A Hitchhiker's Guide to Invasion Biology

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Biological Sciences Theses & Dissertations Biological Sciences

Spring 2016

A Hitchhiker’s Guide to Invasion Biology: Describing the Ecological Mechanisms Underlying the Range Expansions of Two Ixodid Tick Species

Robyn M. Nadolny Old Dominion University, [email protected]

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Recommended Citation Nadolny, Robyn M.. "A Hitchhiker’s Guide to Invasion Biology: Describing the Ecological Mechanisms Underlying the Range Expansions of Two Ixodid Tick Species" (2016). Doctor of Philosophy (PhD), Dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/cs2c-sx53 https://digitalcommons.odu.edu/biology_etds/11

This Dissertation is brought to you for free and open access by the Biological Sciences at ODU Digital Commons. It has been accepted for inclusion in Biological Sciences Theses & Dissertations by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. A"HITCHHIKER’S"GUIDE"TO"INVASION"BIOLOGY:"DESCRIBING"THE"ECOLOGICAL"

MECHANISMS"UNDERLYING"THE"RANGE"EXPANSIONS"OF"TWO"IXODID"TICK"SPECIES"

! by! ! ! Robyn!M.!Nadolny! B.S.!May!2008,!Beloit!College! M.S.!August!2011,!Old!Dominion!University! ! ! A!Dissertation!Submitted!to!the!Faculty!of!! Old!Dominion!University!in!Partial!Fulfillment!of!the!! Requirements!for!the!Degree!of! ! ! DOCTOR!OF!PHILOSOPHY! ! ECOLOGICAL!SCIENCES! ! OLD!DOMINION!UNIVERSITY! May!2016" " " " " " " " " " " Approved!by:! ! Holly!D.!Gaff!(Director)! ! David!T.!Gauthier!(Member)! ! Wayne!L.!Hynes!(Member)! ! Elsa!N.!Schaefer!(Member)! ! Eric!L.!Walters!(Member)! " " " " " " " " ! ! ! ABSTRACT"

A!HITCHHIKER’S!GUIDE!TO!INVASION!BIOLOGY:!DESCRIBING!THE!ECOLOGICAL! MECHANISMS!UNDERLYING!THE!RANGE!EXPANSIONS!OF!TWO!IXODID!TICK!SPECIES! ! Robyn!M.!Nadolny! Old!Dominion!University,!2016! Director:!Dr.!Holly!D.!Gaff!" !

Increasing!incidence!of!many!tickZborne!diseases!have!been!linked!to!recent! expansions!of!tick!species!distributions.!Many!tick!species!are!expanding!their!ranges! because!of!anthropogenic!changes!in!the!landscape,!shifting!climatic!variables,!and! increasing!populations!of!suitable!host!species!and!tick!habitat.!Few!empirical!studies!have! been!performed,!however,!investigating!the!ecological!mechanisms!underlying!these!range! expansions.!Ticks!are!parasitic!organisms!that!disperse!across!landscape!by!hitchhiking!on! hosts,!but!must!then!survive!in!the!environment!for!long!periods!of!time!between! bloodmeals.!Two!species!of!ixodid!tick,!Ixodes'affinis!and!Amblyomma'maculatum,!are! simultaneously!expanding!their!ranges!throughout!the!MidZAtlantic!region!of!the!United!

States,!and!provide!a!case!study!from!which!to!examine!the!relative!importance!of!host! choice!and!apparent!habitat!preference!in!the!resulting!patterns!of!range!expansion.!

The!first!objective!of!this!research!was!to!use!field!studies!to!describe!the!life!history! of!each!of!these!species!in!Virginia,!and!determine!important!host!and!habitat! characteristics!for!the!survival!and!expansion!of!these!species!in!the!MidZAtlantic.!Tick! surveillance!data!were!collected!over!five!years!in!a!variety!of!habitats!throughout! southeastern!Virginia.!These!data!were!used!to!answer!basic!natural!history!questions! about!these!ticks!in!novel!habitats,!including!determining!tick!abundance!and!phenology,!as! well!as!habitat!and!host!preferences.!Although!both!tick!species!parasitized!many!of!the!

same!host!species,!differences!in!the!habitat!conditions!necessary!for!tick!population! establishment!resulted!in!different!patterns!of!invasion.!Understanding!how!and!where! these!ticks!establish!is!useful!in!understanding!the!public!health!risks!associated!with!areas! being!invaded.!

The!second!objective!of!this!research!was!to!determine!the!ancestry!of!the!northern! populations!of!these!ticks,!using!genetic!connectivity!among!populations!to!determine!the! mostly!likely!pathways!for!dispersal!from!ancestral!populations!to!the!MidZAtlantic.!

Despite!overlapping!host!preferences!throughout!ontogeny,!each!species!exhibited!very! different!genetic!and!geographic!patterns!of!population!establishment!and!connectivity.!

Genetic!evidence!suggests!that!these!species!may!rely!on!different!key!life!stages!to! disperse!successfully!into!novel!environments,!and!that!host!vagility,!habitat!stability!and! habitat!connectivity!all!play!critical!roles!in!the!establishment!of!new!tick!populations.!

The!third!objective!of!this!research!was!to!use!metrics!derived!from!field!and! genetic!studies!to!parameterize!agentZbased!models,!simulating!tick!range!expansions! under!different!habitat!and!host!conditions.!Incorporating!parameter!values!specific!to!I.' affinis!and!A.'maculatum!life!history,!host!ranges,!habitat!preferences,!and!genetic!diversity! allowed!for!hypothesis!testing!on!whether!habitats!or!hosts!have!greater!influences!on!the! invasions!of!these!species!in!the!MidZAtlantic.!This!research!describes!the!first!comparative! case!study!of!two!tick!species!with!unique!host!and!habitat!preferences!dispersing! simultaneously!across!a!landscape,!and!increases!our!understanding!of!the!relative! importance!of!hosts!and!habitat!in!hitchhiker!invasions. iv

! !

This!dissertation!is!dedicated!to!my!husband,!Tucker,!for!his!unwavering!support,! encouragement!and!love.!Also!to!my!mother,!father!and!sister,!for!always!believing!in!me,! and!for!always!making!time!to!listen.!! vi

ACKNOWLEDGMENTS"

This!research!would!not!have!been!possible!without!the!tireless!assistance!and! guidance!of!many!mentors!and!friends!along!the!way.!First,!I!would!like!to!thank!my! advisor,!Dr.!Holly!Gaff,!for!her!countless!hours!of!patience,!friendship,!and!support!in!the! field,!lab,!and!beyond.!She!has!gone!above!and!beyond!in!every!way!to!help!me!succeed,! and!I!am!forever!grateful.!Dr.!David!Gauthier!was!my!mentor!in!the!lab,!and!the!population! genetics!component!of!this!dissertation!would!have!been!impossible!without!his!guidance.!

His!humor!and!thoughtful!commentary!have!been!invaluable!throughout!this!process,!and! he!is!also!deserving!of!special!thanks.!Thanks!also!to!my!stalwart!committee!members;!Dr.!

Wayne!Hynes,!microbiologist!extraordinaire;!Dr.!Eric!Walters,!ecological!theory!expert,! maker!of!wisecracks,!and!statistician;!and!Dr.!Elsa!Schaefer,!modeler,!mathematician,!and! cheerleader,!for!their!patience,!insight,!and!diligent!red!pens.!Special!thanks!also!to!my!ad' hoc!committee!member,!Ellen!Stromdahl,!who!continues!to!be!a!wonderful!professional! mentor!and!friend.!Gratitude!also!to!the!man!himself,!Dr.!Dan!Sonenshine,!for!being!willing! to!answer!strange!questions!about!tick!biology!at!odd!hours,!and!for!showing!us!all!how!it’s! done.!

My!grad!student!family!at!ODU!was!also!instrumental!in!helping!me!persevere! through!this!doctoral!adventure.!Thank!you!to!my!lab!mates!Chelsea!Wright,!Lexi!White,!

Pam!Kelman,!Amy!Johnson,!Erin!Heller,!and!Lindsey!Bidders!for!their!friendship,! camaraderie,!and!general!mischiefZmaking!in!the!lab!and!way,!way!beyond.!Special!thanks! to!fellow!ecology!grad!students!Ben!Gutzler,!Anna!Brownson,!Natasha!Hagemeyer,!and!

Carly!York,!for!always!being!willing!to!listen,!laugh,!or!grab!a!beverage!over!the!last!five! vii years.!!

This!PhD!was!made!possible!by!the!generous!support!of!the!SMART!(Science,!

Mathematics,!and!Research!for!Transformation)!scholarship!through!the!American!Society! for!Engineering!Education!and!the!Department!for!Defense,!specifically!my!supporting! institution,!the!Army!Public!Health!Center.!This!research!was!also!supported!by!the!

National!Institutes!of!Health,!grant!number!K25AI067791.!Thanks!also!to!the!Virginia!

Academy!of!Science,!the!Entomological!Society!of!America,!and!the!Jayne!Koskinas!and!Ted!

Giovannis!Foundation!for!Health!Policy!for!supporting!this!research.!A!final!thank!you!to! the!army!of!undergraduates!and!others!who!came!out!to!the!field!to!help!collect!data!–! without!you,!none!of!this!would!be!possible.!

! ! viii

TABLE"OF"CONTENTS!

" Page! ! LIST!OF!TABLES!...... !x! ! LIST!OF!FIGURES!...... !xii" ! INTRODUCTION!...... !1! ! ECOLOGY!AND!PUBLIC!HEALTH!RELEVANCE!OF!IXODES'AFFINIS!...... !5! ! ECOLOGY!AND!PUBLIC!HEALTH!RELEVANCE!OF!! ! AMBLYOMMA'MACULATUM!...... !8! !!!!!!!!!! BIOLOGICAL!INVASIONS!...... !11! !!!!!!!!!! TOOLS!FOR!MEASURING!BIOLOGICAL!INVASIONS!...... !14! ! RESEARCH!AIMS!...... !18! ! DEFINITION!OF!KEY!TERMS!...... !20! " IXODES'AFFINIS!IN!SOUTHEASTERN!VIRGINIA!AND!IMPLICATIONS!! FOR!THE!SPREAD!OF!BORRELIA'BURGDORFERI,'THE!AGENT!OF!! LYME!DISEASE!...... !23! ! INTRODUCTION!...... !23! !!!!!!!!!! MATERIALS!AND!METHODS!...... !24! !!!!!!!!!! RESULTS!...... !26! !!!!!!!!!! DISCUSSION!...... !29! ! NATURAL!HISTORY!OF!IXODES'AFFINIS!IN!VIRGINIA.!...... !32! ! INTRODUCTION!...... !32! !!!!!!!!!! MATERIALS!AND!METHODS!...... !33! !!!!!!!!!! RESULTS!...... !37! !!!!!!!!!! DISCUSSION!...... !45! ! NATURAL!HISTORY!OF!AMBLYOMMA'MACULATUM!IN!VIRGINIA.!...... !62! ! INTRODUCTION!...... !62! !!!!!!!!!! MATERIALS!AND!METHODS!...... !64! !!!!!!!!!! RESULTS!...... !68! !!!!!!!!!! DISCUSSION!...... !75! !!!!!!!!!! ANTHROPOGENIC!DISTURBANCE!AND!SUBSEQUENT!RESTORATION!! EFFORTS!FACILITATE!BIOLOGICAL!INVASIONS!AND!VECTORZBORNE!! DISEASE!...... !91! ! INTRODUCTION!...... !91! !!!!!!!!!! MATERIALS!AND!METHODS!...... !94! !!!!!!!!!! RESULTS!...... !100! !!!!!!!!!! DISCUSSION!...... !105! ! ix

!!!!!!!! COMPARATIVE!POPULATION!GENETICS!OF'IXODES'AFFINIS'AND!! AMBLYOMMA'MACULATUM'!...... !115! ! INTRODUCTION!...... !115! !!!!!!!!!! MATERIALS!AND!METHODS!...... !119! !!!!!!!!!! RESULTS!...... !125! !!!!!!!!!! DISCUSSION!...... !140! !!!!!!!!!! MODELING!THE!EFFECTS!OF!HABITAT!AND!HOST!PARAMETERS!ON!! TICK!INVASIONS!...... !150! ! INTRODUCTION!...... !150! !!!!!!!!!! BACKGROUND!INFORMATION!AND!MODELING!CONSIDERATIONS!...... !153! !!!!!!!!!! MODEL!DESCRIPTION!...... !156! !!!!!!!!!! MODEL!EVALUATION!...... !166! ! MODEL!APPLICATION:!HOST!DENSITY!VS.!HABITAT!QUALITY!...... !173! ! MODEL!APPLICATION:!INFLUENCE!OF!HABITAT!CONNECTIVITY!! ON!GENETICS!...... !175! ! DISCUSSION!...... !179! ! FUTURE!DIRECTIONS!...... !181! ! GENERAL!DISCUSSION!...... !183! ! CONCLUSIONS!...... !189! ! REFERENCES!...... !192! ! APPENDICES! ! TABLE!OF!IACUC!PROTOCOL!APPROVAL!NUMBERS!...... !231! ! PERMISSIONS!FOR!REPRINTING!PUBLISHED!MATERIALS!...... !232! ! VITA!...... !233! ! ! ! ! x

LIST"OF"TABLES" ! ! Table! ! ! ! ! ! ! ! ! ! !!!!!!!!!!!!!!!!Page! ! ! 1.! SequenceZconfirmed!presence!of!I.'affinis!in!southeastern!Virginia!...... !28! ! ! 2.! Ixodes'affinis!adults!collected!by!flagging,!2010Z2014!...... !38! ! ! 3.! Adult!tick!species!composition!of!sites!where!I.'affinis!was!collected,!! ! 2010Z2014!...... !39! ! ! 4.! Mammalian!hosts!of!immature!Ixodes'affinis'in!southeastern!Virginia'!...... !44! ! ! 5.! Hosts!of!adult!I.'affinis!in!southeastern!Virginia!...... !45! ! 6.! Percentage!of!hosts!infested!with!Ixodes'affinis!each!month,!2010Z2014!...... !49! ! ! 7.! Published!host!records!of!Ixodes'affinis!in!the!United!States!and!Canada!...... !51! ! ! 8.! Dispersal!potential!of!hosts!of!Ixodes'affinis!in!southeastern!Virginia!...... !57! ! ! 9.! Amblyomma'maculatum'adults!collected!by!flagging,!2010Z2014!...... !71! ! ! 10.! Amblyomma'maculatum'immatures!collected!by!flagging,!2010Z2014!...... !71! ! 11.! Adult!tick!species!composition!of!sites!where!A.'maculatum!was!collected,!! ! 2010Z2014!...... !73! ! ! 12.! Hosts!infested!with!Amblyomma'maculatum'in!southeastern!Virginia!...... !74! ! ! 13.! Dispersal!potential!of!hosts!of!Amblyomma'maculatum!in!southeastern!! ! Virginia!...... !81! ! ! 14.! Amblyomma'maculatum'and!A.'americanum'ticks!collected!from!! ! mainland!successional!site,!2010Z2014!...... !101! ! ! 15.! Small!mammal!minimum!number!alive!(MNA)!throughout!succession,!! ! 2005Z2012!...... !101! ! 16.! Hog!Island!collections!of!adult!A.'maculatum!and!A.'americanum!...... !103! ! ! 17.! Ticks!sequenced,!by!year!and!site!...... !120! ! ! 18.! Pairwise!ΦST!and!false!discovery!rate!(FDR)!adjusted!q!values!for!! ! I.'affinis!...... !131! xi

! ! 19.! Ixodes'affinis'mitochondrial!rRNA!SAMOVA!population!groupings.!...... !133! ! ! 20.! Changing!haplotype!frequencies!of!I.'affinis'and!A.'maculatum'ticks!! ! sampled!over!time!...... !133! ! 21.! Pairwise!ΦST!and!false!discovery!rate!(FDR)!adjusted!q!values!for!! ! A.'maculatum.!...... !138! ! 22.! Amblyomma'maculatum'mitochondrial!rRNA!SAMOVA!population!! ! groupings.!...... !139! ! ! 23.! Baseline!parameter!values!used!in!model!...... !161! ! ! 24.! Parameters!varied!in!sensitivity!analyses.!...... !168! ! ! 25.! Spearman!rank!correlation!tables!from!sensitivity!analyses!...... !170! ! ! 26.! Partial!rank!correlation!coefficients!(PRCC)!measuring!effects!of!host!! ! density!and!habitat!quality!on!tick!invasions!...... !173! ! ! ! ! ! ! ! xii

LIST"OF"FIGURES" " " Figure!! ! ! ! ! ! ! ! ! ! !!!!Page! ! 1.! Range!map!of!Ixodes'affinis!in!the!United!States!...... !7! ! ! 2.! Range!map!of!Amblyomma'maculatum'in!the!United!States!...... !10! ! ! 3.! Map!of!tick!collection!sites!in!southeastern!Virginia!...... !19! ! ! 4.! Map!of!Ixodes'affinis!collection!sites,!2010!...... !25! ! ! 5.! Ixodes'affinis!and!Ixodes'scapularis'collections!from!southeastern!! ! Virginia,!2010!...... !29! ! 6.! Map!of!Ixodes'affinis'collection!sites,!2010Z2014!...... !35! ! ! 7.! Ixodes'affinis'collected!per!m2!at!flagging!sites,!2010Z2014!...... !41! ! ! 8.! Phenology!of!all!life!stages!of!Ixodes'affinis!in!Virginia!...... !42! ! ! 9.! Map!of!Amblyomma'maculatum!collection!sites,!2010Z2014!...... !66! ! ! 10.! Phenology!of!adult!A.'maculatum'in!Virginia!over!five!years!...... !69! ! 11.! Amblyomma'maculatum'adults'collected!per!m2!at!each!site,!2010Z2014!...... !70! ! ! 12.! Tick!and!mammal!densities!throughout!ecological!succession!...... !103! ! ! 13.! Hog!Island!map!and!barrier!island!habitat!effects!on!A.'maculatum'' ' density!...... !104! ! ! 14.! Range!map!of!A.'maculatum'in!the!United!States,!with!barrier!! ! islands!highlighted!...... !111! ! ! 15.! Haplotype!rarefaction!curves!for!I.'affinis!and!A.'maculatum!...... !126! ! 16.! 16S!haplotype!accumulation!curves!for!I.'affinis!and!A.'maculatum!...... !126! ! ! 17.! Phylogenetic!relationships!among!16S!rRNA!mitochondrial!haplotypes!! ! for!I.'affinis'and!A.'maculatum!...... !128! ! ! 18.! Minimum!spanning!trees!for!I.'affinis!and!A.'maculatum!...... !129! ! ! ! xiii

19.! Maps!of!I.'affinis!and!A.'maculatum!haplotypes!across!the!eastern!! ! United!States!...... !132! ! 20.! Flow!diagram!for!processes!for!each!time!step!of!agentZbased!model!...... !162! ! 21.! Seasonal!phenology!of!A)!larvae,!B)!nymphs,!and!C)!adult!ticks!in!a!! ! generalized!model!...... !167! ! ! 22.! Models!testing!the!influence!of!habitat!quality!and!host!density!! ! on!tick!invasions!...... !172! ! ! 23.! Boxplots!comparing!means!between!four!treatments!groups!testing!! ! influence!of!host!density!and!habitat!quality!on!invasion!metrics!...... !174! ! ! 24.! Models!for!testing!the!influence!of!habitat!connectivity!on!genetic!! ! connectivity,!and!reproducing!patterns!exhibited!by!I.'affinis!(left)! ! and!A.'maculatum'(right)!...... !176! ! 25.! Boxplots!depicting!differences!between!means!of!four!different!invasion!! ! metrics!for!I.'affinis!and!A.'maculatum!models!...... !178! "

"" " ! 1

INTRODUCTION"

The!ecological!effects!of!biological!invasions!are!myriad!and!manifest!across! populations,!communities,!and!landscapes.!Arthropod!vectors!of!pathogens!are!expanding! their!ranges!because!of!climate!change!and!shifting!patterns!of!land!use,!resulting!in! dramatic!and!often!unpredictable!effects!on!human!and!wildlife!health!(Kilpatrick!and!

Randolph,!2012).!In!the!past!decade,!an!increasing!incidence!of!tickZborne!diseases!has! been!linked!to!a!recent!expansion!of!tick!species!distributions!(Ogden!et!al.,!2013).!Many! tick!species!are!expanding!their!ranges!because!of!anthropogenic!changes!in!the!landscape,! shifting!climatic!variables,!and!increasing!populations!of!suitable!host!species!and!suitable! tick!habitat!(Mixson!et!al.,!2006;!Ogden!et!al.,!2006;!Ogden!et!al.,!2008a).!Climate!change! has!been!forecasted!to!increase!tick!habitat!in!the!coming!years,!and!is!already!facilitating! tick!range!expansions!worldwide,!leading!to!increasing!disease!risks!(Cumming!and!Van!

Vuuren!2006;!George!2008;!Léger!et!al.,!2013).!While!the!results!of!some!range!expansions! have!been!documented!and!mapped,!such!as!increasing!cases!of!tickZborne!diseases!and! emerging!pathogens,!there!has!been!little!attention!paid!to!the!mechanisms!by!which!these! organisms!colonize!across!a!landscape.!It!is!essential!to!understand!factors!that!limit!tick! distribution!in!order!to!predict!tickZborne!disease!emergence!(Léger!et!al.,!2013).!!

To!understand!the!movement!patterns!of!ticks!across!a!landscape,!both!abiotic!and! biotic!factors!must!be!considered.!Ticks!have!a!complex!life!history!involving!differing!host! preferences!throughout!ontogeny,!and!must!be!able!to!find!suitable!hosts!to!parasitize!as! well!as!survive!in!the!environment.!Ticks!depend!on!the!largeZscale!movements!of!their! hosts!to!facilitate!dispersal!across!the!landscape,!and!are!particularly!vulnerable!to! 2 environmental!pressures,!such!as!desiccation,!when!they!are!freeZliving!offZhost!(Léger!et! al.,!2013).!Host!specificity!is!key!to!any!parasite’s!ability!to!disperse!across!a!landscape!and! invade!new!areas!(Kruse!et!al.,!2012).!However,!because!many!humanZbiting!species!of! ticks!are!generalists!and!can!feed!on!a!variety!of!avian,!mammalian,!and!reptilian!taxa! throughout!ontogeny,!tick!range!expansions!may!be!limited!more!by!climatic!variables!

(Cumming!and!Van!Vuuren,!2006).!Ticks!are!strongly!dependent!on!both!host!availability! and!environmental!factors!for!their!survival!and!reproduction!in!any!habitat!(Léger!et!al.,!

2013),!but!the!relative!importance!of!hosts!and!habitats!in!tick!range!expansions!has!not! been!fully!explored.!!

Ticks!are!a!highly!diverse!group!of!ectoparasites,!with!899!recognized!species!in! three!families,!of!which!713!species!comprising!13!genera!are!classified!in!the!family!

Ixodidae,!the!hardZbodied!ticks!(Barker!and!Murrell,!2004).!Each!tick!species!possesses!a! unique!life!history!consisting!of!a!number!of!discrete!and!distinctive!life!stages.!Most!tick! species!require!a!single!blood!meal!from!a!set!of!preferred!host!species!specific!to!life!stage!

(Sonenshine,!1991).!The!ecology!of!hardZbodied!ticks!often!changes!as!individuals!develop! from!one!life!stage!to!the!next!(Sonenshine,!1991).!Ixodid!species!progress!from!egg!to! larval,!nymphal,!and!finally!adult!reproductive!stages!throughout!ontogeny.!During!each! life!stage,!a!tick!will!attempt!to!seek!out!a!single!host!and!will!remain!attached!to!that!host! for!2Z14!days!(Sonenshine,!1991).!After!the!tick!is!fully!engorged,!it!will!drop!off,!bury!into! substrate,!and!become!quiescent!(Sonenshine,!1991;!Kuhnert!et!al.,!1995).!The!quiescent! period!lasts!from!several!months!to!one!year!depending!on!the!species,!life!stage,!time!of! year,!and!climate!(Sonenshine,!1991).!During!this!time,!larval!and!nymphal!ticks!develop! into!their!next!life!stage.!Adult!male!ticks!of!many!ixodid!species!often!do!not!feed!or!take! 3 only!a!small!blood!meal!from!their!final!host!before!seeking!out!and!mating!with!females! feeding!on!the!same!host!(Sonenshine,!1991).!Engorged!adult!females!drop!off!their!final! host!after!mating!and!then!lay!a!brood!of!up!to!several!thousand!eggs!(Sonenshine,!1991).!

The!search!for!a!suitable!host,!an!activity!known!as!questing,!is!a!seasonal!activity!in!ticks,! and!juvenile!and!adult!ticks!may!quest!at!different!times!of!the!year!(Sonenshine,!1991).!

