensionof B. bassianawas responsible A. for a mortality of 34% in adult R appendiculatus, although it failed to kill any A bassiana variegatum. In field trials, Ix 10' conidia ml-1suspensions of M anisopliae and

causedmortalities of 83% and 77% respectively when applied to R appendiculatusnaturally

infesting zebu cattle (Kaaya et al., 1996). In vitro experimentsconducted by Maranga el al. bassiana (2005) showedthat 100% mortality can be achievedwith both M anisopliae and B. levels againstA. variegatum. Metarhizium anisopliae has also been shown to induce high of

infection in the cattle louse, Bovicola bovis (L. ) both in vitro and in vivo (Briggs et al.,

2006); 71% of lice exposed to aIx 108conidia ml" suspensionof M anisoplide in vitro

becameinfected as did 73% of lice exposedto the sameconcentration in vivo (Briggs et al.,

2006).

The first in vivo trial conducted in the present study with a Danish strain of M. from anisopliae found only 2 infected mites out of 291 recovered. Both of these mites came

the samesheep in a wash sampletaken 14 days after application of M anisopliae. Hence,an

in vitro trial was set up to assessthe relative pathogenicity of the isolate of M anisopliae

used in this in vivo trial in comparison with another isolate (the French strain). The Danish

73 Chapter3- Effectsof fungalpathogens on Psoropiesmites

isolatewas much lesspathogenic than the Frenchisolate with proportionsof infectedmites

of 0.38and 0.91 respectively.Hence it would appearthat the reasonfor the lack of infected

mitesfound could havebeen that the isolateused was not as pathogenicas other isolatesof

M. anisopliae.

The secondin vivo trial used the French strain of M. anisopliaerather than the

Danishstrain and also tried a differentapproach with the methodof exposure.As with the

previoustrial, the potentialfor M anisopliaeto treatan establishedinfestation was tested but

also its potential to act as a preventativetreatment. However, no infected mites were

recoveredfrom any of the sheeptreated with the fungal pathogen.Possible explanations for

this were that the temperatureat the skin surfacemay have beentoo hot for the conidiato

germinateand infect the mites. All of the sheepwere housedindoors and the trial was run

duringthe summer.

Severalstudies have shownthat grasshoppersinoculated with M. anisoplideand B.

bassianashow reduced levels of mycosiswhen allowedto baskat temperaturesof 35 - 40

'C (Inglis et al., 1996). It has been suggestedthat grasshoppersand locusts induce a

behaviouralfever, raising their body temperatureto 42 'C in responseto fungal infection

(Blanford et al., 1998). The increasedbody temperaturebecomes too hot for the fungal

pathogen,inhibiting its growth within the insect and thus increasingthe survival of the

infected individual (Inglis et al., 1996,1999; Blanford et al., 1998). It is not known whether

or not behavioural responsesmay be inducedby fungal infection in A ovis. However, it may not be necessaryto induce a behavioural responsesuch as basking in P. ovis if the body temperatureis already high enough to inhibit the infection process.

Another explanation for the lack of infection seen in the in vivo trials was consideredto be the possibility that there is something present on the skin or fleece of sheep that might inhibit the action of the conidia. The skin secretions consist of two main components;the water insoluble wool wax and the water soluble suint. The wool wax, also referred to as lanolin, is made up of many different compounds including fatty esters

(branchedand unbranched),free alcohols and alkanols, sterols and ceramides(Alzaga et al.,

74 Chapter3- Effects of fungal pathogenson Psoroplesmites

1999; Eychenne et al., 2001; Coderch el al., 2004). Suint consists of a complex mixture of

metallic ions, peptides, weak bases and organic acids such as acetic and propionic acid

(Deaneand Truter, 1955). Some skin secretionscan inhibit the adherenceof fungal sporesto

epidermal cells (Meyer et al., 2001). It has been shown that saccharideresidues that inhibit

the binding of fungal sporesto epidermal cells are present in the skin secretionsof sheepand

goats (Meyer et al., 2001). It might be that these chemicals are able to inhibit the action of

M. anisopliae and B. bassianaagainst P. ovis in vivo.

The in vitro sheepskinbioassay was designedto test whether or not there was any

intrinsic inhibition by the skin and fleece. The results show that 52% of mites exposedto

sheepskinand fleece treated with M. anisopliae for 24 h, becameinfected. For mites kept in

chamberswith fungal treated cloth 34% of mites became infected. Although the difference

was not significant, the lower level of infection found in the mites exposedto cloth treated

with the fungal pathogencould be due to the ability of the mitesto escapethe pathogenby

climbing on the walls and lid of the chamber. The mites exposedto the pathogenon

sheepskinhad less free space in the chamber. Clearly therefore, it was not intrinsic

inhibition by fleececomponents that inhibitedinfection in the first two in vivo trials.

The temperatureat the skin surfaceof a sheephas been estimated to be between31 -

37 IC (Wall et al., 1992). It hasalready been shown that the Frenchisolate of M anisopliae,

also used here, can infect a high percentage(48%) of mites in vitro at 37.5 'C (Brooks,

2004). Bioassayswere conductedin the presentstudy using isolatesof M anisoplidethat had beenisolated from insectsinhabiting hotter climatesand thus would be expectedto be more tolerant of higher temperatures. However, none of these isolates showedany pathogenicitytowards P. ovis whentested at 37 IC; hencethe Frenchisolate would appearto be unusual.

The strain of B. bassianathat was isolatedfrom a Psoroplesmite removedfrom a sheepat the CSL was foundto be highly pathogenicagainst P. ovis in the in vitro trials with

97% of mites becoming infected at a concentrationof Ix 105 conidia ml*'. At a concentrationof Ix 10' conidiaml" all exposedmites became infected. However,the initial

75 Chapter3- Effects of fungal pathogenson Psoroptesmites

bioassayto assessthe pathogenicityof B. bassianausing aIx 108conidia ml" suspension

only caused58% of mites to becomeinfected. The ability to produceconidia with a

consistentlevel of pathogenicityis an importantfactor if enoughappropriately pathogenic

conidiaare to be producedto makefungal pathogensa viable alternativetreatment for scab.

Theuse of fungalpathogens as a treatmentfor scabwill be discussedlater in the chapter.

A third possibilityfor the lack of infectedmites found in the earlierin vivo trials was

that the fleece was acting as a physical barrier preventing the conidia from coming into

contact with the mites. The conidia of the fungal pathogen were applied onto the sheep

either in suspension or as dry, unformulated conidia. The conidia would have to pass

through the fleece to the surface of the skin where the mites are. The scab itself may also act

as a barrier.

Given that the in vitro work suggestedthat fleece was likely to have had no, or little

effect, on the pathogenicity of M anisopliae, a third in vivo trial was planned. However,

rather than simply trying to treat a scab infested sheep as with the previous trials a more

controlled approachwas used. By using rubber arenasattached to the surfaceof the sheepit was possible to contain a known number of mites in vivo for a set period of time. This method also allowed for a controlled exposureto the fungal pathogensand ensuredthat the mites came into contact with the conidia as the fleece had been sheared. The double

exposureto conidiathat somemites were given was usedso that somemites would already be infectedwith the fungi prior to beingplaced on the sheep.This wasdone so that possible points of inhibition could be identified. If the mitesthat had the doubleexposure failed to show signs of infection it would have demonstratedthat inhibition was due to something inherentto the sheepskin environment.

There was no significant differencebetween the proportionof mites infectedfrom the groupsthat had the doubleexposure and the groupsthat were only exposedto the fungi in vivo. This thereforedemonstrates that mites are able to acquirelethal infectionswhen exposedto conidia on a live animal. Mites must havebeen infected internally as they were surfacesterilised on removalto kill off conidia presenton the externalcuticle. However,

76 Chapter3- Effects of fungal pathogenson Psoroplesmites

mites were only on host for 48 h so signs of mycosis were only seen after they were

removed. No mite with external hyphaewas ever found on a live sheep. Hence, it is still not

known whether or not the mites that became infected on the host would have developed

lethal mycosesif they had remainedon the host.

The two new strains of M. anisopliae tested in the third in vivo trial were mixed

togetherto minimisethe numberof arenasthat would be used. Undertile guidanceof CABI this seemedto be acceptablemethod. If relativelyhigh levelsof infectionwere obtained, tile two strainscould be testedseparately at anothertime. A high level of infectionwas obtained exceptfor the groupsexposed to the doubleexposure regime, where an inexplicablylow

level of infection was seen. However,all fungal strainstested performed well and so no further investigationswere carriedout on eitherof the mixed strains. However,mixing two or more fungal strainsmay not be the bestapproach to use as it could be possiblethat any infectionsfound arethe resultof only one of the strains. The additionof anotherstrain that maybe of lower virulenceor pathogenicitywould dilute the effect seen.

In the fourth in vivo trial (the secondtrial to utilise the rubberarenas) mites were exposedto conidiafor 5 dayson a host. Despitethe longertime on host,there were still no mites that showed signs of advancedinfection when removed from the arenas. A significantly greater proportion of mites exposed to conidia formulated in diatomaceous earth were dead on removal than for any other formulation. Unfortunately, due to the experimental design, it is not possible to determine if this effect was due to an interaction betweenthe conidia and the diatomaceousearth or the diatomaceousearth alone. Also, care needsto be taken when interpreting the data, as a lower number of mites were removed from the arenastreated with diatomaceousearth. There were also fewer mites recoveredfrom the arenasthat containedM anisopliae. This can be explainedby the physicalnature of the fungal treatment.The formulationscontaining M. anisoplidewere muchdarker and thicker than the B. bassianaformulations making it much harderto find and recoverthe mites.

Althoughthe differencein numberswas taken into accountby the statisticaltests, the effect

77 Chapter3- Effectsof fungalpathogens on Psoroplesmites

of treatmenton the numberof mites recoveredmay well have been due to the physical

propertiesof the mediumin which the funguswas formulated rather than its pathogenicity.

In both of the in vivo trials that usedthe rubberarenas, there was a significanteffect

of formulationon the proportionof infectedmites. The first of thesetrials found that the

conidiaformulated in diatomaceousearth induced the lowest level of infection. The second

of the arenabased trials found a high proportionof infection in the mitesexposed to conidia

in diatomaceousearth. A possiblereason for the increasedmortality of the mitesexposed to

conidiain diatomaceousearth could be dueto the actionof the earth. Diatomaceousearth is

an inert dust which, when applied to an arthropod,adheres to the cuticle and causes

desiccation(Ebeling, 1971). It could be that the action of diatomaceousearth requiresa

longerexposure period to affect enoughdamage to the mite for it to becomesusceptible to

infection from the conidia. Until this thresholdis reachedthe diatomaceousearth may

actually hinder the conidia by binding to the cuticle of the mite and occupyingpotential

binding sitesof the conidia. The conidiaof B. bassianaare muchsmaller than M anisopliae

and may not be so stronglyaffected by the decreasedsurface area. This may well explain

the differentresults between the two trials.

Otherstudies have found that whendiatomaceous earth is combinedwith pathogenic

fungi to control storedproduct pests,greater mortality is achievedat higher temperatures,

providedthe lethal limit of the fungi is not reached(Lord, 2005; Michalaki et al., 2006;

Vassilakoset al., 2006; Michalaki et al., 2007). A study looking at the effects of diatomaceousearth and B. bassianaon the lessergrain borer,Rhyzopertha dominica (F. ) and the rice weevil, Sitophilus oryzae (L.) found that the diatomaceousearth causedgreater mortality at higher temperatures,although B. bassianawas more effectiveat 26 OCthan 30

T (Vassilakoset al., 2006). A similar study comparingthe use of Silicoseceand M. anisopliae,alone and in combinationfound that bothwere more effective in killing larvaeof the confusedflour beetle,Tribolium confusumdu Val at higher temperaturesand that M. anisopliaeachieved the greatestmortality when mixed with Silicosec4D(Michalaki el al., 2006).

78 Chapter3- Effects of fungal pathogenson Psoroptesmitcs

The exact mechanismby which diatomaceousearth acts a dessicantis unclear. A recentstudy examiningthe effectsof threecommercially available diatomaceous earths on

A. siro showedthat althoughthe particlesof the diatomaccousearth had removedcertain cuticularhydrocarbons, there were no signsof abrasionon the mite cuticlewhen examined by scanningelectron microscope after 72 h of exposure(Cook et al., 2008). Despitetile conflicting resultsfrom the two arenabased trials, diatomaceousearth could be an effective mediumfor the formulationof fungalconidia.

The third and fourth in vivo trials showedthat under controlled conditionsthe temperatureat the skin surface of a sheepdoes not inhibit M. anisopliae or B. bassianafrom infecting P. ovis. There is also no inhibition due to any substancespresent on the skin and fleece on the infectivity of the fungal pathogens. However a mite showing signs of infection i. e. a cadaver with conidia or hyphae growing on its surface has not yet been observed in vivo. It may be that mites do not develop mycosis while on the host or it may be that infection develops initially but is then suppressedwithin a short period and thus was not observed in the trials. Alternatively, it may be that infected mites that did develop mature mycosis either decayedrapidly or were lost off the sheepor were otherwise not recovered.

The effects that the fungal pathogenshave on exposed mites prior to death are not known in vivo. In the fourth in vivo trial, live protonymphswere found at the end of the 5 day in vivo period. Larvaeand eggswere also present. This suggeststhat ovipositionand subsequentdevelopment had taken place in the presenceof the fungal pathogens.More work is thereforerequired to find out how the reproductiveoutput and the developmentof juvenile stagesare affectedin vivo. If the fungal pathogendoes not kill or debilitatethe mite, allowing it to continueto developand oviposit,the advantagesof using fungi may be limited. If the reproductiveoutput of the femalesis largely unaffected,along with the subsequentdevelopment of the juvenile stages,the mite populationwill still be able to increase. The scabwould spreadacross the body of the sheepas the infection rate of the fungalpathogen in the miteswill struggleto keeppace.

79 Chapter3- Effects of fungal pathogenson Psoroplesmites

A further complication facing the use of fungal pathogensas a control for sheepscab is the problem of formulating the conidia. Several formulations were tested for their pathogenicity against Psoroptesboth in vitro and in vivo. As has been demonstratedwith the diatomaceousearth formulations, the results can be very variable. A formulation that may be suitablefor R bassianamay not be suitablefor M. anisopliaeand vice versa. In vitro bioassaysshowed that fewer mites became infected when exposedto M. anisopliae suspendedin Outpue and sunfloweroil (29% and 25%, respectively)than when suspended in vegetableoil or Codacide'D(100% and 99%, respectively).Mites exposedto A bassiana however,showed very low levelsof infectionregardless of formulation. The highestlevel of infection was found from conidia suspendedin sunfloweroil (6%). However,when mites were exposedto conidia in Codacidevand in sunfloweroil in vivo, much greaterlevels of infectionwere seen.

In the last two in vivo trials the fleecewas shornto under20 mm in length. This ensuredcontact of the treatmentswith the mites. However,on a scabinfested sheep, the fleecewill be longer,approximately 100 mm. This can act as a physicalbarrier preventing the conidia from reachingthe mites. A suitableformulation and applicationmethod will needto be devisedthat not only takesinto accountthe potentialeffects on pathogenicityand viability of the conidia but also deliversthe conidiato the skin surface. The initial in vivo trials may have failed to producea significantnumber of infectedmites becausethe mites were simply not exposedto enoughconidia.

The inconsistencyseen in the resultsof this chapterare a clear indicationof how manyvariables and problemsare facingthe developmentof a commerciallyavailable fungal treatmentof sheepscab. This will be consideredfurther in Chapter6.

80 Chapter4- Life historyand population dynamics of Acarussiro

CHAPTER4

LIFE HISTORY AND POPULATION DYNAMICS OF

ACARUSSIRO

The primary aim of this thesiswas the studyof the biologicalcontrol of sheepscab and its causative agent, P. ovis. However, as an obligate parasite, this mite is particularly difficult to use in long-term in vitro studies and in vivo studies are also difficult, expensive and compromise animal welfare. Therefore in vivo studies need to be used sparingly. It was decidedthat when attemptingto investigatethe transmissionof entomopathogenicfungi throughpopulations, to usean alternative,model mite species,Acarus siro.

Acarus siro sharessome key similaritieswith P. ovis that make it a useful model species(more detail is provided on A. siro in the following sections). Both speciesof mite are astigmatic and therefore potentially very susceptible to infection with the fungal pathogensused in this study. The life-cyclesof both mites are also very similar with the same number of life stages. However, an important difference is that A sir'Oare free living and relatively easyto rear under laboratoryconditions that closely resemblethe conditions that the mites are naturally found in. This meansthat the work presentedin Chapter5 will be ableto showthe effectsof a fungalpathogen on a healthypopulation of mites.

4.1 Introduction to Acarus siro

The astigmatid flour mite, A. siro is a major pest of grain stores (Solomon, 1946;

Griffiths, 1962;Cunnington, 1965; LAM el al., 2007).It is distributedacross Europe, North

America, Russia and Australia (Cunnington, 1985). The range of the mite is limited to regionsof temperateclimate; the developmentof the mite requirestemperatures of between

81 Chapter4- Life historyand population dynamics of Acarussiro

4 and 32 'C, dependingon relative humidity (Solomon, 1962; Cunnington, 1965).A study of

514 storage units from 147 grain stores in the Czech Republic between 1996 and 1998, showed that 67% were infected with pest mites (LukAg et al., 2007). It is easily cultured and makesa good model species.

4.1.1 Life-cycle

Adult females are morphologically the largest stage. They are approximately 0.5 mrn in length with a rounded appearance. The adult males are slightly smaller and flatter and can also be distinguished by a spur on the trochanter of each fore leg. Tile adult mites have a pale yellow/brown body with dark brown legs. Juvenile stagesare paler and lack the brown colouration in the legs.

There are 5 stagesin the lifecycle: egg, larva, protonymph, tritonymph and adult.

The juvenile stages,apart from the egg, show two phases;an active phaseand a resting

(moulting) phase. The active phaseis the period that the mite is mobile and feeding. The restingphase occurs before the mite moults into the next stageof the lifecycle. It can be distinguishedby the changein behaviouras the mite becomesstill and the distancebetween the anteriortwo pairs of legsand posteriortwo pairsof legs increasesand the distal legsare tuckedunder the body. The mite alsobecomes much more round in appearance.The resting phase,during which apolysisoccurs, usually lasts 48 hours(Cunnington, 1984). At ecdysis the mite then moults into the active phaseof the next stagein the lifecycle. Under certain conditionsit is possiblethat a sixth stagein the lifecyclecan occur. This is the formationof a deutonymph,also known as the hypopus,between the protonymphand the tritonymph stages(Solomon, 1962; KnOlle, 1987;Corente and KnOlle, 2003). The hypopusis a stagein the lifccycle that is physiologicallysuited for dispersalor for resistingconditions that arenot suitablefor continueddevelopment and is particularly common in the astigmata(Knalle,

1987;Corente and KnOlle,2003). However,the productionof a hypopusis very rare in A. siro and not seenat all in P. ovis (Knalle, 1987;Sanders, 2000). Althoughhypopi have been observedand induced in other astigmaticspecies, less successhas been achievedwith

82 Chapter4- Life history and population dynamics of Acarus siro

attemptsto inducethe formationof hypopi inA siro (KnOlle, 1987). Experimentsdesigned

to inducehypopus formation in A siro rearedon poor quality food haveonly sporadically the development hypopus. A hypopusforming yielded of a line of .4. siro has not been establishedas it has been for other astigmaticmites. It may be that the predominant

conditions under which A siro has evolved have not applied strong enough selection

pressuresto retainformation of hypopi(KnUlle, 1987).

Several studies have previously been conducted investigating the life history and

populationdynamics of A. siro (Cunnington,1985; Kawamoto et al., 1991;2dirkova and

Voracek, 1993;Fejt and Zdarkova,2001). However,these studies show a large degreeof

variation in the developmenttimes of the lifecycle stagesand also in the conditionsused to

rear the mites, sincethe developmentof A. siro is highly dependanton the environmental

conditions,particularly temperature and relativehumidity. Under favourableconditions the

completedevelopment can occur in 14days (Fejt and2dArkovA, 2001).

4.1.2 Effects of infestation

Acarus siro infests a wide rangeof food stuffs such as grain, cheeseand various

cereal-basedproducts found in the supermarketand people's homes(Thind and Clarke,

2001). An infestation of mites can cause considerableeconomic loss through the

consumptionof and damageto the food and the treatmentnecessary to removethe mites

(Dunn et al., 2005). Mites infesting grain storesfeed on the wheatgermof the grain, removingit completelybut leavingthe rest of the grain intact (Solomon,1946). The grain hasto be damagedfor the mitesto gain accessto the germas they are unableto penetratethe grain-coat,however, usually less than 10%of storedgrain is undamaged(Solomon, 1946).

The mites also havethe potentialto causeskin and respiratoryproblems in humans and animals. Severalstudies have shownthat extractsfrom storagemites includingA. siro can induceallergic reactionswhen administeredto humansin a skin prick test (Calvo el al.,

2005; Ruoppi, et al., 2005; Vidal et al., 1997).

83 Chapter4- Life historyand population dynamics of Acarussiro

A study in Chile demonstrateda prevalenceof 56% of child asthmasuffcrcrs rcacted to extracts of A. siro (Calvo et al., 2005). A study of university employees involved in animal handling, showed that 14 out of 40 people tested (35%) were sensitive to storage mites (Ruoppi et al., 2005). Of those 14 people scrisitised to storagemites, only one person tested positive for a reaction to house dust mites, suggesting that it is not cross-reactivity causing false positives. Even dogs show sensitisation to A. Siro extracts when injected intradermally; 28% of atopic dogs tested showed positive reactions (Bensignor and Carlotti,

2002). Howeverthese studies were conducted only on peopleor animalsthat wereknown to have respiratory problems or skin disorders at the time of tile trials. There is no evidenceto show that.A. siro or other storagemites were the initial causeof the problem.

A study of the general population of the municipality of A-Estrada in Spain showed that 24.4% of people tested positive for sensitisation to the storage mites Tyrophagus putrescentiae(Schrank) and Lepidoglyphusdestructor (Schrank) even though they had no occupationalexposure to the mites (Vidal el al., 1997). For the people who showed sensitivityto mites (either storagemites or housedust mites), 50% were sensitiveto both typesof mite but 44.6%were sensitiveto storagemites only. Only 5.4% were sensitiveto housedust mitesonly. The studydid not pick test subjectswho werealready suffering from an allergic disorder. It was also found that there was no significant link betweenallergic scnsitisationand profession (Vidal et al., 1997).

4.2 Materials and methods

4.2.1 Longevity,fecundity and developmentof Acarus siro

Previous studies investigating the life history of A. siro, have shown that there can be considerablevariation in the developmenttime, longevity and fecundity under different conditions. The aim of the work presentedin this sectionwas to confirm the development times, longevity, fecundity and mortality rates ofA siro when rearedunder the constant laboratoryconditions to be usedfor laterfungal infectionstudies.

84 Chapter4- Life historyand population dynamics of Acaruss1ro

To measurethe oviposition rates, tritonymplis were collected from culture, placed into individual incubation chambers(Fig. 2.4) and checked every 24 h (Cunnington, 1985).

Once the mites had moultcd and emergedas adults, pairs of adult females and males were placed into incubation chamberswith 0.003g (deemedto be an excess)of food and incubated at 20 OCand 70% r.h. This was recorded as Day 0. Each subsequentday, the pair was checked and carefully removed from the chamber with a paint brush and placed into a new clean chamber with the sameamount of food. This was done so that any eggs that had been laid in the previous 24 h period could be counted. If one of the pair died, the dead individual was replaced with another mite of the samesex, although not necessarilythe sameage. The replacementmite was addedto the chamberso that the remaining original mite was still kept in a pair. No recordings were taken from the replacementmite. The number of eggs laid each day by the females and the day of death for each mite was recorded. In total, recordings were taken from 40 mites of each sex. Adult mites were paired becausea previous study had found that this gave the highest mean egg output per female (Cunnington,

1985).