Many!tick!species'prefer!small!mammals,!birds,!and!reptiles!in!the!juvenile!life!stages!and! larger!mammals!in!the!adult!life!stage!(Sonenshine,!1991;!Apperson!et!al.,!1993;!Barker!et! al.,!2004).!!

Ticks!possess!limited!mobility!because!of!their!small!size!and!their!vulnerability!to! environmental!conditions!such!as!low!humidity,!and!rely!on!their!hosts!for!most!longZ distance!dispersal!(Falco!and!Fish,!1991;!Daniels!et!al.,!1996;!Madhav!et!al.,!2004).!The! dramatically!increasing!populations!of!whiteZtailed!deer!(Odocoileus'virginianus)!and!wild! turkey!(Meleagris'gallopavo)!in!the!eastern!United!States!have!been!blamed!for!increasing! abundance!and!wider!distribution!of!lone!star!ticks!(Amblyomma'americanum)!(Childs!and!

Paddock,!2003).!Migratory!birds!have!been!implicated!in!the!northern!expansion!of!the! range!of!the!blacklegged!tick!(Ixodes'scapularis)!(Ogden!et!al.,!2008b;!Brinkerhoff!et!al.!

2009).!Because!ticks!remain!attached!to!their!hosts!for!up!to!two!weeks,!this!attachment! period!provides!vagile!hosts!ample!opportunity!to!transport!ticks!far!from!where!they!first! attached.!This!is!especially!true!in!the!case!of!migratory!avian!hosts!or!of!wideZranging! large!mammals!such!as!American!black!bears!(Ursus'americanus)!(Madhav!et!al.,!2004).!

Once!a!tick!drops!off!in!a!novel!environment,!there!must!be!both!suitable!environmental! conditions!and!host!species!available!for!ticks!to!establish!new!populations.!Engorged! 4 females!may!lay!eggs!immediately!upon!arrival!in!a!new!area!if!they!have!mated!onZhost,! but!mating!opportunities!must!also!occur!for!a!population!to!become!established.!

Two!tick!species!that!have!recently!established!populations!outside!of!their! historical!ranges!are!Amblyomma'maculatum!(the!Gulf!Coast!tick)!and!Ixodes'affinis'(no! common!name),!both!of!which!have!expanded!northward!through!North!Carolina!and!more! recently!into!Virginia!(Harrison!et!al.,!2010;!Fornadel!et!al.,!2011;!Nadolny!et!al.,!2011;!

Wright!et!al.,!2011;!VarelaZStokes,!2011).!The!extent!of!their!colonization!of!southeastern!

Virginia!has!been!documented!since!2009,!and!both!species!are!infected!with!agents! capable!of!causing!human!disease!(Wright!et!al.,!2011;!Nadolny!et!al.,!2014;!Heller!et!al.,!

2015).!The!effects!of!these!invasions!on!the!tick!community,!host!community,!and!on! human!disease!incidence!in!the!MidZAtlantic!are!unknown,!but!irreversible!changes!are! likely.!It!is!important!to!understand!the!mechanisms!underlying!tick!range!expansions!in! order!to!determine!where!human!and!animal!populations!are!most!at!risk!of!contracting! tickZborne!disease.!

Because!both!of!these!tick!species!are!undergoing!simultaneous!range!expansions! into!the!MidZAtlantic,!they!provide!a!unique!opportunity!to!investigate!the!influences!of! differing!host!and!habitat!preferences!on!patterns!of!tick!invasions!and!subsequent!human! health!risks.!This!dissertation!will!focus!on!exploring!the!underlying!mechanisms!of! hitchhiker!invasions!and!will!use!I.'affinis'and!A.'maculatum!as!model!species!to!investigate! the!relative!importance!of!hosts!and!habitat!in!tick!invasions.!

" " 5

ECOLOGY"AND"PUBLIC"HEALTH"RELEVANCE"OF"IXODES!AFFINIS"

Ixodes'affinis!is!a!hardZbodied!tick!known!to!be!a!competent!vector!for!Borrelia' burgdorferi,!the!agent!of!Lyme!disease!(Oliver,!1996).!Ixodes'affinis!has!been!historically! reported!from!Mexico,!Panama,!Costa!Rica,!Guatemala,!and!Brazil,!as!well!as!in!the!United!

States!from!Florida,!Georgia,!South!Carolina,!and!recently!throughout!coastal!North!

Carolina!(Oliver!et!al.,!1987;!Harrison!et!al.,!2010;!Kohls!and!Rogers,!1953).!The!first! specimens!of!I.'affinis!north!of!Mexico!were!collected!in!Florida!in!1953,!suggesting!that! this!species!has!been!moving!northward!over!the!last!60!years!(Kohls!and!Rogers,!1953).!

Ixodes'affinis'was!confirmed!in!North!Carolina!in!2010,!and!established!populations!have! now!been!found!in!the!coastal!plain!of!southeastern!Virginia!north!through!

Northumberland!County!(Harrison!et!al.,!2010;!Nadolny!et!al.,!2011;!Fig.!1).!The!CDC! defines!an!“established!population”!as!a!site!where!more!than!one!life!stage!or!more!than! six!adults!have!been!collected!(Fish!and!Howard,!1999).!Ixodes'affinis!was!uncommon!in!

North!Carolina!prior!to!1988!(Harrison!et!al.,!2010),!which!suggests!a!northward!expansion! into!the!MidZAtlantic!only!in!the!last!two!decades.!It!is!plausible!that!I.'affinis!crossed!from!

North!Carolina!to!Virginia!through!one!of!the!numerous!potential!hosts!that!use!coastal! plain!habitats!in!both!states.!

Ixodes'affinis!is!known!to!parasitize!15!mammal!and!seven!bird!species!in!the!United!

States!(Harrison!et!al.,!2010;!Heller!et!al.,!2015).!Two!important!hosts!linked!to!I.'affinis! abundance!in!Georgia!and!South!Carolina!are!the!cotton!mouse!(Peromyscus'gossypinus)! and!hispid!cotton!rat!(Sigmodon'hispidus)!(Durden!and!Oliver,!1999;!Clark!et!al.,!2001;!

Oliver!et!al.,!2003).!The!hispid!cotton!rat!and!species!closely!related!to!the!cotton!mouse,! such!as!the!whiteZfooted!mouse!(Peromyscus'leucopus),!are!abundant!in!southeastern! 6

Virginia.!Ixodes'affinis!has!been!collected!from!habitats!along!the!Piedmont,!Sandhills!and! coastal!plain!of!North!and!South!Carolina,!as!well!as!forested!and!scrub!habitats!in!Florida! and!Georgia!(Clark!et!al.,!2001;!Clark,!2004).!In!Virginia,!I.'affinis!has!been!collected!from! successional!oldZfield,!forested!and!anthropogenically!disturbed!habitats!throughout!the! coastal!plain!(Nadolny!et!al.,!2011).!Interestingly,!even!in!its!historical!range,!I.'affinis'is! often!collected!in!low!numbers!(Clark,!2004).!Collections!in!North!Carolina!and!Virginia! have!revealed!that!many!sites!support!lowZdensity!populations!of!I.'affinis,!with!occasional! locations!where!these!ticks!are!more!common!(Harrison!et!al.,!2010;!Nadolny!et!al.,!2011).!!

Ixodes'affinis!is!morphologically!very!similar!to!Ixodes'scapularis,!the!primary! human!vector!for!B.'burgdorferi!in!the!eastern!United!States.!Although!all!active!stages!of!I.' affinis!can!be!morphologically!distinguished!from!I.'scapularis!(Keirans!and!Clifford,!1978;!

Oliver!et!al.,!1987;!Durden!and!Keirans,!1996),!specimens!collected!in!the!field!are!often! damaged,!engorged,!or!otherwise!difficult!to!identify.!Additionally,!available!keys!assume! specimens!are!cleared!and!mounted!for!identification,!but!this!process!precludes!molecular! testing!for!pathogens.!The!distributions!of!these!Ixodes!spp.!overlap!in!some!areas,!which! can!make!visual!distinction!very!difficult,!particularly!in!the!juvenile!stages!(Keirans!and!

Clifford,!1978).!Additionally,!there!are!several!other!Ixodes!species!that!parasitize!the!same! wild!mammal!and!avian!hosts,!including!I.'muris,'I.'brunneus,'I.'minor,'I.'cookei,!and!I.' dentatus'(Keirans!et!al.,!1999).!The!morphological!similarities!between!the!juvenile!Ixodes' have!complicated!the!ability!to!determine!species!distributions!without!the!use!of! molecular!diagnostics.!!

Understanding!the!role!of!these!species,!particularly!I.'affinis!and!I.'scapularis,!in! disease!vector!ecology!provides!a!need!to!confirm!the!identity!of!any!Ixodes!species!in!areas!! 7

Figure"1."Range!map!of!Ixodes'affinis!in!the!United!States.!Dark!blue!indicates!the!known! range!of!Ixodes'affinis.!Inset!shows!counties!with!reported!populations!of!I.'affinis!in!North! Carolina!and!Virginia,!with!counties!colored!by!date!of!first!confirmation!of!I.'affinis.!Grey! counties!were!sampled,!but!no!I.'affinis'were!collected.!Data!included!in!the!main!map!are! from!Oliver!et!al.!1987,!data!for!the!inset!map!are!from!Harrison!et!al.!2010!(North! Carolina),!Nadolny!et!al.!2011,!Gaff!lab,!unpublished!data!2010!–!2014!(Virginia)."

! where!ranges!overlap!(Goddard,!1992).!The!recent!population!expansion!of!I.'affinis!in!

North!Carolina!was!infected!with!B.'burgdorferi!at!a!rate!of!63.2%,!(Maggi!et!al.,!2010).!

Ixodes'affinis!are!not!known!to!bite!humans!but!are!considered!more!important!than!the! 8 human!biting!vector,!I.'scapularis,!in!maintaining!the!enzootic!spirochete!cycle!among! mammalian!host!species!in!the!southern!United!States!(Oliver,!1996;!Oliver!et!al.,!2003).!

Ixodes'affinis!feed!on!many!common!species!of!mammals!and!may!amplify!the!prevalence! of!B.'burgdorferi!and!B.'bissettii!in!these!reservoir!hosts!(Clark,!2004).!This!could!increase! the!number!of!infected!animals!that!could!in!turn!transmit!the!pathogen!to!I.'scapularis.'

ECOLOGY"AND"MEDICAL"RELEVANCE"OF"AMBLYOMMA!MACULATUM"

The!Gulf!Coast!tick!(Amblyomma'maculatum)!has!been!routinely!collected!from!the!

Gulf!Coast!and!South!America!since!the!early!1900s!(Teel!et!al.,!2010).!The!range!of!A.' maculatum!throughout!the!Caribbean!and!South!and!Central!America!is!extensive,! including!records!from!Mexico,!Jamaica,!Belize,!the!West!Indies,!Columbia,!Venezuela,!and!

Peru!(Teel!et!al.,!2010).!The!southern!extent!of!the!South!American!range!of!A.'maculatum! is!in!question!because!of!the!morphological!similarity!of!A.'maculatum!to!A.'tigrinum'and!A.' triste'(Mertins!et!al.,!2010).!Gulf!Coast!ticks!have!historically!been!established!in!the! southeastern!United!States!from!Texas!north!along!the!Gulf!Coast!to!South!Carolina,!with! incidental!collections!of!individual!ticks!further!north,!generally!along!migratory!bird! pathways!(Teel!et!al.,!2010).!The!cattle!industry!is!blamed!for!the!establishment!of!an! inland!A.'maculatum!population!in!Kansas!and!Oklahoma!in!the!1950sZ1980s!(Semtner!and!

Hair,!1973;!Teel!et!al.,!2010).!Researchers!in!Arkansas!collected!over!200!A.'maculatum! between!2006!and!2009,!and!now!consider!A.'maculatum!to!be!established!in!that!state!

(Trout!et!al.,!2010).!Between!2009!and!2011,!A.'maculatum!populations!reached!sufficient! levels!to!be!considered!established!in!North!Carolina,!having!previously!been!known!only! from!sporadic!records!(Harrison!et!al.,!2010;!VarelaZStokes!et!al.,!2011).!At!around!the! 9 same!time,!established!A.'maculatum!populations!were!discovered!in!southeastern!Virginia! and!northern!Virginia!(Fornadel!et!al.,!2011;!Wright!et!al.,!2011).!Recently,!these!ticks!have! been!discovered!in!Delaware!and!Maryland,!although!the!extent!of!their!establishment!in! these!states!is!uncertain!(Florin!et!al.,!2014).!Although!A.'maculatum!is!clearly!expanding! its!range!northward,!it!appears!to!be!doing!so!in!isolated!populations,!with!large!areas!with! few!to!no!A.'maculatum!between!established!populations!(Fig.!2).!!

Along!the!Gulf!Coast!and!in!the!inland!Midwest,!A.'maculatum!are!common!in!grassZ dominated!habitats!such!as!prairies,!scrublands!and!oak!savannahs!(Scifres!et!al.,!1988;!

Teel!et!al.,!2010).!Amblyomma'maculatum!is!a!threeZhost!tick!and!has!been!collected!from! over!71!species!of!birds!and!mammals,!including!humans,!in!the!United!States!(Teel!et!al.,!

2010).!Rodents!and!birds!are!most!often!infested!with!larvae!and!nymphs,!while!adult!ticks! generally!parasitize!larger!mammals.!There!are!no!reports!of!A.'maculatum!being!collected! from!reptile!hosts.!In!Virginia,!the!largest!populations!of!A.'maculatum!have!been!collected! from!successional!old!fields!reverting!to!forested!swamp!habitat,!and!a!landfill!(Fornadel!et! al.,!2011;!Wright!et!al.,!2011).!In!the!absence!of!prairies,!A.'maculatum!may!be!establishing! in!areas!where!there!has!been!enough!human!disturbance!to!temporarily!establish!grassy! habitats.!It!remains!to!be!seen!whether!populations!of!A.'maculatum!will!survive!at!sites! undergoing!succession!from!grass!to!forest,!but!survival!may!be!unlikely!given!the!shift!of! small!mammal!and!bird!species!composition!during!succession!(Langley!and!Shure,!1980).!

Amblyomma'maculatum!is!a!vector!of!the!agents!of!numerous!human,!veterinary,! and!wildlife!diseases!including!Rickettsia'parkeri,!which!causes!a!spotted!fever!in!humans!

(Paddock!et!al.,!2004).!Other!pathogens!these!ticks!carry!include:!the!agent!of!Panola! 10 mountain!disease!Ehrlichia'species;!Hepatozoon'americanum,!the!principal!agent!of!

American!canine!hepatozoosis;!Rickettsia'felis,!the!agent!of!feline!rickettsiosis;!Leptospira'!

Figure"2."Range!map!of!Amblyomma'maculatum'in!the!United!States.!Dark!blue! indicates!the!known!range!of!Amblyomma'maculatum.!Inset!map!shows!counties!with! reported!populations!of!A.'maculatum!in!North!Carolina,!Virginia,!and!Delaware.!The! data!included!in!the!main!map!are!from!Paddock!and!Goddard!2015,!data!for!the!inset! map!is!form!David!Gaines,!personal!communication!(western!Virginia!population),! Florin!et!al.!2014!(Delaware),!Nadolny!et!al.!2014,!Wright!et!al.!2011,!Gaff!lab! unpublished!data!2009!–!2014!(Virginia),!VarelaZStokes!et!al.!2011!(North!Carolina).! ! pomona,!causal!agent!of!leptospirosis!in!livestock;!and!Ehrlichia'ruminatum,!the!agent!of! heartwater!in!ruminants!(Paddock!and!Goddard,!2015).!In!addition!to!vectoring!microbial! pathogens,!A.'maculatum!are!large,!aggressive!ticks,!and!their!bites!can!cause!inflammation,! 11 edema,!abscesses,!and!predisposition!to!myiasis,!anemia,!and!secondary!infections,! especially!in!livestock!(Teel!et!al.,!2010).!The!A.'maculatum!collected!in!southeastern!

Virginia!were!infected!with!R.'parkeri'at!a!level!of!43%!in!2010!and!55%!in!2011!and!2012!

(Wright!et!al.,!2011;!Nadolny!et!al.,!2014).!The!aspects!of!the!ecology!and!physiology!of!A.' maculatum!in!southeastern!Virginia!that!result!in!such!a!high!infection!prevalence!are!not! well!understood!(Wright!et!al.,!2011).!Recent!studies!suggest!that!R.'parkeri'can!be! transmitted!from!invading!A.'maculatum'to!more!common!MidZAtlantic!tick!species,! including!the!lone!star!tick!Amblyomma'americanum'and!the!American!dog!tick!

Dermacentor'variabilis!(Henning!et!al.,!2014;!Wright!et!al.!2015).!!

BIOLOGICAL"INVASIONS"

To!understand!how!ticks!are!expanding!their!ranges,!and!why!parasite!invasions! may!have!unique!properties!from!other!species,!a!general!understanding!of!biological! invasions!is!important.!A!range!expansion!occurs!when!an!organism!establishes! populations!in!an!area!where!that!organism!has!rarely!been!encountered!before,!and!is!a! type!of!biological!invasion.!Invasions!and!range!expansions!are!common!in!nature!and!have! happened!throughout!Earth’s!history!without!human!assistance!(Vermeij,!1991;!Ludsin! and!Wolfe,!2001).!However,!damage!caused!by!humanZintroduced!invasive!species!is!well! documented,!and!the!economic!and!ecological!impact!of!these!events!is!severe!and!growing!

(Pimentel!et!al.,!2005).!Human!environmental!disturbance!creates!opportunities!for! invaders!to!exploit!temporarily!vacant!niches,!and!anthropogenically!induced!climate! change!can!permanently!change!landscapes,!allowing!invaders!to!thrive!(D’Antonio!and!

Meyerson,!2002;!Cumming!and!Van!Vuuren,!2006).!Ticks!have!taken!advantage!of!such! 12 opportunities!in!the!US!and!around!the!globe,!changing!local!species!assemblages!and! influencing!pathogen!dynamics!(Léger!et!al.,!2013).!The!effects!of!any!invasion!can!be! myriad,!and!manifest!at!the!genetic,!individual,!population,!community,!and!ecosystem! levels!in!microhabitats!and!across!landscapes!(Vermeij,!1991;!Kenis!et!al.,!2009).!!

It!is!imperative!to!describe!important!ecological!parameters!in!a!biological!invasion,! so!that!invasions!may!be!predicted,!modeled,!controlled,!and!even!prevented!(Hengeveld,!

1988;!Lodge,!1993;!Lounibos,!2002;!Mack!et!al.,!2000;!Ludsin!and!Wolfe,!2001;!Barney!and!

Whitlow,!2008;!Blackburn!et!al.,!2011).!It!has!been!difficult!historically!to!identify!all!of!the! attributes!of!a!successful!invader,!but!some!major!tenents!that!are!broadly!supported! include:!arrival!or!transport,!establishment,!spread,!equilibrium,!and!effects!(Lodge,!1993;!

Mack!et!al.,!2000;!Ludsin!and!Wolfe,!2001;!Blackburn!et!al.,!2011).!Frequency!of!arrival!of! the!invader,!characteristics!of!the!invaded!and!source!habitats,!the!ecology!of!the!invader,! and!the!time!since!the!introduction!are!the!most!important!areas!to!consider!(Barney!and!

Whitlow,!2008).!An!invader!must!overcome!barriers!of!geography,!survival,!reproduction,! dispersal,!and!environment!(Blackburn!et!al.,!2011).!Invaders!generally!have!high! dispersal,!fast!reproductive!rates,!high!genetic!variability,!phenotypic!plasticity,!large! native!ranges,!are!generalists,!and!are!commensal!with!humans!(Lodge,!1993).!

Communities!that!are!invaded!are!often!climatically!matched!with!an!invader’s!native! habitat,!in!an!early!stage!of!succession,!have!low!diversity!of!native!species,!exhibit!an! absence!of!predators,!and!are!disturbed!(Lodge,!1993).!Once!an!ecosystem!has!been! changed,!invasions!may!beget!more!invasions,!creating!a!cycle!of!depauperate!and! weakened!ecosystems!(Vermeij,!1991;!Parker!et!al.,!1999;!Heger!and!Trepl,!2003).! 13

To!invade!an!area,!a!species!must!arrive!and!establish!a!population!of!sufficient!size! to!avoid!extinction!(MacArthur!and!Wilson,!1967).!Different!dispersal!mechanisms,!such!as! avian!or!mammalian!hosts,!lead!to!different!probabilities!of!ticks!reaching!locations!at!a! given!distance!from!a!source!area!(As,!1984;!Hengeveld,!1988;!Léger!et!al.,!2013;!Ogden!et! al.,!2013).!Propagule!pressure,!or!the!frequency!at!which!a!species!arrives!at!a!new!area,!is! also!important,!as!species!with!more!chances!to!establish!successfully!are!more!likely!to! invade.!For!example,!infrequent!visits!by!dispersing!hosts!would!be!less!likely!to!seed!a! new!population!of!ticks!than!frequent!visits!during!an!annual!migration!by!birds!from!the! same!point!of!origin.!To!be!colonized!successfully!by!invaders,!a!new!area!must!have! suitable!biotic!and!abiotic!factors,!such!as!agreeable!climate,!and!the!presence!of!reliable! food!sources.!New!invaders!must!also!be!able!to!successfully!find!a!mate!(Lodge,!1993).!

Adult!ticks!of!many!species!congregate!and!mate!on!host,!which!minimizes!the!challenges! associated!with!reproduction!after!initial!colonization.!The!negative!genetic!effects!that! often!hinder!small!populations,!called!Allee!effects,!as!well!as!propagule!pressure!affect!the! probability!of!population!establishment,!with!high!immigration!compensating!for!Allee! effects!(Drake!and!Lodge,!2006).!It!is!also!important!to!consider!that!range!expansions!can! be!“peripheral!populations”,!and!may!be!stochastic,!ephemeral,!and!differ!in!their!genetic! signatures!from!central!populations!(Lesica!and!Allendorf,!1995;!GarciaZRamos!and!

Kirkpatrick,!1997;!Bunnell!et!al.,!2004).!

Generalist!tick!species!have!high!propagule!pressure,!a!track!record!of!previous! success!as!an!invader,!and!adaptations!to!disturbed!environments,!all!characteristics!of! species!able!to!invade!successfully!(Ludsin!and!Wolfe,!2001;!Lounibos,!2002).!Because!of! their!ability!to!feed!on!many!different!hosts,!generalist!tick!species!are!likely!to!survive! 14 where!mammals!or!birds!are!abundant.!Generalist!tick!species!also!have!a!great!capacity! for!both!shortZ!and!longZdistance!dispersal,!whether!as!immatures!feeding!on!migratory! birds,!or!adults!feeding!on!large!mammals!(Cumming!and!Van!Vuuren,!2006).!Because!of! their!ability!to!capitalize!on!many!hosts!for!food!and!transport,!it!is!challenging!to!predict! how!ticks!will!move!across!a!landscape.!Climatic!variables!and!other!environmental! pressures!may!provide!more!significant!constraints!on!tick!dispersal!than!host!abundance.!!

TOOLS"FOR"MEASURING"BIOLOGICAL"INVASIONS"

Enough!data!for!a!rigorous!analysis!of!an!invasion!are!hard!to!find,!and!there!are!few! experimental!studies!of!invasion!(Lodge,!1993;!Kenis!et!al.,!2009).!Important!tools!for! quantifying!and!characterizing!biological!invasions!include!fieldwork,!molecular!methods! including!population!genetics,!and!mathematical!modeling!(Léger!et!al.,!2013).!Field! collections!provide!empirical!data!on!the!presence!or!absence!of!species,!as!well!as! information!on!the!ecology!of!invading!species,!the!rate!at!which!they!are!invading,!and!the! circumstances!under!which!populations!can!establish.!A!solid!understanding!of!the!ecology! and!natural!history!of!invading!organisms,!both!in!their!historic!range!and!in!newly! invaded!areas,!is!the!cornerstone!upon!which!all!other!knowledge!must!build!(Holway!and!

Suarez,!1999).!

The!use!of!molecular!methods!to!trace!the!ancestry!of!invading!species!is!well! established!and!has!been!used!for!many!species,!including!ticks!(Templeton!et!al.,!1995;!

Ibrahim!et!al.,!1996;!Le!Roux!et!al.,!2009).!Molecular!markers,!such!as!maternally!inherited! mitochondrial!haplotypes,!are!used!to!seek!out!geographical!patterns!in!the!genetic! material!of!organisms!(Ibrahim!et!al.,!1996).!The!timing!and!directionality!of!the!movement! 15 of!an!organism!can!be!identified!through!patterns!of!haplotypes!in!the!ancestral!and! resulting!populations.!Populations!separated!by!long!distances!tend!to!be!more!genetically! isolated!than!those!that!are!close!together,!because!of!spatially!limited!gene!flow!