To determinethe developmenttimes for each stageof the life-cycle, mites from each of the juvenile stageswere taken from the generalculture and placed individually into incubationchambers (Fig. 2.4) with approximately0.003g of food. Daily observationswere made. Mites to be observedwere placedinto chambersduring the life-cycle stageprior to the stageto be examinedand the day that the mitesemerged into the requiredlife-cycle stage was notedas Day 0. The time spentin the active phaseand the restingphase was recorded for eachstage. The day that the mitesentered the restingphase was recordedwhen the mites showedno signs of movementwhen touchedwith a paintbrush. Dead mites could be distinguishedfrom mites in the restingphase by their appearance.In tile restingphase, each mite becomescharacteristically more rounded and the legs curl underneathtile ventral surfaceof the mite (Solomon,1962). Deadmites do not showthe curling up of tile legsand also appeardesiccated. Each stageof the life-cycle was deemedto have endedat ccdysis, i.e. once the mites had completedthe moult into the next life-cycle stage. Eggs for

85 Chapter4- Life historyand population dynamics of Acarussiro observationwere collected from the paired adults. Observationswere taken from 40 mites of each stageof the lifecycle incubatedat 20 T and 70% r. h. Sex was not determined for any of thejuvenile stagessince there are no descriptionsfor morphological differences.

4.2.2 Thepopulation dynamics of Acarus siro

Sampling technique

A technique for sampling laboratory populations was developed and tested for its accuracy before being used to sample the experimental populations. Cuvettes (Fig. 4.1) containing known numbers of mites in 0.3 g of wholemeal flour and yeast in a 3: 1 (Nv/w) ratio were sampled using a capillary tube with an internal diameter of 4 mm which was gently pushed through the culture of food and mites until the tube reachedthe bottom of the cuvette. The content of the capillary tube was then tapped out into a worm egg counting chamber (Improved McMaster, Weber Scientific International Ltd. Teddington, U. K. ). A saturatedsalt solution (NaCl) was then added to the chamber to float the mites up from the food and allow them to collect on the underside of a coverslip that was placed over the chamber. The mites were then countedunder a binocularmicroscope. Each cuvettewas sampled5 timesand the samplespooled. The amountof substrateremoved from the cuvette was recordedeach time a samplewas takenby measuringthe weight of the cuvetteafter the samplewas takenand subtractingit from the weight of the cuvcttebefore the sample.The numberof mites in eachsample with a known weight could then be multiplied to give the numberof mites presentin the cuvette(0.3g). Sampleswere taken from 5 replicatesof 5 cuvettesseeded with 100,200,300,400or 500 mites.

Populationdynamics

To examinethe populationdynamics of A. siro, groups of mites were set up in cuvettes,each containing 0.3 g of wholemealflour andyeast in a 3: 1 (w/w) ratio. Tile initial populationconsisted of 50 adult femalesand 50 adult males,all of a similar age,taken from the samestock culture and placed into the food in the cuvette. The cuvetteswere then sealed

86 Chapter4- Life historyand population dynamics of Acarussiro with non-absorbentcotton wool and incubatedat 20 T and 70% r.h. The cuvcttcswere agitated every 48 h by inverting the cuvette 3 times in quick successionto prevent the food from clumping and to ensure similar conditions were present in all the cuvcttcs. The cuvettes were sampled using the technique described and mites of all life-cycle stageswere counted. Sex was also recorded for the adult mites. Only mites that appearedto be alive were counted. These were mites which showed movement or had no signs of the discolouration of the cuticle or desiccationassociated with death. Over an 80 day period, 5 different cuvetteswere sampledevery 48 h. Once a cuvette had been sampledit was removed from the experiment.

87 Chapter4- Life historyand population dynamics of Acarussiro

Fig. 4.1. Cuvetteused to housepopulations ofAcarus siro into which 0.3 g of wholemcal flour andyeast (3: 1, w/w) wasplaced.

40 mm

) mm 10 mm

88 Chapter4- Life historyand population dynamics of Acarussiro

4.2.3Modelling the population dynamics ofAcarus siro

The populationdynamics of Acarus siro has beenstudied previously, usually with the aim of creatingmodels, yet very few of thesestudies have usedcomplete sets of data.

Hence,the analysispresented here, uses data gathered in the previoussection of this chapter allowing parameterswhich are consistentwith eachother to be used. Thesedata were used to createa simpleLeslie matrix model(Leslie, 1945;Begon and Mortimer, 1981;Wall et al.,

1999)to simulatethe dynamicsof A. siro populations.The aim of using the Leslie matrix approachto modelling was not to createa comprehensivesimulation but rather to allow basic hypothesesto be generatedrelating to expectedmite abundanceagainst which tile experimentaldata could be compared.This approachhas beensuccessfully employed with mite populationspreviously (Wall et. al., 1999).

A Leslie matrix model (Fig. 4.2) consistsof a column vector that describesthe starting age structure of the population at time ti. A suitable transition matrix is then constructedusing the rules of matrix multiplication to create values for the age-structureat time 12. The parametersused in the transition matrix are the survival rates for each stageof the life-cycle and the birth rate. The transition matrix is then multiplied by tile age distribution at t, to give the age distribution at time t2. The values used in the transition matrix for this study are the proportion of mites surviving at each stage of the lifecycle and the number of eggs laid per adult over a6 day period. A time-step of 6 days was chosenfor

duration juvenile 4. (see Results the model as an approximation of the of each stage of . siro section 4.3.1). The stage survival values used for the Leslie matrix model were: 75% for eggs, 65% for larvae, 82.5% for protonymphs and tritonymphs and 51.6% for adults. The

survival value for the adults representsthe survival rate for adult mites of both sexesover a6 day period. The number of eggs laid pcr day per adult female was 1.25, thus the number of

female eggsper time step per adult female mite was 3.75.

Once the model had been created,using tile data gathered previously in this chapter, the model was run with a range of different adult reproductive outputs. The increasesin the reproductive output were made by multiplying the original value by 2,3 and 4 times. To

89 Chapter4- Life historyand population dynamics of Acarussiro checkthe fit betweenthe modelsand the laboratorypopulation an index of estimationratio

was obtainedfor eachmodel/population comparison. The Q value is similar to the ý valueused in regressionand has been used in previousstudies that havecompared models of

A. siro with actualpopulation data (Kawamoto et al., 1991; Pekarand Mrkovi, 2004).

The equationfor Q is:

(Yobs Yest)2 / 2: Ynull)2, - (Yobs -

"'--log (Nobs +1) Y,, log (N,., 1). Y,, log E Nobs/ whereyobs and t = t + ý,,,= n which actsas tile null model. Nobsis the numberof observedmites in the population.N,, t is the numberof mitesestimated by the model. AQ valueof I indicatesa perfectfit betweenthe modeland the population.Q cannotbe greaterthan I but canbe negativewhich indicatesthat the estimatedvalues from the modelgive a worsefit to the datathan the null model(Kawamoto et al., 1991; Pekarand 2ddrkovd, 2004).

90 Chapter4- Life historyand population dynamics of Acarussiro

Fig. 4.2. Leslie matrix modelforAcarus siro fed on a diet of wholegrainflour andyeast in a

3: 1 (w/w) ratio andmaintained at 20 *C and70% r. h. The columnvector (first setof brackets)shows the initial agedistribution of the population.The transitionmatrix (second setof brackets)shows the agespecific survivorship and birth rate. The third setof brackets showshow the transitionmatrix is appliedto the columnvector to give the agedistribution at time t2(fourth setof brackets).Each time step,t, representssix days.

Eggs ti 0 0 0 0 0 3.75ý Larvae ti 0 0.75 0 0 0 0 Protonymph ti 0 x 0 0.65 0 0 0 Tritonymph ti 0 0 0 0.825 0 0 Adult Lt, 1001 0 0 0 0.825 0.516

(0 x 0) + (0 x 0) + (0 x 0) + (0 x 0) + (3.756 x 100) 42 375.6 (0.75 x 0) + (0 x 0) + (0 x 0) + (0 x 0) + (0 x 100) t2 0 (0 x 0) + (0.65 x 0) + (0 x 0) + (0 x 0) + (0 x 100) 42 0 (0 x 0) + (0 x 0) + (0.825 x 0) + (0 x 0) + (0 x 100) t2 0 L(Ox 0) + (0 x 0) + (0 x 0) + (0.825 x 0) + (0.51 x IOU) Lt2 51.6

91 Chapter4- Life historyand population dynamics ofAcarus siro

4.2.4 Analysis

All data were tested for normality using the Kolmogorov-smirnov test prior to statistical analysis and subjectedto loglo transformation when required.

4.3 Results

4.3.1 Longevity,fecundity and developmentof Acarus siro

There was little adult mortality over tile first 5- 10 days of adult life but subsequently mortality occurred at a constant rate over time (Fig. 4.3). Adult females survived for a mean (± S.D. ) of 23 ± 10.5 days and adult males survived for a mean of 26 ±

10.2 days (Table 4.1). However, there was a large degree of variation in survival times as the shortest survival period was I day for an adult male and 2 days for an adult female. The longest survival times were 43 days and 40 days for an adult male and adult female respectively.

Adult females produced a mean (± S.D. ) of 29 d: 17.1 eggs. The egg production reacheda peak at day 7 with 2.18 ± 1.2 eggs being laid per female with a mean (± S.D. ) egg output per female per day of 1.25 ± 0.4 eggs. Egg production gradually declined with the age of the female (Fig. 4.4). Unsurprisingly, females which lived longer laid more eggs(FI, ?= 36= 412.02, P<0.001, 92%).

The length of eachjuvenile life-cycle stage varied significantly (ANOVA; F3,jig

24.7, P<0.001; Fig. 4.5). Tukey post-hoctests showed that although there was no significantdifference in the durationof egg and larval stages,both took significantlylonger than both the protonymphand tritonymph stages(P < 0.001). There was no significant differencebetween the durationof the protonymphand tritonymph stages.

The durationof the active phasewas significantlydifferent betweenthe variouspre- adult life-cycle stages(ANOVA; F2,gg = 39.8, p<0.001; Fig. 4.5) with larvaehaving a significantlylonger active phase than both protonymphsand tritonymplis(P < 0.001). There was no significant difference in the duration of the two nymphal stages. There was no

92 Chapter4- Life historyand population dynamics of Acarussiro significantdifference in the time spentin the resting phasebetween any of the lifc-cycle stages.

Using the mean values for longevity of each stage of the life-cycle, the mean (±

S.D. ) egg to adult time is 25.64:h5.05 days. The quickestpossible egg to adult time is 17 daysand the longestpossible time is 38 days.

Of the 40 eggs examined, 10 failed to hatch (25% mortality). The mean levels of mortality for larvae, protonymphs and tritonymphs were: 35%, 17.5% and 17.5% respectively.The cumulativelevel of mortality suggeststhat only 33.2%of eggsare likely to hatchand mature into adult mites.

93 Chapter4- Life historyand population dynamics of Acarussiro

Table. 4.1. The mean duration (± S.D. ), range of duration and stage-specificsurvival for eachstage of the life-cycleof Acarussiro. For eachstage in the lifc-cycle andfor bothsexes of adult mites,40 individualswere maintained on wholemealflour andyeast (3: 1,w/w) at 20

IC and70% r. h.

Life-cycle stage Mean duration Range Stage-specific (days)± S.D. (days) survival Adult female 23 ± 10.53 2-40

Adult male 26 110.22 1-43 - Tritonymph 5.55 ± 1.39 4-10 0.825 Protonymph 5.33± 1.16 4-7 0.825 Larva 7.73:1: 1.28 5-11 0.65 Egg 7.03 d: 1.22 4-10 0.75

94 Chapter4- Life historyand population dynamics of Acarussiro

Fig. 4.3. The numberof adult female(solid circles)and adult male(open squarcs). 4carus siro alive overtime (days)when maintained on wholemealflour andyeast (3: 1, w/w) at 20

T and 70% r. h.

45

40

35

0 30 low, E a) 25

0 20 P m 15

10

5 1%

0 0 10 20 30 40 50

Time (days)

95 Chapter4- Life history and population dynamics of Acarus siro

Fig. 4.4. Meannumber of eggs(b S.D. ) laid per day per mite, by 40 adult femaleAcarus siro rearedon wholemealflour andyeast (3: 1, w/w) at 20 IC and70% r. h. Fitted line is a orderpolynomial (y = 0.0002x3 - 0.0158X2 + 0.2788x+0.3616). Dashedline showstile meanegg output per femalefor the 40 day period(1.25 10.4 1).

4.0

3.5

3.0

2.5 E

2.0

CL V 1.5 I; 0 tm 1.0 LUtm

0.5

0.0

0 10 20 30 40

Time (days)

96 Chapter4- Life historyand population dynamics of Acarussiro

Fig. 4.5. Meanduration (± S.D. ) of the larval,protonymph and tritonymph stages of Acarus siro split into time spentin the activephase (solid bars)and time spentin the restingphase

(open bars) when maintained on wholemeal Ilour and yeast (3: 1, w/w) at 20 *C and 70% r. h.

9

8

7

6

5

3

2

0 Larva Protonymph Tritonymph

Lifecycle stage

97 Chapter4- Life historyand population dynamics of Acarussiro

4.3.2 Thepopulation dynamics ofAcarus siro

Sampling technique

In the calibration of the capillary tube sampling techniquethere was a highly significantrelationship between the calculatednumber of mites and the actual numberof

that had beenplaced into (F, 146.2, P<0.001,86.4%; Fig. 4.6). mites a cuvctte , 23' This demonstratesthat the sampling technique used gives an appropriate estimation of population size.

Population dynamics

The laboratory populations show a steady and synchronousrise throughout the life- cycle stagesuntil approximately day 60 Whenthe population begins to rapidly decline. This decline is due to a shortageof food and spaceand increasedcompetition (Fig. 4.7). For this reason,analysis of the population was conductedup to day 60.

The laboratorypopulations showed no significantperiodicity in the meanabundance of any stageof the lifecycle when analysedwith an autocorrelation.Eggs were the most abundantstage throughout the growth of the population(Fig. 4.7). The eggsper female peakedat day 18 with a meanof 33.3 (± 22.7) eggscounted per femaleand a maximumof

58 eggsper female(Fig. 4-8). The numberof eggsper femalethen decreasedover time after reachingthe peakat day 18.

The mean sex ratio for the 60 day period was 1.3:1, female: male (Fig. 4-9).

However the ratio fluctuated in cycles of approximately 20 to 25 days ranging from 0.8: 1, female:male to 2.1:1, female:male.

4.3.3 Comparisonofthe modelledand observedpopulationdynamics of Acarussiro

The model estimatesthat there shouldbe a peakin mite numbersat day 48 with a total numberof mites,of all life-cycle stages,of 8965mites (Fig. 4.10). The estimatedmean number of mites taken from samples of the populations reared under laboratory conditions

98 Chapter4- Life historyand population dynamics of Acarussiro showthat the peakpopulation is 10080mites at day 54. Tile modelpredicts that at day 54 the populationshould be 8827mites.

When the various life-cycle stagesare distinguished,the final model indicatesthat the initial cohortof adultswould be expectedto declinesteadily during the first 24 days(Fig.

4.8). However,oviposition followed by the developmentof thejuvenile stageswould leadto an increasein adult numbersby day 30 (Fig. 4.10). The populationwould then be expected to move through a period of unstablegrowth until a stable age structure is reached, approximately300 days after the introductionof mites to the food source. However,the laboratorypopulations only ran for 80 days,reaching a peakof 10080mites at day 54. The stableage structure was not reachedby the laboratorypopulation.

Comparisonbetween the model and the laboratorypopulation was conductedup until day 60. The original Leslie matrix modelparameterised using the datacollected from the current study was found to underestimatethe total number of mites observedin the laboratorypopulations. Sensitivity analysis showed that overall,tripling the numberof eggs laid per adult in the model was the single changethat gave the best improvementin fit betweenthe total numberof mites predictedby the modeland the laboratorypopulations (Q

= 0.76;Fig. 4.11). However,while the overallfit was better,tripling the numberof eggsdid not always give the best fit when each stageof the life-cycle was consideredseparately

(Table 4.2). Nevertheless,for the purposeof this study, the model populationwith a reproductiveoutput of 11.25eggs per femaleper six day period was usedfor comparison with the laboratorypopulations.

Comparingthe distribution of mites acrossthe life-cycle stages(Fig. 4.12) showed that while the generalpattern of abundancewas similar there was a significantdifference betweenthe predictionof the final modeland the laboratorypopulation 797,P<0.01; Fig. 4.13).

99 Chapter4- Life history and population dynamics of Acarus siro

Fig. 4.6. Numberof adult femalcAcaruss1ro estimated in 0.3gof wholcmealflour andyeast

(3: 1, w/w) at 20 T and70% r.h. whenknown numbers of miteswere introduced.Samples weretaken from 5 replicatesat densitiesof 100,200,300,400and 500 mitesper cuvctte.

Y=0.75x + 17.95;r2 = 86.4%.

500 . 450 .

400 S 350 " V . 300 7 250 " .7. . 7 200 Sr V . 150

100

S 50

o I I I I 0 100 200 300 400 500 600

Number of mites

100 Chapter4- Litle history and population dynamics oflicarus siro

Fig. 4.7. The 3 point sliding mean of the log number of mites +1 for each stage of the Id'e- cycle of Acarus siro sampled at different times from initial populations of 50 adult 1einalc and 50 adult male mites maintained on 0-3g of wholemeal flour and yeast (3: 1, w/w) at 20 OC and 70% r. h. Eggs are shown by the pink line; larvae, green, protonymphs, blue-, tritonymphs, red and adults, black. Dashedline indicates the point ot'decline for the total number of mites.

1000

100

10

1 0 20 40 60 80

Time (days)

101 Chapter4- Life historyand population dynamics of Acaruss1ro

Fig. 4.8. Number of eggs per adult female Acarus siro sampled from 5 replicates of 40 populations of various ages reared on wholemeal flour and yeast (3: 1, w/w) at 20 IC and

70% r. h. The fitted line is the 3 point sliding mean.

70

60 .

50

. E 40

0 0 00 U) 30 w 0 0008 20 0 0 00 10 0 08 00§0 880 08,0 .t o- 0 20 40 60

Time (days)

102 Chapter4- Life history andpopulation dynamics of Acarussiro

Fig. 4.9. Mean sex ratio (± s.d. ) for adult Acarus siro sampled from 5 replicates of 40 populations of various agesmaintained on wholemcal flour and yeast (3: 1, w/w) at 20 IC and

70% r. h.

4.5

4.0

3.5

3.0

A 2.5 m E 2.0

1.5

1.0 'H

0.5

0.0

0 10 20 30 40 50 60

Time (days)

103 Chapter4- Lifle history and population dynamicsol'Acurussiro

Fig. 4.10. The number of Acarus siro of all lifecycle stages as predicted by a Leslic matrix model over time. Eggs are shown by the pink line; larvae, green; protonyrnphs, blue; tritonymphs, red and adults, black.

5000

4500

4000

3500

3000 E 4- 0 2500

E 2000 z

1500

1000

500

0 12 18 24 30 36 42 48 54 60

Time (days)

104 Chapter4- Life history andpopulation dynamics of Acarussiro

Fig. 4.11. The number of Acarus siro as predicted by a Leslie matrix model over time (solid circles) and the mean number of mites sampledat different times from initial populationsof

50 adult female and 50 adult male mites maintained on 0.3g of wholcmeal flour and yeast

(3: 1, w/w) at 20 *C and 70% r. h. (open squares).

12000

10000 I'R /  / 'S " / _" 7 ' 8000 /

/ / / 0 / 6000 / / E z 4000

2000 -% A %.

0 ýý2-- -11 -- -11 06 12 18 24 30 36 42 48 54 60

Time (days)

105 Chapter4- Life historyand population dynamics of Acarussiro

Table 4.2. Index estimation ratio showing the goodnessof fit for the numbersof mites of each life-cycle stagebetween populations of A. siro maintained at 20 T and 70% r. h. and a

Lesliematrix modelwhen the eggoutput per adult is unadjusted,doubled, tripled and quadrupled.

Goodnessof fit (Q) betweentile modeland populations

Life-cycle stage Unadjusted x2 x3 x4

Egg -2.46 -0.31 0.09 -0.02 Larva 0.69 0.79 0.62 0.40 Protonymph 0.72 0.88 0.88 0.84

Tritonymph 0.43 0.62 0.68 0.70 Adult 0.38 0.003 0.14 0.18

All mites -0.23 0.74 0.75 0.52

106 Chapter4- Life historyand population dynamics of Acarussiro

Fig. 4.12. The number of Acarus siro predicted by the adjusted Leslie matrix model over time (solid circles) and the mean number of mites sampledat different times from initial populations of 50 adult female and 50 adult male mites maintained on 0.3g of wholemcal flour and yeast (3: 1, w/w) at 20 *C and 70% r. h. (open squares).

2000

Adults / / 1600

Içl" I 1200 I I 0 / / 800 I I E I 0 I z 400 I -_I. Z - / J. - / _D_ 0 06 12 18 24 30 36 42 48 54 60 Time (days) '7

3000 I Tritonymphs 0 2500 ", CL E : 2000 r'IN 1500 0 m looo

500

0 06 12 18 24 30 36 42 48 54 60

Time WaYs)

107 Chapter4- Life historyand population dynamics of Acarussiro

2500 ----1 Protonymphs 2000 E C 1500

0 1000

E dm0 500 #0 z 0 06 12 18 24 30 36 42 48 54 60

Time (days) 5000 Larvae 4000 0 m 2.1 3000 //\\ 04 2000 -0E

1000

WO

-- o 1 06 12 18 24 30 36 42 48 54 60

Time (days) 5000 Eggs 4000 / S, im /\ p im I ,0 3000 I/- I/ I/ \ I/ \' 0 -0 2000 I0 E / Z / 1000

00 -II 0- -cf 06 12 18 24 30 36 42 48 54 60

Time (days) 108 Chaptcr4- Life historyand population dynamics of Acarussiro

Fig. 4.13. The mean proportion of mites at eachstage of the lifccycle ofAcarus siro during the 60 day period post establishmentof an initial population of 50 adult femalesand 50 adult males. Solid bars representmean values (:L S.D. ) of tile adjusted Leslie matrix model and open bars representmean values (± S.D. ) of 5 populations maintained on 0.3g of wholemeal flour and yeast (3: 1, w/w) at 20 IC and 70% r.h.

0.8

0.7

0.6

0.5

0 1-- 0 0.4 tE 0 CL 0 Oý 0.3

0.2

0.1

0 Egg Larva Protonymph Tritonymph Adult

Life-cycle stage

109 Chaptcr4- Life historyand population dynamics of Acarussiro

4.4 Discussion

In this study the meantime requiredto go from egg to adult was 25.6 ±5 days.

Howeverthere was a largeamount of variationas the minimumtime calculatcdfor compicte developmentwas 17 daysand the maximumtime requiredfor compictcdevelopment was 38 days. The resultsof the presentstudy support the findings of previousstudies that foundthat the protonymphsand tritonymphs had similar durations,which wereshorter than tile eggand the larval stages(Fejt and2ddrkovd, 2001). Howeverthe previousstudies found that tile egg andnot the larvawas the longeststage.

In the presentstudy there was no significant differencebetween adult femaleand adult male longevity. However,a previousstudy had found that adult maleslived longer than adult femalesacross all the temperatureand humidity regimesexamined (Cunnington,

1985). At 20 'C and 70% r.h. it wasfound that the meanduration of adult life for maleswas

52 daysbut was only 36.2 daysfor females(Cunnington, 1985). Similar resultswere found at 15 'C and 90% r.h. with adult males sometimesliving up to 90 days longer than tile females(Parkinson et aL, 1991). However,when the diet was changedfrom yeastand wheatgerm(considered to be optimal) to a fungal diet the disparity betweenthe sexes decreasedas the femaleswere able to live longerthan on the control diet whereasthe male longevitydecreased.

As expected,the length of the ovipositionperiod increasedwith female longevity.

However,females did not lay eggsup until the day they died but stoppeda few daysbefore.

The numberof eggsa femalelaid in her lifetime was determinedmore by the lengthof time availableto the femaleto oviposit ratherthan the ability to lay more eggsper day. It was also found in this study that there is a meanpre-oviposition time of 2 days (4: 1.4 S.D. ) beforethe female lays her first egg regardlessof the longevity of the female. A previous study showedthat at low temperatures(5 and 10 "Q the mean numberof eggs laid per femaleper day stayedat a lower and moreconsistent rate throughoutthe ovipositionperiod whereasat highertemperatures a sharpincrease in egg productionduring the first week led

110 Chapter4- Life history andpopulation dynamics of Acarussiro to a peak at approximately 7 days (Cunnington, 1985). This study also shows the sharp rise over time in mean eggs laid per day per female (Fig. 4.2) with the peak rate being achieved7 days into the adult female's life, approximately 5 days into the oviposition period.

The number of eggs laid each day by females paired with a male and kept in chambers,was much lower in the presentstudy than that found in previousstudies. Also, a much higher numberof eggs per femalewas countedin the laboratorypopulations, than when mites were kept in pairs (Figs. 4.4 and 4.8). It is possiblethat somedead eggs were countedas these cannot be distinguishedfrom live eggs.

The numberof eggsper femaledecreased as the numberof mitesincreased. It could be that the presenceof conspecificsat high densitiesinhibits oviposition. However,the decreasein ovipositionat the higher densityseen in the presentstudy could be due to the diminishedresources rather than the density of the mites. Previousstudies have been conductedon variousfoodstuffs but havenot usedthe samefood as the work presentedhere.

The most similar food used in a previousstudy is wheatgermand yeastbut tile studythat usedthis food sourcewas conductedat 15 'C and 90% r.h. (Parkinsonet al. 1991). Another study,conducted on wheatgermflakes found that at 20 IC and 70% r.h. the meanfecundity was 133 eggsin a female's lifespan(Cunnington, 1985). This finding was supportedby a later study also using wheatgermat 20 'C and 70% r.h. which found similar results,with femaleslaying a mean of 140 eggs in a lifetime (Fejt and 2dArkovA,2001). The lower fecundityof the mites usedin the presentstudy could have beendue to the different food substratethat the miteswere reared on or a straindifference.

In many naturally occurring populationsof arthropodsit might be expectedthat a cyclical patternof growth and abundanceof the lifecycle stageswould be observed. The periodicityexplained by the developmenttime of thejuvenile stagesand the longevityof the adult stages.An initial populationof a few adultswill lay eggswhich hatchinto larvae,the larvaemoult into protonymphsand so on until the next generationof adultsemerge, at which point the initial populationof adultshas died. As eachstage of the lifecycle progressesinto the next, the compositionof the populationchanges in a synchronous,periodical manner as

III Chapter4- Life history andpopulation dynamics of Acarussiro the generationsrarely overlap, although this would only happen initially. Tile Leslie matrix model created in the present study is a clear example of how this process leads to a stable age structure and exponential growth after an initial unstable period of growth as the population establishes itself. The original model, constructed using the life-history data collectedpreviously in this study,shows that a stableage structurewill not be reachedfor approximately162 daysafter the introductionof 50 adult femalesand 50 adult male mitcs.

Howeverwhen the eggoutput is tripled the populationdoes not beginto stabiliscuntil about

306 dayspost establishment.

Early on in the growth of the laboratorypopulations, the emergenceand abundance of the lifecycle stages are synchronised, as expected, and there is a slight periodicity noticeable in the adult females. However, there was no significant periodicity when analysed by autocorrelation. This lack of periodicity is largely due to the adult females living for a long period of time, up to 40 days found in the present study. This meansthere is a steady production of eggs and a consistenttransition of mites between life-cycle stages.

The populationsin the presentstudy were maintainedfree from predationand diseaseand early on in the studythere would havebeen an abundanceof resources.As shownby the life-history data collected in this study and confirmed in the population dynamics experiment,A. siro can survive for a long time as adults and continueto lay eggsfor tile majority of their adult life. Adult femalelongevity is not the only likely causeof the lack of periodicity seenin this study. There is largevariation in the developmenttimes of the life- cycle stagesand the time takento reachadulthood ranged from 17to 38 days

The synchronicitybetween the lifecycle stagesis largely maintained. Howeverthe abundanceof the tritonymphswas greaterthan the protonymphs.This could be due to the difficulty in identifyingthe nymphalstages when floating in a salt solution. Howeverdue to the relatively low mortality of protonymphsand tritonymphsit would be expectedthat the abundancefor the nymphalstages and even the adultstages would be fairly similar.

As the resourcesof spaceand food startto diminish,the populationbegins to decline

(Fig. 4.7). This is seenfirst in the larvaeand closely followed by the eggs. The larvaeare

112 Chapter4- Life history andpopulation dynamics of Acarussiro morphologically the smallest stage in the life-cycle and may be more likely to suffcr tile effects of starvation and therefore less likely to survive in a highly competitive environment.

The egg abundancestarts to decline as the female abundancebegins to plateau(Fig. 4.7). At the same time, resourcesare beginning to run low. Fewer females may be laying eggs and those females that are still able to, may not be laying as many eggs as they potentially could, due to the lack of resources. The decreasein the number of eggs will also contribute to the decreasein the abundanceof larvae and both lines representing the eggs and larvae in Fig.

4.7 show parallel rates of decline from day 50 onwards. The adult and nymphal stagesdo not show any signs of decline until much later, around day 70. However growth in the abundanceof each stage stops between day 48 and day 54. Once these stages begin to decline, it is the smaller life cycle stage, the protonymph, that decreasesin abundancethe most rapidly. This could be for similar reasonsthat the decline is first noticed in the larval stage; that the smaller stages are not able to compete for resources as well as the larger stages. The larger stagesmay also be more tolerant of unfavourable conditions such as the build up of waste products as well as starvation.

In previousstudies, there has been some debate about the sex ratio in populationsof

A. siro. One study calculatedthat femalesmade up 64.5% of populationsmaintained on wheatgermand yeast (1: 3) at 15 'C and90% r.h. andused the assumptionof 60% femalesin a simplemodel of populationgrowth (Berreen,1974). However,it was found that a model using46.8% femalesgave the bestestimation for the datacollectcd by Solomon(1969) for

15 IC and 70% r.h. (Berreen,1974). A sex ratio of 1:1, was found by Ignatowicz(1986,

1987),and was used for the constructionof the populationmodel by Pekarand 2ddrkovd

(2004). The findings from the presentstudy demonstratethat the sex ratio is not always constantthroughout the growth of the population. Here the meansex ratio for the 60 day periodwas 1.3:1, female:male. Howeverthe ratio fluctuatedin cyclesof approximately20 to 25 days ranging from 0.8:1, female:male to 2.11, female:male (Fig. 4.9). The initial populationof 50 femalesappear to survivefor longerthan tile initial populationof 50 males.

As the populationbegins to decline the sex ratio becomesmore variable but also biased

113 Chaptcr4- Life history andpopulation dynamics of Acarussiro

towardsfemales. It could be that the largerfemales are ableto survivepoorer conditions for

longer than the males. They may be more resistantto starvationor the build of waste

products.A studyon Hemisarcoptescoccophagus Meyer, a mite parasiticto armouredscale

insects,showed that femalessurvived longer than malesin the absenceof food (lzraylevich

and Gerson, 1995). Another study showedthat the spider mite Tetrallychusmcdanielli

McGregor, producesmore female offspring in responseto deterioratingconditions and

extremetemperatures (Roy et al., 2003). The authorsof this studysuggested that it wasan

evolutionaryresponse as the femalesare better suited to dispersalthan the malesdue to their

ability to develop into a mobile hypopusduring times of poor conditions. Acarus siro,

however,very rarely develophypopi and none were found in the presentstudy. Telranychus

mcdanielli mites are arrhenotokous,therefore females can producemale progenyby the

ovipositionof unfertilisedeggs (Roy el al., 2003). Sex determinationin A siro is currently

unknown, although arrhenotokyhas not been observed in this mite. This does not 4. from necessarilypreclude . siro adjustingthe sex ratio of its progeny. Many animals,

particularlyinvertebrates, are able to adjustthe sex ratio of their progenyregardless of the

methodof sex determination(Toyoshima and Amano, 1998;Roy et al., 2003; West el al.,

2005). However,it is not possiblefrom the presentstudy to determineif the fluctuationin

the sex ratio has beencaused by adjustmentof the sex ratio in tile offspring or by different

mortality levelsbetween the sexesor a combinationof thesetwo factors. It can howeverbe

seenin this studythat the sexratio doesfluctuate and favours a femalebias. ity-dependant Dens effectswere not takeninto accountwith the Leslie matrix model in used the presentstudy. This meansthat oviposition was consistentat all population densitieswhich is unlikely to be the casefor the laboratorypopulations. The lower egg day countper adult at 6 is due simply to therebeing fewer eggs in the laboratorypopulations by than predicted the model even thoughthe numberof adults presentwere very similar. by day However, 12 there were a greaternumber of eggsin the laboratorypopulation but also a greaternumber of adultsthan predictedby the original model. From day 12 up until day 72, laboratory the population consistentlyhad a much greater number of eggs than

114 Chaptcr4- Life history andpopulation dynamics ofAcarus siro predicted by the model and between days 30 and 42 had a greater number of eggs per adult female.

Multiplying the number of eggs laid per adult in the model by a factor of 3 gave a better fit betweenthe model and the population with regards to the number of eggs counted.

This increase in fecundity still falls within the range found in previous studies (Boczck and 2dArkovd, Davis, 1985; Cunnington, 1985; and Fejt and 2001) and could be a more accurate representationof the fecundity of A. siro within the laboratory populations than tile paired mites in chambers.

The model used in this study, although useful for generating broad hypotheses, seems to be too simplistic to accurately predict the dynamics of A. siro populations.

Relatively good fits were achieved between the final model (when the egg output was tripled) and the laboratory populations for the larval, protonymph and tritonymph stagesbut were not so good for the egg and adult stages(Table 4.2). However if the final model is lagged by 2 time stepsthe fit betweenthe model and the laboratory populations for the adults is greatly improved, Q=0.58. Lagging the final model did not improve the fit for the other pre-adult stages.

The model, even with adjustments, is unable to cope with variation over time or density-dependant effects. The model relies upon mean data collected from life-history studieswhich themselvesshowed a wide rangeof variation. Factorssuch as the sex ratio, egg productionand developmenttime are not alwaysconstant over time in real populations.

A more complex model may be neededto accuratelypredict the dynamics of A siro populations.The simpleLeslie matrix modelused in the presentstudy was howevera useful tool for interpretingand highlighting the complexitiesof populationsofA siro.

The resultspresented in this chapterare similar to thosefound in previousstudies.

However,clearly A. siro is very sensitivewhen rearedunder different conditionsand even when rearedunder the sameconditions in the samestudy. Temperature,relative humidity and the food substrateon which the mitesare rearedon are the major factorsin determining the growth and compositionof populations. When all of thesefactors are considered,it is

115 Chapter4- Life historyand population dynamics orAcarus siro evidently important to refer the life history parametersfor A. siro to the spccific conditions under which they are maintained.

116 Chapter 5- Effects of entomopathogenicfungi on Acarus siro

CHAPTER 5

EFFECTS OF ENTOMOPATHO GENIC FUNGI ON

ACARUSSIRO

5.1 Introduction

The entornopathogenicfungus M. anisopliaehas beenused before to control many pest speciesbut has yet to be testedon A siro. Researchhas focusedon the effectsthat fungal pathogenshave on individualsor on the reductionin the amountof damagedone to the crop by the targetpest. Not so muchis knownabout the effectsof an introducedfungal pathogenon populationdynamics. Key questionsinclude whether or not the funguscan be transmittedthrough the mite population,whether dead infected mites will act as a reservoir of diseaseand whetherthe pathogenand host will establishstable or cyclical co-existence patternsor will the diseasedie-out.

A recent study using the entomopthoraleanfungal pathogen,Neozygiles lanajoae

Delalibera,Hajek and Humber, against the cassavagreen mite, Mononychelluslanajoa

Bondar, found that the fungal pathogencan spreadthroughout a population from an inoculurnof live infectedmites (Hountondjiet al., 2007). It was shown in the samestudy that the numberof miteson plantsinoculated with the fungal pathogenwas lower thanon the uninoculatedcontrol plants and that there was a decreasedgrowth rate (Hountondjiel al.,

2007). However,the fungal pathogenused was an entomophthoraleanfungus which, onceit hassporulated on an infectedhost, forcibly dischargesthe spores.This would help to spread the pathogenand increasethe chancesof a mite coming into contact with the infective spores. The entomopathogenicfungi M. anisopflaeand B. bassianado not dischargetheir conidia but rely upon direct contactbetween the infectedmites and uninfectedmites. It has

117 Chapter5- Effects of entomopathogenicfungi on Acarus siro already been shown that M. anisopliae is pathogenic to astigmatid mites with high levels of infection achievedagainst Psoroptes mites (Smith et aL, 2000; Brooks and Wall, 2002; Chapter 3).

Hence,the work presentedin this chapteraimed to investigatethe pathogenicityof,

B. bassianaand M anisopliae againstA. siro when mites were directly exposedto unformulatedconidia and conidia suspendedin siliconeoil. The chapteralso examinestile transmissionof infection betweenmites under controlled conditions and then, following on from this, the effect a fungalpathogen may have when introducedto nalivepopulations.

5.2 Materials and methods

5.2.1 Pathogenicity of Metarhizium anisopliaeand Beauveria bassiana

To test the pathogenicity of M anisopliae and A bassiana against A siro, adult femalemites were takenfrom the stockculture and divided into 5 groupsof 20 mites. The groups were then treated, using the method describedin Chapter 2, with one of the following: silicone oil, conidia of M. anisopliae(IM1386697) formulated in silicone oil, unformulatedM anisopliae(IM1386697), unformulated B. hassiana(CSLI) conidiaand no treatment(control). Mites were exposedto eachtreatment for I minute. The M anisopliae formulatedin silicone oil was at a concentrationof Ix 10' conidia ml*' and 2ml of this suspensionwas used to inoculatethe mites.The samevolume, 2ml, was usedfor tile silicone oil only treatment. The unformulatedconidia of M. anisopliae and B. bassianawere weighedso that the miteswould be exposedto a similarnumber of conidiaas when the fungi were formulatedin 2 ml of siliconeoil at Ix 108conidia ml*'. Thereare approximately1.8 x

1010conidia per gram for M. anisopliaeand 1.2 x 1011conidia per gram for A bassiana. 0.011 Therefore, g was measuredfor M anisopliae and 0.01 7g was measuredfor B. bassiana. Following exposure,the miteswere placedinside clean chambers (Fig. 2.4) with

0.03gof food and incubatedat 20 OCand 70% r.h. The chamberswere inspectedevery 24 h anddead mites were removed,surface sterilised and then placed into individualwells of a 96

118 Chapter5- Effects of entomopathogenicfungi on Acarus siro well microtitre plate with a drop of distilled water. Dead mites were checked every 24 li for signs of infection.

5.2.2 Horizontaltransmission offungal infection

Prior to the horizontaltransmission experiments, a trial was carriedout to determine the effects,if any,of a permanentblack ink appliedto the idiosomaof the mites. For this, 20 adult femalemites were selectedfrom the stockculture. A small spotof black ink (Stacdticr,

Germany)was appliedto the dorsalsurface of the idiosomausing a fine gradepaintbrush.

The ink was applied so that it coveredapproximately one quarterto a third of the dorsal surfaceof the idiosoma. The miteswere then placedinto chamberswith 0.03gof food and incubatedat 20 'C and 70%r. h. The controlmites had no ink appliedbut weretouched with a fine gradepaintbrush that had beendipped in distilled water. The experimentwas repeated

5 times. Chamberswere inspected every 24 h for the presenceof deadmites.

To find whether transmissioncan occur between mites treated with a fungal pathogenand live untreatedmites, adult femaleswhich had beenexposed to dry conidiaof

M anisopliae(IM1386697) were placedinto incubationchambers (Fig. 2.4) containing0.03g of food alongwith live unexposedadult femalesin variousratios. Five groupsof 10 treated adult femalesand five groupsof 5 treatedadult femaleswere placedinto chambers.Each chamberthen had live naYvemites added to make a total of 20 mites per chamber.

Therefore,in the chamberscontaining 10 treatedmites, 10 na7fve,adult, femalemites were addedand for the chamberscontaining 5 treatedmites, 15 na*fve,adult, femaleswere added.

The mitesthat had beentreated with the fungal pathogenwere markedon the dorsalsurface of the idiosomawith the black permanentink as describedabove, to distinguishthem from the mitesthat had not beeninoculated. As a control, 10 morechambers containing mites in the sameratios were set up, replacingthe inoculatedmites with mites that had only been markedwith the permanentblack ink. Each chamberwas inspectedevery 24 h for tile presenceof deadmites, which were treatedand checkedfor signsof infection,as described in Chapter2. This experimentwas then repeatedbut with the treatedlive, mites replaced

119 Chapter 5- Effects of entomopathogcnicfungi on Acaruv siro with deadmites. Cadaverswith 5 to 10 day-old infectionsofXf anisopliacwere usedand placedinto the chambersin set spatialpatterns that were constantfor all the chambers(Fig.

5.1). Each cadaverwas markedon the idiosomawith permanentblack ink. Uninfectcd cadaverswere used as controls.

120 Chapter5- Effects of entomopathogenicfungi on Acarus sim

Fig. S.1. Position of Acarus siro cadaversplaced into the experimental chambcrsused in experimentsto testhorizontal transmission between cadavers infected with Afelarhizium anisopliaeand live, naYvemites when in the ratio of 5: IS, infectedcadavers: live, na*fvc mites(top) and 10:10, infected cadavers: live, naYvemites (bottom).

pot-- 1ý06-Fý; kc

121 Chapter5- Effects of entomopathogenicrungi on Acartis siro

5.2.3 Effect of afungalpathogen on thepopulation dynamics of Acarus siro

Small-scale,high density

To find out whether or not a fungal pathogen could transmit between mites within one generationin a small but densepopulation, populations consisting of 50 adult female and

50 adult male, A. siro were set up in 4 incubation chambers (Fig. 2.4) each containing 0.03g

of food. The mites were selectedat random from the stock cultures. To thesechambers, 20 cadaversmarked with permanentblack ink (as describedpreviously) and with 5- 10 day old

infections of M anisopliae QM1386697)were added. The chamberswere then incubatedat

20 'C and 90% r.h. for 4,8,12 or 16 days. At the end of this period, eachchamber was placedinto a freezerat - 12 (± 0.5) OCfor 48 h. It had previouslybeen confirmedthat

freezing would not alter the subsequentexpression of mycosisof an infected mite. The chamberswere then removedfrom the freezerand the adult mites were removedfrom the

chamber,surface sterilised and checkedfor signs of infection every 24 11as previously

described. Five replicateswere used for each time period. For controls, five chambers

receivedonly uninfectedcadavers.

Large-scale,low density

This experimentwas set up to find out if a fungal pathogencould transmitbetween mites and persistwithin larger populationsof A. siro over severalgenerations. Populations of A. siro were set up in cuvettes,(Fig. 4.2) in the sameway as describedin Chapter4. Tile cuvetteswere sealedwith non-absorbentcotton wool and incubatedat 20 'C and 70%r. h. for

20 days before the addition of 20 A. siro cadaverswhich had been infected with Af anisopliae(IMI3 86697). The infectionfor eachcadaver was between5 and 10 daysof age.

A previousstudy which had examinedthe infectivity of cadaversof A ovis foundthat 5 day- old infectionswere the most infectiveto live mites (Brooks and Wall, 2002). The cuvcttes were agitatedevery 48 h by invertingthem 3 times in quick succession,to preventthe food from clumpingand to ensuresimilar conditionswere maintained in all the cuvettes.

122 Chapter5- Effects of entomopathogenicfungi on Acarus s1ro

Samples were taken from the cuvettes and the mites were counted as described in

Chapter4. The numberof live mites,life-cycle stageand sex of the adultswere recorded.

Over an 80 day period, 5 differentcuvettes were sampledevery 4 days. Five sampleswere takenfrom eachcuvette and the sampleswere pooled. Oncea cuvcttehad beensampled it was removed from the experimentand frozen at -12 (± 0.5) OC. At the end of the experiment,cuvettes containing populations aged 40 and 60 days post-establishmentwere defrosted,79 and 71 days after they were frozen, respectively. A saturatedsalt solution

(NaCl) was addedto the cuvettesto float the remainingmites from the food. Thesemites were carefully removedusing a paint brush,surface sterilised and then placedonto a damp piece of filter paper in a 100 mm diameterpetri dish. The petri dish was sealedwith parafilm and incubatedat 20 IC. The mites were checkedevery 24 h for signs of fungal infection.

5.2.4 Analysis

Data were checked for normality using the Kolmogorov-Smirnovtest and

proportions were arcsine transformed before analysis. However, for clarity the untransformedmean (I S.D. ) dataare plotted in the figurespresented.

5.3 Results

5.3.1Pathogenicity of Metarhiziumanisopliae and Beauveriabassiana

There was a significant effect of treatmenton tile arcsineproportion of infected mites (ANOVA; F4,25= 204.3, P<0.001; Fig. 5.2). As expected, there were no infected mites in the control group or the group that was treated with silicone oil only. All tlIrcc groups exposedto conidia were significantly different to each other (Tukey post hoc test; P<

0.01; Fig. 5.2).

There was also a significant effect of treatmenton the LT5ovalues (ANOVA; F4,25"'-

12.62, P<0.001; Fig. 5-3). There was no significant difference in the LT50 between the

123 Chapter5- Effects of entomopathogenicfungi onAcarta siro groups treated with silicone oil, silicone oil and M. anisopliac, unfort-nulatcdAt anisopliae or unformulated B. bassiana at between 3-5 days (P > 0.05). However, there was a significant difference betweenall of the groups and the untreatedcontrols (P < 0.05) with tile untreatedcontrols having a significantly longer LT50than all the other groups at 9.5 days (I

2.8).

124 Chapter5- Efrccts of entomopathogcnicrungi on Acarus siro

Fig. 5.2. Proportionof adult femaleAcarus siro infected (± S.D. ) after cxposurcto no treatment(1), siliconeoil only (2), Metarhiziumanisoplide in siliconeoil (3), unrormulatcd

Metarhizium anisopliae (4), or unformulated Beauveria hassiana(5) at 20 IC and 70% r.h,

1.0

0.8

E M

JOR 0.6 C z_- 0 c 0 tE

0 a 0.4 0

0.2

0.0 III 1

Treatment

125 Chapter 5- Effects of entomopathogenicfungi on Acartis x1ro

Fig. 5.3. Mean LT5o values (+ S.D. ) for adult female Acarus siro af1cr cxposure to no treatment (1), silicone oil only (2), Metarhizium anisopliac in silicone oil (3), unformulatcd

Metarhizium anisopliae (4), or unformulated Beauveria bassiana (5) at 20 T and 70% r. h.

Letters indicate groups betweenwhich there is no significant difference.

14

12

10 a

8 cc Q E 0 " b b

4

b 2

0 I I I I I 1

Treatment

126 Chapter 5- Effccts of cntomopathogcnicfungi on Acarus siro

5.3.2 Horizontaltransmission offungal infection

For the experiment testing for any effects of the black ink on A. siro survival, there was no significant difference for the LT5obetween the mites marked with black ink (5 days) and the control mites (6 days) (ANOVA; Fi, s=0.20, P>0.05).

There was a significantly greater proportion of infections (0.33 -4-0.16) in the nSive mites exposedto M anisopliae (either via infected cadaversor exposedlive mites), than the

M FI, P< nave mites that were not exposedto anisopliae (controls), (ANOVA; 3822 81 -159 0.001; Fig. 5.4).

There was no significant difference in the proportion of infected mites regardlessof whetheror not the fungal pathogenwas introducedvia live, exposedmites or via infected cadavers(two-way ANOVA; F1,19= 2.49, P=0.13; Fig. 5.4) with 0.38 (± 0.18 S.D. ) and

0.28 (± 0.11 S.D. ) of the mites becominginfected, respectively. 0.34 (± 0.15 S.D. ) of mites becameinfected when exposedto M. anisopliaein the ratio of 15:5 (na*fvc:exposed) and

0.32 (± 0.17 S.D. ) of mites becameinfected when exposedin the ratio of 10:10. Therewas no significantdifference in the proportionof infectedmites, regardlessof the ratio that the mites or cadaverswere introducedto the naYvemites (two-way ANOVA; F1,19= 0.61,P

0.81;Fig. 5.4). Therewas no significantinteraction (FI, 19= 0.77,P=0.39).

When groupedtogether, mites exposedto M anisopliaehad a significantly lower meanLT50 of 12.7(± 8, S.D. ) dayswhereas the controlmites had a meanLT50 of 18.4(± 7.5,

S.D. ) days(ANOVA; F1,39= 5.4, P=0.03; Fig. 5.5).

Mites exposedto M. anisopliaein the ratio 15:5 had a meanUso of 12.5(: L 6) days andshowed no significantdifference to the mitesexposed to Af. anisopliaein tile ratio 10:10, which had a meanLT50of 12.9(± 10)days (two-way ANOVA; F1,19= 0.02,P=0.89; Fig.

5.5). There was also no significant differencefor the LT50between the groupsof mites housedwith live, exposedmites (15.1 days:L8.2 S.D. ) and the mites housedwith infected cadavers(10.3 daysd: 7.4 S.D. ) (two-wayANOVA; F1,19= 2.9, P=0.105; Fig. 5.5). There was howevera significantinteraction between ratio andthe type of introducedmites (FI, 19 12.3,P=0.003).