(GorrochoteguiZEscalante!et!al.,!2000).!The!life!history!of!a!species,!or!the!life!history!of!the! hosts!on!which!parasites!feed,!determines!the!minimum!geographic!distance!at!which!gene! flow!can!occur.!!!

Species!with!small!home!ranges!and!limited!dispersal!ability,!such!as!small! mammals!and!mosquitoes,!have!shorter!distances!over!which!they!can!interbreed!(Hartl,!

1988).!Animals!that!can!travel!long!distances,!such!as!migratory!birds,!often!have!fewer! barriers!to!gene!flow!(Hartl,!1988).!Species!that!exhibit!longZdistance!dispersal!have! different!genetic!patterns!than!those!that!only!disperse!shorter!distances!(Hartl,!1988).!

These!differences!are!typically!measured!using!the!fixation!index!(FST),!which!measures!the! variance!of!allele!frequencies!in!different!populations!(Hartl,!1988).!When!there!is!high! gene!flow!between!two!populations,!the!FST!will!be!closer!to!one,!with!one!indicating! panmixis,!or!that!two!populations!are!interbreeding!freely!(Hartl,!1988).!A!value!closer!to! zero!indicates!that!the!populations!are!not!interbreeding,!with!zero!indicating!no!genetic! overlap.!Tick!gene!flow!at!different!spatial!scales!is!often!dependent!on!the!host!species! exploited,!but!tick!life!history!and!behavior!can!also!limit!gene!flow!(Lampo!et!al.,!1998;!

McCoy!et!al.,!2003;!ArayaZAnchetta!et!al.,!2015).!

Mathematical!models!have!been!used!for!many!years!to!elucidate!the!complex!life! history!of!ticks,!predict!tickZborne!disease!risk,!and!more!recently!to!understand!tick! invasions.!Empirical!models!based!on!field!data!have!been!used!to!determine!likely!hosts! responsible!for!transporting!ticks!into!new!areas,!such!as!the!importance!of!migratory! 16 birds!in!facilitating!the!intrusion!of!I.'scapularis!into!Canada!(Ogden!et!al.,!2008b;!Leighton! et!al.,!2012).!Local!dispersal!of!ticks!by!resident!hosts!is!also!important!in!tick!population! establishment,!and!warming!temperatures!are!expected!to!increase!rate!of!tick!population! establishment!in!Canada!(Leighton!et!al.,!2012).!Models!have!explored!the!relative!roles!of! different!hosts!in!tick!range!expansions,!and!have!shown!that!animals!with!large!home! ranges!or!that!migrate!long!distances!can!play!crucial!roles!in!tick!range!expansions,!and! that!animals!with!small!home!ranges!can!limit!dispersal!by!diverting!ticks!from!more! mobile!hosts!(Madhav!et!al.,!2004).!Environmental!factors,!such!as!habitat!fragmentation,! may!influence!host!populations!and!affect!tick!range!expansions!and!the!dynamics!of!tickZ borne!disease!risk!(Li!et!al.,!2012).!Environmental!variables!such!as!rainfall!and! temperature!have!also!been!used!to!predict!areas!at!risk!of!tick!invasion,!especially!as! climate!changes!(Sutherst!and!Bourne,!2009).!!

AgentZbased!models,!also!called!individualZbased!models,!can!simulate!the!actions!of! individuals,!and!can!be!used!to!capture!emergent!phenomena!resulting!from!agents! interacting!with!each!other!and!with!the!environment!(Judson,!1994;!Bart,!1995;!Wilson,!

1998;!Grimm,!1999;!Schmitz,!2000;!DeAngelis!and!Mooij,!2005).!Rather!than!using! populationZlevel!measures,!the!actions!of!each!individual!are!recorded,!allowing!for! stochasticity!that!can!help!to!account!for!the!variance!seen!in'situ.!An!agentZbased!model! parameterized!using!field!data!from!A.'americanum!in!south!Texas!modeled!the!expected! increase!of!ticks!and!tickZborne!disease!risk!after!the!addition!of!a!greenbelt,!or!urban! habitat!island!(Wang!et!al.,!2012).!A!more!recent!iteration!of!this!model!showed!that! seasonal!shifts!of!host!populations!resulting!from!climate!change!influences!tick!densities! and!tickZborne!disease!risks!(Wang!et!al.,!2015).!AgentZbased!modeling!is!increasing!in! 17 popularity!as!computational!power!increases,!and!is!increasingly!being!applied!to!the! question!of!tick!range!expansions.!In!a!simple!model!of!tick!invasions,!it!was!shown!that!if! an!area!has!sufficient!host!density,!population!establishment!is!inevitable,!however,! pathogen!establishment!may!require!connectivity!to!other!areas!with!pathogens!(Gaff!and!

Nadolny,!2013).!AgentZbased!models!that!include!tick,!host!and!habitat!parameters!that! accurately!reflect!locally!collected!field!datasets!will!allow!investigators!to!model!realistic! tick!migration!dynamics.!Comparing!such!an!agentZbased!model!with!active!surveillance! data!would!provide!a!new!tool!to!explore!the!mechanisms!underlying!tick!range! expansions.!

" " 18

RESEARCH"AIMS"

! The!purpose!of!this!dissertation!research!is!to!explore!the!ecological!mechanisms! underlying!tick!range!expansions,!with!a!focus!on!describing!the!dynamics!underlying! differences!in!invasion!patterns!of!I.'affinis!and!A.'maculatum!in!southeastern!Virginia.!The! first!objective!is!to!use!field!studies!to!gain!insight!into!the!life!history!of!each!of!these! species!in!Virginia,!and!determine!important!host!and!habitat!characteristics!for!the! survival!of!these!species!and!expansion!of!these!species’!ranges!into!the!MidZAtlantic.!These! tick!surveillance!data!were!collected!over!five!years!at!a!variety!of!habitats!throughout! southeastern!Virginia,!from!2010!through!2014!(Fig.!3).!This!research!provides!answers!to! basic!natural!history!questions!about!these!ticks!in!a!new!habitat,!including!abundance,! phenology,!and!apparent!habitat!and!host!preferences.!!

A!second!research!objective!is!to!determine!the!ancestry!of!the!northern! populations!of!these!ticks,!using!genetic!connectivity!between!populations!to!determine!the! mostly!likely!pathways!for!dispersal!from!ancestral!southern!populations!to!the!MidZ

Atlantic.!A!third!research!objective!is!to!use!parameters!derived!from!field!and!genetic! studies!to!parameterize!agentZbased!models!to!simulate!tick!range!expansions!under!a! series!of!habitat!and!host!conditions.!These!models!investigate!questions!of!broad! ecological!relevance!for!hitchhiking!organisms!under!different!conditions.!Incorporating! parameter!values!specific!to!I.'affinis!and!A.'maculatum!life!history,!host!ranges,!habitat! preferences,!and!genetic!diversity!for!allows!for!hypothesis!testing!on!whether!habitats!or! hosts!have!greater!influences!on!the!invasions!of!these!species!in!the!MidZAtlantic.!This! research!enables!the!first!comparative!case!study!of!two!tick!species!with!unique!host!and! 19 habitat!preferences!dispersing!simultaneously!across!a!landscape,!and!increases!our! understanding!of!the!relative!importance!of!hosts!and!habitat!in!hitchhiker!invasions.!

! Figure"3."Map!of!tick!collection!sites!in!southeastern!Virginia.!There!are!fifteen!tick! collection!sites!throughout!southeastern!Virginia,!where!ticks!were!collected!on! standardized!transects!from!2010Z2014.!Site!names!are!based!on!the!county!or! independent!city!where!ticks!were!collected,!and!will!be!consistent!throughout!this! dissertation.!CAP!=!Cape!Charles!in!Northampton!County,!YRK!=!York!County,!NEW!=! Newport!News,!HAM!=!Hampton,!PT!=!Portsmouth,!NO!=!Norfolk,!CH!=!Chesapeake,!SF!=! Suffolk,!ILW!=!Isle!of!Wight!County,!and!VB!=!Virginia!Beach.!The!inset!is!a!map!of!Virginia,! with!the!enlarged!sampled!area!indicated!by!a!square." " " 20

DEFINITION"OF"KEY"TERMS!

The!following!terms!are!defined!to!ensure!clarity!of!their!meaning!as!it!pertains!to! this!particular!study.!The!author!developed!all!definitions!not!accompanied!by!a!citation.!

AgentFbased"model!–!Also!known!as!an!individualZbased!model.!A!mathematical!model!

that!simulates!the!actions!and!interactions!of!autonomous!agents!to!assess!their!effects!

on!the!system!as!a!whole!(DeAngelis!and!Mooij,!2005).!

Anthropogenic!–!Influenced!by!human!activities,!i.e.!“anthropogenically!disturbed”!would!

indicate!that!human!activity,!such!as!mowing!or!clearZcutting,!resulted!in!the!

disturbance!of!a!habitat.!

Atlantic"coastal"plain!–!A!physiographic!region!of!flat,!lowZlying!land!adjacent!to!the!

Atlantic!Ocean!(Auch,!2014).!

Biological"invasion!–!A!broad!term!that!refers!to!both!humanZassisted!introductions!and!

natural!range!expansions!of!organisms!(Carlton,!2001).!!

Borrelia!burgdorferi!–!An!obligate!intracellular!bacterium!vectored!by!Ixodes'spp.!ticks!

that!is!the!agent!of!Lyme!disease.!Unless!otherwise!stated,!B.'burgdorferi!is!assumed!to!

be!sensu'stricto,!or!of!a!strain!known!to!cause!infection!and!illness!in!humans.!

Connectivity!–!The!degree!to!which!the!landscape!facilitates!or!impedes!movement!among!

habitat!patches!(Taylor!et!al.,!1993).!For!example,!highly!connected!populations!of!ticks!

will!have!few!barriers!impeding!dispersal!and!gene!flow.!

Disturbance!–!A!temporary!change!in!environmental!conditions!that!causes!a!pronounced!

change!in!an!ecosystem,!such!as!clearZcutting!or!burning!that!reverts!a!forest!to!a!field! 21

(Dale!et!al.,!2001).!The!field!may!then!undergo!succession!to!revert!to!a!forest!over!

time.!

Fixation"index"(FST)!–!A!measure!of!population!differentiation!due!to!genetic!structure,!or!

the!patterns!of!differences!in!the!genetic!makeup!of!individuals!within!a!population!

(Hartl,!1988).!

Hampton"Roads!–!The!metropolitan!region!in!southeastern!Virginia!where!the!majority!of!

field!collections!for!this!dissertation!took!place!(see!Fig.!3).!!

Heteroplasmy!–!The!presence!of!more!than!one!mitochondrial!genome!within!an!

individual!animal,!specifically!an!individual!tick!in!this!dissertation.!

Infection!–!The!invasion!of!an!organism’s!body!with!diseaseZcausing!agents.!I!will!use!this!

to!describe!both!human!infections!and!ticks!infected!with!diseaseZcausing!agents.!

Invader!–!An!organism!or!population!of!organisms!that!is!moving!to!establish!a!population!

in!regions!where!it!was!previously!rare!or!unknown.!

Ixodidae!–!A!family!of!ticks!that!have!a!hard!outer!shell!and!usually!feed!on!three!hosts!

during!their!lifetime.!Both!ticks!discussed!in!this!work!are!ixodid!ticks.!

MidFAtlantic!–!A!region!of!the!United!States!comprising!the!area!between!New!England!

and!the!Southeast.!Includes!the!states!and!commonwealths!of!Virginia,!West!Virginia,!

Maryland,!Delaware,!Pennsylvania,!New!Jersey,!and!New!York.!

Old"field!–!A!term!used!in!ecology!to!describe!lands!formerly!cultivated!or!grazed!but!later!

abandoned.!The!dominant!flora!often!includes!grasses!and!herbaceous!plants,!with!

encroaching!woody!vegetation.!

Panmixia!–!Random!mating!(Hartl!,!1988).!Genetically!mixed!organisms!across!a!region!

where!there!are!few!barriers!to!dispersal!would!be!considered!panmictic.!! 22

Phenotype!–!The!physical!expression!of!a!genotype!(Hartl,!1988),!or!set!of!observable!

characteristics!of!an!individual!resulting!form!the!interaction!of!its!genotype!with!the!

environment.!

Population!–!A!group!of!organisms!in!a!specified!geographic!area.!To!be!considered!an!

“established”!population,!six!or!more!adult!ticks!or!ticks!of!two!life!stages!must!be!

collected!from!one!geographic!location!(Fish!and!Howard,!1999).!

Quiescence!–!A!state!of!inactivity,!also!known!as!diapause,!generally!used!by!ticks!after!

feeding!to!molt!to!their!next!life!stage!(Gray,!1998).!

Range"expansion!–!A!species!expanding!its!geographic!range!to!encompass!new!areas,!

either!by!a!series!of!small!encroachments!or!by!longZdistance!dispersal.!A!type!of!

biological!invasion,!usually!by!natural!means!as!opposed!to!through!direct!translocation!

by!humans!(Lodge,!1993).!

Sensitivity"analysis!–!Analysis!of!how!the!uncertainty!in!the!output!of!a!mathematical!

model!can!be!apportioned!to!different!sources!of!uncertainty!in!its!inputs!(Grimm,!

1999).!

Stability!–!When!referring!to!tick!populations,!a!stable!population!is!one!that!is!wellZ

established!at!a!site,!where!individuals!can!be!reliably!collected!over!multiple!years.!

This!term!is!no!used!to!suggest!that!a!population!has!reached!a!steady!state.!

Succession!–!The!observed!process!of!change!in!the!species!structure!of!

an!ecological!community!over!time.!Generally!used!in!this!work!specifically!to!describe!

the!shift!in!vegetation!from!open!to!closed!canopy!woodland!after!a!disturbance!event.!

" " 23

IXODES!AFFINIS"IN"SOUTHEASTERN"VIRGINIA"AND"IMPLICATIONS"FOR"THE"SPREAD"

OF"BORRELIA!BURGDORFERI,"THE"AGENT"OF"LYME"DISEASE"

**This!chapter!is!published,!and!is!reprinted!here!with!permission:! Nadolny,!R.!M.,!C.!L.!Wright,!W.!L.!Hynes,!D.!E.!Sonenshine,!H.!D.!Gaff.!2011.!Ixodes' affinis!(Acari:!Ixodidae)!in!southeastern!Virginia!and!implications!for!the!spread!of! Borrelia'burgdorferi,!the!agent!of!Lyme!disease.!Journal'of'Vector'Ecology.!36(2):! 464Z467.! "

INTRODUCTION"

!!!!!!!!!!!!Ixodes'affinis!Neumann!is!a!hardZbodied!(ixodid)!tick!known!to!be!a!competent! vector!for!Borrelia'burgdorferi,!the!agent!of!Lyme!disease,!and!agents!of!other!human! diseases!(Oliver,!1996).!Ixodes'affinis!has!been!reported!in!Florida,!Georgia,!and!South!

Carolina!and!throughout!coastal!North!Carolina!(Clark!et!al.,!1998;!Harrison!et!al.,!2010).!

Harrison!et!al.!(2010)!indicated!that!I.'affinis!was!established!throughout!the!coastal!plain! of!North!Carolina!up!to!the!Virginia!border!and!suggested!that!I.'affinis!might!occur!in!

Virginia.!

Ixodes'affinis!is!morphologically!very!similar!to!Ixodes'scapularis!Say,!the!primary! vector!for!B.'burgdorferi!in!the!eastern!United!States.!Although!all!active!stages!of!I.'affinis! can!be!morphologically!distinguished!from!I.'scapularis'(Keirans!and!Clifford,!1978;!Oliver! et!al.,!1987;!Durden!and!Keirans,!1996),!fieldZcollected!specimens!are!often!damaged!and! difficult!to!identify.!The!distributions!of!these!Ixodes!spp.!overlap!in!some!areas,!which!can! make!visual!distinction!very!difficult,!particularly!in!juvenile!stages!(Durden!and!Keirans,!

1996).!Understanding!the!role!of!the!two!species!in!disease!vector!ecology!provides!an! incentive!to!confirm!the!identity!of!any!Ixodes!species!ticks!in!areas!where!ranges!overlap!

(Goddard,!1992).!Traditionally!I.'affinis!adults!are!reported!to!actively!quest!during!the!hot! 24 summer!months!when!I.'scapularis!adults!are!dormant!(Harrison!et!al.,!2010).!I.'affinis!are! not!known!to!bite!humans,!but!are!considered!more!important!than!the!human!biting! vector,!I.'scapularis,!in!maintaining!the!enzootic!spirochete!cycle!among!mammalian!host! species!(Oliver,!1996;!Oliver!et!al.,!2003).!I.'affinis!feed!on!many!common!species!of! mammals!and!as!such!may!amplify!the!prevalence!of!B.'burgdorferi!and!B.'bissettii!in!these! reservoir!hosts!(Clark,!2004).!Potentially!this!would!increase!the!number!of!infected! animals!that!in!turn!could!transmit!the!pathogen!to!I.'scapularis.!

!!!!!!!!!!!!As!part!of!an!ongoing!study!to!document!the!presence!and!abundance!of!tick!vectors! of!infectious!diseases!in!southeastern!Virginia,!individual!Ixodes!spp.'ticks!were!identified! by!morphological!criteria!and!confirmed!using!molecular!methods.!This!paper!presents! evidence!of!established!populations!of!I.'affinis!in!Virginia!and!discusses!potential! implications!for!the!spread!of!B.'burgdorferi!infections!in!mammalian!host!reservoirs.!

MATERIALS"AND"METHODS"

!!!!!!!!!!!!Adult!and!nymphal!ticks!were!collected!from!nine!sites!throughout!southeastern!

Virginia!using!flags!made!of!white!denim!attached!to!dowel!rods!and!dragged!through! vegetation.!The!sites!sampled!represent!a!variety!of!habitat!types,!including!forests!and! grasslands,!from!Atlantic!Ocean!beaches!to!James!City!County,!Virginia!(Fig.!4).!All!ticks! found!on!flags!were!removed!with!forceps!and!placed!in!vials!labeled!with!the!time!and! date!of!collection,!as!well!as!the!sampling!location,!temperature!and!weather.!Ticks!were! identified!to!the!lowest!taxonomic!level!possible!using!morphological!criteria!(Keirans!and!

Clifford,!1978)!before!being!frozen!at!Z80°C.!In!order!to!ensure!accurate!identification!of! the!Ixodes!spp.!molecular!techniques!were!employed.! 25

Figure"4.!Map!of!Ixodes'affinis!collection!sites,!2010.!Map!shows!southeastern!Virginia,! with!red!circles!indicating!collection!sites!where!I.'affinis!was!collected!using!flags!during! the!summer!of!2010.!Counties!and!independent!cities!where!I.'affinis!was!collected!are! darker!grey,!and!site!names!correspond!to!cities!and!counties!as!indicated!in!Figure!3.! !

Ixodes!ticks!collected!from!the!same!site!on!the!same!day!were!pooled!into!groups!of! up!to!4!adults!and!8!nymphs.!DNA!was!extracted!using!the!DNeasy!Blood!and!Tissue!Kit!

(Qiagen!Inc.,!Valencia,!CA)!according!to!the!manufacturer’s!protocol!and!stored!at!Z20°C! until!processing.!Samples!were!originally!pooled!to!test!for!the!presence!of!B.'burgdorferi! and!other!pathogens.!Because!multiple!Ixodes!species!were!not!expected!in!the!region,!all! 26 pools!were!sequenced!to!determine!species!only!after!one!pool!suggested!the!presence!of!I.' affinis:!subsequently!two!individual!ticks!were!sequenced.!In!the!future,!all!ticks!collected! that!cannot!be!morphologically!identified!to!species!level!will!be!individually!sequenced.!

Species!identification!was!confirmed!by!sequencing.!A!454!base!pair!(bp)!fragment!of!the! tick!mitochondrial!16S!ribosomal!RNA!gene!was!amplified!on!an!iCycler!(BioRad!

Laboratories,!Hercules,!CA)!using!primers!16SZ1!(5’!ZGTCTGAACTCAGATCAAGTZ!3’!)!

(Macaluso!et!al.,!2003)!and!16S+1!(5’!Z!CTGCTCAATGATTTTTTAAATTGCTGTZ3’!).!All!PCR! products!for!sequencing!were!purified!by!using!Wizard!PCR!Preps!DNA!Purification!System!

(Promega,!Madison,!WI,!USA),!and!sequencing!reactions!were!performed!by!using!the!

BigDye!Terminator!v.3.1!Cycle!Sequencing!Kit!(Applied!Biosystems,!Foster!City,!CA,!USA)!as! described!by!the!manufacturer!and!using!16S!primers.!Sequence!similarities!were! identified!by!a!BLAST!search!(http://blast.ncbi.nlm.nih.gov).!

RESULTS"

Tick'species'identification!

!!!!!!!!!!!!All!Ixodes'ticks!collected!were!tentatively!identified!morphologically!using! microscopic!examination!before!pooling;!molecular!methods!were!used!to!confirm!species.!

A!total!of!94!Ixodes'spp.!ticks!were!collected!during!the!summer!of!2010.!DNA!was! extracted!and!a!fragment!of!the!16S!rRNA!was!amplified!from!each!pool.!Sequencing! analysis!revealed!that!of!the!55!Ixodes!pools!tested,!23!pools!shared!maximum!identities! with!the!I.'scapularis!16S!ribosomal!RNA!gene!while!32!pools,!including!four!nymphal! pools,!shared!maximum!identities!with!the!I.'affinis!ribosomal!RNA!gene.!Maximum! identities!ranged!from!93%!to!100%!for!I.'affinis!and!I.'scapularis.!In!addition!to!pooled! 27 samples,!two!individual!I.'affinis!were!sequenced!and!a!BLAST!search!revealed!a!97%! maximum!identity!to!Ixodes'affinis!16S!ribosomal!RNA!gene!(Genbank!accession!number!

U95879.1).!An!alignment!showed!these!individually!sequenced!samples!to!be!99.8%! identical!to!the!consensus!sequence!of!the!I.'affinis!pools.!

Geographic'and'temporal'occurrence'of'I.'affinis'collections!

TwentyZeight!adult!and!four!nymphal!tick!pools!confirmed!as!I.'affinis!were! collected!during!the!summer!of!2010!from!eight!of!the!sampled!sites!representing!six! independent!cities!and!counties!in!southeastern!Virginia!(Fig.!4).!Ticks!from!a!variety!of! habitats!were!collected!from!three!sites!in!City!of!Virginia!Beach,!and!one!site!each!in!the!

City!of!Chesapeake,!City!of!Portsmouth,!Northampton!County,!York!County!and!Isle!of!

Wight!County!(Table!1).!I.'affinis!specimens!were!collected!between!April!and!November!

2010,!with!the!greatest!number!between!midZMay!and!midZJune!(Fig.!5).!There!was! considerable!overlap!in!the!times!of!year!that!both!species!were!active!with!the!exception! of!late!August.!To!determine!temporal!distribution!of!I.'affinis,!all!sites!were!pooled.!While! it!is!known!that!tick!seasonal!dynamics!vary!with!habitat!type,!pooling!the!sites!in!this!way! provides!a!broad!picture!of!I.'affinis!activity!in!the!Hampton!Roads!region.!Although!I.' affinis!was!collected!from!sites!representing!grassland!and!numerous!forest!types,!the!most! productive!collection!site!bordered!the!Great!Dismal!Swamp!in!Chesapeake!and! represented!an!oldZfield!habitat!undergoing!secondary!succession.!!

!!!!

Table&1.!Sequence(confirmed!presence!of!I.#affinis!in!southeastern!Virginia.!Results!of!sequencing!tests!for!Ixodes#spp.#ticks! collected!using!flags!at!each!study!site!over!the!summer!of!2010.!All!ticks!were!adults!except!as!noted!at!one!site.!Basic!habitat! information!for!each!site!is!also!provided.!See!Figure!4!for!identification!and!location!of!each!site.! ! PT2& PT2& Site& CHS& ILW& YRK& CAP& VB3& VB1& VB2& Total& (Adults)& (Nymphs)& Mixed! Mixed! Successional! Successional! Mixed!pine( Habitat! pine( Deciduous! pine( Deciduous! Dunes/scrub! Deciduous! old!field! old!field! deciduous! !! Type! deciduous! forest! deciduous! forest! forest! forest! (grassland)! (grassland)! forest! forest! forest! Pools! with!I.# 11! 5! 3! 1! 0! 4! 6! 1! 1! 32! affinis! present! ! Total! Ixodes# 12! 12! 3! 1! 3! 14! 7! 2! 1! 55! spp.! pools! Total!! Ixodes# 18! 15! 3! 2! 3! 32! 13! 6! 2! 94! spp.! ticks# 28

28 29

Figure'5.!Ixodes'affinis!and!Ixodes'scapularis'collections!from!southeastern!Virginia,!2010.! Total! number! of! I.' affinis! (solid! line,! blue! diamonds)! and! I.' scapularis! (dotted! line,! red! squares)!collected!from!all!sites!in!southeastern!Virginia!from!March!through!November! 2010.!This!graph!assumes!that!all!ticks!in!a!given!pool!are!of!the!same!species.! !