127 Chapter 5- EiTcctsotentomopithogenic fungi on Acarus siro

There were no significant differences in the LT50 for the controls regardless of treatment.

128 Chapter 5- Effccts of entomopathogenicfungi on Acarus siro

Fig. 5.4. Mean proportion of naYveAcarus siro (I S.D. ) infected with Afelarld.-lum anisopliae (IM1386697) when 5 or 10 exposedlive mites, or infected cadavers,were introducedto IS or 10 nalve live mites, to give a total of 20 mites in an experimental chamber(Fig. 2.4) with 0.03g of food. Open squaresshow the mean proportion of nalive mites infected when housedwith mites which had been exposedto or infected with the fungal pathogen. Solid circles show the mean proportion of mites infected when housed with mites that had not been exposedor infected with the fungal pathogen(controls).

0.7

0.6

(A 0.5 E

0.4

0 c 0 0.3 %E 0 CL H 16. CL0 0.2

0.1

I

15 live 15 live 10 live 10 live na*fve: na*fve: naTve: na'(ve: 5 live 5 dead 10 live 10 dead exposed infected exposed Infected

Treatment

129 Chapter 5- Effccts of cntomopathogcnicfungi on_Acaru,v sira

Fig. 5.5. Mean LT50of naYveAcarussiro (4- S.D. ) when 5 or 10 exposedlive mites or infected cadaverswere introduced to 15 or 10 naYvelive mites to give a total of 20 mites in an experimentalchamber (Fig. 2.4) with 0.03gof food. Opensquares show the meanLTSO of naYvemites when housed with miteswhich hadbeen exposed to or infectedwith the fungalpathogen. Solid circlesshow the meanLT50 of miteswhen houscd with mitesthat hadnot beenexposed or infectedwith the fungalpathogen (controls).

35

30

25

20

0 E 15

10

5

0 15 live 15 live 10 live 10 live na*fve: nalve: nal've: na'(ve: 5 live 5 dead 10 live 10 dead exposed infected exposed Infected

Treatment

130 Chapter 5- Effects of entomopathogenicrungi on Acarussim

5.3.3 Effect ofafungal pathogen on thepopulation dynamics ofAcarus siro

Small-scale,high density

There was no significant difference in the number of infected mites regardlessof tile exposuretime (4,8,12,16 days) to the infected cadavers(ANOVA; F3,13- 0.62, P-0.6 1;

Fig. 5.6). The mean number of infected mites (± S.D. ) found in tile groups of 100 mites exposedto 20 infected cadaversfor 4,8,12 and 16 days, were: 8 (± 6.2), 8.2 (± 4.8), 4.4

4.6) and 6 (± 5.6) respectively, i. e. less than 10%.

However, one replicate, exposedto infected cadaversfor 4 days, had 51 mites which became infected (Fig. 5.6). This was consideredas an outlier for the purpose of statistical analysis. There were no infected mites found in the control groups.

Large-scale,low density

The abundanceof mites increasedover time, in a similar mannerto the pathogen- free population(Fig. 5.7). Using the index of estimationratio (Q) there was a strong correlationbetween both populations(0.90). When the separatelife-cycle stageswere comparedthere were closefits betweenall stages(Table 5.1). However,in the later stages of population growth there were a greater number of mites in the population with the introducedpathogen than the pathogen-freepopulation (Fig. 5.7). When the distributionof mitesacross the life-cycle stagesover the 60 day periodwas analysedwith Chi square,there wasa significantdifference between the two populations(ý = 636, P<0.01; Fig. 5.8). This differencewas mainly due to the much greaternumber of eggscounted in tile population with the pathogen.The peaknumber of eggscounted, were 7612at day 52 and 4493at day

46 for the populationwith pathogenand the pathogen-freepopulation, respectively. Tile meannumber of eggsper femalewas slightly greaterin the populationwith tile pathogen

(11.1 ± 10.2, S.D. ) than the pathogen-freepopulation (9-7 + 7.3) althoughthis was not a difference log significant after a transformationto normalizethe data (ANOVA; Fl, ss 0.234,P=0.63; Fig. 5-9).

131 Chapter 5 -Effects of cntomopathogenicfungi on Acarusslro

The mean sex ratio for the 60 day period was 0.93:1, fcmalc: male (Fig. 5.10).

Howeverthe ratio rangedfrom 0.75:1, female:male to 1.5:1, female:male. The meansex ratio for the pathogenfree populationwas 1.4:1, female: male (Fig. 5.10). There was a significant difference in the mean sex ratio between the cxposcd and pathogen-frce

F1,38 populations(ANOVA; = 17.03,P<0.00 1).

132 Chapter 5- Effccts of entomopathogenicfungi on Acarus siro

Fig. 5.6. NumberofAcarus siro infectedwith Metarhiziumanisopliae (IM1386697) aftcr varioustime periodsof exposureto 20 infectedcadavers when maintained on 0.03gof wholemealflour andyeast (3: 1,w/w) at 20 T and70% r.h. Eachpoint rcprcscntsthe numberof infectedmites from onereplicate consisting of approximately100 adult mites.

60

Outlier 50

40

30

20

10

0 12 16

Time (days)

133 Chapter 5- EfTectsof entomopathogcnicfungi on Acarus s1ro

Fig. 5.7. The calculated mean number of mites sampled at diffcrcnt times post-cstablislinictit from populations of Acarus siro initially consisting of 50 adult female and 50 adult male mites maintained on 0.3g of wholcmeal flour and yeast (3: 1, w/w) at 20 IC and 70% r. h.

Open squares represent populations which had 20 cadavers infcctcd with Afelarldzium anisopliae added at day 20, soild circles represent pathogen-frec populations which were not exposed to the fungal pathogen. Each data point is the mean of 5 replicates (standard deviations not shown for clarity).

14000

C3 13 12000 13

10000 E3

E 6000 0 L.4) m E 6000 z

4000

2000 00 13 13 0 0 10 20 30 40 50 60

Time (days)

134 Chapter 5- Effects of entomopathogcnicfungi on Acarux siro

Table 5.1. Index estimationratio showingthe goodnessof fit for tile numbersof mitesof eachlife-cycle stagebetween the populationthat was exposedto Afetarhiziumanisopliae at day 20 and the pathogen-freepopulation that was not exposedto Af anisopliac. Both populationswere maintained on wholemealflour andyeast, 3: 1 (w/w) at 20 T and70% r.h.

Life-cycle stage Goodnessof fit All stages 0.89 Egg 0.82 Larva 0.76 Protonymph 0.92 Tritonymph 0.92 Adult 0.89

135 Chapter 5- Effccts of entomopathogcnicfungi on Acarussiro

Fig. 5.8. The mean number of mites at each stageof the lifccycle of. 4carus siro during the

60 day period post-establishmentof an initial population of 50 adult females and 50 adult

males. Solid bars representmean values (± S.D. ) of 5 pathogen-freepopulations that had no

exposureto Metarhizium anisopliae and open bars representmean values (k S.D. ) of 5

populations which had 20 cadaversinfected with Metarhizium anisopliae added20 days post

establishment. Populationswere maintained on 0.3g of wholemeal flour and yeast (3: 1,

w/w) at 20 *C and 70% r. h.

7000

6000

5000

4000 0

E 3000 z

2000

1000

0 Egg Larva Protonymph Tritonymph Aduft

Life-cycle stage

136 Chapter5- Effects of entomopathogcnicfungi on Acarin sim

Fig. 5.9. The meannumber of eggsper adult fcmaleAcarussiro calculatcdfrom sampics takenat varioustimes, post-establishment, from initial populationsof Soadult femaleand 50 adultmale mites maintained on 0.3gof wholemealflour andyeast (3: 1, w/w) at 20 'C and

70%r. h. Solid circlesrepresent pathogen-free populations that were not exposedto

Metarhiziumanisopliae; open squares represent populations that had20 cadaversinfcctcd with M anisopliaeadded 20 dayspost-establishment. Each point is the meanof 5 replicates

(standarddeviations not shownfor clarity).

100

I]*

0 E:1 00 [30 0 E 13 13 11 0 13 1:3 10 000 0 ec] V 13 11 0 Jmý0 tm 0 tm 0 13 0 00 0 13 0 -i 00

00 Do oo 0 0 0 10 0

1"- 0 20 40 60 80

Time (days)

137 Chapter5- Effccts of entomopathogenicfungi on Acarus sim

Fig. 5.10. Meansex ratio (± S.D. ) for adultAcarus siro maintainedon wholcmcalflour and yeast(3: 1, w/w) at 20 T and70% r. h. Opensquares represent populations that had20 cadaversinfected with Metarhiziumanisopliae QM1386697) introduced at day 20. Solid circlesrepresent pathogen-free populations that werenot exposedto Afetarhiziumanisoptlae.

Eachpoint representsthe meanof 5 replicates.

4.50 -

4.00 -

3.50 -

3.00 -

,E0 50 .22cc . E %.Ja- 2.00 010 x 1.50 (D

1.00 V 13 0.50

0.00

-0.50 0 10 20 30 40 50 60

Time (days)

138 Chapter 5- Effects orentomopathogcnic fungi on Acarty siro

5.6 Discussion

The results from the present study show that A. siro is susceptible to inrcction by both of the fungal pathogensM anisopliae and B. bassiana. However, in contrast to the data shown in Chapter 3 on the effect of fungal pathogens on Psoroptes mites, inrcctcd A. siro survive for longer after infection than P. ovis. Acarus siro had a LT50 of approximately 5 days when directly exposedto conidia whereasP. ovis had a LTso of about 3 days (Brooks and Wall, 2001; Chapter 3). However, the Uso for mites not exposedto fungal conidia was also higher for A. siro than A ovis, approximately 10 and 6 days respectively. This difference could be due to the conditions at which the experiments were conducted in relation to the conditions that the mites arc adaptedfor.

Previous studieshave shown that A. siro can act as a vector of non-pathogcnic fungi, carryingconidia in their gut and on the surfaceof their cuticle (Hubert et al., 2003,2004).

The resultsfrom the presentstudy showthat horizontaltransmission of AL anisopliaefrom exposedlive mitesand infectedcadavers to live, na.I. ve mites is possible. A greaternumber of infectionswas achievedin the na*fvemites that were housedwith the exposedlive mites than those housedwith infected cadavers,particularly when the ratio was 15:5, naliveto exposedmites. Howeverthere was no significanteffect of ratio, or the type of introduced miteson the survivalof the naYvemites. The mitesexposed to the conidia but still alive are able to move around the chamberand would come into contact with the nalfvcmites, transferringconidia as they touch. The contactrate betweenthe infectedcadavers and live, nave mites would not be as high. However,there was no significanteffect of ratio on tile level of infectionwhich would be expectedif this wasthe case.

The fungusM anisopliaeis clearly pathogenicto. 4. siro and able to transmitfrom onemite to anotherbut with varying degreesof success.The levelsof infection foundin the horizontaltransmission part of the presentstudy are relatively low (33%) in comparisonto a previousstudy using P. ovis (Brooksand Wall, 2001). It was shownthat when5 cadaversof

A ovis infectedwith the samestrain of M. anisoplidewere introducedto 15 na*fvemites,

139 Chaptcr5- Effcctsof entomopathogcnicfungi on Acaritssiro over 50% of mitesbecame infected. This increasedto over 60% when 10 infectedcadavers wereintroduced to 10 naYvemites (Brooks and Wall, 2001).

It could be that the speciesor strainsof fungi used are more pathogenicto P. ovis thanA. siro but high levelsof infectionwere recorded in A. siro that weredirectly cxposcdto conidia. However,it could be that the high levelsof infection seenin the Brooksand Wall

(2001)paper are partly attributableto the Psoroptesmites being removedfrom thcir natural environmentand thus more susceptibleto infection. The levels of infection and survival seenin the presentstudy with A. siro, may be moreindicative of what might be seenwith A ovis when exposedto a fungal pathogenon host. The resultspresented in Chapter3 show that while P. ovis is highly susceptibleto infectionin vitro, the mitesarc still ableto lay eggs and developto at leastthe protonymphstage while on a sheepeven when exposedto fungal pathogens.

The small-scale,high densitypopulations showed significant prevalence of infection

in the populationsexposed to the fungal pathogen. However, surprisinglythere was no

significanteffect of exposuretime on the prevalenceof infection. This suggeststhat after4 days of exposurethe fungal conidia are either no longer infectious or there are no more

susceptiblemites left to becomeinfected. It is unlikely that thereis a lack susceptiblemites,

as the previouswork has shown that most mites exposedto conidia becomeinfected. It could be that the conidia only remainviable for a few days and that contactwith conidia after this point does not lead to infection. A previousstudy on the pathogenicityof Af anisopliaeto P. ovis showedthat the mostinfectious conidia were those that were 5 days-old andpathogenicity decreased at 10 daysand older (Brooks and Wall, 2001).

Although the smaller populationswere allowed to reproduceand developwithout

interference,only the initial populationof 100adults was usedfor analysisand thereforethe

effectsof the fungal pathogenwere only studiedfor one generation. The large-scale,low

density populationswere studied for several generationsand all life-cycle stageswere

examined. However, there seemedto be no effect of the introducedpathogen on tile

dynamicsof those populations. Only one infected mite was found from all tile mites

140 Chapter5- Effects of entomopathogenicfungi on Acaria x1ro removedfrom the cuvettesat the end of the bioassay. This mite camefrom a population sampled40 dayspost-establishment (20 daysafter the introductionof tile infectedcadavers).

Apart from this infectedmite, the fungalpathogen did not showsigns of persistingwithin tile populationof A. siro.

The growth of the populationfollowed the sametrends as for the patliogen-frce populationwith no significant periodicity. The mean egg output per female peaksat a similar time and the samerelationships are seen betweeneggs laid per female and tile numberof adult mitesas well asthe changesin sexratio. However,there was lessvariation

in the sex ratio in the populationsexposed to the fungal pathogenthan in tile pathogen-frce population. There was also a significant difference in the mean sex ratio of tile two populationswith femalebias in the pathogen-freepopulation but a moreeven sex ratio in the populationwith the pathogen.However, there was considerablevariation in the sex ratio of

bothpopulations across the samplingperiod.

Although there was a greaternumber of mites in the populationwith the pathogen

than in the pathogen-freepopulation, the changein abundanceover time for the variouslife-

cycle stageswere very similar when analysedwith the index of estimation ratio (Q).

However,Chi squareanalysis on the meanabundance of eachlife-cycle stageshowed that

therewas a significantdifference between the two populations.This seemsto be duemainly

to the increasednumber of eggs countedin the populationwith the pathogen. Previous

studieshave found that fungal pathogenssuch as M anisopliaeand A basslanacan lower

the fecundityof infectedfemales (Wekesa et al., 2006;Roy et al., 2008). However,no such

effectwas seenin this study.

The numbersof larvae and nymphal stagesmatch very closely betweenthe two

populations. This would suggest that although more eggs are being laid in the population

with the added pathogen,there is also a greater mortality for the eggs. Conversely, it seems

that the mortality of tritonymphs is less in the population with the added pathogen as the

number of tritonymphs is very similar between the two populations, yet there are a greater

number of adults in the population with the added pathogen. A previous study on the use of

141 Chapter5- EiTectsof cntomopathogenicftingi on Ararin sira

M anisoplideon P. ovis showedthat there was no significant differencefor the levels or infection betweenthe different life-cycle stagesand also that the exposureor eggsto the conidiahad no effect on hatching(Brooks and Wall, 2005).

Giventhe lack of any othersignificant effect of the fungal pathogenon the dynamics of the population and the lack of infection present in the population exposedto At. anisopliae,it could be that the differencesseen between the two populationsarc due to naturalvariation for a speciesof mite that hashighly variablelifc-history parameters.

It may be that the conditionswithin the cuvettcsare not suitablefor the pathogento remain viable and therefore failed to infect enough mites to elicit a noticeableeffect.

Another possibility is that the fungusdid infect the mites but did not inducemycosis and thereforedid not inhibit the normalbehaviour or effect the reproductionand longevityof the mite to a significant extent. Infectedmites that did not exhibit signs of mycosismay not haveproduced conidia after the deathof the mite and thereforethc propagationof infection from mite to mite would not occurand the populationwas ableto grow in a normalmanncr.

The fungal pathogenused in the presentstudy relies upondirect contactbetween the conidiaand the mitesto transmit infection. The small-scalepopulations would havehad a greatercontact rate between mites and the infectedcadavers than the large-scalepopulations.

Althoughthe large-scalepopulations increased in densityover time, it may be that the fungal pathogenwas no longer viable and was not able to infect mites once the contactrate had level increasedto a comparable to the small-scalepopulations. It would seemprobable that density, there is a threshold below which the pathogenis not able to transmit and persist within a population. The small-scalepopulations may have cxcccdedthis thrcsholdand achievedhorizontal transmission, while the large-scalepopulations did not.

142 Chapter6- Discussion

CHAPTER6

DISCUSSION

The work presentedin this study showsthat under tightly controlledconditions it Is possibleto infectPsoroptes ovis on a live animalwith Af anisopliaeand D. basslana.I Iowcvcr, thereare still manyproblems to overcomeif eitherof thesetwo pathogensare to be usedas a practicaltreatment for sheepscab.

Psoroptesmites live on the skin surfaceof the host wherethey arc protectedby the fourth in fleece. In the third and vivo trials conductedin the presentstudy where a high level or fleece infection Wasachieved, the was removed or reduced in length. This meant that it was possibleto apply the fungal conidia in a position that ensuredcontact with the mites. Although here the resultspresented suggestthat there is no intrinsic inhibition by the fleece,it can still act as a physical barrier preventingthe conidia from reachingthe mites. It is unlikely to be practical or desirable to simply shear sheep that are infested with scab on ethical or wclrarc grounds. is be Therefore,a treatment requiredthat can applied to a fully-flecced sheep. This placesa high degreeof importanceon the formulation and application of the conidia.

in The work the presentstudy demonstratedthat different cxcipients had difTcrcnteffects M on the conidia of anisopliae and B. basslana. A large decreasein the dricacy of At, anisopliae was observedwhen conidia were formulated in sheepdip cxcipicnt and diatomaccous earth, compared to the unformulated conidia, which gave the highest level of infection.

Although the same trend was observed for B. basslana there was no signiricant difference high betweenthe treatmentsand a level of infection was achieved for all formulations. Thcrc

less betweenthe formulations was variation tested, although one formulation. Codacidc", gave fewer infections. However, significantly when the sameformulations were testedIn vilro, thm were significant differences in the proportion of infected mitcs between the different formulations and the two fungal pathogens. When AL anisopliae was formulated in Codacidc"'

143 Chaptcr6- Discussion and vegetableoil, a large proportion of mites becameinfected but few mitcs bccamc infcctcd when exposedto conidia in Outpue and sunflowcr oil. When the sameformulations wCrC tcstcd with B. basslanainstead of M. anisopliae,the proportion of infected mitcs was so low that thcrc was no significant difference to the controls that had no infected mites. The only formulation that gave a significantly greater proportion of infected mites than the controls, was conidia In sunflower oil with 6% of mites infected. Clearly fortnulation can makc a significant difrcrcnce to the pathogenicityof conidia.

The different exposuretimes may explain the difference observedfor the formulation or diatomaccousearth that was tested in the in vivo trials. In one In vivo trial, the diatomaccous earth formulation was highly pathogenicwith both fungal pathogensbut was less so in another, especially with M anisopliae. It could be, as discussedin Chapter 3, that diatomaccouscarth requires a longer period of exposure to the mites before having an effect. Until then, the competition between the diatomaceousearth particles and conidia for attachmentsites on the cuticle, inhibits the pathogenicityof the formulation.

The in vivo trials describedhere have shown that for most of the formulations there is no significantdifference in the proportionof mites infected,provided that the mites come into contactwith the conidiafor a long enoughtime period. This suggeststhat the most important aspectof the excipientis whetheror not it will be able to penetratethe fleeceand carry the conidiato the mites. Therefore,an excipientthat maynot be as cffcctiveas othersIn vitro, may bejust aseffective or evenmore so in vivo,if it is betterable to penetratethe fleece.

Of note was the large degreeof variation betweenthe pathogenicityor the various formulations of the two fungal pathogensin vitro and in vivo, but the reasonsfor this difrcrcnce are unclear. One possibleexplanation is the longer time of exposureto the formulations In vivo.

The fourth in vivo trial had an on host exposureperiod of 5 days. Thcrcforc, Icss cfricacious formulationswould have a longer time to infect mites. The In vitro trials cxposcdthe mitcs for 1

144 Chaptcr6- Discussion

min to the fungal pathogen. This might not be long enoughfor the lesspathogenic formulations

to infect all of the mites.

Another possibleexplanation for the variation seenbetween the trials could be variation betweenthe batchesof conidia used. The propertiesof M. anisopliae and B. bassianacan be highly variable betweenbatches, even when rearedunder the sameconditions (Dr Belinda Luke, personalcommunication). The nutritional propertiesof the culture media that fungal pathogens are reared on have been shown to affect the growth and virulence of the conidia (Shah et al.,

2005; Safavi et al., 2007). Repeatedsubculturing has also been shown to affect the growth and virulence of fungal pathogens, with repeated subculturing weakening the virulence of the pathogen(Shah et al., 2007). It could be possiblethat even slight changesin the culture media or the storageof conidia could affect the level of mycosisproduced in the target organism.

Conidia can be damagedby exposureto high temperatures,low humidities and ultra- violet light (McClatchie et al., 1994;Alves et al., 1998;Arthurs and Thomas,200 1; Bragaet al.,

2001a,b, c). Therefore,the storageand formulation of conidia will be particularly important to minimise the effects of exposureto sub-optimalconditions.

Conidia of B. bassiana and M. anisopliae applied to cowpea foliage (Ceratosanihes hilarlana Cogniaux)and exposed to sunlightin Brazil,were found to havelost viability afterone week(Daoust and Pereira,1986a). Conidiathat were protectedfrom sunlightremained viable for up to 3 weeks(Daoust and Pereira, 1986a). Conidia of B. basslanapresent on the surfaceof cadaversof the cowpeapest, Chalcodermus aeneus Boheman, were able to survivefor at least

16 weeksin PVC cylindersin outdoorconditions when protected form direct sunlightand rain

(Daoustand Pereira,1986b). Cadaverskept in cylindersthat were not protectedform direct sunlightor rain hadmuch fewer conidia present although there was no lossin the viability of the remainingconidia (Daoust and Pereira,1986b). The damagingeffects of sunlightare likely to be due to UV-A and UV-B radiation. Thesehave been shown to causedelays in germination

(Bragaet at., 2001a,b). A Ih exposureof M anisopliaeconidia to UV-B radiationcaused a

145 Chapter6- Discussion delay in germination, with only 15% of conidia germinating after 12h (Braga et al., 2001a).

However, 95% of conidia germinatedafter 48h (Braga et al., 2001a). Certain isolatesmay be more tolerant of UV radiation than others. A previous study found that there was a correlation betweenlatitude and toleranceto UV-B exposure(Braga etal., 2001c). Conidiaof strainsofM anisopliae from increasinglatitudes were more susceptibleto damageby UV-B radiation than thosefrom nearerthe equator(Braga et al., 2001c).

Suspendingthe conidia in oils and chemicalsunscreens can increasethe toleranceto UV exposure(Moore et al., 1993; Alves et al., 1998). A previous study found that 22% of conidia of M. anisopliae suspendedin peanut oil, germinated24h after a 6h exposureto UV radiation comparedto 0.5% germination for conidia suspendedin 0.05% Tween 80 (Alves et al., 1998).

Another study used sunscreenadded to a groundnut oil and Shellsol K mixture to suspend conidia of MetarhIzIum flavoviride Gams and Rozsypol (Moore et al., 1993). The use of sunscreenwas found to increasethe germinationof conidia after UV exposure,with oxybenzone providing the most protection; 82% of conidia germinated48h after a 3h exposure(Moore et al.,

1993). Conidia formulated in oil without the sunscreenonly had 28% germination,48h after a

3h exposureto UV radiation (Moore et al., 1993).

Formulation can also be important in limiting the effects of temperatureon the viability ofconidia. The addition of silica gel to conidia storedin oil gave a germination of 81% at361C,

5h after a Ih exposureto 60 *C (McClatchie et al., 1994). In contrast, only 25% of conidia germinatedwhen suspendedin oil without the silica gel (McClatchie et al., 1994). The same study also found that increasingtemperatures decreased the viability of conidia stored in oil and silica gel over time. Only 20% of conidia storedat 55 *C remainedviable after 42 days whereas

50% remainedviable at 45 'C and 80% were viable at 17 OC(McClatchie et al., 1994).