DISCUSSION'

!!!!!!!!!!!!These!results!confirm!the!suggestion!by!Harrison!et!al.!(2010)!that!I.'affinis!has! extended!its!range!north!beyond!the!North!Carolina!border!and!into!the!coastal!plain!of! southeastern!Virginia.!The!discovery!of!both!adults!and!nymphs!indicates!that!breeding! populations!of!I.'affinis'are!now!established!in!Virginia.!Ixodes'affinis!was!uncommon!in!

North!Carolina!prior!to!1988!(Harrison!et!al.,!2010),!which!suggests!a!recent!northward! expansion!into!Virginia!only!in!the!last!two!decades.!It!is!plausible!that!I.'affinis!crossed! from!North!Carolina!to!Virginia!through!one!of!the!numerous!potential!hosts!that!utilize!

30 coastal!plain!habitats!in!both!states!(Webster!et!al.,!1985).!Two!important!hosts!linked!to!I.' affinis!abundance!in!Georgia!and!South!Carolina!are!the!cotton!mouse!and!cotton!rat!

(Durden!and!Oliver,!1999;!Clark!et!al.,!2001;!Oliver!et!al.,!2003).!These!and!related!species! are!abundant!in!southeastern!Virginia!and!are!common!at!the!Chesapeake!site!where!11!of! the!12!pools!of!adult!ticks!contained!at!least!one!I.'affinis.!!!!!!!!!!!!!!

The!discovery!of!I.'affinis!in!Virginia!has!implications!for!the!disease!vector!ecology! of!ticks!in!the!genus!Ixodes!in!Virginia.!Summer!time!activity!of!I.'affinis!adults!has!been!a! key!behavioral!trait!that!differentiates!them!from!I.'scapularis,!which!has!been!reported!to! quest!more!actively!in!the!cooler!months!(Clark!et!al.,!1998;!Goddard,!1992).!While!this! report!found!that!I.'affinis'peak!in!midSMay!to!midSJune,!the!continued!questing!activity!of!I.' affinis'until!November!and!the!persistent!activity!of!I.'scapularis!in!late!spring!and!early! summer!complicates!the!distinction!of!species!by!their!seasonal!questing!behaviors.!

Because!of!the!difficulty!of!differentiating!between!I.'affinis!and!I.'scapularis!relying!on! morphology!alone,!it!is!possible!that!I.'affinis!is!more!widespread!in!Virginia!than! previously!thought.!The!northward!expansion!of!this!tick!throughout!the!coastal!plain!of! the!southeastern!United!States!suggests!that!areas!further!north!that!share!coastal!plain! characteristics!may!also!be!vulnerable!to!invasion!by!this!tick.!

!!!!!!!!!!!!Although!I.'affinis!rarely!bites!humans!and!thus!plays!little!if!any!role!in!direct! transmission!of!pathogens!to!humans,!I.'affinis!has!been!reported!to!be!more!important! than!I.'scapularis!in!maintaining!the!enzootic!cycles!of!B.'burgdorferi!and!B.'bissettii!(Oliver,!

1996;!Oliver!et!al.,!2003;!Harrison!et!al.,!2010;!Maggi!et!al.,!2010)!in!the!southeastern!

United!States.!The!discovery!of!established!populations!of!I.'affinis!along!with!I.'scapularis' in!areas!of!Virginia!with!many!competent!host!species!could!have!implications!for!the!

31 amplification!of!B.'burgdorferi!and!B.'bissettii!cycles!in!wild!animal!hosts.!Continued! monitoring!of!the!tick!populations!is!needed!to!determine!what!effect!a!second!competent!

B.'burgdorferi!vector!may!have!on!Lyme!borreliosis!in!southeastern!Virginia.!

! This!paper!serves!to!document!widespread!populations!of!I.'affinis!in!a!variety!of! habitats!throughout!southeastern!Virginia.!Adult!and!nymph!I.'affinis!in!Virginia!appear!to! be!actively!questing!throughout!spring,!summer,!and!autumn!in!this!region.!These! populations!were!discovered!through!comprehensive!surveys!of!tick!populations!at!over!

15!sites!in!southeastern!Virginia.!As!tick!collection!efforts!in!this!region!continue,!Ixodes! ticks!should!be!individually!sequenced!if!necessary!for!identification!to!species.!

Perpetuation!of!sampling!efforts!in!this!region!will!provide!more!information!about!the! ecology!of!I.'affinis!at!the!northern!extent!of!its!range,!including!which!habitats!support!the! most!abundant!adult!and!nymph!populations!and!how!habitat!impacts!life!history! parameters!and!interactions!with!I.'scapularis.!!

! !

NATURAL'HISTORY'OF'IXODES'AFFINIS'IN'VIRGINIA'

INTRODUCTION!'

! Ixodes'affinis!is!a!nonShumanSbiting!hard!tick!known!for!its!role!in!the!sylvatic!cycle! of!Borrelia'burgdorferi!in!the!southeastern!United!States!(Oliver!et!al.,!2003;!Maggi!et!al.,!

2010;!Rudenko!et!al.,!2013).!Ixodes'affinis'is!native!to!Central!and!South!America!as!far! south!as!Brazil,!with!the!first!US!specimens!collected!in!Florida!in!1953!(Kohls!and!Rogers,!

1953).!Ixodes'affinis!has!since!expanded!its!range!through!Florida,!Georgia,!and!South!

Carolina,!with!new!populations!discovered!in!North!Carolina!and!Virginia!in!the!last!decade!

(Harrison!et!al.,!2010;!Nadolny!et!al.,!2011).!Genetic!evidence!supports!the!hypothesis!that! the!populations!in!North!Carolina!and!Virginia!are!more!recently!established!than!those!in! more!southern!states,!including!South!Carolina!and!Georgia,!and!that!the!northern!and! southern!ticks!represent!two!distinct!populations!with!a!clear!geographic!break!along!the! border!of!North!and!South!Carolina!(Nadolny!et!al.,!2015).!North!Carolina!and!Virginia!ticks! are!closely!related!in!a!single!genetic!clade!based!on!16S!mitochondrial!haplotype!data.!In! order!to!create!the!observed!connectivity!in!the!North!Carolina!and!Virginia!northern! population,!these!ticks!are!likely!being!dispersed!as!adults!via!mammalian!hosts!(Nadolny! et!al.,!2015).!

Ixodes'affinis!is!known!to!parasitize!15!mammal!and!seven!bird!species!in!the!United!

States!and!Canada,!and!has!been!collected!from!habitats!along!the!piedmont,!sandhills,!and! coastal!plain!ecoregions!of!North!and!South!Carolina,!as!well!as!in!forested!and!scrub! habitats!in!Florida!and!Georgia!(Clark!et!al.,!2001;!Clark,!2004;!Nelder!and!Reeves,!2005;!

Harrison!et!al.,!2010;!Heller!et!al.,!2015).!At!the!northernmost!extent!of!its!range!in!Virginia,!

I.'affinis!has!been!collected!from!successional!oldSfield,!forested,!and!anthropogenically! disturbed!habitats!throughout!the!coastal!plain!(Nadolny!et!al.,!2011).!When!they!are! present,!these!ticks!are!generally!found!in!low!densities!in!any!suitable!riparian!woodland! habitat!along!the!coastal!plain!(Gaff,!unpublished!data;!Clark,!2004);!the!possibility!of! further!northward!expansion!to!other!coastal!plain!habitats!with!appropriate!host! communities!cannot!be!discounted.!

To!understand!how!I.'affinis'will!influence!the!existing!community!of!ticks!and! pathogens!in!northern!states,!it!is!important!to!understand!any!differences!in!life!history! characteristics!this!tick!may!be!exhibiting!at!the!northern!extent!of!its!range,!such!as! altered!habitat!and!host!preferences,!or!altered!phenology.!Such!differences!could!have! implications!for!the!sylvatic!cycle!of!B.'burgdorferi,!and!for!human!health!in!areas!where! both!I.'affinis!and!the!humanSbiting!Ixodes'scapularis!are!abundant.!This!study!presents!the! results!of!five!years!(2010S2014)!of!field!surveillance!on!I.'affinis!populations!in! southeastern!Virginia!and!commentary!on!the!influence!of!ecological!factors!including!host! preference,!tick!phenology,!and!habitat!preference!on!the!northward!range!expansion!of! this!tick.!

MATERIALS'AND'METHODS' '

Collection'of'questing'ticks'

Questing!adult!I.'affinis!were!collected!from!public!and!privately!owned!lands! throughout!southeastern!Virginia!from!2010!to!2014.!Fifteen!sites!representing!a!variety!of! woodland,!grassland,!disturbed,!and!successional!habitats!were!sampled!weekly!during!the! summer!months!and!at!least!monthly!throughout!the!year!(Fig.!6).!All!transects!were!

34 marked!with!surveyors!flags!so!that!the!same!transect!was!walked!and!flagged!during!each! sampling!event.!Ticks!were!collected!using!1m2!white!denim!flags!attached!to!wooden! dowel!rods!swept!over!the!ground!and!through!low!vegetation.!All!ticks!found!clinging!to! the!flag!material!were!removed!from!the!flag!with!forceps!and!placed!in!vials!labeled!with! the!time!and!date!of!collection,!as!well!as!the!sampling!location,!temperature,!and!weather! data.!Ticks!were!frozen!at!S20!C!until!processing,!morphologically!identified!(Keirans!and!

Clifford,!1978;!Oliver!et!al.,!1987;!Durden!and!Keirans,!1996),!and!stored!frozen!at!S80!C.!

! ! Collection'of'ticks'from'hosts'

HostStargeted!sampling!techniques!were!employed!throughout!2010S2014,! including!collection!of!ticks!from!large!mammals!at!hunt!check!locations,!collaboration! with!veterinarians!to!determine!tick!presence!on!companion!animals,!serendipitous! sampling!of!roadSkilled!mammals,!monthly!and!quarterly!small!mammal!liveStrapping,! weekly!to!monthly!avian!sampling!using!mistSnets,!and!opportunistic!handScapture!of! reptiles!at!flagging!sites!(IACUC!#s:!11S012,!13S018,!13S008).!!

From!2010!to!2014,!large!wild!mammals!were!also!sampled!for!ticks!through! collaborations!with!local!hunters,!game!managers,!and!state!biologists,!and!through! checking!roadSkilled!animals!for!ticks.!Any!ticks!found!on!dead!mammals!were!removed! with!forceps,!placed!in!vials,!and!returned!to!the!lab!where!they!were!frozen!at!S20!C!until! processed.!Domestic!animals!were!sampled!through!collaborations!with!veterinarians!with! practices!in!southeastern!Virginia.!If!any!ticks!were!found!on!an!animal!patient,!ticks!were! removed,!labeled,!and!frozen!until!they!could!be!passed!to!the!Old!Dominion!University!tick! lab!for!identification!and!processing.!

! Figure'6.!Map!of!Ixodes'affinis'collection!sites,!2010S2014.!Map!shows!southeastern! Virginia!counties!and!independent!cities,!with!circles!marking!sites!that!were!flagged! weekly!or!biSweekly!from!2010!to!2014.!For!years!of!sampling!and!brief!habitat! descriptions!of!these!sites,!see!Table!2.!Sites!where!mist!netting!also!took!place!are! surrounded!by!a!square,!and!sites!where!small!mammal!trapping!also!took!place!are! marked!with!a!diamond.!! !

Small!mammal!communities!were!sampled!at!two!primary!flagging!sites!(CHS!and!

CH2)!with!established!populations!of!I.'affinis!(Fig.!6,!IACUC!#:!11S012).!These!sites! represented!mid!to!late!successional!habitats!adjacent!to!the!Great!Dismal!Swamp!in!

Chesapeake,!VA,!and!were!dominated!by!grasses!and!forbs!with!mixed!coniferous!and!

36 deciduous!trees!5S15!years!old.!Transects!or!grids!of!modified!Fitch!live!traps!(Rose,!1994)! were!laid!at!each!site.!Trapping!was!conducted!monthly!or!quarterly!from!2010!to!2014.!

Traps!were!baited!with!birdseed!and!sunflower!seeds,!were!set!in!the!evening!and!were! checked!for!three!consecutive!mornings.!During!winter!months,!polyester!fiberfill!provided! insulation!for!trapped!animals.!Small!mammals!were!sexed,!weighed,!examined!for! reproductive!status,!tagged!using!numbered!metal!ear!tags,!and!then!released!after! recording!the!data!and!trap!coordinate.!Trapped!mammals!were!examined!for!ticks,!and! any!ticks!were!removed!using!forceps,!placed!in!vials,!and!returned!to!the!lab!to!be!frozen! until!identification!and!processing.!A!subset!of!ticks!from!mammals!trapped!by!other! researchers!near!the!CAP!flagging!site!on!the!Eastern!Shore!was!donated.!All!mammals! were!handled!according!to!the!guidelines!set!forth!by!the!American!Society!of!

Mammalogists!(Sikes!and!Gannon,!2011).!!

Avian!hosts!were!sampled!from!2013!to!2014!at!a!subset!of!the!flagging!sites!(Fig.!6,!

IACUC!#!13S018)!through!weekly!or!biSweekly!mist!netting,!conducted!as!previously! described!(Heller!et!al.,!2015).!From!2013!to!2014,!local!herpetofauna!was! opportunistically!sampled!at!the!weekly!flagging!transect!sites,!with!an!emphasis!on! collecting!ticks!from!native!lizards,!snakes,!and!turtles!(IACUC!#!13S008).!Reptiles!were! captured!using!standard!collection!techniques,!including!the!implementation!of!artificial! cover!objects!and!visual!encounter!surveys,!and!examined!for!ticks!before!being!released!

(McDiarmid!et!al.,!2012).!Ticks!were!removed!from!reptiles!with!forceps!and!placed!in! vials,!and!placed!in!the!lab!and!frozen!until!identification!and!processing.!

Molecular'methods'and'identification'

! All!adult!ticks!collected!from!flags!and!hosts!were!identified!to!species!using! morphological!characteristics!(Keirans!and!Clifford,!1978;!Oliver!et!al.,!1987;!Durden!and!

Keirans,!1996).!If!morphological!identification!was!impossible!because!of!uncertain! morphological!characteristics!or!damage!to!the!specimen,!molecular!diagnostic!tests!were! employed.!While!nonSengorged!adult!I.'affinis!are!morphologically!distinct!from!other!ticks! in!the!genus!Ixodes,!immatures!of!these!species!are!notoriously!difficult!to!tell!apart,! especially!when!engorged.!To!distinguish!between!species,!a!SybrGreen!real!time!PCR! assay!that!reliably!differentiates!I.'affinis'from!other!common!Ixodes!ticks!in!the! southeastern!US,!including!I.'scapularis,!was!employed!(Wright!et!al.,!2014).!This!assay!was! used!to!determine!the!species!of!immature!ticks!removed!from!hosts!or!collected!on!flags.!

Confirmatory!sequences!using!the!16S!rRNA!mitochondrial!gene!were!produced!for!a! subset!of!samples.!The!16S!gene!of!any!samples!that!resulted!in!ambiguous!PCR!data!was! also!sequenced!following!the!protocol!outlined!by!Nadolny!et!al.!(2011).!!

RESULTS'

Presence'and'abundance'

From!2010!to!2014,!743!questing!adult!I.'affinis!were!collected!from!12!sites! representing!a!variety!of!habitat!types!in!southeastern!Virginia!(Table!2).!Sites!varied! widely!in!the!number!of!I.'affinis!collected,!from!1!to!113!ticks!per!year!and!from!<1%!to! more!than!25%!of!the!relative!abundance!of!adult!ticks!collected!at!a!site,!with!I.'affinis! collected!alongside!numerous!other!tick!species!(Table!3).!Flagging!effort!was!standardized! using!transect!length!as!a!denominator,!allowing!the!number!of!I.'affinis!collected!per!100!

38 m2!at!each!site!to!be!estimated.!!The!density!of!I.'affinis'varied!from!less!than!one!to!more! than!26!adults!per!100!m2!(Table!2).!!

Immature!I.'affinis!were!unlikely!to!be!collected!by!flag!sampling.!Of!159!immature!

Ixodes!spp.!collected!from!2010!to!2012!and!identified!using!PCR,!150!were!I.'scapularis! nymphs!and!one!was!an!I.'scapularis!larva.!Only!three!I.'affinis!nymphs!and!four!I.'affinis! larvae!were!collected!using!flags!during!that!time.!

! ! ! Table'2.!Ixodes'affinis!adults!collected!by!flagging!sites,!2010S2014.!Site!codes!correspond! to!those!mapped!in!Fig.!6.!General!habitat!types!(determined!by!visual!survey)!and!total! mean!density!per!100m2!are!included!for!each!site.!NS!indicates!the!site!was!not!sampled!in! that!year,!zeroes!indicate!no!I.'affinis'(IA)!were!collected.! ! IA' IA' IA' IA' IA' Total' County' Site' 2010' 2011' 2012' 2013' 2014' IA' Habitat'Type' Density' Late!successional!old! Chesapeake! CHS! 17! 8! 22! 22! 61! 130! field! 1.7639! MidSsuccessional!old! Chesapeake! CH2! NS! NS! NS! 2! 23! 25! field! 3.7963! Hampton! HAM! NS! 0! 3! 4! 10! 17! Edge/Deciduous!forest! 4.6058! Isle!of!Wight! ILW! 5! 4! 0! 0! 0! 9! Longleaf!pine!forest! 1.0909! Newport! News! NEW! NS! NS! NS! NS! 1! 1! Edge/Deciduous!forest! 0.2857! Northampton! CAP! NS! NS! 14! 13! 15! 42! Dunes/scrub!forest! 5.3407! Portsmouth! PT2! 0! 6! 19! 13! 87! 125! Pine/Deciduous!forest! 7.4271! Suffolk! SF1! NS! NS! 1! 8! NS! 9! Edge/Deciduous!forest! 2.6667! Suffolk! SF2! NS! NS! NS! NS! 4! 4! Edge/Deciduous!forest! 1.5000! Virginia!Beach! VB1! 3! 0! 0! 0! 0! 3! Dunes/scrub!forest! 0.6818! Virginia!Beach! VB3! 32! 113! 103! 17! 56! 321! Edge/Deciduous!forest! 26.966! York! YRK! 6! 13! 14! 11! 13! 57! Edge/Deciduous!forest! 15.205! Total! 63! 144! 176! 90! 270! 743! !! !! ! ' '

Table'3.!Adult!tick!species!composition!of!sites!where!I.'affinis!was!collected,!2010S2014.! Species!composition!of!adult!ticks!collected!at!the!standard!southeastern!Virginia!sampling! sites.!Site!codes!correspond!to!those!mapped!in!Fig.!6.!Combined!2010S2014!collections!of! questing! adult! Ixodes' affinis! (IA),! Dermacentor' variabilis' (DV),! Amblyomma' americanum! (AA),!Amblyomma'maculatum'(AM),!and!Ixodes'scapularis!(IS)!are!included!for!each!site,!as! well!as!the!percentage!IA!collected!compared!to!all!other!adult!ticks.!

IA' DV' AA' IS' AM' Total' %'IA' County' Site' adults' adults' adults' adults' adults' adults' Chesapeake! CHS! 130! 879! 1360! 50! 270! 2689! 4.83%! Chesapeake! CH2! 25! 234! 202! 1! 24! 486! 5.14%! Hampton! HAM! 17! 112! 586! 41! 9! 765! 2.22%! Isle!of!Wight! ILW! 9! 8! 148! 39! 0! 204! 4.41%! Newport! 1! 0! 45! 7! 0! 53! 1.89%! News! NEW! Northampton! CAP! 42! 65! 448! 17! 1! 573! 7.33%! Portsmouth! PT2! 125! 8! 2374! 151! 0! 2658! 4.70%! Suffolk! SF1! 9! 12! 205! 0! 0! 226! 3.98%! Suffolk! SF2! 4! 0! 12! 0! 0! 16! 25.00%! Virginia!Beach! VB1! 3! 83! 298! 9! 91! 484! 0.62%! Virginia!Beach! VB3! 321! 25! 857! 77! 1! 1281! 25.00%! York! YRK! 57! 27! 1423! 54! 14! 1575! 3.62%! Total! !! 743! 1453! 7958! 446! 410! 11010! 6.75%!

Habitat'preference'

Ixodes'affinis!were!collected!from!a!variety!of!habitat!types,!from!midSsuccessional! fields!dominated!by!tall!grass!to!forest!patches!surrounded!by!suburban!development!to! longleaf!pine!(Pinus'palustris)!forest!preserves!(Table!2).!The!largest!populations!were!all! found!in!mixed!deciduous!forested!sites!near!water!(VB3,!YRK,!CHS!and!PT2),!although!the! particular!vegetation!composition!varied!from!site!to!site.!No!I.'affinis!were!collected!at!two! of!the!primary!collection!sites,!both!of!which!are!very!small!forest!fragments!that!are!highly! disturbed!and!surrounded!by!urban!areas,!and!which!support!very!few!ticks!of!any!species!

(PT1!and!NO1,!Fig.!6).!Interestingly,!very!few!I.'affinis!were!collected!at!the!ILW!and!VB1! habitats,!both!of!which!are!large!nature!preserves!that!support!other!tick!species.!!

Population'permanence'

While!I.'affinis!were!collected!in!many!habitats,!population!stability!varied!between! sites!(Fig.!7).!Three!sites!(YRK,!VB2!and!CHS)!had!individuals!collected!during!the!first!year! of!sampling!in!2010!that!persisted!as!populations!through!all!five!years!of!collection.!Other! sites!showed!evidence!of!a!newly!establishing!population,!that!then!persisted!after!the! initial!year!when!individuals!were!collected!(PT2,!CAP,!HAM).!There!was!evidence!of! populations!increasing!over!time!at!PT2!and!CHS.!Only!a!few!adults!were!collected!in!2010S

2011!at!the!nature!preserves!ILW!and!VB1,!and!I.'affinis!have!not!been!collected!at!either!of! these!sites!since!the!initial!few!ticks.!These!sites!fall!short!of!the!six!adults!needed!to!be!an! established!a!population!of!ticks!(Fish!and!Howard,!1999).!The!most!productive!site!for! sampling!I.'affinis,!VB3,!yielded!large!numbers!in!2011!and!2012,!but!dropped!off!in!2013! and!then!recovered!in!2014.!

Phenology'!

! Adult!I.'affinis!were!collected!on!flags!from!April!through!October,!with!a!peak!in!

May,!large!numbers!collected!in!June!through!August,!and!then!a!rapid!drop!with!only!a!few! individuals!collected!in!September!and!October!(Fig.!8).!Although!not!regularly! encountered!on!flags,!immature!I.'affinis!were!frequently!collected!from!birds!and!small! mammals,!which!enabled!us!to!determine!the!phenology!of!larvae!and!nymphs!in! southeastern!Virginia.!Ixodes'affinis!larvae!peaked!in!winter,!but!were!collected!from!May!

41 onward!in!low!numbers.!Ixodes'affinis!nymphs!were!collected!from!April!through!

November,!with!a!peak!in!April!(Fig.!8).!

Figure'7.!Ixodes'affinis!adults!collected!per!m2!at!flagging!sites,!2010S2014.!Numbers!of! adult!ticks!per!m2!are!low!because!every!sampling!effort!at!each!site!is!included!in!the! denominator,!including!collection!trips!during!times!of!years!when!adults!were!not!active.! N!indicates!no!sampling!was!conducted!in!those!years,!zeroes!indicate!that!the!site!was! sampled!but!no!I.'affinis!were!collected.!Sites!codes!correspond!to!Fig.!6.!! ' ' '

Figure'8.!Phenology!of!all!life!stages!of!Ixodes'affinis'in!Virginia.!Larval,!nymphal,!and!adult! Ixodes'affinis!(IA)!were!collected!in!southeastern!Virginia!from!2010S2014.!The!number!of! I.'affinis!larvae!and!nymphs!collected!is!reported!on!the!left!axis,!while!adults!are!reported! on!the!right!axis.!

Host'preferences''

Eleven!Ixodes!spp.!larvae!were!collected!from!reptiles!in!southeastern!Virginia!and! sequenced,!including!ticks!from!common!fiveSlined!skinks!(Plestiodon'fasciatus),!ground! skinks!(Scincella'lateralis),!and!a!black!racer!(Coluber'constrictor).!A!fragment!of!the!16S! rRNA!gene!was!successfully!sequenced!from!nine!of!these!ticks,!and!all!were!confirmed!to! be!I.'scapularis.!Although!this!represents!a!very!small!sample!size,!there!is!currently!no! evidence!that!I.'affinis!feed!on!reptiles.!

Immature!I.'affinis!were!collected!from!six!species!of!passerine!birds,!summarized! by!Heller!et!al.!(2015),!and!four!species!of!small!mammal,!whereas!adult!I.'affinis!were! collected!from!four!species!of!medium!and!large!mammal!(Tables!4S5).!The!infestation!rate!

43 of!each!host!species!changed!throughout!the!year!as!tick!phenology!changed!(Table!6).!Out! of!544!ticks!found!crawling!on!or!attached!to!humans!that!were!submitted!to!the!Old!

Dominion!University!tick!lab!between!2011!and!2014,!three!I.'affinis'adults'were!identified.!

However,!there!was!no!evidence!that!any!of!these!three!ticks!were!biting!humans.!!

Competition'and'hostCsharing'

Ixodes'affinis!and'other!tick!species!were!collected!at!all!life!stages!from!the!same! individual!hosts!on!a!number!of!occasions!(Tables!4S5).!Immature!I.'affinis!were!also! observed!hostSsharing!with!I.'scapularis!on!two!house!mice!(Mus'musculus)!and!three! shortStailed!shrews!(Blarina'carolinensis)!(Table!4).!Adult!I.'affinis!were!found!hostSsharing! on!every!species!of!large!mammal!they!were!collected!from!in!this!study,!including! domestic!dogs!(Canis'lupus'familiaris),!domestic!cats!(Felis'catus),!whiteStailed!deer!

(Odocoileus'virginianus),!and!one!coyote!(Canis'latrans)!(Table!5).!Adult!I.'affinis!were! found!coSfeeding!with!Amblyomma'americanum,!Dermacentor'variabilis,'and!Ixodes' scapularis!(Table!5).!Host!sharing!on!birds!in!southeastern!Virginia!is!summarized!by!

Heller!et!al.!(2015).!

' '

Table'4.!Mammalian!hosts!of!immature!Ixodes'affinis!in!southeastern!Virginia.!This!table! includes!observations!of!I.'affinis'(IA)!host!sharing!with!Ixodes'scapularis!(IS).!Mammalian! species!were!hispid!cotton!rats!(Sigmodon'hispidus),!eastern!harvest!mice! (Reithrodontomys'humulis),!house!mice!(Mus'musculus),!and!southern!shortStailed!shrews! (Blarina'carolinensis).! ! Host!species! Site! Month! IA!larvae! IA!nymphs! IS!larvae! IS!nymphs! Total! Eastern!harvest!mouse! CH2! November! 1! 1! Hispid!cotton!rat! SF1! May! 1! ! ! ! 1! Hispid!cotton!rat! CHS! October! 1! ! ! ! 1! Hispid!cotton!rat! CHS! December! 1! ! ! ! 1! House!mouse*! CAP! July! 2! ! 5!! ! 7! House!mouse*! CAP! September! 1! ! 1! ! 2! ShortStailed!shrew*! CH2! January! 19! ! 1! ! 20! ShortStailed!shrew! CH2! January! 2! ! ! 2! ! ! ! ShortStailed!shrew! CH2! January! 1! 1! ShortStailed!shrew! CH2! March! 1!! ! ! 1! ShortStailed!shrew! CH2! April! 3!! ! ! 3! ShortStailed!shrew*! CH2! April! 6! ! 1!! ! 7! ShortStailed!shrew! CH2! April! 10! ! ! 10! ShortStailed!shrew! CHS! April! ! 1! ! ! 1! ShortStailed!shrew! CHS! September! 3!! ! ! 3! ! ! ! ShortStailed!shrew! CHS! September! 1! 1! ShortStailed!shrew! CHS! October! 1! ! ! ! 1! ShortStailed!shrew! CHS! November! 2! ! ! ! 2! ShortStailed!shrew! CHS! December! 1! ! ! ! 1! ! ! ! ShortStailed!shrew! CHS! November! 1! 1! ! ! ! ShortStailed!shrew*! CHS! December! 1! 1! 2! ShortStailed!shrew! CHS! January! 1!! ! 1! ShortStailed!shrew! CHS! February! 1! ! ! ! 1! ShortStailed!shrew! CH2! December! 35! ! ! ! 35! Total!ticks! !! !! 83! 14! ! 8!! 1!! 106! *!denotes!mammal!with!both!I.'affinis!and!I.'scapularis!! ! ! ! !

Table'5.!Hosts!of!adult!I.'affinis!in!southeastern!Virginia.!Hosts!of!adult!Ixodes'affinis!(IA)!in! southeastern!Virginia!and!observations!of!host!sharing!with!Dermacentor'variabilis!(DV),! Amblyomma'americanum!(AA),!and!Ixodes'scapularis!(IS).!! ! Host!! Collection!Date! City/County! DV! AA! IS! IA! Total! Cat! 4/23/11! Virginia!Beach! 1! 1! Cat! 5/30/14! Williamsburg! ! ! ! 1! 1! Cat*! 6/25/14! Virginia!Beach! ! 3!! ! 1! 4! Coyote**! 5/25/14! Smithfield! 3!! 34! ! 1! 38! Deer! 10/28/11! Virginia!Beach! ! 1! 1! Deer*! 6/2/14! Franklin! ! 10! ! ! 10! 20! Deer*! 6/2/14! Franklin! ! 56! ! 24! 80! Deer*! 7/2/14! Hampton! ! 1! ! 5! 6! Dog*! 5/15/14! Suffolk! ! 9! ! 2! 11! Dog**! 5/28/14! Virginia!Beach! 1!! 7! ! 1! 9! Total!ticks! 4! 120! 10! ! 47! 171! *denotes!I.'affinis'! hostSsharing!with!one!othe! r!tick!species! **denotes!I.'affinis'hostSsharing!with!more!than!one!tick!species!

DISCUSSION'

Ixodes'affinis!has!been!spreading!northward!since!the!original!report!of!specimens! in!Florida!in!1948!and!1951!(Kohls!and!Rogers,!1953).!In!the!ensuing!decades,!I.'affinis! established!and!become!widespread!in!Florida,!Georgia,!and!South!Carolina!(Oliver!et!al.,!

1987).!This!tick!was!uncommon!or!unknown!in!North!Carolina!before!1999,!but!surveys!in!

2009!indicated!that!I.'affinis'had!become!widely!distributed!throughout!coastal!North!

Carolina!(Harrison!et!al.,!2010).!Harrison!et!al.!(2010)!suggested!that!I.'affinis!might!be! present!in!Virginia!because!the!geographical!distribution!in!North!Carolina!abutted!Virginia!

–!this!suggestion!was!confirmed!with!the!report!of!established!I.'affinis!in!Virginia!in!six! independent!cities!and!counties!in!southeastern!VA!in!a!variety!of!habitats!in!2010!

(Nadolny!et!al.,!2011).!This!documentation!of!the!ecology!of!populations!in!southeastern!

Virginia!sheds!light!on!the!conditions!needed!for!the!survival!and!further!spread!of!this! tick.!

Ixodes'affinis!is!common!in!certain!locations!in!coastal!counties!of!North!Carolina!

(Harrison!et!al.,!2010),!and!similar!trends!were!found!in!surveys!of!coastal!Virginia.!While!I.' affinis!was!widespread!at!low!densities!throughout!southeastern!Virginia,!it!was!only!found! to!be!common!at!certain!locations,!presumably!where!habitat!and!host!availability!were! suitable!for!population!explosions.!Each!of!the!sites!where!I.'affinis!was!found!supported! diverse!tick!assemblages,!including!the!established!tick!species!Dermacentor'variabilis,!

Amblyomma'americanum,!and!I.'scapularis!as!well!as!occasional!coSoccurrence!with! another!new!invader,!Amblyomma'maculatum!(Wright!et!al.,!2011).!Ixodes'affinis'was!never! the!most!numerous!tick!species!at!any!site,!but!in!select!locations!it!was!second!in! abundance!to!the!ubiquitous!A.'americanum.!Only!two!sites!(ILW!and!VB1,!both!nature! preserves),!where!a!few!I.'affinis!had!been!collected!in!previous!years,!exhibited!extirpation! in!later!years,!suggesting!that!established,!I.'affinis'populations!tend!to!persist.!!!

Habitat'

The!sites!where!populations!of!I.'affinis!were!consistently!found!(YRK,!PT2,!CAP,!

VB3!and!CHS)!were!of!a!number!of!different!habitat!types,!including!edge/deciduous!forest,! pine/deciduous!forest,!dune/scrub!forest,!and!late!successional!old!field.!However,!each!of! these!locations!was!located!adjacent!to!a!water!source,!indicating!that!proximity!to!moist! environments!may!be!an!important!factor!in!I.'affinis'population!establishment.!In!addition,! each!of!the!locations!with!abundant!I.'affinis!was!anthropogenically!disturbed!in!some!way,! possibly!indicating!that!disturbed!secondary!woodlands!located!near!water!are!preferred!I.'

47 affinis!habitat.!Disturbed!secondary!woodland!is!common!in!the!MidSAtlantic,!and!habitat! connectivity!may!play!a!role!in!facilitating!the!range!expansion!of!this!tick!species.!The!least! disturbed!sites,!ILW!and!VB1,!yielded!very!few!I.'affinis,!and!there!may!be!a!barrier!to!I.' affinis!establishment!in!intact!habitats.!The!ILW!site!is!a!longleaf!pine!restoration!area!that! is!burned!biSannually,!so!managed!fires!may!not!be!equivalent!to!other!types!of!human! disturbance!for!providing!good!habitat!conditions'for'I.'affinis!establishment.!!

In!North!Carolina,!I.'affinis!is!found!in!coastal!plain!counties!but!not!in!piedmont!or! mountain!counties,!which!is!similar!to!its!reported!presence!in!coastal!plain!habitats!in!

Georgia!and!South!Carolina!(Harrison!et!al.,!2010).!One!I.'affinis'tick!parasitizing!a!rabbit! has!been!reported!in!the!piedmont!ecoregion!of!South!Carolina!(Nelder!and!Reeves,!2005).!

Despite!extensive!sampling!efforts!in!the!central!piedmont!region!of!Virginia!(Brinkerhoff,! personal!communication),!I.'affinis!was!only!found!in!coastal!plain!habitats.!The! environmental!conditions!common!to!this!physiographic!region!may!be!germane!to!I.' affinis!survival,!enabling!the!steady!northward!expansion!of!I.'affinis!in!these!habitats.!The!

Atlantic!coastal!plain!physiographic!region!extends!from!Florida!north!to!Massachusetts!

(Auch,!2014),!and!further!spread!of!I.'affinis!into!northern!states!may!be!possible!if!coastal! plain!habitat!is!the!limiting!factor!in!I.'affinis'survival.!

Phenology'

Ixodes'affinis!in!Virginia!exhibits!an!almost!identical!phenology!to!that!found!in!

Georgia!(Oliver!et!al.,!1987),!with!larval!peaks!in!the!winter,!nymphal!peaks!in!the!spring,! and!adult!peaks!in!the!summer.!Adult!I.'affinis'in!North!Carolina!were!also!found!to!quest! during!April!through!November!(Harrison!et!al.,!2010),!a!characteristic!that!can!be!used!to!

48 help!differentiate!adult!I.'affinis!from!I.'scapularis,!the!latter!of!which!quests!in!winter.!

Because!the!adults!of!both!species!are!easily!collected!on!flags,!flagging!suspected!I.'affinis' habitats!in!the!summer!months!is!the!simplest!way!to!determine!if!this!tick!species!has! been!established!in!a!new!area.!!

Oliver!et!al.!(1987)!suggest!that!I.'affinis!can!complete!a!full!life!cycle!in!one!year!in! the!southern!states.!The!lack!of!any!discernable!difference!between!I.'affinis!phenology!at! the!northern!extent!of!their!range!and!that!of!more!established!populations!in!southern! states!is!unexpected,!as!Virginia!experiences!somewhat!more!severe!winters!and!less! severe!summers!than!states!further!south.!It!is!possible!that!temperature!limits!for!this! species!are!more!plastic!than!previously!assumed,!and!are!not!a!limiting!factor!for!I.'affinis! population!establishment!given!access!to!appropriate!host!and!habitat!conditions.!

Host'preferences'

As!I.'affinis!is!not!a!very!abundant!tick!species,!unlike!A.'americanum,!it!is!not! surprising!that!I.'affinis!was!not!found!in!large!numbers!on!hosts.!Also,!small!mammals!are! easier!to!trap!during!winter!when!food!is!scarce!and!large!mammals!such!as!deer!are! easiest!to!sample!during!the!autumn!hunt!season,!so!some!times!of!year!may!have!been! underSsampled.!However,!I.'affinis'was!found!infesting!from!<1%!to!50%!of!individual! hosts!examined,!with!fluctuations!depending!on!the!time!of!year!and!subsequent!shifting! phenology!of!the!species!(Table!6).!Ixodes'affinis!was!documented!on!numerous!hosts!that! had!been!previously!reported,!but!this!tick!was!also!found!parasitizing!several!new!hosts! that!may!be!important!for!population!establishment!as!this!tick!moves!north!(Table!7).!!

Table&6."Percentage"of"hosts"infested"with"Ixodes'affinis'each"month,"201072014."Percentage"of"animal"hosts"infested"with" Ixodes'affinis"(IA)"each"month,"with"the"number"of"hosts"with"ticks"over"the"number"of"hosts"checked"in"parentheses."Data" shown"includes"all"avian"and"mammalian"species"hosting"IA"from"which"any"ticks"were"collected"from"201072014."Zeroes" indicate"no"hosts"of"that"species"were"sampled"during"that"month."Annual"infestation"rates"(IR)"are"also"indicated."

Jan& Feb& Mar& Apr& May& Jun& Jul& Aug& Sep& Oct& Nov& Dec& Annual&IR& " Adult&IA& 100%" 9.09%" 20%" 13.63%" Domestic"Cat" 0" 0" (0/1)" (0/3)" 0" 0" 0" (0/1)" 0" (1/1)" (1/11)" (1/5)" (3/22)" 100%" Coyote" 0" 0" 0" 0" (0/1)" 0" 0" 0" 0" 0" 0" 50%"(1/2)" (1/1)" White7tailed" 25%" 50%" 1.86%" 3.33%" (0/1)" 0" 0" 0" (0/1)" (0/1)" (0/1)" (0/42)" (0/10)" Deer" (2/8)" (1/2)" (1/54)" (4/120)" 3.45%" 1.63%" Domestic"Dog" (0/2)" (0/1)" (0/1)" (0/10)" (0/17)" (0/23)" (0/1)" (0/2)" (0/2)" (0/5)" (0/1)" (2/58)" (2/123)" Nymphal&IA" 5.26%" 1.10%" Carolina"Wren" (0/3)" (0/8)" (0/7)" (0/9)" (0/13)" (0/12)" (0/31)" (0/24)" (0/12)" (0/8)" (0/17)" (2/38)" (2/182)" White7throated" 2.85%" 0.63%" (0/24)" (0/23)" (0/19)" (0/26)" (0/1)" 0" 0" 0" 0" (0/1)" (0/30)" Sparrow" (1/35)" (1/159)" Short7tailed" 25%" 16.67%" 16.67%" 9.09%" 5.56%" (0/12)" (0/5)" (0/7)" (0/4)" (0/6)" (0/2)" (0/3)" (0/18)" Shrew" (1/4)" (2/12)" (1/6)" (1/11)" (5/90)" Larval&IA" 11.11%" 0.59%" American"Robin" 0" (0/1)" (0/4)" (0/17)" (0/16)" (0/17)" (0/8)" (0/4)" (0/44)" (0/50)" 0" (1/9)" (1/170)" 5.56%" 6.25%" 2.67%" Brown"Thrasher" (0/2)" (0/2)" (0/4)" (0/5)" (0/4)" (0/3)" (0/6)" (0/11)" (0/3)" (0/1)" (1/18)" (1/16)" (2/75)" 2.63%" 4.17%" 8.33%" 12.5%" 1.65%" Carolina"Wren" (0/3)" (0/8)" (0/7)" (0/9)" (0/13)" (0/12)" (0/31)" (0/17)" (1/38)" (1/24)" (1/12)" (1/8)" (3/182)" 16.67%" 5.56%" Eastern"Towhee" 0" 0" (0/1)" (0/3)" 0" (0/2)" (0/2)" (0/2)" 0" 0" (0/2)" (1/6)" (1/18)" Northern" 3.85%" 0.33%" (0/8)" (0/19)" (0/8)" (0/15)" (0/21)" (0/48)" (0/51)" (0/41)" (0/36)" (0/19)" (0/10)" Cardinal" (1/26)" (1/302)" 6.67%" 20%" 2.56%" 1.33%" Hispid"Cotton"Rat" (0/31)" (0/31)" (0/4)" (0/23)" (0/9)" (0/5)" (0/9)" (0/2)" (0/52)" (1/15)" (1/5)" (1/39)" (3/225)" " " " " " " " " " " " " " " 49

Table&6,&continued.& " " " " " " " " " " " & " " " " " " " " " " " " Jan" Feb" Mar" Apr" May" Jun" Jul" Aug" Sep" Oct" Nov" Dec" Annual&IR" Larval&IA,&continued" 20%" 33.3%" 12.5%" House"Mouse" (0/4)" (0/4)" (0/1)" (0/5)" 0" 0" 0" 0" 0" (0/2)" (1/5)" (1/3)" (3/24)" Eastern"Harvest" 16.67%" 1.08%" (0/15)" (0/8)" (0/6)" (0/24)" (0/3)" (0/5)" (0/2)" (0/5)" (0/5)" 0" (0/14)" Mouse" (1/6)" (1/93)" Short7tailed" 33.33%" 20%" 16.67%" 66.67%" 5.56%" 16.67%" 18.18%" 14.44%" (0/4)" (0/7)" (0/4)" (0/6)" (0/2)" Shrew" (4/12)" (1/5)" (2/12)" (2/3)" (1/18)" (1/6)" (2/11)" (13/90)" "" "

5050

Table&7.!Published!host!records!of!I.#affinis!in!the!United!States!and!Canada.!Hosts! for!adults!(A),!nymphs!(N),!and!larvae!(L)!are!indicated!separately.!

Common Name Scientific Name A N L Location Source Passeriformes

Carolina Wren Thyrothorus ludovicianus X X GA Oliver et al., 1987 Swainson’s Thrush Catharus ustulatus X X Canada Scott et al., 2012 American Robin Turdus migratorius X VA Heller et al., 2015

Brown Thrasher Toxostoma rufum X VA Heller et al., 2015

Eastern Towhee X Pipilo erythrophthalmus VA Heller et al., 2015 White-throated Zonotrichia albicollis X VA Heller et al., 2015 Sparrow Northern Cardinal Cardinalis cardinalis X VA Heller et al., 2015 Didelphimorphia

Virginia Opossum Didelphia virginiana X GA Oliver et al., 1987 Lagomorpha

Nelder and Reeves, Cottontail rabbit Sylvilagus floridanus X SC 2005 Rodentia

Cotton mouse Peromyscus gossypinus X X GA, VA Oliver et al., 1987 Eastern harvest Reithrodontomys humulis X VA This study mouse House mouse Mus musculus X VA This study Durden and Oliver, Marsh rice rat Oryzomys palustris X X GA 1999 Wood rat Neotoma floridana X GA Oliver et al., 1987 Oliver et al., 1987 Hispid cotton rat Sigmodon hispidus X X GA (N); This study (L) Gray squirrel Sciurus carolinensis X GA Oliver et al., 1987 Soricomorpha

Southern short- Oliver et al., 1987 Blarina carolinensis X X GA, VA tailed shrew (L); This study (N) Carnivora

American black Ursus americanus X FL Yablsey et al., 2009 bear Kohls and Rogers, Bobcat Lynx rufus X FL 1953 Domestic cat Felis catus X NC Harrison et al. 2010 Raccoon Procyon lotor X X GA Oliver et al., 1987 Coyote Canis latrans X VA This study Kohls and Rogers, Domestic dog Canis lupus familiaris X FL 1953 Artiodactyla

Kohls and Rogers, White-tailed deer Odocoileus virginianus X X GA, FL 1953 (A); Oliver et al., 1987 (L)

Competition#and#host#sharing#

Newly!documented!hosts!include!the!eastern!harvest!mouse!

(Reithrodontomys#humulis),!the!ubiquitous!house!mouse!(Mus#musculus),!and!the! coyote!(Canis#latrans).!Five!new!species!of!birds!have!also!been!recently!reported!as! hosts!for!I.#affinis!in!southeastern!Virginia!(Heller!et!al.,!2015),!indicating!the!host! preferences!of!this!tick!species!may!be!more!varied!than!previously!thought.!These! generalist!tendencies!may!influence!the!ability!of!this!tick!to!spread!through!a! variety!of!habitats!using!many!different!possible!routes.!!

In!previous!studies,!the!presence!and!abundance!of!I.#affinis!has!been! associated!with!the!distribution!of!three!rodent!species:!the!cotton!mouse!

(Peromyscus#gossypinus),!the!hispid!cotton!rat!(Sigmodon#hispidus),!and!the!eastern! wood!rat!(Neotoma#floridana)#(Durden!and!Oliver,!1999;!Clark!et!al.,!2001;!Harrison! et!al.,!2010).!Southeastern!Virginia!is!outside!the!range!for!the!eastern!wood!rat!

(Wiley,!1980),!and!is!at!the!very!northernmost!extent!of!the!range!for!the!cotton! mouse!(Wolfe!and!Linzey,!1977),!which!is!easily!confused!with!the!whiteWfooted! mouse!(Peromyscus#leucopus),!with!which!its!range!in!Virginia!overlaps!(Lackey!et! al.,!1985).!In!this!study,!of!these!three!mammals!only!the!hispid!cotton!rat!was! present!and!found!to!host!I.#affinis,!although!Peromyscus!spp.!mice!were!captured! and!examined!for!ticks.!

By!far!the!most!dominant!small!mammal!host!for!I.#affinis!in!southeastern!

Virginia!was!the!southern!shortWtailed!shrew.!Oliver!et!al.!(1987)!found!an!average! of!two!I.#affinis!larvae!per!shrew!they!examined!in!Georgia,!the!highest!infestation! rate!of!any!small!mammal!examined,!but!had!only!a!small!sample!size!of!four!

53 shrews.!Southern!shortWtailed!shrews!are!likely!an!important!host!for!immature!I.# affinis!in!Virginia,!with!an!annual!larval!infestation!rate!of!over!14%!(Table!4),!and! that!the!closely!related!northern!shortWtailed!shrew!(Blarina#brevicauda),#which! have!overlapping!ranges!in!Virginia!(George!et!al.,!1986;!McCay,!2001),!could!be!a! potential!host!as!this!tick!continues!to!move!northward.!One!potential!bias!to!this! dataset!was!an!increased!risk!of!trap!mortality!for!captured!shrews!in!traps! designed!to!capture!rodents!(Do!et!al.,!2013);!dead!animals!were!returned!to!the!lab! and!any!attached!ticks!were!allowed!to!detach,!enabling!a!more!thorough!census!of! ticks!on!shrews!than!of!any!other!small!mammal.!!!

! A!typical!life!history!pattern!for!an!I.#affinis!tick!in!southeastern!Virginia! might!start!by!larvae!feeding!on!a!small!mammals!or!birds!in!the!fall!and!winter! months.!Once!the!nymphs!became!active!in!the!spring,!shrews!are!the!most!likely! source!of!a!bloodmeal,!although!other!small!mammals!and!birds!are!also! parasitized.!In!the!summer!months,!an!adult!I.#affinis#would!need!to!find!a!large! carnivore!or!ungulate!to!obtain!a!large!bloodmeal;!these!large!animals!would!also! provide!an!easy!mode!of!dispersal!across!the!landscape!between!optimal!riparian! habitat!patches.!

! Interesting,!I.#affinis!was!commonly!found!on!domesticated!animals,! especially!cats!(Felis#catus).!In!its!native!Central!and!South!America,!I.#affinis!feeds! on!ocelots!(Felis#pardalis),!pumas!(Puma#concolor),!and!jaguars!(Panthera#onca)!

(Kohls!and!Rogers,!1953),!so!it!is!not!surprising!that!domesticated!felines!would!be! an!appealing!option!for!I.#affinis!that!have!ventured!further!north!to!where!wild! felines!are!less!abundant.!Areas!of!North!America!where!wild!cats,!such!as!bobcats!