Freezedrying and spray-drying have also been investigatedfor the storageof conidia.

Spray-dryinguses a flow of compressedair to dry the conidia at temperaturesbetween 60 and 80

IC (Stephanand Zimmerman, 1998; Horaczezkand Viernstein, 2004). A study investigatingthe

146 Chapter6- Discussion

effect of spray-dryingon M. anisopliae and B. basslanafound that there was no significant loss

in viability of conidia after they had beenspray-dried (provided that skimmedmilk powder was

addedas a protective agent)compared to the controls that had not beenspray-dried (Stephan and

Zimmerman, 1998). Skimmed milk acts as membranestabiliser during cryopreservationand

also acts as an emulsification agent (Horaczezkand Viernstein, 2004). However, when other protective agentswere used there was a significant decreasein germination after spray-drying

(Stephanand Zimmerman, 1998). Another study using skimmed milk as a protective agent observed93% germinationfor spray-driedM. anisopliae (Kassaet al., 2004). However, another study found that spray-dryingcaused a loss in viability, even when skimmedmilk was usedas a protective agent(Horaczezk and Viemstein, 2004). Conidia of M anisopliae that had not been spray-dried had a germination of 96% after 24h incubation, whereasonly 34% of spray-dried conidia germinated. The sametrend was observedfor Beauverla brongniartil (Saccardo)Petch; control conidia had 96% germination while spray-dried conidia had 37% germination

(Horaczezkand Vierristein, 2004).

Freeze-dryinguses liquid nitrogen to freeze the conidia (approximately -80 *C) before they are placed into an oven at temperaturesbetween -35 T and 40 T (Horaczek and

Viernstein, 2004; Kassa et al., 2004). As with spray-drying, mixed results have been found.

Germination levels of 68% were found for B. hrongniartli but only 4% for M. anisopliae

(Horaczek and Viernstein, 2004). Another study found a germination level of 66% for freeze- dried M. anisopliae (Kassaet al., 2004).

Conidia that have been spray-driedor freeze-driedsuccessfully, can be stored for long periods of time at low temperatures(14oraczek and Viernstein, 2004; Kassa et al., 2004).

Freeze-driedconidia of A brongniartli showed78% germination after 8 weeks of storageat 6

T, although the spray-dried conidia only had 28% germination under the same conditions

(Horaczek and Viemstein, 2004). Freeze-dried conidia of M anisopliae had a lower germination level than B. brongniartil; 35% after 8 weeks at 6T (Horaczek and Viemstein,

147 Chaptcr6- Discussion

2004). However, another study found that over 75% of freeze-dried M. anisopliae conidia

germinatedafter 45 days storageat 10 T (Kassaet al., 2004).

Freeze-drying or spray-drying conidia may provide a potential storage method for conidia but more work needsto be done to identify suitable strains of fungi and appropriate methodsof drying that will not damagethe conidia. The ability for long-term storageof conidia will be an important feature for a potential sheepscab treatment. A product that can be easily stored by farmers or vets that will remain viable through the autumn and winter months, if not severalyears, would be required so that treatmentcould be readily applied when necessary.A product with a short shelf-life would probably not appeal to most farmers. A long shelf-life would also preventthe misapplicationof a product that has lost viability.

There have beenmany studiesconducted on the effect that fungal pathogenscould have on non-targetorganisms (Burgner et al., 1998;Revankar et al., 1999; Dromph and Vestergaard,

2002; Ginsberget al., 2002; Milner et al., 2002; Stolz et al., 2002; Zimmerman,2007a, b).

The isolate of M anisopliae used in the presentstudy for the control of P. ovis has also been found to be pathogenic against the cattle louse B. bovis (Briggs et al., 2006) and the blowfly Lucilia serricata (Meigen) (Wright et al., 2004). This could be beneficial for a treatment of sheep scab as it may be able to provide protection against other ectoparasites.

However, the potential for these fungal pathogensto infect non-target organisms could also cause problems if large quantities are releasedinto the environment via run-off. Both M. anisopliae and B. bassianaare naturally presentin the environment. However, their use in the control of sheepscab could considerablyincrease the number of conidia in certain parts of the environmentat certain times of the year.

A strain of M anisopliae usedto infect the tick, L scapularis was also pathogenicto the ladybird beetle, Hippodamia convergens Guerin-Meneville and the house cricket, Acheta domesticusL. (Ginsberg et al., 2002). Infection of hymenopteranparasitoids exposed to the same strain of M anisopliae used in the commercial product Green Muscle4Dhas also been

148 Chaptcr6- Discussion shown (Stolz et al., 2002). The survival time for adult femalesof the wasp,Apoanagyrus lopezi

De Santis,was reducedby 24% when exposedto conidia of M. anisopliae (Stolz et al., 2002).

However, direct exposure for 10s, of three collembolan species to various isolates of M. anisopliae and B. basslanaconidia at a concentrationof IX 107conidia ml*' had no effect on mortality (Dromph and Vestergaard,2002). Continual exposure to M. anisopliae conidia in sphagnummoss resulted in an increasedmortality for the collembolan, Folsomlafimetaria (L. ) after 14 days, with 42% mortality for exposedF. fimetarla and only 17% for controls (Dromph and Vestergaard,2002).

A study conducted on the risk of M anisopliae to aquatic ecosystemsfound no significant difference betweenthe mortality of mayfly nymphs (Ulmerophlebia spp.) exposedto

2x 10' conidia ml" and nymphs that were not exposedto conidia (Milner et al., 2002). There was also no effect of conidia on 8 week-old rainbow fish (Melanotaenia duboulayl Casteinau), althoughit was not reportedin the paperwhat parameterswere being tested(Milner el al.,

2002). At concentrationsof 1.3 x 10' conidia ml", 100% mortality of the cladoceran,

Ceriodaphniadubla Richardoccurred within 48h of exposure(Milner et al., 2002). However theseconcentrations are likely to be much greaterthan they would experiencein the field.

Samplestaken from six water bodies, 48h after field spraying, showed low levels of contamination,with only 18 of 96 agar platesinoculated with the sampledwater, growing coloniesof M. anisopliae(Milner et al., 2002). Howeverthe size of the waterbodies or their locationin relationto the field sprayingwas not reported.

The effectof entornopathogenicfungi on vertebrateshas also been investigated. A study that fed laboratoryreared mice with food inoculatedwith M. anisopliaeconidia, found no clinical signs of infection or harm to the mice (Toriello et al., 2006). Conidia of M. anisopliae and B. basslanaalso appearto be harmlessto reptiles, amphibiansand birds when ingestedor via skin contact (Zimmerman, 2007a,b). However, a case of a domestic cat with sinusitis was shown to have an infection of M. anisopliae (Muir et al., 1998). The cat was successfully

149 Chapter6- Discussion

treatedwith itraconizole, administeredorally (Muir et al., 1998). Two human casesof sinusitis

have also been attributed to M anisopliae (Revankaret al., 1999). One occurred in a 36 year-

old male and the other casewas a 79 year-old female. Both humans were immunocompetant

and were treated surgically without the use of antifungal therapy (Revankaret al., 1999). One

case exists of an immunocopromisedchild with a disseminated infection of M. anisopliae

(Burgner et al., 1998). Lesions were found in the patient's lungs, skin and brain. Although the patient's deathwas not causedby the infection, it was concludedthat the infection contributedto his deathby preventingfurther chemotherapeutictreatment (Burgner et al., 1998).

Although it has been shown that both of the fungal pathogensused in the presentstudy are able to infect non-target organisms, it is unlikely that these organisms would become infected under field conditions. The studiesmentioned previously have all concludedthat while it was possible to infect the study organism in the laboratory, the concentrationsof conidia required to induce those infections are much greater than that which the organism would be exposedto in the wild. The fungal pathogensM. anisoplide and B. basslanaare still considered to be safe to vertebratesand non-target organisms and are unlikely to pose a threat to the environment.

It has been shown that non-targetorganisms could even be utilised as dispersalagents of entornopathogenicconidia (Butt et al., 1998). Pollen beetles (Meligethes aeneus (F.)) were maintainedon flowers in cages,in the presenceof honeybees(ApIs mellifera L. ). Trays of M. anisopliae conidia were placed at the entranceof the hive so that the beeswould pick up conidia as they walked acrossthe surface. Mortality levels for pollen beetleskept in cageswith beesand conidia reached99% for samplestaken 7 days after the start of the trial (Butt et al., 1998). The mortality of pollen beetlesmaintained in the presenceof beesbut no conidia were only 23%, 7 days post-establishment(Butt et al., 1998). The authors concluded that honey bee mediated transportof conidia to flowers were pollen beetlesare present,could be incorporatedinto a crop managementstrategy (Butt et al., 1998).

150 Chapter6- Discussion

Although the work in Chapter3 has shown that it is possible to infect Psoroptesmites

on a live animal, it has yet to be seenif mycosiswill fully develop while the mite is on the host.

All infected mites were removed from the sheep before they showed any exterior signs of

mycosis. It was observedin the fourth in vivo trial that oviposition continuedin the presenceof

the conidia as did the subsequentdevelopment of the eggs up to the protonymphstage. At this

point the trial endedso it is not known whether or not the mites would continueto developinto

reproductiveadults.

In vitro experiments using Psoroptes mites exposed to conidia, produced low LT50

values. This was due to the sub-optimal conditions for the Psoroptesmites that are unableto

survive for long periods off host at the conditions used in the bioassays(30 OC, 90% r.h. ).

However, the in vitro bioassaysconducted with A. siro rather than P. ovis had larger LT5o

values. Thesebioassays were conducted at conditionsthat werehighly suitablefor the survival h. anddevelopment of the mites(20 IC, 70%r. ). Althoughthe conditionsthat P. ovisand '4. siro are exposedto are considerablydifferent, the 4. sirolfungalpathogen system provides useful

informationas to what couldbe expectedin populationsof P. ovis exposedto a fungalpathogen in vivo. The laboratorypopulations did not reacha stableage structure, which waspredicted to occurapproximately 300 dayspost-establishment. However, this is not a problemwhen using the systemto gain an insightinto what might occurwhen a fungal pathogenis introducedto a populationof P. ovis on a live host. The infestationof P. ovis will rapidly grow but is unlikely to reacha stable-agestructure before the point at which interventionis required. Thereforethe transientstate of the A. siro populationsshould give a closerepresentation of a growingP. ovis populationat a point at whichthe fungalpathogens would be applied.

Previousstudies, investigating the horizontaltransmission of fungal infection from infectedcadavers to live, nafve Psoroptesmites, showeda high level of transmissionand subsequentinfection; 50 - 60%(Brooks and Wall, 2001). The currentstudy (Chapter 5) showed that levelsof transmissionand infection from infectedcadavers to live, naYveA. s1rowere lower,

151 Chapter6- Discussion

only 33%. When the pathogen was introduced via infected cadavers to small but dense

populations it was able to induce between 22% and 42% infection within the adult mites.

However, when the pathogenwas introduced to larger, less dense populations,there were no

signs of infection or of any effect on the developmentof the populations. This suggeststhat

there could be a density threshold, below which the contact rate is not sufficient to transmit the

diseasethrough the population. Although M. anisopliae induces high levels of infection in A.

s1ro with controlled exposures, it seems that the fungus lacks enough pathogenicity and

transmissibility to persist within growing populations of A. siro. The initial density of the

population is too low for a pathogenthat is unableto infect every individual that makescontact

with conidia or infected cadavers. By the time the A. siro populations reacheda density that

could support the transmissionof the pathogen,the conidia were no longer viable. It could also be that any individual that becameinfected was not killed before reproducingand ovipositing.

The transmissionand persistenceof pathogensin many casesdepends on host density, with important implications for the control of pests,parasites and disease(Swinton et al., 1998;

McCallum et al., 2001). The effect of density is complicated in populations that are not constrainedto particular geographicalareas. Many pest speciesare characterisedby their ability to disperseand colonisenew areas.This migrationoffers the populationa chanceto escapethe pathogen(Loehle, 1995;Bradley and Altizer, 2005). If the proportionof infectedindividuals within a populationis low, it is unlikely that any new colonythat is formedby migrationwill containenough infected individuals for the pathogento be maintainedwithin the newpopulation

(Andersonand May, 1979). Someviruses are unable to persistat sub-thresholdhost densities

(Swinton et al., 1998) and the sameprinciple might apply to fungal pathogens. Even if the level of infection is high within an original population,migration would still provide an opportunityto escapethe disease. New populations formed from migration of a few individuals would most likely exist, initially, at low densitieswhich would be below the density threshold requiredfor the persistenceof the disease(Anderson and May, 1979). Therefore, populations,such as 4.

152 Chapter6- Discussion

siro, infectedwith a pathogenlike M. anisopliae,would be able to evadeinfection through

migration.This couldalso be the casefor P. ovis. Populationsof A ovisgrow rapidly on a host

and spreadfrom the initial point of infestationto cover a large part of the host body. This

growthmay occurtoo rapidly for a fungalpathogen to persistwithin the expandingelement of

the population.Even if an infectedmite is ableto migratewith the expandingpopulation, it will

not becomeinfectious to othermites until it hasdied and sporulated. The growthof conidiaon

the surfaceof a deadmite doesnot usuallyoccur until 48h after death. By this point the restof

the developingpopulation may have moved on (Bradleyand Altizer, 2005).

One of the possiblebenefits of biologicalcontrol using entomopathogenic fungi is the

potentialthat only one treatmentneeds to be appliedfollowing which the infectionwill spread

andproduce new conidia. This wouldreduce the initial volumeof treatmentrequired. The work

in this thesishas shown that this is not necessarilythe case. It may be necessaryto reapplythe

treatmentat certaintimes after the initial applicationto ensurethat there is a fresh supplyof

viable conidiain an areain contactwith the pest. It may also be necessaryto ensurethat all

pestscome into direct contactwith the treatmentand not rely on the transmissionfrom one

individualto another.

There is still potentialfor use of entornopathogenicfungi for the treatmentof sheep

scab. However, the work presentedin this thesis suggeststhat it is unlikely that either M.

anisopliae or B. bassianawill provide an effective treatmentfor sheepscab in the near future.

Although it has been shown that both fungal pathogenstested can infect P. ovis on a live animal,

it remains to be seen whether or not these infections are lethal to the mites in vivo. The work using A. siro as a model specieshighlights the problemsof using a pathogenwith low virulence

in populationsof a low density or an ability to migrate. The work presentedin Chapter 3 also

demonstratesthe difficulties in the application of a fungal treatment to control scab. Even if a highly pathogenicstrain of fungus is found that is highly efficacious againstPsoroptes mites in vivo, a suitablemethod of application is still needed.

153 Chaptcr6- Discussion

Perhapsthe use of fungal pathogensagainst scab mites of cattle or other ectoparasites

such as lice, where fleece is not a barrier to the treatment,would be more effective. However, a

previous study on the use of M. anisopliae against A bovis showed that although there was a

high degreeof infection in the lice exposedto conidia in vivo, not all lice exhibited signs of

infection (Briggs et al., 2006). When conventionalinsecticides are usedto treat againstpest or

parasite species, complete control is usually the expected outcome. However, the use of

alternative treatments such as entornopathogenicfungi, will require different goals and

expectationswill need to change. Rather than using biological control as a replacementfor conventionalcontrol methods,they could be usedin conjunction with eachother. The biological controls could be used for suppression,to maintain infestationsat levels below a predetermined welfare and economic threshold (Wall, 2007). If the diseaseor infestation rises above that threshold then conventional treatments could be used selectively. Although this strategy

incorporates the use of chemical pesticides, it would decreasethe amount used and thus minimise the exposureof potentially harmful chemicalsto humansand the environment.

Entornopathogenicfungi have been combined with chemical pesticides in previous studies. The addition of imidacloprid (a neonicotinoid insecticide) to B. bassianasignificantly increasedthe mortality of the aphids My= persicae (Sulzer) and Macrosiphoniella sanborni

(Gillette) (Ye et al., 2005) compared to the unexposedcontrols and aphids exposed to A bassiana only. The volume of imidacloprid added to the conidia was below the lethal dose required for imidacloPrid alone. The authors concluded that the combination would be an effective treatment and reduce the volume of pesticide used to control these pests (Ye et al.,

2005). The same trend was observed in another study combining imidacloprid with M. anisopliae (Jaramillo et al., 2005). The combinationof the insecticidewith the fungal pathogen increasedmortality comparedto the use of the conidia alone when applied to the subterranean burrower bug, Cyrtomenusburgi Froeschner(Jaramillo et al., 2005). Metarhizium anisopliae has also been combined with the pyrethroid, deltamethrin for the control of the cattle tick,

154 Chapter6- Discussion

Boophilus m1croplus (Canestrini) (Bahiense et al., 2006). As with the previous studies, it was

found that mortality was greater for the ticks exposed to the conidia combined with deltamethrin

than for the ticks exposed to conidia alone (Bahiense et al., 2006). It was also found that the

combination of conidia and deltamethrin induced greater mortality than deltamethrin only

(Bahiense et al., 2006). Clearly there is the potential for the combination of entomopathogenic

fungi and conventional pesticides for the control of sheep scab. A combination like this would

potentially increase the efficacy of the fungal pathogen while decreasing the volume of

insecticide used. Further research into the effects of combining entomopathogenic fungi with the conventional treatments of sheep scab such as diazinon and the macrocyclic lactones would provide useful data. Combination of the fungal pathogens with a dip or a pour-on treatment would probably involve a straight-forward mixture. However, use of the pathogens with the

injectable avermectins would require two separate applications of treatment to the sheep; an

injection for the avermectins and a dip or pour-on for the fungal pathogen. This may prove to be too time consuming and too costly to be a practical treatment.

Future work must identify an excipient which can deliver conidia to the skin surface, where the mites are present. This might preclude many of the excipients currently used in agriculture and veterinary medicine, and require the developmentof untestedcompounds which would needto be testedfor the safety to humansand other non-targetorganisms. Alongside the developmentof new formulations, in vivo trials examining the efficacy of fungal pathogensto

Psoroptesmites need to be conducted. Theseshould progressfrom the small-scalearena trials describedhere to larger-scaletests, perhapsusing larger arenason the skin of sheepwith fleece that has not been sheared. Lower concentrationsof conidia should also be tested to find the minimum threshold of conidia required to produce infected mites. Most importantly, trials conductedover a longer time period need to be carried out to see whether infected mites will develop mycosis on a host. Using a careful, stagedapproach, trials could eventually move onto

155 Chaptcr6- Discussion whole sheeptrials using a potential fungal-pathogen-basedtreatment to control an infestationof sheepscab.

There is still potential for use of fungal pathogensfor the control of sheepscab but there are many problems that need to be overcome before a marketable product can be produced.

Although M anisopliae and A bassiana have been shown to be effective against many pest arthropods,doubts still remain about it's efficacy againstPsoroptes mites in vivo.

156 Refcrcnccs

References

Alves,R. T., Bateman,R. P., Prior,C. andLeather, S. R. (1998). Effectsof simulatedsolar radiationon coniclialgermination of Metarhiziumanisopliae in differentformulations. CropProtection 17: 675- 679.

Alves, S. B., Tarnai, M. A., Rossi,L. S. and Castiglioni, E. (2005). Beauveriahassiana pathogenicityto the citrus rust mite Phyllocoptruta oleivora. Experimentaland AppliedAcarology 37: 117- 122.

Alzaga, R., Pascual,E., Effa, P. and Bayona,J. M. (1999). Developmentof a novel supercriticalfluid extractionprocedure for lanolin extraction from raw wool. Analytica Chimica Acta 381: 39- 48.

Anderson,R. M., May, R. M. (1979). Populationbiology of infectious d iscascs: part 1. Nature280: 361 -3 67.

Ansari,M. A., Shah,F. A., Whittaker,M., Prasad,M. andButt, T. M. (2007). Controlof westernflower thrips (Frankliniella occidentalis) pupae with Melarhiziumanisopliac in peat and peat alternativegrowing media. Biological Control 40: 293 - 297.

Arruda,W., Lubeck,I., Schrank,A. andVainstein, M. H. (2005). Morphologicalalterations of Metarhiziumanisopliae during penetration of Boophilusmicroplus ticks. Experimental and Applied AcaroloSy 37: 231 - 244.

Arthurs,S. andThomas, M. B. (2001). Effectsof temperatureand relative humidity on sporulationof Metarhiziumanisoplide var. acridumin mycosedcadavers of Shictocercagregaria. Joumal ofInvertebratePathology 78: 59 - 65.

Babcock,O. G. andBlack, W.L. (1933).The commonsheep scab mite andits control.Texas Agricultural ExperimentStation Bulletin 479: 1-34.

Bahiense,T. C., Fernandes,E. K. K. andBittencourt, V. R. E. P. (2006). Compatibilityof the fungusMetarhizium anisopliae to control a resistantstrain of Boophilus microplus tick. Veterinaryparasitology 141: 319 - 324.

157 Rcfcrcnces

Barreto, R. S., Marques,E. J., Gondim Jr., M. G. C., de Oliveira, J. V. (2004). Selectionof Beauveriabasslana (Bals. ) Will. andMetarhizium anisopliae (Mctsch. ) Sorok.for the control of the mite Mononychellustanajoa (Bondar). ScientiaAgricola 61: 659 - 664.

Bateman.R. P, Douro-Kpindou,0. K., Kooyman,C., Lomer,C. andOuambarna, Z. (1998). Someobservations on the dosetransfer of mycoinsecticdesprays to desertlocusts. Crop Protection 17: 151- 158.

Bates,P. (I 997a).Insecticide resistance in sheepectoparasites. Veterinary Practice: 6-8.

Bates,P. (I 997b). The pathogenesisand ageingof sheepscab lesions- part 1. State VeterinaryJournal 7: 11 - IS.

Bates,P. (1999). Inter- and intra-specificvariation within the genusPsoroples (Acari: Psoroptidae).Veterinmy Parasitology 83: 201-217.

Bates,P. (2004). Therapiesfor ectoparasitism.in sheep. In PracticeNovember/December: 538-547.

Batta,Y. A. (2004). Controlof the rice weevil(Sitophilus or)zae L., Coleoptera: Curculionidae)with variousfonnulations of Metarhiziumanisoplide. Crop Protection 23: 103- 108.

Begon, M. and Mortimer, M. (1981). Population Ecology: A Unified Study ofAnimals and Plants. Chapter 3: 31 - 50. BlackwellScientific Publications, London.

Berreen,J. M. (1974). Developmentand validation of a simplemodel for populationgrowth in the grainmite, Acarus siro L. JournalofStored Products Research. 10,147 - 154.

Berriatua,E., French,N. P., Wall, R., Smith,K. E. andMorgan, K. L. (1999). Within-flock transmissionof sheepscab in naYvesheep housed with singleinfested sheep. VeterinaryParasitolojT 83: 277- 289.

Berriatua, E., French,N. P., Broster, C.E., Morgan, K. L. and Wall, R. (2001). Effect of infestationwith Psoroptesovis on the nocturnalrubbing and lying behavourof housed sheep.AppliedAnimal Behaviour Science71: 43-55.

158 Refcrcnccs

Bensignor,E. and Carlotti, D. N. (2002). Sensitivity patternsto housedust mites and forage in dogs: mites atopic 150cases. Veterinary Dematology 13:39 - 44.

Bisdorff, B, Milnes, A. and Wall, R. (2005). Prevalenceand regional distribution of scab,lice in andblowfly strike GreatBritain. VeterinaryRecord 158: 749- 752.

Blake,B. H., Bay,D. E., Meola,S. M. andPrice, M. A. (1978).Morphology of themouthparts of the sheepscab mite, Psoroptesovis. Annals of the Entomological SocietyofAinerica 71: 289-294.

Blanford, S., Thomas,M. B. Langewald,J. (1998). Behavioural fcvcr in tile Senegalese grasshopper,Oedaleus senegalensis, and its implications for biological control using pathogens.Ecological Entomology23: 9- 14.

Boczek, J. H. and Davis, R. (1985). Effects of alternatingtemperatures onAcarus siro L. (Acari: Acaridae). ExperimentalandAppliedAcarology 1: 213 - 217.

Boucias,D. G. and Pendland,J. C. (1991). Attachmentof mycopathogensto cuticle: the initial eventof mycosesin arthropodhosts. In: Fungalspores and disease initiation in plants andanimals. Ed. G.T. Coleand H. C. Hoch.London, Plenum Press: 101-127.