(Lynx#rufus),!or!even!feral!cats!are!abundant!may!provide!preferred!host! communities!for!I.#affinis.!!

Ixodes#affinis!was!found!coWfeeding!with!other!tick!species!at!every!life!stage,! in!every!month!of!the!year!(Tables!4W5).!Heller!et!al.!(2015)!reported!similar!results! on!avian!hosts!in!southeastern!Virginia,!with!immature!I.#affinis#coWfeeding!alongside! five!other!species!of!tick.!This!is!unsurprising,!given!the!diverse!assemblage!of!ticks! in!southeastern!Virginia,!but!it!does!raise!questions!of!how!a!newly!arrived!species! like!I.#affinis!interacts!with!other!established!ticks.!Could!competition!with!other! ticks!for!hosts!inhibit!I.#affinis!from!establishing!in!an!environment!already!

“saturated”!with!ticks?!

Previous!studies,!and!the!observations!of!frequent!hostWsharing!presented! here,!indicate!that!competition!for!space!on!a!host!is!not!likely!to!be!a!limiting!factor! for!tick!survival.!One!study!of!two!specialist!tick!species!sharing!the!same!lizard!host! found!no!evidence!of!resource!competition!(Bull!et!al.,!1989).!Another!study!of!a! nidiculous!(nestWdwelling)!specialist!and!a!fieldWdwelling!generalist!tick!species! sharing!a!host!suggested!that!the!specialist!tick!was!densityWdependent!so!as!not!to! overburden!its!host,!but!the!generalist!tick!species!was!not!(Pfäffle!et!al.,!2011).!As!I.# affinis!is!a!generalist,!nonWnidicolous!tick!species,!capable!of!sharing!hosts!with!at! least!six!other!tick!species,!there!is!little!evidence!that!competition!for!hosts!is!a! deterrent!to!I.#affinis!population!establishment!or!maintenance.!

! Time!of!year!does!influence!other!tick!species!with!which!I.#affinis!may!share! hosts.!Like!I.#affinis,#A.#americanum#and!D.#variabilis#adults!are!active!and!questing! during!the!summer!months,!whereas!opportunities!for!host!sharing!with!adult!I.#

55 scapularis!ticks!were!during!spring!and!fall!when!both!species!are!regularly! encountered!in!the!field.!Immature!I.#affinis!were!collected!alongside!immature!I.# scapularis!on!small!mammals;!peaks!for!questing!and!feeding!I.#scapularis!nymphs! and!larvae!were!simultaneous!with!those!of!I.#affinis,!in!May!and!June,!with!declining! numbers!collected!through!the!rest!of!the!summer.!Immature!I.#affinis#collected!from! birds!by!Heller!et!al.!(2015)!were!found!coWfeeding!with!Ixodes#dentatus,!I.#brunneus,#

I.#scapularis,#A.#americanum,!and!Haemophysalis#leporispalustris.!

Movement#and#population#establishment#

Ixodes#affinis#needs!less!than!10!years!to!arrive!and!establish!in!an!area! where!appropriate!habitat!and!host!communities!are!abundant!(Harrison!et!al.,!

2010).!Because!I.#affinis!is!a!generalist!tick!species,!feeding!on!many!avian!and! mammalian!hosts!and!living!in!a!variety!of!habitats!(Tables!2,!4W6),!there!are!many! possible!pathways!that!might!facilitate!such!rapid!range!extensions.!To!closely! examine!these!possibilities!and!determine!which!hosts!could!be!most!important!in! range!expansion!and!new!population!establishment,!a!table!of!relevant!ecological! and!life!history!characteristics!of!each!of!the!hosts!of!I.#affinis!in!the!US!was!compiled!

(Table!8).!Tick!dispersal!ability!depends!on!the!behaviors!of!these!hosts!during!the! months!when!their!life!stages!are!questing.!

Larvae!of!I.#affinis#are!actively!questing!and!feeding!from!July!through!April! with!a!peak!in!December!in!Virginia,!and!have!been!found!on!six!species!of!birds,! seven!rodents,!southern!shortWtailed!shrews,!the!Virginia!opossum!(Didelphis# virginiana)!and!whiteWtailed!deer!(Table!7).!Percentages!of!animals!infested!with!

56 ticks!per!month!indicate!that!birds!and!small!mammals!are!all!infested!with!larvae! at!a!rate!of!2!W!33%,!with!shrews!having!the!highest!infestation!rate.!No!immature!I.# affinis#were!found!on!deer!or!the!few!opossums!sampled,!suggesting!that!larvae!are! more!likely!to!feed!regularly!on!birds!and!small!mammals,!at!least!during!the!times! that!deer!and!opossums!were!opportunistically!sampled.!In!the!rare!event!of!larvae! feeding!on!a!larger!mammal,!opossums!are!not!known!to!disperse!in!winter!months,! so!their!ability!to!move!ticks!is!limited!to!their!typical!home!range!of!around!4!ha!

(Sutherland!et!al.,!2000).!WhiteWtailed!deer!may!move!I.#affinis!larvae!longer! distances,!as!they!have!large!home!ranges!of!60W520!ha,!and!yearlings!disperse!15!or! more!km!from!their!natal!site!in!late!summer!when!larvae!are!beginning!to!quest!

(Table!8).!One!neotropical!migrant,!the!Swainson’s!Thrush,!is!known!to!transport!I.# affinis!larvae!north!during!the!spring!migration,!when!it!briefly!stops!in!Virginia! before!continuing!to!its!summer!breeding!grounds!in!Canada!(Scott!et!al.,!2012).!!

All!other!birds!known!to!host!I.#affinis!are!residents!in!Virginia!during!the!winter! months,!and!are!unlikely!to!transport!I.#affinis!larvae!farther!than!their!home!ranges,! generally!less!than!a!few!hectares!(Table!8).!The!small!mammal!hosts!of!I.#affinis!are! not!known!to!disperse!long!distances,!and!only!move!a!few!hundred!meters!from! their!home!ranges,!which!are!generally!<1!ha!(Table!8).!These!mammals!provide! little!opportunity!for!larval!I.#affinis#dispersal,!but!high!densities!may!be!critical!for! population!establishment!of!I.#affinis,!given!that!more!larvae!of!I.#affinis!are!found!on! small!mammals,!especially!shrews,!than!any!other!host!group.!Many!small!mammals! are!found!at!extremely!high!densities!in!the!fall,!after!population!explosions!during! the!summer!months!when!food!is!abundant!(Table!8).!These!high!densities!of!!

Table&8.&Dispersal)potential)of)hosts)of)Ixodes'affinis)in)southeastern)Virginia.)Ecological)information)on)the)known)hosts)of) Ixodes'affinis,)specifically)in)southeastern)Virginia)if)data)was)available.)NA)indicates)not)applicable;)unknown)indicates) insufficient)data)in)the)literature.)This)table)is)color)coded)and)organized)by)dispersal)potential,)as)determined)by)animal)home) range)size)and)dispersal)patterns.) ) Home&range& Main&life&stage&that& %&of&animals& Common&name& Species& Migratory& Resident& (ha)& disperses& that&disperse& Small&home&ranges,&non@migratory:&limited&dispersal&potential&of&immatures& Thyrothorus' Carolina)Wren) No) Yes) 0.7) None) 0) ludovicianus' Brown)Thrasher) Toxostoma'rufum' No) Yes) 1) None) Unknown) Pipilo' Eastern)Towhee) No) Yes) 1.6) Unknown) Unknown) erythrophthalmus' Peromyscus' Cotton)mouse) No) Yes) 0.45) Unknown) Unknown) gossypinus' Eastern)harvest) Reithrodontomys' No) Yes) 0.095) Unknown) Unknown) mouse) humulis' House)mouse) Mus'musculus' No) Yes) 0.02) Unknown) Unknown) Marsh)rice)rat) Oryzomys'palustris' No) Yes) 0.3) Unknown) Unknown) Wood)rat) Neotoma'floridana' No) Yes) 0.2) Breeding)males) Unknown) Hispid)cotton)rat) Sigmodon'hispidus' No) Yes) 0.35) Unknown) Unknown) Southern)shortStailed) Blarina'carolinensis' No) Yes) 0.96) Unknown) Unknown) shrew) Small&to&medium&home&ranges,&moderate&dispersal&potential&of&immatures& American)Robin) Turdus'migratorius' No) Yes) 0.03) Immatures) Unknown) Northern)Cardinal) Cardinalis'cardinalis' No) Yes) 10) Immatures) 10%) Virginia)Opossum) Didelphis'virginiana' No) Yes) 4.65) None) 0) Cottontail)rabbit) Sylvilagus'floridanus' No) Yes) 1) Unknown) Unknown) Gray)squirrel) Sciurus'carolinensis' No) Yes) 2.75) Immatures) Unknown) Raccoon) Procyon'lotor' No) Yes) 49) Immatures) Males) ) ) )

Table&8,&continued.& ) Dispersal& Habitat& Common&name& distance& Dispersal&timing& preferences& Average&host&density& Sources& Small&home&ranges,&non@migratory:&limited&dispersal&potential&of&immatures& Haggerty)et)al.,)2014;)Pardieck)et) Carolina)Wren) None) None) Generalist) 0.33)birds/km) al.,)2015) Cavitt)and)Haas,)2014;)Pardieck)et) Brown)Thrasher) None) Summer) Generalist) 0.1)birds/km) al.,)2015) Greenlaw,)2015;)Pardieck)et)al.,) Eastern)Towhee) Unknown) Unknown) Generalist) 0.325)birds/km) 2015) Woods,)swamps,) Wolfe)and)Linzey,)1977;) Cotton)mouse) 1000)m) Unknown) 3.5)per)ha)(South)Carolina)) oldfields) Sutherland)et)al.,)2000) Eastern)harvest) 85.5m)) Unknown) Oldfields) 8.75S44.4/ha)) Stalling,)1997) mouse) House)mouse) None) Unknown) Generalist) Unknown) Zeilinski)et)al.,)1992;)Brown,)1953)) Woods,)swamps,) Marsh)rice)rat) 80)m) Unknown) 1S109)indiviuals)per)ha) Wolfe,)1982) oldfields) Woods,)swamps,) Wood)rat) 300)m)max) Unknown) 0.51)per)ha) Wiley,)1980) oldfields) 13)m)(average) Cameron)and)Kincaid,)1982;) Hispid)cotton)rat) Unknown) Grassy)habitats) 8S100)per)ha,)peaks)in)autumn) daily)movement)) Cameron)and)Spencer,)1981) Southern)shortS 3.8)per)ha,)ranges)from)1S13,) George)et)al.,)1986;)Genoways)and) 603.7)m)) Unknown) Generalist) tailed)shrew) peaks)in)autumn) Choate,)1998) Small&to&medium&home&ranges,&moderate&dispersal&potential&of&immatures& Vanderhoff)et)al.,)2014;)Pardieck)et) American)Robin) 40)km) Summer) Generalist) 0.55)birds/km) al.,)2015) Northern) Halkin)and)Linville,)1999;)Pardieck) 65)km) Summer) Generalist) 1)bird/km) Cardinal) et)al.,)2015)) McManus,)1974;)Sutherland)et)al.,) Virginia)Opossum) 4)km)max)average) NA) Generalist) 0.625)per)ha) 2000;)Schmidly,)2004) Chapman)et)al.,)1980;)Sutherland) Cottontail)rabbit) 3)km) Unknown) Generalist) 8.9)per)ha)(fall,)Wisconsin)) et)al.,)2000) 100)km) Woods,) 3/ha)in)woods,)up)to)21)per) Gray)squirrel) Autumn) Koprowski,)1994) (maximum)) generalist) ha)in)urban)parks) 3.2)to)266)km,) .1)per)ha)(up)to)4/ha,)usually) Raccoon) generally)less)than) Fall) Generalist) Lotze)and)Anderson,)1979) between).2)and)0.02)) 1.6)km)

5858

) Table&8,&continued. & ) Common& Main&life&stage& %&of&animals&that& name& Species& Migratory& Resident& Home&range&(ha)& that&disperses& disperse& Large&home&ranges,&possible&long&distance&dispersers&of&adults& American)black) Ursus'americanus' No) Yes) 6500) Yearlings) All)males,)5%)females) bear) Bobcat) Lynx'rufus' No) Yes) 4500) Yearlings) All) Coyote) Canis'latrans' No) Yes) 2500) Immatures) Most) WhiteStailed) 80%)yearling)males,)13%) Odocoileus'virginianus' No) Yes) 290) Yearlings) deer) yearling)females) Migratory,&possible&long&distance&dispersers&of&immatures& Swainson's) Catharus'ustulatus' Yes) No) NA) NA) NA) Thrush) WhiteSthroated) Zonotrichia'albicollis' Yes) Yes,)winter) 0.01) NA) NA) Sparrow) & & & & & & ) & & & & & & ) Common& Dispersal& Habitat& Average&host& name& Dispersal&distance& timing& preferences& density& Sources) Large&home&ranges,&possible&long&distance&dispersers&of&adults& American)black) 127)km)max)average)(varies) Summer) Generalist) 1/5.15)km2) Sutherland)et)al.,)2000;)Larivière,)2001) bear) from)13S219)km)) 1)bobcat/11)km2) Bobcat) 119)km)max)average) Summer) Generalist) Larivière)and)Walton,)1997) (Oklahoma)) Fall)and) Coyote) 5)km)up)to)160)km) Generalist) 0.3)per)km2) Bekoff,)1977;)Sutherland)et)al.,)2000) winter) WhiteStailed) 15)km) Summer) Generalist) 25)per)km2) Smith,)1991;)Sutherland)et)al.,)2000) deer) Migratory,&possible&long&distance&dispersers&of&immatures& Swainson's) 119)km)per)day)(during) NA) Forests) NA) Mack)and)Yong)2000;)Pardieck)et)al.,)2015) Thrush) migration)) WhiteSthroated) Here)in) Piper)and)Wiley,)1990;)Falls)and)Kopachena,) NA) Generalist) Unknown) Sparrow) winter) 2010)

5959

60 mammals%corresponding%to%I.#affinis%larval%activity%may%create%an%ideal%situation%for% questing%ticks,%and%may%be%integral%to%population%establishment.%

Nymphs%were%collected%at%low%densities%in%the%summer%and%fall,%but%peak%nymphal% activity%levels%were%observed%in%spring.%Ixodes#affinis%nymphs%have%been%found%on%three% bird%species,%including%a%common%resident,%the%Carolina%Wren,%and%two%migratory%species%–% the%Swainson’s%Thrush%and%WhiteBthroated%Sparrow.%Both%migrants%harbor%the%potential%to% move%nymphs%and%larvae%of%these%species%north%during%their%migrations%in%the%spring%(as% documented%by%Scott%et%al.%2012).%The%WhiteBthroated%Sparrow%arrives%in%Virginia%in%the%fall% and%is%a%resident%throughout%the%winter,%which%may%result%in%greater%opportunity%to%become% infested%with%I.#affinis%nymphs%as%they%first%begin%to%quest%and%then%transport%them%north.%It% is%important%to%note%that%population%establishment%via%longBdistance%dispersal%by%birds%may% be%prevented%by%high%offBhost%mortality%for%immature%ticks%(Needham%and%Teel,%1991).%

Nymphs%are%more%commonly%found%on%mammalian%hosts,%including%cotton%mice,% marsh%rice%rats,%hispid%cotton%rats,%shrews,%and%raccoons%(Table%7).%These%mammals%are% nonBmigratory,%and%generally%have%small%home%ranges%of%less%than%1%ha,%and%have%limited% dispersal%ability%(Table%8).%Densities%of%small%mammals%tend%to%be%lower%during%spring,% because%the%populations%have%not%yet%had%a%chance%to%recover%from%mortality%during%the% winter.%However,%even%lower%densities%of%a%number%of%appropriate%small%mammal%species% may%provide%sufficient%food%for%a%small%number%of%nymphs%to%feed%and%persist.%%

Adult%I.#affinis%quest%from%April%through%October,%and%feed%exclusively%on%larger% mammals%during%these%summer%months%(Tables%5B6).%Large%mammals%travel%much%longer% distances%than%their%smaller%mammalian%counterparts,%and%are%the%more%likely%to%be% responsible%for%the%spread%of%I.#affinis%across%the%landscape.%Yearlings%of%many%larger%

61 mammals,%including%bears,%bobcats,%and%whiteBtailed%deer%all%disperse%during%the%summer% months,%and%move%distances%up%to%hundreds%of%kilometers%away%from%their%natal%area.%

Other%large%mammals,%including%raccoons%and%coyotes,%disperse%in%the%fall,%and%may%be% responsible%for%moving%lateBquesting%I.#affinis%over%long%distances.%Even%the%home%ranges%of% these%larger%animals%are%large,%from%50%ha%(raccoons)%to%6500%ha%(black%bears),%so%nonB dispersing%hosts%may%still%move%I.#affinis#ticks%over%long%distances%(Table%8).%It%is%likely%that% any%adult%I.#affinis%that%successfully%quests%and%feeds%will%detach%and%lay%eggs%in%a%different% area%than%the%one%where%it%attached.%The%presence%of%I.#affinis%on%domestic%cats%and%dogs% that%can%be%moved%long%distances%by%humans%is%another%possible%avenue%for%longBdistance% dispersal%for%these%ticks.%Engorged%adults%transported%by%large%mammal%hosts%are%more% likely%to%found%a%new%population%than%immature%ticks,%as%there%are%fewer%barriers%to% successful%reproduction%for%adult%ticks%that%have%already%obtained%a%blood%meal%than%for% immature%ticks%questing%for%their%next%meal.%%

% In%conclusion,%I.#affinis%is%well%established%throughout%southeastern%Virginia%and% appears%to%be%increasing%its%range%north%each%year.%Anthropogenically%disturbed%forest% habitats%and%diverse%host%communities%in%the%Atlantic%coastal%plain%seem%to%be%facilitating% this%expansion,%and%the%phenology%of%I.#affinis#remains%largely%unchanged%from%populations% further%south.%Ixodes#affinis%was%found%on%several%previously%unreported%species%of% mammal,%and%host%sharing%was%documented%between%I.#affinis%and%other%tick%species,% including%I.#scapularis.%Abundant%small%mammal%and%bird%communities%promote%population% establishment%by%the%immature%stages%of%this%tick,%while%adults%feeding%on%large%mammals% furthers%range%expansion%and%dispersal.%Tick%surveillance%efforts%in%Maryland,%Delaware,% and%beyond%should%remain%vigilant%for%this%invader.%

NATURAL'HISTORY'OF'AMBLYOMMA'MACULATUM'IN'VIRGINIA'

INTRODUCTION'

The%Gulf%Coast%tick,%Amblyomma#maculatum,%is%an%aggressive,%humanBbiting%tick%of% increasing%medical%and%veterinary%significance%(Paddock%and%Goddard,%2015).%It%is%a%known% vector%of%several%emerging%pathogens,%including%Rickettsia#parkeri,%the%agent%of%Tidewater% spotted%fever%(Paddock%et%al.,%2004).%Human%infection%with%R.#parkeri#results%in%an%escharB associated%febrile%illness%that%is%milder%than%the%more%familiar%Rocky%Mountain%spotted% fever%caused%by%the%related%Rickettsia#rickettsii%(Paddock%et%al.,%2008).%Veterinary%pathogens% vectored%by%this%tick%include%Hepatozoon#americanum,%the%principal%agent%of%American% canine%hepatozoosis%(Baneth,%2011),%Rickettsia#felis,%the%agents%of%feline%rickettsiosis,%

Leptospira#pomona,%causal%agent%of%leptospirosis%in%livestock,%and%Ehrlichia#ruminatum,%the% agent%of%heartwater%in%ruminants%(Stromdahl%and%Hickling,%2012).%In%addition,%A.# maculatum#is%often%infected%with%spotted%fever%group%rickettsia%of%unknown%pathogenicity,% including%Rickettsia#andeanae%and%other%newly%described%organisms%(Paddock%et%al.,%2010;%

Jiang%et%al.,%2012).%In%addition%to%carrying%various%microbes,%A.#maculatum%are%large%ticks,% and%their%bites%can%cause%inflammation,%edema,%abscesses,%and%predisposition%to%myiasis,% anemia,%and%secondary%infections,%especially%in%livestock%(Paddock%et%al.,%2010;%Teel%et%al.,%

2010;%Jiang%et%al.,%2012).%%As%the%effects%of%these%pathogens%on%human%and%animal%health% have%become%better%understood%over%the%last%decade,%there%has%been%a%renewed%interest%in% understanding%the%ecology%A.#maculatum%in%order%to%prevent%disease%(Paddock%and%

Goddard,%2015).%

The%historical%range%of%A.#maculatum%throughout%the%Caribbean,%South%and%Central%

America%is%extensive,%including%records%from%Mexico,%Jamaica,%Belize,%the%West%Indies,%

Columbia,%Venezuela,%and%Peru%(Teel%et%al.,%2010;%Paddock%and%Goddard,%2015).%The% southern%extent%of%the%range%of%A.#maculatum%is%in%question%because%of%the%morphological% similarity%of%A.#maculatum%to%the%closely%related%A.#tigrinum#and%A.#triste%(EstradaBPeña%et% al.,%2005).%In%the%early%1900s,%A.#maculatum%was%common%along%the%Gulf%Coast%of%the%US% from%Louisiana%to%Texas,%invading%into%the%coastal%Atlantic%region%up%through%South%

Carolina%in%the%midB1900s%(Teel%et%al.,%2010).%After%the%1950s,%the%distribution%of%A.# maculatum%drastically%changed,%as%the%cattle%industry%facilitated%the%establishment%of%these% ticks%in%Kansas%and%Oklahoma%in%the%1950sB1980s%(Teel%et%al.,%2010;%Paddock%and%Goddard,%

2015).%Researchers%in%Arkansas%collected%over%200%A.#maculatum%between%2006%and%2009,% and%now%consider%A.#maculatum%to%be%established%in%that%state%(Trout%et%al.,%2010).%Between%

2009%and%2011,%A.#maculatum%populations%reached%sufficient%levels%to%be%considered% established%in%North%Carolina,%having%previously%been%known%only%from%sporadic%records%

(Harrison%et%al.,%2010;%VarelaBStokes%et%al.,%2011).%Newly%established%A.#maculatum% populations%have%also%recently%been%discovered%in%southeastern%and%northern%Virginia%

(Fornadel%et%al.,%2011;%Wright%et%al.,%2011),%and%in%Delaware%(Florin%et%al.,%2014).%Incidental% collections%of%individual%ticks%further%north%are%generally%along%migratory%bird%flyways;%that% and%an%expanding%whiteBtailed%deer%population%have%been%suggested%as%hosts%that%may%be% implicated%in%the%continued%expansion%of%this%tick%(Paddock%and%Goddard,%2015).%

Human%infections%with%R.#parkeri%are%reported%everywhere%these%ticks%are%found%

(Sumner%et%al.,%2007;%Jiang%et%al.,%2012;%Paddock%and%Goddard,%2015).%However,%newly% established%populations%of%these%ticks,%especially%in%MidBAtlantic%states,%have%tended%to%

64 exhibit%higher%infection%prevalences%than%in%areas%where%these%ticks%have%long%been% established%(Fornadel%et%al.,%2011;%Wright%et%al.,%2011).%Some%of%the%first%cases%of%R.#parkeri% infection%were%described%in%Virginia,%which%has%only%had%established%A.#maculatum% populations%for%a%short%while,%indicating%these%ticks%can%start%spreading%disease%as%soon%as% they%arrive%(Paddock%et%al.,%2004;%Whitman%et%al.,%2007).%Recent%evidence%suggests%that% spillover%of%R.#parkeri#to%other%common%tick%species,%including,%the%lone%star%tick#

(Amblyomma#americanum),%may%occur%in%areas%invaded%by%A.#maculatum#(Gaines%et%al.,%

2014;%Henning%et%al.,%2014;%Wright%et%al.,%2015).%As%this%tick%continues%to%expand%its%range,% understanding%the%ecological%mechanisms%by%which%new%populations%arise%and%persist%is% critical%to%protecting%human%and%animal%health.%Here,%the%results%of%five%years%of%field% surveillance%on%newly%established%populations%of%A.#maculatum%in%southeastern%Virginia%are% described,%and%the%implications%of%the%findings%on%the%continued%expansion%of%A.#maculatum# into%the%MidBAtlantic%are%discussed.%

MATERIALS'AND'METHODS'

Collection#of#questing#ticks#

Questing%A.#maculatum#were%collected%from%public%and%privately%owned%lands% throughout%southeastern%Virginia%from%for%five%years%from%2010%to%2014.%Fourteen%sites% representing%a%variety%of%woodland,%grassland,%successional,%and%anthropogenically% disturbed%habitats%were%sampled%weekly%during%the%summer%months%and%at%least%monthly% throughout%the%year%(Fig.%9).%Each%of%the%14%sites%was%sampled%for%a%minimum%of%two% consecutive%years.%All%transects%were%marked%with%surveyor’s%flags%so%that%the%same% transect%was%walked%during%each%sampling%event.%In%order%to%determine%the%m2%area%