Boyce,W. M. andBrown, R. N. (1991). Antigenic characterization of Psoroplesspp. (Acari: Psoroptidae)mites from differenthosts. Journal ofParasitology 77: 675-679.

Boyce, W., Elliott, R. C. and Jessup,D. (1990). Morphometric analysisof Psoroptesspp. mites from bighorn sheep,mule deer,cattle and rabbits. Journal of Parasitology76: 823-828.

Bradley,C. A. andAltizer, S.A. (2005). Parasiteshinder monarch butterfly flight: implications for diseasespread in migratory hosts. Ecology Letters 8: 290 - 300.

Braga,G. U. L., Flint, S. D., Messia,C. L., Anderson,A, J. andRoberts, D. W. (2001a). Effect of UV-B on conidia and germlingsof the entomopathogcnichyphomyccte Metarhizium anisopliae. Mycological Research105: 874 - 882.

159 Refcrences

Braga,G. U. L., Flint, S. D., Miller, C. D., Anderson,A. J. and Roberts,D. W. (2001b). Both solarUVA andUVB radiationimpair conidial culturability and delay germination in the entornopathogenicfungus Metarhizium anisopliae. Photochemistryand Photobiology 74: 734 - 739.

Braga,G. U. L., Flint, S. D., Miller, C. D., Anderson,A. J. andRoberts, D. W. (2001 c). Variability in responseto UV-B amongspecies and strains of Metarhiziumisolated from sites at latitudesfrom 61 *N to 54 "S. Journal ofInvertebrate Pathology 78: 98 - 108.

Briggs, L., Colwell, D. D. and Wall, R. (2006). Control of the cattle louseBovicola bovis with the fungal pathogenMetarhizium anisopliae. VeterinaryParasitology 142: 344 - 349.

Brooks, A. J. (2004). Entornopathogeniceffects of the fungus,Metarhizium anisopliae (MetschnikofT)(Deuteromycotina: Hyphomycetes), on Psoroptesscab mites (Acari: Psoroptidae).Ph. D thesis.

Brooks, A. J., Aquino de Muro, M., Buffee, E., Moore, D., Taylor, M. A. and Wall, R. (2004). Growthand pathogenicity of isolatesof the fungusMetarhizium anisopliae against the parasiticmite, Psoroptes ovis: effectsof temperatureand formulation. Pest ManagementScience 60: 1043- 1049.

Brooks,A. J. andWall, R. (2001). Infectionof Psoroptesmites with the fungusMetarhizium anisopliae. Experimentaland Applied Acarology 25: 869 -880.

Brooks, A. and Wall, R. (2005). Horizontal transmissionof fungal infection by Metarhizium anisopliae in parasiticPsoroptes mites (Acari: Psoroptidae).Biological Control 34: 58-65.

Burgner, D., Eagles,G., Burgess,M., Procopis,P., Rogers,M., Muir, D., Pritchard,R., Hocking,A. andPriest, M. (1998).Disseminated invasive infection due to Metarhiziumanisopliae in an immunocompromisedchild. Journal of Clinical Microhiology 36: 1146-1150.

Butt, T. M, Carreck,L. Ibrahim,L. andWilliams, 1. H. (1998). Honey-bee-mediatedinfection of pollen beetle (Meligethesaeneus Fab. ) by the insect-pathogenicfungus, Metarhizium anisopliae. Blocontrol Scienceand Technology8: 533 - 538.

160 Refercnces

Butt, T. M. and Goettel, M. (1997). Bioassaysof entornogenousfungi. flarpendcn, Herts. IACR-Rothamsted.

Butt, T. M., Ibrahim, L., Ball, B.V. and Clark, S.J. (1994). Pathogenicityof the entomogenous fungi Metarhiziumanisoplide and Beauveria bassiana against crucifer pests and the honeybee. Biocontrol Science and Technology4: 207-214.

Calvo, M., Femandez-Caldas,E., Arellano, P. Marin, F., Carnes,J. and Hormaechea,A. (2005). Mite allergen exposure,sensitization and clinical symptomsin Valdivia, Chile. Journal ofInvestigational, 41lergologyand Clinical Immunology 15: 189- 196.

Chandler,D., Davidson, G. and Jacobson,R. J. (2005). Laboratory and glasshouseevaluation of entornopathogenicfungi againstthe two-spottedspider mite, Telranychusurticae (Acari: Tetranychidae),on tomoato,Lycopersicon esculentum. Biocontrol Science and Technology15: 37- 54.

Charnley,A. K. (1989). Mechanismsof fungal pathogenesisin insects.In: Biotechnology of fungi for improvingplant growth. Ed. W. J.M. andL. R.D. Cambridge,Cambridge University Press:85-123.

Clark, A. M., Stephen,F. B., Cawley, G.D., Bellworthy, S.J. and Groves,B. A. (1996). Resistanceof the sheepscab mite Psoroptes ovis to propetamphos.Veterinary Record 139:451.

Clarkson,J. M. andCharnley, A. K. (1996).New insightsinto the mechanismsof fungal pathogenesisin insects.Trends in Microbiology 4: 197-203.

Coderch,L., Fonollosa,J., Martf, M. andParra, J. L. (2004). Ceramidesfrom wool wax. Journalof1he American Oil ChemistsSociety 81: 897- 898.

Coles, G. C. and Stafford, K. A. (1999). The in vitro responseof sheepscab mites to pyrethroidinsecticides. Veterinary Parasitology 83: 327- 330

Cook, D. A., Wakfield, M., E. and Bryning, G. P. (2008). The physical action of three diatomaceousearths against the cuticleof the flour miteAcarus siro L. (Acari: Acaridae). PestManagement Science 64: 141- 146.

161 Refcrences

Cooke,C. M., Shaw,G., Lester, J. N. and Collins, C. D. (2004). Determinationof solid-liquid partitioncoefficients (K-d) for diazinon,propetamphos and cis-permethrin: implications for sheepdip disposal. Scienceof the Total Environment329: 197- 213.

Corente,C. H. andKnalle, W. (2003). Trophicdeterminants of hypopusinduction in the stored-productmite Lepidoglyphus destructor (Acari: Astigmata).Experimental and Applied. 4carology 29: 89 - 107.

Corke, M. J. and Broom, D. M. (2000). SheepScab: a questionnairesurvey of sheepfarmers. Proceedingsof theSheep Veterinary Society 24: 115-121.

Cunnington,A. M. (1965). Physical limits for the completedevelopment of the grain mite Acarussiro L. (Acarina:Acaridae), in relationto its world distribution. TheJournal of Applied Ecology 2: 295 -3 06.

Cunnington,A. M. (1984). Resistanceof the grain mite, 4carussiro L. (Acarina: Acaridae)to unfavourablephysical conditions beyond the limits of its development.Agricullure, EcosystemsandEnvironment 11: 319 - 339.

Cunnington,A. M. (1985). Factorsaffecting oviposition and fecundity in the grainmite Acarussiro L. (Acarina:Acaridae), especially temperature and relative humidity. ExperimentalandAppliedAcaroloSy 1,327 - 344.

Daoust,R. A. andPereira, R. M. (1986a).Stability of entomopathogcnicfungi Beauveria hassianaand Metarhizium anisoplide on beetle-attractingtubers and cowpea foliage in Brazil. Environmental Entomology15: 1237- 1243.

Daoust,R. A. andPereira, R. M. (1986b).Survival of Beauveriabassiana (Deutermycetes: Moniliales)conidia on cadaversof cowpeapests stored outdoors and in laboratoryin brazil. EnvironmentalEntomology 15: 642- 647.

Deane,K. R. andTruter, E. V. (1959). Theconstitution of suint. Part11. Organic acids. Journal ofthe ChemicalSociety: 2746 - 2749.

162 Refcrenccs

De Oliviera, R. C., Neves, P. M. 0. J. and Alves, L. F. A. (2004) Entornopathhogenicfungi selectionto control Ofigonychusyothersi (McGregor) (Acari: Tetranychidae) in Paraguaytea crops (flexparaguariensis St Hill. ). Netropical Entomology 33: 347 - 351.

Downing,W. (1936a).The life-historyof Psoroptescommunis var. oviswith particular referenceto latentor suppressedscab. Journal of ComparativePathology and Therapeutics49: 163 - 180.

Downing,W. (1936b).The life-historyof Psoroptescommunis var. oviswith particular referenceto latent or suppressedscab. Journal of ComparativePathology and Yherapeutics49: 183-209.

Dromph, K. M. and Vestcrgaard,S. (2002). Pathogenicityand attractivenessof entornopathogenichyphomycete fungi to collembolans. Applied Soil EcoloV 21: 197

-210.

Dunn,J. A., Prickett,A. J. andThind, B. B. (2005). Assessmentof monitoringmethods for earlydetection of threespecies of storagemite in bulk oilseedrape. Experimentaland Applied.4carology 37: 131- 140.

Ebeling,W. (1971). Sorptivedusts for pestcontrol. AnnualReview ofEntomology 16: 123- 158.

Eychenne,V., Sdiz,S., Trabelsi, F. andRecasens, F. (2001).Near-critical solvent extraction of wool with modified carbon dioxide - experimentalresults. Journal ofSupercrifical Fluids 21: 23 -31.

Fejt,R. and2dArkovd, E. (2001). BionornicsofAcarus siro L. (Acarina:Acaridae) on oilseeds.Plant ProtectionScience 37: 110- 113.

Ferron,P. (1981). Pest Control by the fungi Beauveriaand Metarhizium. In: MicrobialControl of Pestsand Plant Diseases1970-1980. Ed. H.D. Burges. London, Academic Press: 465-482.

Fichi., G., Flarnini, G., Zaralli, L. J. and Perrucci,S. (2007). Efricacy of an esscntialoil of Cinnamomumzeylanicum against Psoroptes cuniculi. Phylomedicine14: 227 - 231.

163 Refcrcnces

Fletcher,T. and MacLehose,R. (2005). Effects of OP exposurein people. VeterinaryRecord 157: 99.

Forbes,A. B., Pitt, S. R., Baggott, D. G., Rehbien,S., Barth, D., Bridi, A. A., Carvalho,L. A. andO'Brien, D. J. (1999). A reviewof the useof a controllcd-rclcase formulation of ivcrmcctinin thetreatment and prophylaxis of Psoroptesovis infestationsin sheep. VeterinaryParasitology 83: 319 - 326.

Frazzon,A. P. G., Vaz, I. D., Masuda,A., Schrank,A. and VAnistein, M. H. (2000). In vitro assessmentof Metarhiziumanisoplide isolates to controlthe cattletick Boophilus microplus. VeterinaryParasitology 94: 117- 125.

French,N. P., Berriatua, E., Wall, R., Smith, K. and Morgan, K. L. (1999). Sheepscab outbreaksin Great Britain between1973 and 1992:spatial and temporal patterns. VeterinaryParasitology 83: 187-200.

Gervais,M. P. (1841). Sur quelquesesp6ces de Pordredes Acariens. Annales desSciences Naturelles Partie Zoologique 15: 5- 10.

Gillespie,A. T. andMoorhouse, E. R. (1989)Theuse of fungi to controlpests of agricultural and horticulotural importance. In: Biotechnologyoffungifor improvingplant growth. Ed. J. M. Whippsand R. D. Lumsden.Cambridge, Cambridge University Press. Chapter 4: 55 - 83.

Gillespie,J. P., Burnett,C. andCharnley, A. K. (2000). The immuneresponse of thedesert locust Schistocercagregaria during mycosisof the entornopathogenicfungus, Metarhizium anisopliae var acridum. Journal of Insect Physiology 46: 429 - 437.

Gindin,G., Samish,M., Zangi,G., Mishoutchenko,A. andGlazer, I. (2002). flie susceptibility of differentspecies and stages of ticks to entornopathogenicfungi. Experimental and AppliedAcarology 28: 283-288.

Ginsberg,H. S., Lebrun, R.A., Heyer, K. and Zhioua, E. (2002). Potential nontargeteffects of Metarhizium anisoplide (Deuteromycetes)used for biological control of ticks (Acari: Ixodidae). Environmental Entomology31: 1191-1196.

164 Refercnces

Griffiths, D. A. (1962). Flour mite, Acarus siro L., 1758, as a speciescomplex. Nature 196: 908

Hirst, S. (1922). Mites injurous to domesticanimals. In: British Museum (AraturalHistory) EconomicSeries No. 13. London

Hountondji, F. C. C., Hanna,R., Cherry, A. J., Sabelis,M. W., Agboten, B. ad Korie, S. (2007). Scaling up testson virulence of the cassavagreen mite fungal pathogen Neozygitestanajoe (Entomophthorales:Neozygitaceae) under controlled conditions: first observationsat a population level. ExperimentalandAppliedAcaroloSy 41: 153

-168.

Horaczek,A. and Viernstein, H. (2004). Comparisonof three commonly useddrying technologieswith respectto activity and longevity of aerial conidia of Beauveria brongniartii and Metarhizium anisopliae. Biological Control 31: 65 -71.

Hubert,J., Stejskal,V., Kubatova,A., Munzbergova,Z., Vanova,M. and2dirkov4, E. (2003). Mites asselective fungal carriers in storedgrain habitats. Experimental andApplied Acarology 29: 69 - 87.

Hubert, J., Stejskal,V., Munzbergova,Z., Kubatova,A., Vanova, A and 2dirkovd, E. (2004). Mites andfungi in heavilyinfested stores in the CzechRepublic. Journal ofEconomic Entomology97: 2144- 2153.

Hunter,D. M., Milner, R. J., Scanlan,J. C. andSpurgin, P. A. (1999) Aerial treatmentof the migratory locust, Locusta migratoria (L. ) (Orthoptera:Acrididae) with Metarhiziun, anisoplide (Deuteromycotina:Hyphomycctes) in Australia. Crop Protection 18: 699 - 704.

Huntleyj. F., Machell,J., Nisbet, A. J.,Van DenBroek, A., Chua,K. Y., Cheong,N., Hales,B. J. andThomas, W. R. (2004). Identificationof tropomyosin,paramyosin and apolipopborin/vitellogeninas three major allergens of the sheepscab mite, Psoroples ovis. Parasite Immunology26: 335 - 342.

165 Refcrenccs

Huntley, J. F., van den Broek, A., Machell, J., Mackellar, A., Pettit, D., Meikle, L., Barcharn, G., Meeusen,E. N. T. andSmith, D. (2005). The effectof immunosuppressionwith cyclosporin A on the developmentof sheepscab. VeterinaryParasitology 127: 323 - 332.

Huxham,1. M., Lackie,A. M.,McCorkindale, N. J. (1989). Inhibitory effectsof cyclodepsipeptidcs,destruxins, from the fungusMetarhizium anisopliae, on cellular- immunity in insects. Journal ofInsect Physiology35: 97 - 105.

Inglis, G. D., Duke,G. M., Kawchuck,L. M. andGoettel, A S. (1999). Influenceof oscillating temperatureson the competitive infection and colonization of the migratory grasshopperby Beauveria bassianaand Metarhizium anisopliae. Biological Control 14: 111- 120.

Inglis, G.D., Goettel, M. S., Butt, T. M. and Strasser,H. (2001). Use of hyphomycetousfungi for managinginsect pests.In: Fungi as Biocontrol AgentsProgress, Problems and Potential. Ed. T. M. Butt, C. Jacksonand N. Magan. Wallingford, Uk, CABI Publishing.

Inglis, G. D., Johnson,D. L. andGoettel, M. S. (1996). Effectsof temperatureand thermoregulationon mycosisby Beauveriabassiana in grasshoppers.Biological Control7: 131- 139. lzraylevich, S. and Gerson,U. (1995). Sexratio of Hemisarcoptescoccophagus, a mite parasiticon insects:density-dependent processes. Oikos 74: 439- 446.

Jaramillo,J., Borgerneister,C., Ebssa,A., Gaigl,A., Tob6n,R. andZimmcrman, G. (2005). Effect of ombinedapplications of Metarhiziumanisoplide (Metsch. ) Sorokin (Deuteromycotins:Hyphomycetes) strain CIAT 224 anddifferent dosages of imidaclopridon the subterraneanburrower bug Cyrtomenus berg! Frocschner (Herniptera:Cydnidae). Bioogical Control 34: 12- 20.

Kaaya, G.P., Mwangi, E.N. and Ouna, E.A. (1996). Prospectsfor biological control of livestockticks, Rhipicephalus appendiculatus andAmblyomma variegalunt, using tile entomogenousfungi Beauveriabassiana and Metarhizium anisopliae. Journal of Invertebrate PalholoU 67: 15-20.

166 Refcrcnces

Kaaya, G.P. and Okech,M. A. (1990). Horizontal transmissionof mycotic infection in adult tsetse,Glossina morsitans morsitans. Entomophaga 35: 589-600.

Kanga, L. H. B., Jones,W. A. and James,R. R. (2003). Field trials using the fungal pathogen, Metarhiziumanisoplide (Dcutcromycetes: Hyphomycetes) to controlthe ectoparasitic mite, Varroadestructor (Acari: Varroidae) in honeybee, Apis mellifera (Hymenoptera:Apidae) colonies.Journal ofEconomic Entomology96: 1091-1099.

Kassa,A., Stephan,D., Vidal, S. and Zimmerman,G. (2004). Productionand processingof Metarhiziumanisopliae var. acridumsubmerged conidia for locustand grasshopper control. Mycological Research108: 93 -100.

Kavallieratos,N. G., Athanassiou,C. G., Michalaki, M. P., Batta, Y. A., Rigatos,H. A., Pashalidou,F. G., Balotis, G. N., Tomanovic,Z. and Vayias, B. J. (2006). Effect of the combineduse of Metarhizium anisoplide(Metschnikoft) Sorokin and diatomaceousearth for the control of three stored-productbeetle species. Crop Protection 25: 1087- 1094.

Kawamoto,H., Sinha,R. N., Muir, W. E. andWoods, S. M. (1991). Simulationsof. 4carus siro (Acari:Acaridae) in storedwheat. Environmental Entomology 20: 1381 - 1386.

Kershaw,M. J., Moorhouse,E. R., Bateman,R., Reynolds,S. E. andCharnley, A. K (1999). The role of destruxinsin thepathogenicity of Metarhiziumanisopliae for threespecies of insect. Journal ofInvertebrate Pathology 74: 213 - 223.

Kirkland, B. H., Choi E. M. and Keyhani, N. 0. (2004). Differential susceptibility of Amblyommamaculatum and Amblyomma americanum (Acari: Ixodidae)to the entomopathogenicfungi Beauveriabassiana and Metarhizium anisopliae. Biological Control31: 414- 421.

Kirkwood, A. C. (1980). Effect of Psoroptesovis on the weight of sheep.7be Veterinary Record107: 469-470.

Kirkwood,A. C. (1985).Some observations on the biologyand control of the sheepscab mite Psoroptesovis (Hering) in Britain. VeterinaryParasitology 18: 269-279.

167 Refcrcnces

Kirkwood, A. C. (1986). History, biology and control of sheepscab. Parasitology Today2: 302-307.

Kirkwood,A. C. andQuick, M. P. (1981). Diazinon for the controlof sheepscab. Veterinary Record108: 279-2 80.

Kirkwood,A. C. andQuick, M. P. (1982).Propetamphos for the controlof sheepscab. VeterinaryRecord 111: 367.

KnOlle,W. (1987). Geneticvariability andecological adaptability of hypopusformation in a stored product mite. Experimental and Applied Acarology 3: 21 - 32.

Lekimme, M., Mignon, B., Tombeux, S., Focant, C., Mardchel, F and Losson, B. (2006). In vitro cntomopathogenicactivity of Beauveria hassianaagainst Psoroples spp. (Acari: Psoroptidae). Veterinary Parasitology 139: 196- 202.

Leslie,P. H. (1945). On the useof matricesin certainpopulation mathematics. Biometrika 33: 183 - 212.

Lewis, C. (1997). Sheepscab - an updateon the history,control andpresent situation. Proceedingsof the Sheep Veterinary Society 21: 99 - 102.

Liu, C.M., Huang,S. S., Tzeng, Y. M. (2004). Purificationand quantitative analysis of destruxinsfrom Metarhiziumanisopliae by HPLC. Journal ofLiquid Chromtography and Related Technologies27: 1013-1025.

Loehle,C. (1995). Socialbarriers to pathogentransmission in wild animalpopulations. Ecology76: 326- 335.

Lord, J. C. (2005). Low humidity,moderate temperature, and desiccant dust favor efficacy of Beauveriabassiana (Hyphomycetes: Moniliales) for the lessergrain borer, Rhyzoperthadominica (Coleoptera: Bruchidae). Biological Control34: 180- 186.

Loxam,J. G. (1974).Sheep scab epidemic: January 1973. State Veterinary Jouriial 29: 1-10.

168 Refcrcnces

Lubilosa. (2001). Biolgical control with Green Muscle4D.CABI Bioscicnce, International Instituteof TropicalAgriculture (IITA), CILSS/AGRHYMET/DFPV.Available from: http://www. lubilosa.org/index. htm (Accessed25/4/2008).

LAM, J., Stejskal,V., Jarogfk,V., Hubert,J. and 2dArkovA,E. (2007). Differentialnatural performanceof four Cheyletuspredatory mite speciesin Czechgrain stores.Journal ofStoredProducts Research 43: 97 - 102.

Mapstone, S. C., Beasley,A. and Wall, R. (2002). Structure and function of the gnathosoma of the mangemite, Psoroptesovis. Medicaland VeterinaryEntomology 16: 378- 385.

Maranga, R. 0., Kaaya, G. P., Mueke, J. M. and Hassanali,A. (2005). Effects of combining the fungi Beauveria bassianaand Metarhizium anisopliae on the mortality of the tick Amblyomma variegatum (ixodidae) in relation to seasonalchanges. Mycopathalogia 159: 527 - 532

Mathieson,B. R. F. andLehane, M. J. (1996). Isolationof the gram-negativebacterium, Serrata.marcescens, from the sheepscab mite, Psoroptes ovis. VeterinaryRecord 138: 210 - 211.

McCallum,H., Barlow,N. andHone, J. (2001). How shouldpathogen transmission be modelled?Trends in Ecologyand Evolution 16: 295- 300.

McClatchie,G. V., Moore,D. M., Bateman,R. P. andPrior, C. (1994). Effectsof temperatureon the viability of the conicliaof Metarhiziumflavoviride in oil formulations. Mycological Research98: 749 - 756.

Megnin,J. P. (1877).Monographie de la tribu desSarcoptides Psoriques qui comprendtous les acariensde la galede Mommeet desanimaux. Revue et Magasinde Zoologie:46-13 9.

Meyer, W., Neurand,A. andTanylog, A. (2001). Generalanti-microbial properties of the integument in fleece producing sheepand goats. Small Ruminant Research41: 181 -190.

169 Refercnccs

Michalaki,M. P., Athanassiou,C. G., Kavallieratos,N. G., Batta,Y. A. andBalotis, G. N. (2006). Effectivenessof Metarhiziumanisopliae (Metschnikoft) Sorokin applied aloneor in combinationwith diatomaccousearth against Tribollum confusum Du Val larvae: Influenceof temperature,relative humidity andtype of commodity.Crop Protection25: 418 - 425.

Michalaki, M. P., Athanassiou,C. G., Steenberg,T., Buchelos, C. Th. (2007). Effect of Paecilomycesfumosoroseus(Wise) Brown and Smith (Ascomycota: Ilypocrcales) alone or in combination with diatomaceousearth against TrIbolium confusum Jacquelin du Val (Coleoptera:Tenebrionidae) and Ephestia keuhniella Zeller

(Lepidooptera: Pyralidae). Biological Control 40: 280 - 286.

Milner, R. J., Lim, R. P. and Hunter, D. M. (2002). Risks to the aquatic ecosystem from the

application of Metarhizium anisopliae for locust control in Australia. Pest

ManagementScience 58: 718 - 723.

Moore, D. M., Bridge, P. D., Higgins, P. M., Bateman,R. P. and Prior, C. (1993). Ultra-violet radiationdamage to Metarhiziumflavovirideconidia and the protectiongiven by vegetableand mineral oils andchemical sunscreens. Annals of, 4ppliedBiology 122: 605-616.

Muir, D., Martin, P., Kendall,K. andMalik, R. (1998). Invasivehyphomycotic rhinitis in a cat dueto Metarhiziumanisopliae. Medical Mycolpýy 36: 51 - 54.

Nahar,P., Ghormade,V. andDeshpande, M. V. (2004). The extracellularconstititutive production of chitin deacetylasein Metarhizium anisopliae: possibleedge to entomopathogenicfungi in the biological control of insect pests. Journal of InvertehratePathology 85: 80 - 88.

Nieuwhof,G. J. andBishop, S. C. (2005). Costsof the majorendemic diseases of sheepin Great Britain and the potential benefitsof reduction in diseaseimpact. AnimalScience 81: 23 - 29.

O'Brien, D. J. (1999). Treatmentof psoroptic mangewith referenceto epidemiologyand history. VeterinaryParasitology 83: 177-185.

170 Refcrcnccs

O'Brien, D. J., Forbes,A. B., Pitt, S. R. and Baggott,D. G. (1999). Treatmentand prophylaxis of psoropticmange (sheep scab) using an ivermectinintraruminal control Icd-re lease bolus for sheep. VeterinaryParasitology 85: 79 - 85.

O'Brien,D. J., Gray,J. andO'Reilly, P. F. (1993).Control of sheepscab by subcutaneous injectionof ivertnectin.Irish VeterinaryJournal 46: 99-101.

O'Brien, D.J., Gray, J.S. and O'Reilly, P.F. (1994). The useof moxidectin 1% injectablefor the control of psoroptic mangein sheep.Veterinary Parasitology 52: 91-96.

O'Brien, D. J., Parker,L. D., Menton, C., Keaveny,C., McCollum, E. and O'Laoide, S. (1996). Treatmentand control of psoropticmange (sheep scab) with moxidectin. Veterinary Record 139: 437-439.

Ochs,H., Siegfried, E. and Deplazes,P. (2002). Differences in host immune responseand behaviourof mitesin experimentalcross infestations of sheepand rabbits with Psoroptesovis andPsoroptes cuniculi. Cost Action 833:Mange and Myiasis of livestock,University of Bari, Italy, EuropeanCommunities, Belgium.

Parkinson,C. L., Jamieson,N., Eboral1, J., Armitage,D. M., (1991). Comparisonof the fecundity of 3 speciesof grain store mite speciesin Czech grain stores. Journal of StoredProducts Research. 43,97 - 102.

Pegler,K. R., Evans,L., Stevens,J. R. andWall, R. (2005). Morphologicaland molecular comparisonof host-derivedpopulations of parasiticPsoroptes mites. Medicaland Veterinary Parasitology 19: 392 - 403.

Pekar,S. and2dirkovd, E. (2004). A modelof of the biologicalcontrol of. 4carussiro by Cheyletuseruditus (Acari: Acaridae,Cheyletidae) on grain. Journal ofPest Science. 77,1-10.

Perrucci,S. andMacchioni, G. (1995). Structure/activityrelationship of somenatural monoterpenesas acaricidesagainst Psoroptes cuniculi. Journal ofNalural Producls 58: 1261- 1264.

Pilkington, A., Buchanen,D., Jamal, G. A., Gillharn, R., Hansen, S., Kidd, M., Hurley, J. F.

and Souter,C. A. (2001). Occupationaland Environmental Medicine 58: 702-7 10.

171 Refcrcnccs

Rafferty, D. E. and Gray, J. S. (1987). The feeding behaviour of Psoroptes spp. mites on rabbitsand sheep.Journal ofParasitology73: 901 - 906.

Rath, A. C., Worledge, D., Koen, T. B. and Rowe, B. A. (1995). Long-term efficacy of tile entomogenousfungus Metarhizium anisoplide against the subterraneanscarab, Adoryphoruscouloni. BiocontrolScience and Technology5: 439 - 451.

Revankar, S. G., Sutton, D. A., Sanche,S. E., Rao, J., Zervos, M., Dashti, F. and Rinaldi, M. G. (1999). Metarrhizium anisoplide as a causeof sinusitis in immunocompctcnt hosts.Journal of Clinical Microbiology37: 195- 198.

Roberts,D. W. (1981). Toxins of entomopathogenicfungi. In: Microbial control of Pests and Plant Diseases1970 - 1980. Chapter 23: 441- 459. Ed. H. D. Burges. London. Academic Press

Roy, H. E., Brown, P. M. J., Rothery,P., Ware,R. L. andMajerus, M. E. N. (2008). Interactionsbetween the fungalpathogen Beauveria bassiana and the threespecies of coccinellid: Harmoniaaxyridis, Coccinella septempunctata and Adalia bipunciata. BioControl53: 265 - 276.

Roy, M., Brodeur, J. and Cloutier, C. (2003). Temperatureand sex allocation in a spider mite. Oeclogia135: 322 - 326.

Ruoppi, P., Koistinen, T. and Permanen,S. (2005). Sensitisationto mites in laboratory animalworkers with rhinitis. Occupationaland EnvironmentalMedicine 62: 612- 615.

Safavi,S. A., Shah,F. A., Pakdel,A. K., Rasoulian,G. R., Bandani,A. R. andButt, T. M. (2007). Effect of nutrition on growthand virulence of the cntomopathogenicfungus Beauveriahassiana.FEMS Microbiology Letters 270: 116- 123.

Samish,M. andRehacek, J. (1999). Pathogensand predators of ticks andtheir potentialin biological control. Annual Review ofEntomology 44: 159 - 182.

Sanders,A., Froggatt,P. Wall, R. and Smith,K. E. (2000). Life-cycle stagemorphology of Psoroptes mange mites. Medical and Veterinary Entomology 14: 131 - 141.

172 Rcfcrences

Scholte,E. J., Ng'Habi, K., Kihonda,J., Takken,W., Paaijmans,K., Abdulla, S.,Killeen, G. F. andKnols, B. G. J. (2005). An entornopathogenicfungus for the controlof adult African malariamosquitoes. Science 308: 1641- 1642.

Scholte,E. J., Takken,W. andKnols, B. G. J. (2007). Infection of Aedesaegypli andAe. albopictusmosquitoes with the entornopathogenicfungus Metarhizium anisopliae. Acta Tropica 102: 151- 158.

Seddon,H. R. (1964). Eradicationof sheepscab from New South Wales. Australiwi VeterinaryJournal 40: 418-421.

Shah,F. A., Allen, N., Wright, C. J. and Butt, T. M. (2007). Repeatedin vitro subculturing alters spore surfaceproperties and virulence of Metarhizium anisopliae. FEMS Microbiology Letters 276: 60 - 66.

Shah,F. A., Wang,C. S. andButt, T. M. (2005). Nutrition influencesgrowth and virulence of the insect-pathogenicfungus Metarhizium anisoplide. FEMS Microbiology Letters251: 259 - 266.

Shaw,K. E., Davidson,G., Clark, S. J., Ball, B. V., Pell, J. K., Chandler,D. and Sunderland, K. D. (2002). Laboratory bioassaysto assessthe pathogenicity of mitospsoric fungi to Varroadestructor (Acari: Mesostigmata),an ectoparasiticmite of the honeybee, Apis mellifera. Biological Control24: 266 - 276.

Shilston,A. W. (1915). Sheep scab. Observatons on the Iife-history of Psoroplescommunis var ovis, and somepoints connectedwith the epizootiology of the diseasein SouthAfrica. TheThird and FourthReports ofthe Directorof VeterinaryResearch. Pretoria. Departmentof Agriculture,Union of SouthAfrica. 70-98

Siegfried,E., Ochs,H. andDeplazes, P. (2004). Clinical developmentand serological antibodyresponses in sheepand rabbits experimentally infested with Psoroplesovis andPsoroptes cuniculi. VeterinaryParasitology 124: 109- 124.

Sinclair, A. N and Filan, S. J. (1989). Lipid ingestion from sheepepidermis by Psoroples ovis (Acam Psoroptidae).Veterinary Parasitology 31: 149- 164.

173 Refcrcnces

Sinclair,A. N. andFilan, S. J. (1991). Confirmationof degcncrativecffccts on Psoroplic mitesfrom scablesions. Veterinaryrecord 129:492.

Sinclair, A. N. and Kirkwood, A. C. (1983). Feeding behaviour of Psoroples ovis. VeterinaryRecord 112: 65.

Smith, K. E., Wall, R., Berriatua, E. and French, N. P. (1999). The effects of temperatureand humidity on the off-host survival of Psoroptesovis and Psoroptescuniculi. Veterinary Parasitology 83: 265-27S.

Smith,K. E., Wall, R. andFrench, N. P. (2000). The useof entomopathogenicfungi for the control of parasitic mites, Psoroptesspp. Veterinary Parasitology 92: 97 - 105.

Smith, W. D., Bates,P., Pettit, D. M., Van den Brock, A. and Taylor, M. A. (2002). Attempts to immunize sheepagainst the scabmite, Psoroptesovis. Parasite Immunology 24: 303- 310.

Smith.W. D. andPetit, D. M. (2004). Immunizationagainst sheep scab: preliminary identificationof fractionsof Psoroptesovis which conferprotective effects. Parasite Immunology 26: 307-314.

Solomon,M. E., (1946). Tyroglyphoidmites in storedproducts. Nature and amount of damageto wheat. AnnalsofApplied BioloSy33: 280- 289.

Solomon,M. E. (1962). Ecologyof the flour mite,Acarus siro L. (=Tyroglyphusfarinae DeG.). Annals of, 4pplied Biology 50: 178- 184.

Stephan,D. andZimmerman, G. (1998). Developmentof a spray-dryingtechnique for submergedspores of entomopathogenicfungi. BloconfrolScience and Technology8: 3-11.

Stephens,R., Spurgeon,A., Calvert,1. A., Beach,J., Levy, L. S., Berry, 11.and Harrington, J.M. (1995). Neurophysiological effects of long-term exposureto organophospliates dip. in sheep Lancet 345: 1135 - 1139.

174 Refcrences

St.Leger, R. J., Charnley, A. K. and Cooper, R. M. (1986a). Cuticle-degradingenzymes of entomopathogenicfungi: Mechanismsof interactionbetween pathogen enzymes and insect cuticle. Journal ofInvertebratePathology 47: 295- 302.

StIeger, R. J., Charnley,A. K. andCooper, R. M. (1986b). Cuticle-degradingenzymes of entomopathogenicfungi: Synthesisin cultureon cuticle. Journal ofInvertebrate Patholqy 48: 85 - 95.

St.Leger, R. J. (1995). The role of cuticle-degradingproteases in fungal pathogenesisof insects.Canadian Journal ofBotany 73: 1119- 1125.

Stockman,S. (1910).Some points on theepizootiology of sheepscab in relationto eradication. Journal of ComparativePathology and Therapeutics23: 303 -3 04.

Stockman,S. and Berry, A. H. (1913). The Psoroplescommunis ovis: Someobservations on ova and ovipositing. Journal of ComparativePathology and Therapeutics26: 45-50.

Stolz, I., Nagel, P., Lorner, C. and Peveling, R. (2002). Susceptibility of the hymenopteran parasitoidsApoanag7yus (=Epidinocarsis) lopezi (Encyrtidae) and Phanerotomasp (Braconidae)to the entornopathogenicfungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes). Biocontrol Scienceand Technology12: 349 - 360.

Sweatman,G. K. (1958). On the life historyand validity of the speciesin Psoroples,a genusof mangemites. CanadianJournal ofZoology 36: 905-929.

Swinton,J., Harwood,J., Grenfell,B. T. andGilligan, C. A. (1998). Persistencethresholds for phocinedistemper virus infectionin harbourseal Phoca vitulina metapopulations. Journal ofAnimal Ecology67: 54 - 68.

Synge,B. A., Bates, P. G., Clark, A. M. and Stephen,F. B. (1995). Apparent resistanceof P. ovis to flumethrin. VeterinaryRecord 137: 5 1.

Tarry, D. W. (1974). Sheepscab -its diagnosisand biology. VeterinaryRecord 95: 530- 532.

175 Referenccs

Toriello, C., Perez-Torres, A., Burciaga-Diaz, Navarro-Barranco, H., Pcrez-Mcjia,

Lorenzana-Jimenez, M. and Mier, T. (2006). Lack of acute pathogenicity and

toxicicty in mice of an isolate of Metarhizium anisopliae var. anisopliae from

spittlebugs. Ecotoxicology and Environmental Safety 65: 278 - 287.

Toyoshima,S. andAmano, H. (1998). Effect of prey densityon sexratio of two predaceous mites, Phytosciuluspersimilis andAmhlyseiuswomersleyi (Acari: Phytosciidae).

Experimental and. 4pplied.4carology 22: 709 - 723.

Thind, B. B. andClarke, P. G. (2001). The occurrenceof mitesin cereal-basedfoods destinedfor humanconsumption and possible consequences of infestation. Experimental andAppliedAcarology 25: 203 - 215.

Vassilakos, T. N., Athanassiou,C. G., Kavallieratos, N. G. and Vayias, B. J. (2006). Influence of temperatureon the insecticidal effect of Beauveria basslana in combinationwith diatomaceousearth against Rhyzopertha dominica and Silophilus oryzae on storedwheat. Biological Control 38: 270 - 28 1.

Vidal, C., Chomon,B., Perez-Carral,C. andGonzalez-Quintela, A. (1997). Scnsitizationto Lepidoglyphusdestructor, Tyrophagusputrescentiae, and Acarus siro in patients allergic to housedust mites (Dermalophagoidesspp. ). Journal ofAllergy and Clinical ImmunoloU 100: 716 - 718.

Wall, R. (2007). Ectoparasites: Future challengesin a changing world. Veterinary

Parasitology 148: 62 - 74.

Wall, R. and Kolbe, K. (2006). Taxonomic priority in Psoroples mange mites: P. ovis or P. equi? Experimentaland. 4pplied. 4carology 39: 159- 162.

Wall, R., French,N. andMorgan, K. (1992).Effects of temperatureon the developmentand abundanceof the sheepblowfly Lucilia sericata. Bulletin ofEntomological Research 82: 125-131.

Wall, R., Smith, K. E., Berriatua, E. and French, N. P. (1999). Simulation analysisof the population dynamics of the mite, Psoroptesovis, infesting sheep. Veterinary Parasitology 83: 253 - 264.

176 Referenccs

Walstad, J. D., Anderson, R. F. and Stambaugh,W. J. (1970). Effects of environmental conditionson two speciesof muscardinefungi (Beauveriabassiana and Metarhizium anisopliae). Journal ofInvertehrate Pathology 16: 221 - 226.

Wekesa,V. W., Knapp,M., Maniania,N. K. andBoga, H. 1.(2006). Effectsof Beauveria basslanaand Metarhizium anisopliae on mortality, fecundityand egg fertility of Tetranychusevansi. Journal ofApplied Entomology 130: 155- 159.

West, S. A., Shuker, D. M. and Sheldon,B. C. (2005). Sex-ratio adjustmentwhen relatives interact: A test of constraintson adaptation.Evolution 59: 1211- 1228.

Wright, F. C., Riner, J. C. and guillot, F. S. (1984). Cross-mating studies with Psoroples ovis (Hering) and Psoroptes cunicull Delafrond (Acarina; Psoroptidae). Journal of

Parasitology 69: 696 - 700.

Wright, C., Brooks, A. and Wall, R. (2004). Toxicity of the entornopathogenicfungus, Metarhizium anisopliae (Deuteromycotina:Hyphomycetes) to adult females of the blowfly Lucilia sericata (Diptera: Calliphoridae). Pest ManagementScience 60: 639

-644.

Ye, S. D, Dun, Y. H. and Fang, M. G. (2005). Time and concentrationdependent interactionsof Beauveriahassiana with sublethalrates of imidaclopridagainst the aphidpests Macrosiphoniella sanborni and Myzuspersicae. Annals ofApplied Biology 146: 459 - 468.

Zacharuk, R. Y. (1970a). Fine structure of fungus Metarrhizium anisopliae infecting 3 speciesof larval elateridae(coleoptera). 1. Dormantand germinatingconidia. Journal ofInvertebratePathology 15: 63 - 80.

Zacharuk, R. Y. (1970b). Fine structure of fungus Metarrhizium anisopliae infecting 3 specicsof larval clateridae(colcoptera). 2. Conidialgcrm tubcs and appresoria. Journal ofInveriebratePatholov 15: 81 - 91.

Zacharuk,R. Y. (1970c). Fine structureof fungusMetarrhizium anisopliae infecting 3 speciesof larval elateridae(coleoptera). 3. Penetrationof host integument. Journal of

Invertebrate Pathology 15: 372 - 396.

177 Rcfercnces

Zahler,M., Essig,A., Gothe,R. andRinder, H. (1998).Genetic evidence suggests that Psoroptesisolates of differentphenotypes, hosts and geographic origins are conspecific.International Journalfor Parasitology28: 1713-1719.

ZdarkovA,E. andVoracek, V. (1993). The effectsof physicalfactors on survivalof stored food mites. ExperimentalandAppliedAcarology 17: 197- 204.

Zimmerman, G. (1993). The entornopathogenicfungus Melarhizium anisopliae and its potential as a biocontrol agent. Pesticide Science37: 375 - 379.

Zimmerman, G. (2007a). Review on the safety of the entornopathogenicfungus Metarhizium anisopliae. Biocontrol Scienceand Technology17: 879 - 920

Zimmerman, G. (2007b). Review on the safety of the entomopathogenicfungus Beauveria bassianaand Beauveria brongniariii. Blocontrol Scienceand Technology17: 553 - 596.

178 Availableonline at ww. sciencedirect.com veterinary ScienceDirect parasitology ELSEVIER VeterinaryParasitology 148 (2007) 310-317 www.clscvicr. comAocate/vctpar

Control of the sheep scab mite Psoroptes ovis in vivo and in vitro using fungal pathogens bb S. Abolins", B. Thind V. Jackson B. Luke', D. Moore, , , R. Wall a, M. A. Taylor b 'Schoolof BiologicalSciences, University of Bristol,Woodland Road, Bristol BS8 JUG, UK bCentral Science Laboratory, Sand Hutton, York Y041 JL4 UK 'CABI Europe-UK,Silwood Park, Buckhurst Road, Ascot SLJ 7TA,UK Received5 April 2007;received in revisedform I June2007; accepted 4 June2007

Abstract

As part of a researchprogramme designed to identify biological agentsfor the control of sheepscab, the pathogenicityof the fungus Metarhizium anisopliae to Psoroptesmites in the presenceof sheepskinand wool was examined in the laboratory.No inhibitory effects of skin and wool were observedand high levels of infection were recorded.Subsequently the pathogenicityof formulations of both M. anisopliae and Beauveria bassianato Psoroptesovis was studied in vivo. For this, 36 batchesof 20 adult female Psoroptesmites were confined in 25 mm diameter chamberswhich were attachedto the backs of 6 scab-naivesheep. In some treatments,mites were exposedto the fungal pathogensfor 48 h in vitro prior to being placed on the host, while other involved treatments mites with no prior exposureplaced directly onto the skin of a host treatedwith a fungal pathogen.After 48 h on the host, mites were removed,incubated individually and all fungal infections were recorded.Fungal infection was observedin all treatments,except untreated controls. However, B. bassianainfected a significantly greaternumber of mitesthan M. anisoplidewith formulations all the examined.Infection rates were highest when mites were exposedto dry conidia (>90%) and lowest with M. in diatomaceous anisopliae earth. Overall, the infection rate was not affectedby whetheror not the mites were given prior exposure before being to the conidia, placed on the sheep. The results demonstratethat Psoroptes mites can become infected by fungi entornopathogenic: on the skin of sheepand provides a first demonstrationof the potential of this technology for the control of sheepscab. (D 2007 Elsevier BX All rights reserved.

Psoroptes Sheep Fungus; Keywords: ovis; scab; Metarhizium anisoplide; Beauverjabassiana; Biological control

1. Introduction breaksof the diseasein that year (Frenchet al., 1999).In 1991,when the final compulsorydip wasabolished, there In theUK, in 1989when compulsory national plunge- were approximately 120 outbreaks(French et al., 1999). dipping for the control of psoropticmange in sheep Following that year,sheep scab was treated by farmerson (sheepscab) was reducedfrom two to one annual a case-by-casebasis. However, by 1997 up to 3000 treatment,there were approximately40 reportedout- outbreaks were estimated to have occurred nationally (Lewis, 1997)and by 2004 a nationalprevalence of 9% of flocks was established,equating to approximately 6750 of scab per year (Bisdorff 2006). This * Correspondingauthor. Tel.: +44 1179287489; cases et al., fax: +44 1179257374. representsa steadyand concerningincrease. The disease E-mail address:stephen. abolins@bristol. ac. uk (S. Abolins). appearsto be most prevalent in Wales, Scotland and

0304-4017/$- seefront matter (D 2007 Elsevier B.V. All rights reserved. doi: 10.101 6/j. vetpar. 2007.06.008 S. Abolins et al. lVeterinary Parasitology148 (2007) 310-317 311 England Northern (Bisdorff et al., 2006), suggestingan quantitativetrials of the pathogenicityof the fungus associationwith upland farms, the movement of stock undercarefully controlledexperiments on live hosts. and the use of common grazing (O'Brien, 1999). Throughoutmost of this period, the only insecticides 2. Materials and methods registered for use in dips were the organophosphates propetamphos and diazinon, and the pyrethroids 2.1. Sheepskin bioassay flumethrin and later high cis-eypermethrin. However, because of environmental concernsthe pyrethroids are The skin of a freshlykilled lamb with no historyof for in currently unavailable scab control the UK. treatmentfor P ovis wasobtained from an abattoir.A Propetamphos for was withdrawn economic reasons seriesof 20 mm diametercircles were cut from theskin following and temporary withdrawal to allow the andthe fleece was cut to a lengthof 20 mm.These discs developmentof safer packaging, diazinon remains the of skin were placed into chambersconstructed from only available insecticide for use in dips in the UK, 6 mm x 25 mm x 75 mm glassblocks. Each block had although pressurefor its removal remains becauseof a 20 mm.diameter hole drilled throughits centrc.The human concerns over exposure. Currently, only the bottomof the block wassealed with a fine gradecotton injectable avermectins (moxidectin, ivermectin and cloth glued to the glasswith epoxy resin (AralditeO, doramectin) are available as therapeutic alternativesto BostikFindlay Ltd., Leicester,UK). Oncea discof skin dips, although their use is constrained by the relative and fleece,with its appropriatetreatment, had been application difficulty by farmers, lack of residual placedinto the chambercreated in theseblocks, the (ivermectin) activity and expense(Bates, 2004). chamberwas sealed using a glassmicroscope slide with As a result, there has been considerable recent a5 mm diameterhole drilled throughits centre,which in interest the developmentof alternative approachesto wasalso closed with cottoncloth. The holein theupper scab treatment, particularly the use of entomoPatho- glassslide was to allow for regulationof thehumidity of genic fungi (Brooks and Wall, 2001; Brooks et al., the air within the chamber. 2004). High levels of infection and mortality in Four treatmentgroups were used:untreated fleece Psoroptes ovis (Herring) (Acari: Psoroptidae)has been only, fleece to which conidia of M. anisopliae demonstrated in vitro, using the fungus Metarhizium suspendedin silicone oil had been added,fleece to (Metschnikoff) anisopliae Sorokin (Deuteromycotina: whichonly siliconeoil hadbeen added and, as a positive Hyphomycetes) following immersion in a1x IC control, cotton cloth to which conidia suspendedin conidia ml-1 suspension(Smith et al., 2000). Subse- siliconeoil had beenadded. quently, the ability of mites to acquire a fatal infection Treatmentscontaining conidia used a suspensionof following contact with a treated surface (Brooks and Ix 108 conidia ml-1 of M. anisopliae (isolate Wall, 2001) and the ability of infected mites to transfer IMI386697) in silicone oil (dimethylpolysiloxane infection to other in-contact individuals, were demon- hydrolyzate,Sigma-Aldrich, Dorset, UK). The isolate (Brooks Wall, 2005). strated and However, preliminary had beencultured on potato dextroseagar plus yeast attemptsto use the fungal pathogen applied to infested (PDAY).Conidia were collected from 10to 14day-old initially sheep gaveunpromising results (Brooks, 2004). plates, by scraping a culture of sporulating M. One possibleproblem was that the lack of initial success anisopliaewith a sterile loop, into 4ml of silicone in have been vivo might due to naturally occurring oil. The concentrationof the conidial suspensionwas fungicides in present the sheep fleece. A second calculatedusing a haernocytometerand dilutedfurther is possibility that the temperature at the sheep skin with siliconeoil to producethe desiredconcentration. is high; surface too at the skin temperaturefound on a The silicone oil or silicone oil with its suspended fully fleeced 35 'C, sheep, around most strains of M. conidia was appliedusing a householdplant sprayer lethal anisopliae are close to their upper limits (Brooks (HozelockGroup Ltd., Aylesbury,UK) fixed 20 cm 2004; Polar 2005). A et al., et al., third alternative was directly abovethe skin or cloth using a clamp stand. failed that the method of application to deliver fungal This distancebetween the nozzleof the sprayerand the The sporesto the skin surface. aims of the work reported discs of skin or cloth was chosento give an even here, first were to assessthe two possible obstaclesto in applicationof the treatmentacross the skin or cloth and by vivo application examining the pathogenicity of the to simulatea possibleapplication method for use on fungal M. two pathogens, anisoplide and B. bassiana sheep.The sprayerdelivered a standard0.08 ml of (Balsamo) Willemin (Deuteromycotina: Hyphomy- conidia suspendedin silicone oil to each surface to cetes), when applied sheep skin and undertaking resultingin the applicationof 8x 105conidia per disc. 312 S. Abolins et aLIVeterinary Parasitology148 (2007) 310-317