65 sampled%at%each%site,%GPS%coordinates%were%taken%along%each%transect.%ArcGIS%was%used%to% draw%a%line%connecting%the%GPS%points%from%each%transect%and%to%draw%a%two%meter%buffer% around%the%line.%Ticks%were%collected%using%1m2%white%denim%flags%attached%to%wooden% dowel%rods%swept%over%the%ground%and%through%low%vegetation.%All%ticks%found%clinging%to% the%flag%material%were%removed%from%the%flag%with%forceps%and%placed%in%containers%labeled% with%the%time%and%date%of%collection,%as%well%as%the%sampling%location,%temperature,%and% weather%data.%Ticks%were%frozen%at%B20%C%until%morphologically%identified;%they%were%then% stored%frozen%at%B80%C%(Keirans%and%Durden,%1998).%Only%sites%where%more%than%six%adult% ticks%or%multiple%life%stages%were%collected%were%considered%to%be%established%populations%

(Fish%and%Howard,%1999).%

Collection#of#ticks#from#hosts#

HostBtargeted%sampling%techniques%were%employed%from%2010%to%2014,%including% collection%of%ticks%from%large%mammals%at%hunt%check%locations%during%the%fall%of%each%year,% serendipitous%collaboration%with%veterinarians%to%determine%tick%presence%on%companion% animals,%serendipitous%sampling%of%roadBkilled%mammals,%monthly%or%quarterly%small% mammal%liveBtrapping,%weekly%avian%sampling%using%mistBnets,%and%opportunistic%handB capture%of%reptiles%at%flagging%sites%(IACUC%#s:%11B012,%13B018,%13B008).%%

Large%wild%mammals%were%sampled%for%ticks%through%collaborations%with%local% hunters,%game%managers,%and%state%biologists,%and%through%checking%roadBkilled%animals%for% ticks.%Any%ticks%found%on%dead%mammals%were%removed%with%forceps,%places%in%vials,%and% returned%to%the%lab%where%they%were%frozen%at%B20%C%until%processing.%Domesticated%animals% were%sampled%through%collaborations%with%veterinarians%with%practices%in%southeastern%

Virginia.%If%any%ticks%were%found%on%an%animal%patient,%ticks%were%removed,%labeled,%and% frozen%until%they%could%be%passed%to%the%Old%Dominion%University%tick%lab%for%identification% and%processing.%

Figure'9.%Map%of%Amblyomma#maculatum%collection%sites,%2010B2014.%Fourteen%sampling% sites%in%southeastern%Virginia%were%sampled,%eight%of%which%yielded%at%least%one%A.# maculatum#in%five%years%of%sampling%from%2010B2014.%Blue%sites%yielded%no%A.#maculatum,# yellow%sites%indicate%where%fewer%than%six%A.#maculatum%were%collected,%and%red%sites% indicate%established%populations%of%more%than%six%A.#maculatum.% % %

Small%mammal%communities%were%sampled%at%two%primary%flagging%sites,%CHS%and%

CH2,%with%established%populations%of%A.#maculatum.%These%sites%represented%mid%to%late%

67 successional%habitats%adjacent%to%the%Great%Dismal%Swamp%in%Chesapeake,%VA,%and%were% dominated%by%grasses%and%forbs%with%mixed%coniferous%and%deciduous%trees%5B15%years%old.%

Transects%or%grids%of%modified%Fitch%live%traps%(Rose,%1994)%were%laid%at%each%site.%Trapping% was%conducted%monthly%or%quarterly%from%2010%to%2014.%Traps%were%baited%with%birdseed% and%sunflower%seeds,%were%set%in%the%evening%and%were%checked%for%three%consecutive% mornings.%During%winter%months,%polyfill%provided%insulation%for%trapped%animals.%Small% mammals%were%sexed,%weighed,%examined%for%reproductive%status,%tagged%using%numbered% metal%ear%tags,%and%then%released%after%recording%the%trap%coordinate%and%other%data.%

Trapped%mammals%were%examined%for%ticks,%and%any%ticks%were%removed%using%forceps,% placed%in%vials,%and%returned%to%the%lab%to%be%frozen%until%identification%and%processing.%All% mammals%were%handled%according%to%the%guidelines%set%forth%by%the%American%Society%of%

Mammalogists%(Sikes%and%Gannon,%2011).%

Avian%hosts%were%sampled%from%2013%to%2014%at%a%subset%of%the%flagging%sites%(Fig.%6)% through%weekly%or%biBweekly%mist%netting,%conducted%as%previously%described%(Heller%et%al.,%

2015).%Local%herpetofauna%were%sampled%at%the%weekly%flagging%transect%sites,%with%an% emphasis%on%collecting%ticks%from%native%lizards,%snakes,%and%turtles.%Reptiles%were%captured% using%standard%collection%techniques,%including%the%implementation%of%artificial%cover% objects%and%visual%encounter%surveys,%and%examined%for%ticks%before%being%released%

(McDiarmid%et%al.,%2012).%Ticks%were%removed%from%reptiles%with%forceps%and%placed%in% vials,%and%were%returned%to%the%lab%and%frozen%until%identification%and%processing.%

Molecular#methods#and#identification#

% All%ticks%collected%from%flags%and%from%hosts%were%identified%to%species%using% morphological%characteristics%(Clifford%et%al.,%1961;%Keirans%and%Durden,%1998).%If% morphological%identification%was%impossible%because%of%uncertain%morphological% characters%or%damage%to%the%specimen,%a%fragment%of%the%16S%mitochondrial%rRNA%gene%was% sequenced%using%previously%established%methods%(Nadolny%et%al.,%2015)%to%determine%tick% species.%While%adult%A.#maculatum%are%morphologically%distinct%from%other%US%ticks%in%the% genus%Amblyomma,%immatures%can%be%difficult%to%tell%apart%from%other%Amblyomma#and%

Dermacentor%species,%especially%when%engorged.%Unengorged%immatures%from%flags%were% identified%morphologically,%and%engorged%immature%ticks%were%identified%via%sequencing%or% via%restriction%fragment%length%polymorphism%(RFLP)%assay%(Fornadel%et%al.,%2011).%

RESULTS'

% Questing%adult%A.#maculatum%ticks%were%collected%from%eight%of%14%sites%sampled% from%2010B2014%(Fig.%9,%Table%9).%Out%of%those%sites%where%A.#maculatum#was%collected,%five% yielded%sufficient%numbers%of%adults%to%be%considered%a%population%(Fig.%9).%Adults%were% collected%only%during%the%summer%months,%from%April%through%September,%with%peak% activity%in%June%(Fig.%10).%While%immature%A.#maculatum%were%infrequently%collected%on% flags,%a%small%number%were%collected%over%the%five%years%(Table%10).%Amblyomma#maculatum% immatures%were%flagged%up%almost%exclusively%after%grassy%fields%had%been%mowed%at%sites% where%large%numbers%of%adults%were%regularly%collected.%Larvae%were%collected%in%spring% while%nymphs%were%collected%from%spring%through%late%summer.%Insufficient%numbers%of% immatures%were%collected%to%establish%a%clear%phenology%of%these%life%stages%in%Virginia.%

% Amblyomma#maculatum%adults%were%collected%from%a%variety%of%habitat%types%over% the%five%years%(Table%9),%but%all%of%the%most%productive%sites%where%A.#maculatum#could%be% reliably%collected%were%characterized%by%open%habitat,%including%anthropogenically% maintained%fields%(YRK,%HAM,%and%parts%of%VB1),%farmlands%undergoing%ecological% succession%to%forest%(CHS%and%CH2),%and%grassy%dunes%(VB1).%Only%sites%with%large%swaths%of% open%habitat%had%high%densities%of%A.#maculatum%per%m2%(Fig.%11),%and%maintained%those% populations%of%A.#maculatum%over%multiple%years%(Table%9).%%

Figure'10.%Phenology%of%adult%A.#maculatum%collected%in%Virginia%over%five%years.%

Figure'11.%Amblyomma#maculatum%adults%collected%per%m2%at%each%site,%2010B2014.%The% numbers%of%ticks%collected%per%m2%sampled%are%low%because%all%sampling%events,%including% during%times%when%A.#maculatum%were%not%active,%are%included%in%the%denominator.%

Table&9.!Amblyomma(maculatum(adults!collected!by!flagging,!201052014.!All!sites!were!in!southeastern!Virginia.!Site!codes! correspond!to!those!mapped!in!Figure!9,!and!habitat!types!and!A.(maculatum!density!are!included!for!each!site.!NS!indicates! that!that!site!was!not!sampled!in!those!years.!General!habitat!types!were!determined!by!observing!vegetation!communities! along!transects.!

AM& AM& AM& AM& AM& Total& AM/& County& Site& Habitat&Type& 2010& 2011& 2012& 2013& 2014& AM& 100&m2& Chesapeake! CHS! 54! 104! 88! 17! 7! 270! Late!successional!old!field! 2.66! Chesapeake! CH2! NS! NS! NS! 6! 18! 24! Mid5successional!old!field! 4.44! Hampton! HAM! NS! 2! 2! 0! 5! 9! Edge/Deciduous!forest! 2.16! Northampton! CAP! 0! 0! 1! 0! 0! 1! Dunes/scrub!forest! 0.57! Portsmouth! PT1! 3! 0! 0! 0! 0! 3! Urban!park! 0.31! Virginia!Beach! VB1! 9! 6! 27! 31! 18! 91! Dunes/scrub!forest! 8.55! Virginia!Beach! VB3! 0! 1! 0! 0! 0! 1! Edge/Deciduous!forest! 0.13! York! YRK! 0! 1! 13! 0! 0! 14! Field/Edge/Deciduous!forest! 2.14!

Table&10.!Amblyomma(maculatum(immatures!collected!by!flagging,!201052014.!All!A.(maculatum((AM)!nymphs!and!larvae! were!collected!in!southeastern!Virginia.!Site!codes!correspond!to!those!mapped!in!Figure!9. !

Month& AM&nymphs& AM&larvae& Site& March! 9! VB1! April! 1! 5!! CH2!(n),!YRK!(l)! May! 1! VB1! August! 6! ! VB1! September! 2! ! VB1! ! 71

71 72

Sites&where&populations&of&A.#maculatum&were&established&did¬&maintain&static& numbers&of&ticks&from&year&to&year&(Table&9,&Fig.&11).&In&particular,&the&largest&population&of&

A.#maculatum&at&the&CHS&site&reached&peak&abundance&in&2011,&with&decreasing&numbers& each&subsequent&year,&indicating&the&decline&of&this&population.&This&decline&corresponded& with&the&progression&of&ecological&succession,&and&the&closure&of&the&canopy&at&that&site.&

Another&site,&CH2,&has&yielded&increasing&numbers&of&A.#maculatum&in&the&two&years&it&has& been&sampled,&indicating&that&this&population&may&be&undergoing&an&increase&in&A.# maculatum&numbers,&similar&to&the&first&few&years&of&sampling&VB1&and&CHS.&This&site&is& earlier&in&the&successional&process,&and&will¬&undergo&canopy&closure&for&several&more& years.&While&numbers&of&A.#maculatum#were&lower&than&other&sites&at&VB1&(Table&9),&A.# maculatum&were&the&most&consistently&abundant&per&m2&this&site&(Fig.&11).&The&VB1&site&is&a& nature&reserve&that&comprises&grassy&areas&that&are&anthropogenically&maintained&through& annual&mowing,&as&well&as&preserved&dune&communities.&

& At&each&site&where&A.#maculatum&were&collected,&other&common&species&of&ticks&were& also&found,&including&Dermacentor#variabilis,&Amblyomma#americanum,&Ixodes#affinis,&and&

Ixodes#scapularis&(Table&11).&At&almost&every&site,&A.#americanum&was&by&far&the&most& abundant&tick&species,&with&the&exception&of&D.#variabilis&at&one&site,&PT1.&Amblyomma# maculatum&was&the&second&most&abundant&tick&species&at&only&one&site,&VB1.#

Out&of&370&individual&large&animal&hosts,&from&15&species&sampled,&and&3,293&ticks& removed&only&seven&were&adult&A.#maculatum.&These&seven&ticks&were&collected&from&six& individual&animals,&including&cats&(Felis#catus),&dogs&(Canis#lupus#familiaris),&whiteQtailed& deer&(Odocoileus#virginianus),&and&feral&swine&(Sus#scrofa)&during&the&summer&months& between&2010&and&2014&(Table&12).&Most&A.#maculatum#were&sharing&hosts&with&other&

73 common&ticks&at&the&time&of&collection,&including&Dermacentor#albipictus,&D.#variabilis,&A.# americanum,&and&I.#scapularis&(Table&12).&&

Immature&A.#maculatum&were&more&difficult&to&sample&than&adults.&Of&643&rodents& captured&and&1,087&individual&ticks&collected&from&small&mammals,&only&seven&immature&A.# maculatum#from&five&rodents&were&able&to&be&conclusively&identified&using&molecular& techniques&(Table&12).&Out&of&944&ticks&collected&from&1,888&birds&sampled&yearQround& from&2012Q2014,¬&a&single&A.#maculatum&immature&was&collected&(Heller&et&al.,&2015;&

Walters&lab,&unpublished&data).&Similarly,&11&larval&ticks&collected&from&81&native&reptiles& were&determined,&via&sequencing,&to&be&I.#scapularis.&Out&of&544&ticks&found&crawling&on&or& attached&to&humans&that&were&submitted&to&the&Old&Dominion&University&tick&lab&between&

2011&and&2014,&four&adult&A.#maculatum#were&identified.&&

Table&11.&Adult&tick&species&composition&of&sites&where&A.#maculatum&was&collected,&2010Q 2014.&Site&codes&correspond&to&those&mapped&in&Figure&9.&Combined&2010Q2014&collections& of&questing&adult&Amblyomma#maculatum&(AM),&Dermacentor#variabilis#(DV),&Amblyomma# americanum&(AA),&Ixodes#affinis#(IA),&and&Ixodes#scapularis&(IS)&are&included&for&each&site.&

County& Site& DV& AA& IS& AM& IA& Total&adults& %&AM& Chesapeake& CHS& 879& 1360& 50& 270& 130& 2689& 10.04%& Chesapeake& CH2& 234& 202& 1& 24& 25& 486& 4.94%& Hampton& HAM& 112& 586& 41& 9& 17& 765& 1.18%& Northampton& CAP& 65& 448& 17& 1& 42& 573& 0.17%& Portsmouth& PT1& 135& 25& 2& 3& 165& 1.82%& Virginia&Beach& VB1& 83& 298& 9& 91& & 3& 484& 18.80%& Virginia&Beach& VB3& 25& 857& 77& 1& 321& 1281& 0.08%& York& YRK& 27& 1423& 54& 14& 57& 1575& 0.89%& Total& && 1560& 5199& 251& 413& 595& 8018& 5.15%&

Table&12.!Hosts!infested!with!Amblyomma(maculatum(in!southeastern!Virginia.!!Hosts!were!infested!with!one!or!more!A.( maculatum!(AM)!adult!(A),!nymph!(N),!or!larva!(L).!Immatures!were!identified!using!a!RFLP!assay,!with!*!indicating!sequence! confirmation.!Site!and!month!where!the!host!was!collected!are!included!for!each!host.!For!mammals!hosting!adults,!coGfeeding! ticks!collected!from!the!same!host!are!reported,!including!Dermacentor(variabilis!(DV),!Amblyomma(americanum!(AA),!Ixodes( scapularis((IS),!and!Dermacentor(albipictus((DA).!The!monthly!and!annual!infestation!rate!(IR),!or!percentage!of!hosts!checked! infested!with!AM,!are!included!for!each!host!species!hosting!adults.!CoGfeeding!ticks!and!IR!are!not!reported!for!rodent!hosts,! because!of!challenges!associated!with!identifying!immature!engorged!ticks.!

AM&& AM& AM& DV& AA& AA& AA& IS& DA& Total& Monthly& Annual& Host& Site& Month& (L)& (N)& (A)& (A)& (L)& (N)& (A)& (A)& (N)& ticks& host&IR& host&IR& Felis(catus!(Domesticated!Cat)**! HAM! Jul! 1! 1! 33.33%! 4.35%! Odocoileus(virginianus(! (WhiteGtailed!Deer)! ! ( ! ! ! ! ! ! VB1! Oct! 2! 1! 1! 2! 2! 8! 1.85%! 2.50%! ! CHS! Dec! ! ! 1! ! ! 8! 9! 10.00%! 2.50%! Tappahannock! ! Jun! ! ! 1! ! ! 6!! 7!! ! 14! 12.50%! 2.50%! Canis(lupus(familiaris((Domesticated!Dog)! ! ( ! ! ! ! !!!!!!!NEW! May! 1! 1! 1.72%! 0.81%! Sus(scrofa((Feral!Swine)( ! ! ! ! ! ! ! ! VB1! Jul! 1! 15! 2! 11! 29! 100.00%! 5.26%! Microtus(pennsylvanicus(! (Meadow!Vole)! ! ( ! ! ! CHS! Feb! 1*! 1! NA! NA! ! CHS! Apr! ! 1! ! ! ! ! ! ! ! 1! NA! NA! Sigmodon(hispidus! !(Hispid!cotton!rat)! ! ! ! ! ! ! ! ! CHS! Aug! 2! 2! NA! NA! Oryzomys(palustris! !(Marsh!Rice!Rat)! ! ! ! ! ! ! ! ! CHS! July! 2! 2! NA! NA! !! CHS! Dec! 1! ! ! ! ! ! ! ! ! 1! NA! NA! **The!actual!number!of!ticks!from!this!cat!is!unknown;!this!! ! ! ! A.(maculatum(! ! ! tick!may!have!been!co! Gfeeding!with!A.(americanum! larvae!or!I.(scapularis!adult!ticks!that!were!received!by!the!Old!Dominion!University!tick!lab!from!one!veterinary!clinic!on!the! same!day. 74

74 75

DISCUSSION((

Phenology)

The$seasonal$phenology$of$A.)maculatum$shifts$depending$on$where$the$A.) maculatum$are$established$(Teel$et$al.,$2010).$The$three$life$stages$minimally$overlap$in$ peaks$of$activity,$however$A.)maculatum$from$the$inland$populations$in$Oklahoma$and$

Kansas$are$active$five$months$earlier$than$the$ticks$from$historic$populations$in$Texas$

(Williams$and$Hair,$1976;$Johnson,$1990;$Teel$et$al.,$1998;$Teel$et$al.,$2010).$In$Texas,$ adults$feed$in$September$while$in$Kansas$and$Oklahoma,$adults$feed$from$April$through$ early$June$(Teel$et$al.,$2010).$The$phenology$of$A.)maculatum)adults$in$Virginia$falls$ between$those$described$for$Texas$and$Oklahoma$(Teel$et$al.,$2010),$with$peak$adult$ collections$in$the$month$of$June.$It$is$most$similar$to$populations$described$from$

Mississippi,$where$adults$were$collected$from$March$through$November$with$peaks$in$July$ and$August$(Goddard$and$Paddock,$2005;$Goddard,$2007).$Virginia$ticks$may$have$a$ shorter$amount$of$time$for$questing$due$to$cooler$temperatures$in$Virginia$compared$to$

Mississippi$(Hancok$et$al.,$2011).$Survival$and$duration$in$each$life$stage$is$dependent$on$ environmental$variables,$including$habitat,$temperature,$and$humidity$(Teel$et$al.,$2010).$A$ period$of$fall$and$winter$quiescence$of$adults$is$standard$in$all$areas$in$the$US$where$this$ tick$species$has$been$collected.$

Virginia$nymphs$were$collected$from$March$through$September$and$a$few$larvae$ were$collected$on$flags$in$April,$but$these$small$numbers$were$insufficient$to$establish$ immature$phenology.$In$Texas,$peak$larval$and$nymphal$feeding$seasons$are$in$January$and$

February,$respectively,$while$in$Kansas$and$Oklahoma,$larvae$peak$in$June$and$nymphs$in$

July$(Teel$et$al.,$2010).$Mississippi$A.)maculatum)nymphs$have$been$collected$from$

February$through$August,$and$larvae$have$been$collected$from$June$though$November$

(Paddock$and$Goddard,$2005;$Portugal$and$Goddard,$2015).$If$the$trends$from$these$ populations$hold$true$for$Virginia$A.)maculatum,$peak$larval$abundance$would$be$expected$ several$months$after$the$adult$peak,$followed$closely$by$nymphs,$probably$in$late$summer$ and$early$fall.$Immature$A.)maculatum$are$notoriously$difficult$to$collect$(Paddock,$2007),$ and$additional$work$with$novel$collection$methods$such$as$swabbing$rodent$burrows$will$ continue$to$increase$our$understanding$of$immature$phenology$in$this$tick$species$

(Portugal$and$Goddard,$2015).$

Predictors)of)A.$maculatum)presence)and)absence)

$ Robust$populations$of$A.)maculatum)were$discovered$at$only$five$of$twelve$sites$ sampled$continuously$over$multiple$years,$indicating$that$high$sampling$frequency$does$ not$guarantee$discovery$of$these$ticks.$Unlike$A.)americanum,)these$ticks$are$not$ universally$distributed$throughout$the$landscape$of$southeastern$Virginia.$A$specific$set$of$ abiotic$and$biotic$factors$may$be$necessary$to$support$a$population;$if$these$factors$are$in$ place,$large$numbers$of$A.)maculatum$may$arise$in$just$a$few$years.$The$life$cycle$of$A.) maculatum$is$generally$completed$in$1Y2$years$(Teel$et$al.,$2010).$Engorged$females$lay$an$ average$of$8,000$and$up$to$15,000$eggs$(Drummond$and$Whetstone,$1970;$Wright,$1971),$ demonstrating$the$ability$of$this$tick$species$to$undergo$rapid$population$expansion$after$ arrival$in$a$suitable$new$habitat.$$

In$southeastern$Virginia$and$throughout$their$range,$A.)maculatum$have$been$found$ sharing$habitats$and$hosts$with$other$ticks.$One$key$predictor$of$where$A.)maculatum$ would$be$found$was$the$presence$of$D.)variabilis,$as$the$habitat$and$host$preferences$of$

77 these$ticks$seem$to$strongly$overlap$(Table$11).$Because$of$these$overlaps,$as$well$as$ morphological$similarities$between$these$two$tick$species$at$all$life$stages$(Paddock$and$

Goddard,$2015),$recognizing$when$A.)maculatum)has$invaded$may$require$careful$ surveillance$by$experienced$acarologists.$$

$ In$addition$to$populations$of$D.)variabilis$at$sites$where$many$A.)maculatum)were$ found,$these$sites$had$other$commonalities,$most$notably$particular$habitat$characteristics.$

All$A.)maculatum)sites$comprised$open$habitat$with$little$shade,$dominated$by$grasses$and$ shrubs$(Table$9).$These$xeric$habitats$are$similar$to$other$areas$where$A.)maculatum$have$ been$found$in$other$regions;$along$the$Gulf$Coast$and$in$the$inland$Midwest,$A.)maculatum$ are$common$in$grassYdominated$habitats$such$as$prairies,$scrublands,$oak$savannahs,$and$ mesquite$(Scifres$et$al.,$1988;$Teel$et$al.,$2010,).$Other$newly$identified$populations$of$A.) maculatum$in$the$MidYAtlantic$have$been$found$in$disturbed$grassy$habitats,)including$a$ large$population$at$a$landfill$in$northern$Virginia$(Fornadel$et$al.,$2011)$and$small$numbers$ of$A.)maculatum)from$disturbed$secondary$growth$habitat$(Florin$et$al.,$2014).$In$general,$

A.)maculatum$habitat$has$been$described$as$southern$coastal$habitat$with$high$rainfall,$ temperature$and$humidity,$with$open,$nonYshaded$habitats$where$A.)maculatum)can$quest$ for$hosts$in$the$heat$of$the$day$(Paddock$and$Goddard,$2015).$$

Most$open$habitats$in$Virginia$are$anthropogenically$influenced$through$prescribed$ burning,$tree$clearing,$or$mowing$to$create$fields$or$other$open$spaces$for$human$use.$Once$ these$open$habitats$are$created,$ecological$succession$is$inevitable$unless$they$are$ anthropogenically$maintained,$because$of$the$continual$fire$suppression$along$the$east$ coast$(for$a$review$of$fire$and$is$effects$on$A.)maculatum,$see$Paddock$and$Goddard,$2015).$

Amblyomma)maculatum$populations$appear$in$secondary$successional$habitats$in$Virginia,$