After application, the skin and cloth were placed into giving a total of 36 arenas.Prior to attachment,the the chambersand left for 1h before the addition of 20 fleecewas shorn, close to the skin and the arenaswere adult, femalePsoroptes mites, collected from the earsof attachedwith surgicalglue. The arenashad an internal experimentally infested rabbits on the same day that diameterof 25 mm andwere 10mm high. They were they were used in the experiment. sealedwith fine meshcotton held in placewith a tight In the cloth treatment, 500 jil of ovine serum was fitting rubberwasher placed in the insideof the arena. also added(Sigma-Aldrich, Poole) before the addition Treatmentswere allocated to specificshcep/arenas at of the mites. Five replicates of each treatment were set random,with the constraintthat neither sheep had more up. The mites were then left in their treatmentchambers thanone treatment of anyparticular type. The skin in six for 24 h at 30 *C and 90% relative humidity. After 24 h, arenasacted as controlsand hadno treatmentapplied. all mites were removed from the chambers and The skin of six arenaswas treatedwith 0.01g of dry transferred in their respective treatment groups to sporesof M. anisoplide (isolate IM1386697)at a clean, cloth-basedchambers to which 500 ýLlof sheep concentrationof 1.8 x 1010conidia g-1, threearenas serum had been added. They were then incubated at were treated with 2 ml of the same isolate of M. 30 "C and 90% relative humidity. A further 200 Rl of anisopliaeconidia suspended at 1.5 x 109conidia ml -1 sheepserum was added every 48 h until all mites had in sheepdip excipient(70% octyl oil plus stabilisers, died. diluted to 15%, w/v, in water) and three arenaswere The chambers were inspected every 24 h for the treatedwith 0.01g of the sameisolate of M. anisopliae presenceof dead mites. Death was defined as the point conidiamixed in 0.001g of diatomaceousearth giving a when there was no movement from the mite when concentrationof 1.6 x 1010conidia g-1. A furthersix touchedwith a paintbrush.Different paint brusheswere arenaswere treatedwith 0.01g of a mixture of two used for each of the treatments. The brushes were differentisolates of M. anisopliae(isolate ARSEF3297) washed in 70% ethanol and wiped dry between each and(isolate ARSEF4556) at a concentrationof 8x 109 sample. Any dead mites were removed from the conidiag-1. A secondfungal species, B. bassiana,was chambersand surface sterilised in 2% (w/v) sodium also examinedin this trial. This isolate had been hypochlorite solution for 30 s and then washedin sterile obtainedpreviously from a naturallyinfected Psoroples water for 30 s to remove any conidia that had not mite isolatedfrom a sheepat the CSL and will be penetratedthe mite cuticle before the point of death. referredto as CSLL For this, six arenaswere treated Each individual dead mite was then transferred to a with 0.01g of dry sporesof B. bassiana(1.2 x 1011 damp piece of sterile filter paper (WhatmanO No. 1, conidiag-1), threearenas were treated with 2 ml of B. Whatman International Ltd., Maidstone, UK) at the bassianaconidia suspended at Ix 1010conidia ml- 1in base of a well in a 96-well microtitre plate (Bibby sheepdip excipientand three arenas were treated with Sterilin, Stone, UK). The microtitre plates were then 0.01g of B. bassianaconidia mixed in 0.001g of sealedwith ParafilmO M (Pechiney Plastic Packaging, diatomaceousearth (1.1 x 1011conidiag-1). The skin Chicago,USA) and incubatedat 30 T. The plates were in eachof the arenaswas treated Ih beforemites were inspectedfor mites with fungal infections every day for applied. 2 weeks following the observation of the first infected For the trial, 25-30 adult female Psomplesmites corpse.Fungal infections were recorded when external were placed inside each arena.The mites had been hyphae could be observed protruding through the removedfrom infestedsheep 72 h prior to beingplaced cuticle of a mite. on the experimentalsheep. Prior to applicationto the experimentalsheep, half of the groupsof mites were 2.2. In vivo trial maintainedin cleanchambers at 90%relative humidity and 30 *C andoffered 500 tLI of sheepserum. Six pathogen-freepoll Dorset sheepaged between6 Othergroups of miteswere exposed to formulations and 12 months were used in this trial. The sheepwere of either M. anisopliaeor B. bassiana.Among these maintained indoors as a single group, on straw in groups,the mites to be exposedto dry conidiaon the approvedcontainment facilities, at the Central Science hostwere first dippedin suspensionsof Ix 108conidia Laboratory (CSQ, York. All animal procedures were ml- 1of eitherM. anisopliaeor B. bassianain Tween80 conductedunder UK Home Office licence and approved for I min. Themites were then transferred to a dry piece by appropriateethics committees. of filter paperfor I min to allow anyexcess fluid to run Each sheephad six circular rubber arenasglued to off beforebeing placed into a cleanincubation chamber. the skin on its back, three either side of the midline, Themites to beexposed on thesheep to conidiain sheep S. Abolins et aLlVeterinary Parasitology148 (2007) 310-317 313 0.9 dip excipient or conidia mixed in diatomaceousearth wereplaced onto filter paperin a 45 mm diameterPetri- 0.8 dish which had been treatedwith the samevolume and concentrationof the treatmentto which they were to be 0.7 exposedon the sheepfor 48 h. 0.6 Hence, on the experimental sheep, half the arenas containing dry spores, contained mites that had been 0.5 pre-exposed,while half contained mites that had not. in 2 0.4 All the arenastreated with conidia dip excipient and CL c all the arenastreated with conidia in diatomaceousearth cu (D 0.3 contained mites that had been pre-exposed.Table I surnmarisesthe treatment methods. 0.2 At the end of the 48 h period on host, the sheepwere euthanisedby a scheduleI method and the skin with the 0.1 was excised. The and arenaswere then chambers skin S packed into individual sealable plastic bags and refrigerated over night. The following day the mites were removed from the arenas,surface sterilised with Fleece Fleece Fleece Cloth+ 2% sodium hypochlorite solution, placed into clean only +011 + conidia conidia incubation chamberswith 500 W of sheep serum and Treatment incubatedat 30 "C and 90% relative humidity. A further Fig. 1. Mean mites (±S. D. ) showing signs of fungal 200 48 h proportion of jil of sheepserum was added every until all infection after 24 h exposureto untreatedfleece, fleece treated with mites had died. The chambers were inspected every silicone oil, fleecetreated with silicone oil plus A anisopliaeconidia M. 24 h for the presenceof dead mites. Dead mites were (I x 108 conidia ml-1) and cloth treated with silicone oil plus treated as described previously and fungal infections anisopliae conidia (I x 108conidia ml-1). were recordedwhen external hyphaecould be observed protruding through the cuticle. 3. Results

2.3. Analysis 3.1. Sheepskin bioassay

The proportionof mitesinfected in eachtreatment was Thesetrials aimed to determinewhether mites would arcsin transformed prior to statistical analysis using be infected in vitro, when exposed to conidia on analysis of variance (Statgraphics Plus, Statistical sheepskin.In the groups that received no treatmentor Graphics corporation, VA, USA). For clarity however, silicone oil only, there were no infected mites (Fig. I). figures display non-transformed mean proportions± In the group that was exposedto silicone oil andconidia their standarddeviation. on the skin, 52.2% (±13.8) of mites became infected

Table I Summaryof the fungal pathogens,formulations and concentrations applied to the arenason the experimentalsheep and the exposuremethod used to inoculate adult, female R ovis mites

Fungal pathogen Formulation of fungal pathogen Concentration Exposuremethod

M. anisopliae (IM1386697) Dry conidia 1.8 x 1010conidia S-1 48 h on host M. anisopliae (IM1386697) Dry conidia 1.8 X 1010conidia g-1 48 h in lab and 48 h on host M. anisopliae (IMI386697) Sheepdip excipient 1.47 X 109conidia ml-' 48 h in lab and 48 h on host host M. anisopliae (IM1386697) Diatomaceousearth 1.6 x 10'0 conidia g-' 48 h in lab and 48 h on B. bassiana(CSLI) Dry conidia 1.2 X 1011conidia g-1 48 h on host 11 B. bassiana(CSLI) Dry conidia 1.2 X 10 conidia g- 48 h in lab and 48 h on host 10 1 A bassiana(CSLI) Sheepdip excipient Ix 10 conidia ml- 48 h in lab and 48 h on host 'conidia B. bassiana(CSLI) Diatomaceousearth 1.1 X 101 g-1 48 h in lab and 48 h on host 8x 109 48 h host A anisopliae (ARSEF3297+ ARSEF4556) Dry conidia conidia g-1 on 8x 109conidia 48 h in lab 48 h on host U. anisoplide (ARSEF3297+ ARSEF4556) Dry coniclia g-I and n/a Control (no fungal pathogen) n/a. n1a 314 S. Abolins et at. lVeterinary Parasitology148 (2007) 310-317

and for the group exposedto silicone oil plus conidia on Overall, B. bassiana infected a significantly greater the cloth, 34.1% (±14.1) of mites becameinfected. The number of mites than A anisopliae (IM1386697) when difference between the two groups exposedto conidia compared in different formulations (F2.12 = 9.93, and the two groups not exposed to conidia (control P=0.003; Fig. 3). But formulation also had a level infection groups) was highly statistically significant (F3,19= 8.4, significant effect on the of (FI, 12'= P<0.001; Fig. 1). There was no signiflcant difference 24.42, P<0.001; Fig. 3). Overall, dry conidia were in the proportion of infected mites in the two groups significantly more infectious than conidia formulated exposedto silicone oil and conidia whether on fleeceor in sheep dip excipient or diatomaceous earth for both cloth (Tukey HSD post hoe test, P>0.05). A anisopliae and B. bassiana (Tukey HSD post hoc test, P<0.05). However, this difference was due a] most 3.2. In vivo bioassay entirely to the A anisopliae, because for B. bassiana the level of infectiousness remained high when in both These trials aimed to determine whether (a) mites sheep dip excipient and in diatomaccous earth, whereas infected in vitro could sustain and develop infections if for M. anisopliae the level of infection observed with then placed onto a living host immediately after infection these two formulations was significantly lower. and (b) whether mites could become infected in vivo, after exposure to the skin of treated sheep. The results 4. Discussion showed that there was no significant difference between the group of mites that were exposed to the fungi for 48 h Entomopathogenic fungi have been used success- in vitro followed by 48 h exposure in vivo and the group of fully as treatments for a variety of arthropod pests. mites that only had the 48 h exposure in vivo (Fig. 2). Several studies have investigated the use of U. There was, however, a significant interaction, as those anisopliae and A bassiana against ectoparasites.A mites exposed to the mixed strains of M. anisopliae for Ix 108 conidia ml-1 suspension of U. anisopliae 48 h in vitro prior to exposure for 48 h in vivo showed induced mortalities of 30 and 37% in adult Rhipice- fewer infections than those mites exposed to the fungus phalus appendiculatus Neumann and Amblyomma for only 48 h in vivo (F2,12" 11.15, P=0.002; Fig. 2). variegatum Fabricius (Acari: Ixodidae), respectively,

0.8 0.8 0c 'E s. c e ars 0.6 2 (0 CL C 0.4 0.4

0.2 0.2

0 4 M. anisopfiae M. anisopliae B. bassiana IM1386697 mixedisolates Dry conidia Sheep-dip Diatomaceous Fungal strain exciplent earth Formulation Fig. 2. Mean proportion of mites (±S. D. ) showing fungal infection when treated with M. anisopliae (IM1386697), a mixture of A Fig. 3. Mean proportion of mites (±S. D. ) showing fungal infection anisopliae isolates (ARSEF3297and ARSEF4556)or an isolate of when treated with either M. antsopliae (circles) or B. bassiana B. bassiana(CSLO either for 48 h in vivo only (squares)or 48 h in (squares) in vivo when formulated as dry spores, in sheep dip excipient vitro followed by 48 h exposurein vivo (circles). or with diatomaceous earth. S. Abolins et al. lVeterinary Parasitology148 (2007) 310-317 315

asthey fed on the earsof rabbits (Kaaya et al., 1996).In pathogenicity significantly. Choosing a suitable for. the samestudy (Kaaya et al., 1996), aIx 108conidia mulation will clearly play a key role in maximising the ml-1 suspensionof B. bassianawas responsiblefor a ability of any fungal pathogen to infect mites in vitro. mortality of 34% in adult R. appendiculatusbut failed to Diatomaceousearth has previously been shown to be kill any A. variegatum. In field trials, Ix 109 conidia. able to increasethe pathogenicity of M. anisopliae and ml-1 suspensionsof M. anisopliae and B. bassiana B. bassiana to some pests of stored products. It is causedmortalities of 83 and 77%, respectively, when thought that diatomaceous earth may improve the applied to R. appendiculatus naturally infesting zebu adherenceof the conidia to the cuticle and act as an cattle (Kaaya et al., 1996). In vitro experiments abrasiveand dessicating agent, easing the penetration conducted by Maranga et al. (2005) showed that of the pathogen (Lord, 2005; Michalaki et al., 2006, 100% mortality could be achieved with both 2007; Vassilakos et al., 2006). However, this was not M. anisopliae and B. bassiana against A. variegaturn. observed here. Some studies have shown that the M. anisopliae has also been shown to induce high insecticidal properties of diatomaceousearth decrease levels of infection in the cattle louse, Bovicola bovis with increasing humidity (Cook and Armitage, 1998; (Linnaeus) (Trichodectidae: Ischnocera) both in vitro Fields and Korunic, 2000; Vayias and Athanassiou, and in vivo (Briggs et al., 2006); 71% of lice exposedto 2004; Kavallieratos et al., 2006). It could be possible aIx 108conidia ml-1 suspensionof M. anisopliae in that the high moisture levels at the skin surface of the vitro becameinfected as did 73% of lice exposedto the sheep interfere with the action of the diatomaceous same concentrationin vivo (Briggs et al., 2006). earth, inhibiting the processof infection. In contrast, previous attempts to apply fungal Various forms of sheep dip excipient are widely pathogens to P ovis on living sheep have given available and familiar carriers for insecticides used in disappointing results (Brooks, 2004). In this previous the fanning industry; their ability to spreadacross the work, fungal suspensions were simply pored onto body makes them good potential candidatesas carriers infested hosts but little curative effect was observed. for entornopathogenicfungi. The results presentedhere Here, a much more controlled approach was adopted. show that someexcipients may haveadverse effects; M. First, the laboratory trials were used to confirm that anisopliae in the dip excipient used here perfonned there, was nothing intrinsic in the pelt that inhibited poorly with low levels of infection. In contrast, B. Subsequently, infection. on the living host the experi- bassianaseemed unaffected and evenwhen in sheepdip demonstrated ments that conidia of both M. anisopliae excipient, was able to infect a high proportion of mites. B. bassiana infect and could P ovis on the skin. The In terms of pathogenicity alone, this study hasshown purposeof pre-exposureof some groups of mites, prior that the fungi can induce high levels of mycosis in being to placed on the sheep, was to allow possible Psoroplesmites in vivo, in excessof the level requiredto inhibition infection points, where of might occur to be suppressa mite population (Wall et al., 1999).However, Infection in identified. pre-exposedmites would have it has also been shown that M. anisopliae has sub-lethal been initiated they already when were placed on the effects against ticks by reducing fecundity, egg mass lack infection sheep,so any subsequent of would have and hatchability (Kaaya et al., 1996). It is possiblethat inhibition infection demonstrated of the after penetra- fungal pathogens would also result in sub-lethal by inherent tion of the mite cuticle something to the reductions in fecundity when on an ovine host, and sheep skin environment. However, no such inhibition therefore, increase the effectiveness of the fungal was found and similar levels of infection were observed pathogen further. However, not only must the patho- in the pre-exposedand naive groups of mites. Because genicity of the fungal formulations be taken into mites were surface sterilised when they were collected account, several other key requirements need to be from the skin at the end of the trial, any infections must considered,such as the period of residual activity, the have been acquired before being retrieved from the practicalities of applying the treatmentand the effect on sheep.Hence it can be concludedthat conidia placedon the environment and human safety. the sheep,survived and remained infectious within the P ovis can survive off the host for up to 18 daysand if skin microclimate, attached, germinated and then the treatmentdoes not provide protection for this period successfully penetrated the mite cuticle, allowing the the sheepwill be highly susceptibleto re-infestationand development of subsequentlethal infections. the flock will have to be moved and maintained in a Notably, A anisopliae showed greater variability clean environment after treatment. Previous work has than B. bassiana with the sheep dip excipient suggestedthat M. anisopliae conidia can persist on the and diatomaceous earth formulations reducing its skin or in the fleece of the sheep for up to 4 weeks 316 S. Abolins et AlVeterinary Parasitology 148 (2007) 310-317

(Brooks, 2004). However, as yet, no work has been Psoroples ovis: effects of temperature and formulation. Peat Manag. Sci. 60,1043-1049. determine the level carried out to of pathogenicity Cook, D.A., Armitage, D. M.. 1998.The Dryacide, Inert Nevertheless, efficacy of an remaining after this time. unless sig- dust, against two species of Astigmatic! mites, Glycophagus nificantly greater levels of residual pathogenicity destructor and Acrus s1ro, at nine temperature and moisture extending well beyond this period can be achieved it content combinations on stored grain. Exp. Appl. Acarol. 23, 51-63. is most likely that entornopathogenicfungi would need Fields, P.,Korunic, 4,2000. Ile effect of grain moisturecontent to be used as a therapeutic treatment rather than as a and temperatureon the efficacy of diatomaccousearths from different prophylactic measure. geographicallocations against storcd-productbeetles. J. Stored M. anisopliae and B. bassianaare classified as low- Prod. Res. 36.1-13. risk pathogens to vertebrates and are considered French,N. P.,Berriatua, E., Wall. R., Smith. K., Morgan. K. L., 1999. Sheep In Great Britain between 1973 unlikely to causehuman or animal disease;hence their scab outbreaks and for 1992: spatial and temporal patterns. Vet. Parasitol. 83,187- safety to vertebratesmakes them a good candidate 200. However, pest arthropod control. precautions will still Kaaya,G. P., Mwangi, E.N., Guns,E. A.. 1996.Prospects of biological need to be taken when using the fungal pathogen and control of livestock ticks, Rhipicephalus appendiculatus and further investigations into its safety will have to be Amblyomma variegatum, using the entomogcnousfungi Deau. bassiana J. Invertebr. conducted before it can be used as a commercial veria and Metarhizium anisopliae. Pathol. 67,15-20. In the fungal product. practical application pathogen Kavallicratos,N. G., Athanassiou,C. G., Michalski, M. P.. Batts, Y.A., would be exposed to several constraints, such as Rigatos, H.A., Pashalidou.F. G.. Balotis, U. N.. Tomanovic, Z.. extremes of temperature and humidity, ultra violet Vayias, B.J., 2006. Effect of the combined use of Afetarhizium radiation and rainfall. In the current work, the sheep anisopliae(Metschnikoff) Sorokin anddiatomaccous earth for the beetle Crop Prot. 25, were housedindoors, and therefore,were not exposedto control of three stored-product species. 1087-1094. radiation Future work would also need to solar or rain. Lewis, C., 1997.Sheep scab--an updateon the history. control and addressthese potential problems. presentsituation. Proc. SheepVot. Soc. 21,99-102. Lord, J.C., 2005. Low humidity, moderatetemperature, and c1cssicant dustfavour efficacy of Beauveriabassiana (Hyphornymes: Mon- Acknowledgments iliales) for the lessergrain borer,Rhyzopertha dominica (Colcop- tera: Bruchidae). Biol. Control 34,180-186. We are grateful to the Department for Environment, Maranga,R. O., Kaaya,G. P., Mucke, J. M., Hassanali.A., 2005.Effects fungi Beauverla bassiana Afetarhizium Food and Rural Affairs of the UK government for of combining the and the of the tick Amblyommavvriegatum funding this to Dr. Tariq Butt of the anisopliae on mortality research and (ixodidae) in relation to seasonalchanges. Mycopathologla 159, University of Wales for supplying us with isolates 527-532. ARSEF3297 and ARSF4556. Michalaki, M. P., Athanassiou,C. Q. Steenberg,T. Buchelos,C. Th.. 2007. Effect of Paecilomycesfumosoroseus (Wise) Brown and Smith (Ascomycota:llypocreales) alone or In combinationwith References diatomaccousearth againstTribolium confusumJacquelin du Val (Coleoptera:Tenebrionidae) and Ephista AuchniellaZeller (Lepi- Bates,P., 2004. Therapiesfor ectoparasiticismin sheep.Practice 26, doptera:Pyralidae). Biol. Control 40,280-286. 538-547. Michalski, M. P.,Athanassiou, C. G., Kavallieratos,N. G., Batts, Y.A.. Bisdorff, B., Milnes, A., Wall, R., 2006. Prevalenceand regional Balotis, N., 2006. Effectiveness of Afetarhizium anisopliae distribution of scab,lice and blowfly strike in Great Britain. Vet. (Metschnikoff) Sorokin applied alone or in combination with Rec. 158,749-752. diatomaccousearth against Tribolium coqfusumDu Val larvae: Briggs, L. L., Colwell, D. D., Wall, R., 2006.Control of the cattle louse influenceof temperature.relative humidity and type of commod. Bovicola bovis with the fungal pathogenMetarhizium anisopliae. ity. Crop Prot. 25,418-425, Vet. Parasitol.142,3441349. O'Brien, D.J.. 1999.Treatment of psoroptic mangewith referenceto Brooks, AJ., 2004. Entomopathogeniceffects of the fungus, Metar- epidemiologyand history. Vot. Parasitol.$3,177-195. hizium anisopliae (Metschnikoff) (Deutcromycotina: Hyphomy- Polar,P., Aquino do Muro, M., Kairo, M.T. K., Moore, D., Pegram,R., cetes),on Psoroptesscab mites (Acari: Psoroptidae).Ph. D. Thesis. John, S.A., Roach-Bcnn, C., 2005. Thermal characteristicsof University of Bristol. Metarhizium anisopliae isolates Important for the development Brooks, AJ., Wall, R., 2001. Infection of Psomptes mites with the of biological pesticidesfor the control of cattle ticks. Vet. Pars. fungusMetarhizium anisopliae. Exp. Appl. Acarol. 25,869-880. sitol. 134,159-167. Brooks, A. J., Wall, R., 2005. Horizontal transmission of fungal Smith, K. E., Wall. R., French,N. P., 2000. no use of entomopatho- infection by Metarhizium anisoplide in parasitic Psoroptesmites genic fungi for the control of parasitic mites Psoroples&pp. Vet. (Acari: Psoroptidae).Biol. Control 34,58-65. Parasitol.92,97-105. Brooks, AJ., Aquino de Muro, M., Buffee, E., Moore, D., Taylor, Vassilakos,T. N.. Athanassiou,C. G., Kavallicratos,N. C., Vayias.B. L. M. A., Wall, R.. 2004. Growth and pathogenicityof the isolatesof 2006. Influence of temperature on the insecticidal effect of the fungus Merarhizium anisopliae against the parasitic mite, Beauveria basslana in combination with diatomaceousearth S. Abolins ef al. lVeterinary Parasitology148 (2007) 310-317 317

DuVal Cfop against Rhyzoperthadominica and Sitophilus oryzae on stored confusum (Coleoptera:Tenebrionidae). ProL23,565- wheat. Biol. Control 38,270-281. 573, Vayias, BI, Athanassiou,C. G.. 2004. Factors affecting the insecti- Wall, R., Smith,K. E., Bariatua,R., French,N. P., 1999.Simulation cidal efficacy of the diatomaccousearth formulation SilicoSec ! nalysisof the populationdynamics of the mite,Arorvptes oyhr, beetle Triboliurn againstadults and larvae of the confusedflour infestingsheep. Vct. Parasitol.83,253-264.

I UNWMSITY ?I OF BRJSTOL lr%r% A ý% I