78 when$there$is$tall$grass$with$shrubs,$but$populations$seem$to$disappear$as$the$canopy$ closes$and$field$becomes$forest.$The$landscape$of$the$east$coast$is$extremely$developed,$so$ patches$of$open$habitat$occur$mainly$by$human$design,$such$as$when$an$agricultural$field$is$ left$fallow,$or$an$area$is$cleared$for$construction.$These$sites$are$often$near$areas$with$large$ human$populations,$including$patches$in$urban$or$suburban$developments.$By$creating$and$ maintaining$these$open$habitats,$especially$in$areas$with$lots$of$human$habitation,$humans$ are$inviting$A.)maculatum$to$establish$and$generating$new$human$health$risks.$

Regulation)of)A.$maculatum)populations)

If$open$areas$are$not$maintained$by$natural$fire$or$anthropogenic$influence,$A.) maculatum)populations$seem$to$be$unable$to$last$more$than$a$few$years$in$southeastern$

Virginia$habitats.$This$ephemeral$nature$may$be$unique$to$A.)maculatum$populations$in$the$ southeast,$as$open$habitats$and$rangelands$persist$along$the$gulf$coast$and$in$the$central$US$ where$A.)maculatum)are$well$established.$As$A.)maculatum)ticks$move$northward$into$the$

MidYAtlantic,$more$ephemeral$populations$can$be$expected,$with$shortYlived$open$habitats$ used$as$stepping$stones$to$continue$the$process$of$expansion$into$otherwise$unsuitable$ habitat.$Anthropogenically$disturbed$habitat$islands$seem$to$be$the$only$habitat$able$to$ facilitate$the$establishment$and$spread$of$A.)maculatum$populations$in$the$MidYAtlantic$ region.$Inland$populations$of$A.)maculatum)have$also$been$described$as$“ephemeral”$ because$of$periodic$expansions$and$contractions$associated$with$patterns$of$drought$and$ rainfall$(Teel$et$al.,$2010).$It$is$possible$that$precipitation$patterns$in$the$central$US$may$ regulate$those$populations$as$anthropogenic$activity$regulates$coastal$populations$which$ receive$more$regular$precipitation.$Alternatively,$peripheral$populations$at$expension$

79 fronts$may$be$inhererently$more$fragile$and$prone$to$periodic$extirpation$than$more$ established$populations$in$the$historic$range$of$A.)maculatum.$

Pathways)for)dispersal)and)spread)

Because$MidYAtlantic$populations$of$A.)maculatum)generally$exist$in$habitat$islands,$ they$are$unlikely$to$be$founded$by$contiguous$populations$of$ticks.$In$order$for$a$ population$to$arise,$ticks$must$be$translocated$from$an$existing$population$to$a$suitable$ habitat$with$an$appropriate$population$of$hosts.$Amblyomma)maculatum$has$been$collected$ from$over$71$species$of$birds$and$mammals,$including$humans,$in$the$United$States$alone$

(Teel$et$al.,$2010).$Rodents$and$birds$are$most$often$host$larvae$and$nymphs,$while$adult$ ticks$generally$parasitize$larger$mammals.$There$are$no$reports$of$A.)maculatum$collected$ from$reptile$hosts.$The$wide$host$range$utilized$by$this$tick,$combined$with$its$ability$to$ withstand$xeric$habitats,$has$likely$contributed$to$the$successful$spread$of$A.)maculatum$ over$the$last$few$decades$(Paddock$and$Goddard,$2015).$

Because$A.)maculatum)feed$on$a$large$variety$of$avian$and$mammalian$hosts$with$ varying$dispersal$capabilities,$there$are$many$possible$pathways$to$arrival$at$a$new$site.$

Amblyomma)maculatum$were$not$documented$parasitizing$any$novel$hosts$in$Virginia$

(Table$12),$but$it$is$possible$to$reconstruct$possible$pathways$to$colonization$in$the$MidY

Atlantic$using$previously$reported$hosts$of$A.)maculatum.$Cattle$have$been$implicated$in$ invasions$of$A.)maculatum)in$the$central$US,$and$previous$studies$have$suggested$that$ migratory$birds$might$be$transporting$immature$A.)maculatum,$as$incidental$collections$of$ this$tick$have$historically$been$along$flyways$(Teel$et$al.,$2010).$The$expansion$of$feral$ swine$across$the$southeastern$and$central$US,$and$the$growing$population$of$whiteYtailed$

80 deer$have$also$been$suggested$as$facilitators$of$the$A.)maculatum$expansion$(Paddock$and$

Goddard,$2015).$There$is$no$significant$cattle$industry$in$southeastern$Virginia,$so$ discussion$of$possible$invasion$routes$will$focus$on$wild$hosts$that$are$present$in$the$MidY

Atlantic.$Available$hosts$have$been$divided$into$four$categories$(C1YC4)$based$on$dispersal$ ability,$for$the$purposes$of$this$discussion$(Table$13).

Small$bodied,$nonYmigratory$hosts$with$small$home$ranges$have$limited$dispersal$ ability$for$immature$A.)maculatum)ticks.$This$category$(C1)$includes$rodents$and$small$nonY migratory$birds$with$home$ranges$generally$<1$ha,$that$generally$disperse$<2$km$from$ their$natal$area.$Rodent$hosts$can$become$extremely$abundant$in$suitable$habitat,$with$ some$species$reaching$over$100$animals$per$ha$in$prime$ecological$conditions$(Table$13).$

Although$C1$hosts$probably$do$not$play$much$of$a$role$in$moving$ticks$from$site$to$site,$it$is$ likely$that$high$rodent$densities$are$important$at$sites$where$A.)maculatum)are$dropped$ from$more$wideYranging$hosts.$Large$rodent$populations$may$be$necessary$for$a$ population$of$A.)maculatum$to$establish$in$a$new$area,$because$they$provide$a$critical$food$ source$for$immature$ticks.$If$the$phenology$curves$are$as$expected,$immature$A.)maculatum$ should$be$questing$in$the$late$summer$and$early$fall,$just$as$rodent$populations$reach$ seasonal$peaks$after$reproducing$during$the$spring$and$summer.$Timing$the$arrival$of$A.) maculatum)to$years$in$the$successional$process$when$field$rodents$have$had$time$to$ establish$robust$populations$but$before$the$canopy$closes$may$be$critical$to$enable$ population$establishment.$

Table& 13.! Dispersal! potential! of! hosts! of! Amblyomma( maculatum! in! southeastern! Virginia.! Ecological! information! on! the! known!hosts!of!Amblyomma(maculatum,!specifically!in!southeastern!Virginia!if!data!was!available.!NA!indicates!not!applicable,! unknown! indicates! insufficient! data! in! the! literature.! This! table! is! color! coded! and! organized! by! dispersal! potential,! as! determined!by!animal!home!range!size!and!dispersal!patterns.! ! Migratory&in& Reside Home&range& Main&life&stage&that& %&of&animals&that& Common&name& Species& VA?& nt& (ha)& disperses& disperse& Category!1!(C1)!hosts,!with!home!ranges!<1ha,!dispersal!distances!generally!<2!ha.!Small!home!ranges,!nonHmigratory,!smallHbodied,!limited!dispersal! potential!of!immatures! Blue!Jay! Cyanocitta(cristata( No! Yes! 0.03! Immatures! 71%! Eastern!woodrat! Neotoma(floridana( No! Yes! 0.2! Breeding!males! Unknown! Field!Sparrow! Spizella(pusilla( No! Yes! 0.76! Immatures! All! Brown!Thrasher! Toxostoma(rufum( No! Yes! 1! None! Unknown! Thyrothorus( Carolina!Wren! ludovicianus( No! Yes! 0.7! None! 0! Eastern!Harvest! Reithrodontomys( mouse! humulis( No! Yes! 0.095! Unknown! Unknown! WhiteHfooted!mouse! Peromyscus(leucopus( No! Yes! 0.1! Immatures! Unknown! Hispid!cotton!rat! Sigmodon(hispidus( No! Yes! 0.35! Unknown! Unknown! Norway!rat! Rattus(norvegicus( No! Yes! 1! Adult!males! Almost!none! Rice!rat! Oryzomys(palustris( No! Yes! 0.3! Unknown! Unknown! Roof!rat! Rattus(rattus( No! Yes! 0.5! Unknown! Unknown! ! ! ! ! ! 81

Table&13,&continued:&C1&hosts.& ! Common& Dispersal& name& Dispersal&distance& timing& Habitat&preferences& Average&host&density& Sources& Category!1!(C1)!hosts,!with!home!ranges!<1ha,!dispersal!distances!generally!<2!ha.!Small!home!ranges,!nonHmigratory,!smallHbodied,!limited!dispersal! potential!of!immatures! Smith!et!al.,!2013;!Pardieck!et! Blue!Jay! 2!km! Any!time! Mixed!woodlands! 0.275!birds/km! al.,!2015! Eastern! 300!m!max! Unknown! Woods,!swamps,!oldfields! 0.51!per!ha! Wiley,!1980! woodrat! Field! Best,!1977;!Carey!et!al.,!2008;! Unknown! Summer! Brushy!fields! 0.245!birds/km! Sparrow! Pardieck!et!al.,!2015! Brown! Cavitt!and!Haas,!2014;!Pardieck! None! Summer! Generalist! 0.1!birds/km! Thrasher! et!al.,!2015! Carolina! Haggerty!and!Morton,!2014;! None! None! Generalist! 0.33!birds/km! Wren! Pardieck!et!al.,!2015! Eastern! 85.5!m!(maximum! 8.75H44.4/!ha!(Total! Harvest! distance!between! Unknown! Oldfields! captured!at!CHS!in!2010:! Stalling,!1997! mouse! captures)! 57/ha)! WhiteH Krohne!et!al.,!1984;!Lackey!et! footed! 330!m! Summer! Generalist! 10/ha! al.,!1985! mouse! 8H100!per!ha,!peaks!in! Hispid! 13!m!(average!daily! Cameron!and!Kincaid,!1982;! Unknown! Grassy!habitats! autumn!(Total!captured!at! cotton!rat! movement)! Cameron!and!Spencer,!1981! CHS!in!2010:!104/ha)! Davis!and!Emlen,!1948;! Autumn! Norway!rat! NA! Urban!areas,!generalist! 50/barn! McGuire!et!al.,!2006;!Recht,! and!winter! 1988! 1H109!indiviuals!per!ha;! Rice!rat! 0.08!km! Unknown! Woods,!swamps,!oldfields! (Total!captured!at!ST!in! Wolfe,!1982! 2010:!25/ha)! Whisson!et!al.,!2007;!Recht,! Roof!rat! 0.5!km! Unknown! Urban!areas,!generalist! Unknown! 1988! ! & & 82

Table&13,&continued:&C2&hosts.& ! Migratory&in& Resid %&Animals&that& Common&name& Species& VA?& ent& Home&range&(ha)& Main&life&stage&that&disperses& disperse& Category!2!(C2)!hosts,!with!home!ranges!<50!ha,!and!which!may!disperse!distances!of!<200!km.!Small!to!medium!home!ranges,!small!bodied,!moderate! dispersal!potential!of!immatures.! Eastern! Yes! Meadowlark! Sturnella(magna( No! 3! Immatures! 50%! Pipilo( Eastern!Towhee! erythrophthalmus( No! Yes! 1.6! Unknown! Unknown! RedHwinged! Agelaius( Blackbird! phoeniceus( Sometimes! Yes! 0.2! All! 50%! 25!ha!(summer)!110!ha! Eastern!Bluebird! Sialia(sialis( Sometimes! Yes! (winter)! Immatures! 90%! Cardinalis( Northern!Cardinal! cardinalis( No! Yes! 10! Immatures! 10%! Dumetella( Sometimes! Yes( Gray!Catbird! carolinensis( 0.3! Immatures! 95%! BrownHheaded! Sometimes! Cowbird! Molothrus(ater( Yes! 4H400!ha!(variable)! All! Unknown! Yes,!shortH Yes! Common!Grackle! Quiscalus(quisula( distance! Unknown! Adults!migrate,!immatures!natal! All! Turdus( American!Robin! migratorius( No! Yes! 0.03! Immatures! Unknown! Charadrius( Killdeer! vociferus( Sometimes! Yes! 0.68! No!natal!dispersal,!adults!migrate! Unknown! Northern! Colinus( Immatures,!adults!short!distances! Bobwhite! virginianus( No! Yes! 44! seasonally! All! RedHbellied! Melanerpes( Woodpecker! carolinus( No! Yes! 8.8! Immatures! All! Sylvilagus( Cottontail!Rabbit! floridanus( No! Yes! 1! Unknown! Unknown! Sylvilagus( Marsh!rabbit! palustris( No! Yes! 0.025! Unknown! Unknown! Sciurus( Gray!squirrel! carolinensis( No! Yes! 2.75! Immatures! Unknown! Southern!flying! squirrel! Glaucomys(volans( No! Yes! 1.5! Unknown! Unknown! & 83

Table&13,&continued:&C2&hosts.& ! Common& Dispersal& Habitat& name& Dispersal&distance& timing& preferences& Average&host&density& Sources& Category!2!(C2)!hosts,!with!home!ranges!<50!ha,!and!which!may!disperse!distances!of!<200!km.!Small!to!medium!home!ranges,!small!bodied,!moderate! dispersal!potential!of!immatures.! Eastern! Jaster!et!al.,!2012;!Kershner!et! Meadowlark! <5!km! Summer! Open!habitat! 0.388!birds/km! al.,!2004;!Pardieck,!2015! Eastern! Greenlaw,!2015;!Pardieck!et!al.,! Towhee! Unknown! Unknown! Generalist! 0.325!birds/km! 2015! RedHwinged! 32.1!km!between!nesting!sites,! Fields!and! Yasukawa!and!Searcy,!1995;! Blackbird! 600!km!while!migrating! Any!time! wetlands! 0.462!birds/km! Pardieck!et!al.,!2015! Eastern! 1!km,!unless!migrating! Gowaty!et!al.,!2015;!Pardieck!et! Bluebird! (distance!variable)! Summer! Open!habitat! 0.173!birds/km! al.,!2015! Northern! Halkin!and!Linville,!1999;! Cardinal! 65!km! Summer! Generalist! 1!bird/km! Pardieck!et!al.,!2015!! Summer,! Smith!et!al.,!2011;!Pardieck!et! Gray!Catbird! 0.358!km! annual! Generalist! 0.133!birds/km! al.,!2015! BrownH headed! Summer,! Lowther,!1993;!Pardieck!et!al.,! Cowbird! 300!km! annual! Open!habitat! 0.14!birds.km! 2015! Common! Fall/spring! Open!and! Peer!and!Bollinger,!1997;! Grackle! 20!km!natal,!500!km!migration! for!migration! edge!habitats! 2.02!birds/ha! Pardieck!et!al.,!2015! American! Vanderhoff!et!al.,!2014;! Robin! 40!km! Summer! Generalist! 0.55!birds/km! Pardieck!et!al.,!2015! Fall/spring! Jackson!and!Jackson,!2000;! Killdeer! Unknown! for!migration! Open!habitat! 0.04!birds/km! Pardieck!et!al.,!2015! Northern! Brennan!et!al.,!2014;!Pardieck! Bobwhite! 50!km! Summer! Open!habitat! 0.47!birds/km! et!al.,!2015! RedHbellied! Shackleford!et!al.,!2000;! Woodpecker! Unknown! Summer! Forests! 0.15!birds/km! Pardieck!et!al.,!2015! Cottontail! Chapman!et!al.,!1980;! Rabbit! 3!km! Unknown! Generalist! 8.9!per!ha!(fall,!Wisconsin)! Sutherland!et!al.,!2000! Marshy! Blair,!1936;!Chapman!and! Marsh!rabbit! Unknown! Unknown! wetlands! Unknown! Willner,!1981;,!Payne,!1975! Woods,! 3/ha!in!woods,!up!to!21!per!ha! Gray!squirrel! 100!km!(maximum)! Autumn! generalist! in!urban!parks! Koprowski,!1994! Southern! 35/ha!peaks!in!se!VA!(Rose),! Bendel!and!Gates,!1987;!Dolan! flying!squirrel! 3!km!(homing)! Unknown! Mixed!forests! probably!closer!to!10/ha! and!Carter,!1977! 84

Table&13,&continued:&C3&hosts. & ! Common& Migratory& Home&range& Main&life&stage&that& name& Species& in&VA?& Resident& (ha)& disperses& %&Animals&that&disperse& Category&3&(C3)&hosts,&with&home&ranges&50L6500&ha,&and&which&disperse&distances&of&1L200&km.&Medium&to&large&home&ranges,&medium&and& largeLbodied,&possible&long&distance&dispersers&of&adults.! Raccoon! Procyon(lotor( No! Yes! 49! Immatures! Males! Skunk! Mephitis(mephitis( No! Yes! 350! Yearlings! Unknown! Urocyon( Grey!fox! cinereoargenteus( No! Yes! 215! Immatures! Males! WhiteHtailed! Odocoileus( 80%!yearling!males,!13%!yearling! deer! virginianus( No! Yes! 290! Yearlings! females! Feral!swine! Sus(scrofa( No! Yes! 300! NA! NA! Coyote! Canis(latrans( No! Yes! 2500! Immatures! Most! Black!bear! Ursus(americanus( No! Yes! 6500! Yearlings! All!males,!5%!females! Bobcat! Lynx(rufus( No! Yes! 4500! Yearlings! All! ! ! Common& Dispersal& name& Dispersal&distance& timing& Habitat&preferences& Average&host&density& Sources& Category!3!(C3)!hosts,!with!home!ranges!50H6500!ha,!and!which!disperse!distances!of!1H200!km.!Medium!to!large!home!ranges,!medium!and!largeH bodied,!possible!long!distance!dispersers!of!adults.! 3.2!to!266!km,!generally! 0.1!per!ha!(up!to!4/ha,!usually! Raccoon! less!than!1.6!km! Fall! Generalist! between!0.2!and!0.02)! Lotze!and!Anderson,!1979! WadeHSmith!and!Verts,!1982;! Skunk! 22!km!max! Summer! Forests!and!fields! 3!per!km2! Sutherland!et!al.,!2000! Forests!and!fields,! Fritzell!and!Haroldson,!1982;! Grey!fox! 84!km! Fall! especially!deciduous!forest! 1.5!per!km2! Sutherland!et!al.,!2000! WhiteH Smith,!1991;!Sutherland!et!al.,! tailed!deer! 15!km! Summer! Generalist! 25!per!km2! 2000! Feral! swine! 22!km! NA! Generalist! Variable! Wood!and!Barrett,!1979! Fall!and! Bekoff,!1977;!Sutherland!et!al.,! Coyote! 5!km!up!to!160!km! winter! Generalist! 0.3!per!km2! 2000! 127!km!max!average! Sutherland!et!al.,!2000;! Black!bear! (varies!from!13H219!km)! Summer! Generalist! 1/5.15!km!2! Larivière,!2001! Bobcat! 119!km!max!average! Summer! Generalist! 1!bobcat/11!km2!(Oklahoma)! Larivière!and!Walton,!1997! 85 85

& Table&13,&continued:&C4&hosts.& & Migratory& Home& Main&life&stage&that& %&Animals&that& Common&name& Species& in&VA?& Resident& range&(ha)& disperses& disperse& Dispersal&distance& Category!4!(C4)!hosts,!neotropical!migrants!which!disperse!distances!of!>200!km.!Migratory,!possible!long!distance!dispersers!of!immatures.! Grasshopper! Ammodramus( Yes,! 0.8! Sparrow! savannarum( Yes! summer! NA! All! NA! Lincoln's! Melospiza( No! NA! NA! NA! Sparrow! lincolnii( Yes! All! Melospiza( Yes,! 0.17! NA! NA! Swamp!Sparrow! georgiana( Yes! winter! All! Hylocichla( Yes,! 646H4600!km!during! Wood!thrush! mustelina( Yes! summer! 2.4! All! All! migration! Setophaga( Yes,! Palm!Warbler! palmarum( Yes! winter! Unknown! NA! All! NA! Troglodytes( Yes,! House!Wren! aedon( Yes! summer! 0.5! NA! All! NA! Cistothorus( Yes,! Sedge!Wren! platensis( Yes! winter! Unknown! NA! All! NA! Common! Geothylpis( Yes,! Yellowthroat! trichas( Yes! summer! 0.5! NA! All! NA! & & Common&name& Dispersal&timing& Habitat&preferences& Average&host&density& Sources& Category!4!(C4)!hosts,!neotropical!migrants!which!disperse!distances!of!>200!km.!Migratory,!possible!long!distance!dispersers!of!immatures.! Grasshopper!Sparrow! Migration!spring/fall! Open!habitat! 0.074!birds/km! Vickery,!1996;!Pardieck!et!al.,!2015! Lincoln's!Sparrow! Migration!spring/fall! !Generalist!during!migration! NA! Ammon,!1995! Swamp!Sparrow! Migration!spring/fall! Wetlands! Unknown! Mowbray,!1997! Wood!thrush! Migration!spring/fall! Woods! 0.379!birds/km! Evans!et!al.,!2011;!Pardieck!et!al.,!2015! Palm!Warbler! Migration!spring/fall! Riparian!forest! Unknown! Wilson,!2013! House!Wren! Migration!spring/fall! Generalist! 0.05!birds/km! Johnson,!2014;!Pardieck!et!al.,!2015! Sedge!Wren! Migration!spring/fall! Open!wetlands! Unknown,!low! Herkert!et!al.,!2001! Guzy!and!Ritchison,!1999;! Common!Yellowthroat! Migration!spring/fall! Riparian!forest,!fields! 0.16!birds/km! Pardieck!et!al.,!2015! & 86

86 87

Small%bodied+animals+with+home+ranges+<50+ha+that+disperse+up+to+200+km+have+ moderate+dispersal+potential+for+immature+A.#maculatum.+This+category+(C2+hosts)+ comprises+mammals+including+lagomorphs+and+sciurids+that+may+disperse+from+their+place+ of+birth,+or+birds+that+are+partial+migrants+in+Virginia.+Partial+migrants+do+not+breed+in+

Canada+or+winter+in+the+neotropics,+but+will+sometimes+move+a+few+hundred+km+seasonally.+

While+this+group+has+the+ability+to+disperse+over+long+distances,+they+often+do+not+move+ more+than+a+few+km+from+their+natal+area.+Populations+of+C2+hosts+are+generally+lower+than+ those+of+C1+hosts,+but+the+capacity+of+C2+hosts+to+transport+immatures+over+longer+distances+ is+greater.+Most+movement+of+animals+in+this+group+occurs+during+the+late+summer+and+fall,+ when+immature+A.#maculatum#may+be+questing.+

Medium+and+large+mammals+with+home+ranges+over+50+ha+that+disperse+up+to+and+ beyond+200+km+(C3+hosts)+are+likely+to+be+the+most+important+long+distance+dispersers+of+ adult+ticks.+Adult+A.#maculatum#quest+in+the+summer+months,+which+corresponds+to+the+ timing+when+first+year+offspring+of+these+large+mammals+are+dispersing+from+their+place+of+ birth.+The+natal+dispersal+timing+of+juvenile+striped+skunks+(Mephitis#mephitis),+white%tailed+ deer,+black+bears+(Ursus#americanus),+and+bobcats+(Lynx#rufus)+aligns+particularly+well+with+

A.#maculatum#phenology.+Adult+A.#maculatum#were+collected+into+August+and+September,+so+ it+is+possible+that+dispersing+juvenile+coyotes+(Canis#latrans),+grey+foxes+(Urocyon# cinereoargenteus),+and+raccoons+(Procyon#lotor)+which+generally+move+in+fall+would+also+ have+an+opportunity+to+move+these+ticks+from+habitat+patch+to+habitat+patch.+Male+A.# maculatum+attach+to+hosts+first+and+produce+a+pheromone+that+aggregates+females+to+mate,+ increasing+the+tick+load+on+these+large%bodied+hosts+and+the+likelihood+that+hosts+will+ distribute+multiple+ticks+across+a+landscape+(Gladney,+1971;+Gladney+et+al.,+1974).+Because+

A.#maculatum#mate+on%host,+engorged+females+can+lay+eggs+directly+after+detaching+in+a+ suitable+habitat.+Because+these+mammals+can+host+many+adult+ticks+and+move+them+over+ long+distances+in+short+amounts+of+time,+C4+hosts+translocate+the+ticks+that+are+most+able+to+ found+new+populations.+If+engorged+females+drop+off+in+areas+of+suitable+habitat+with+high+ densities+of+C1+and+C2+hosts,+their+larvae+stand+a+good+chance+of+survival.+

Of+course,+adult+ticks+are+not+the+only+life+stage+that+can+be+moved+long+distances.+

The+final+host+category+(C4+hosts)+comprises+migratory+birds+that+are+known+hosts+for+ immature+A.#maculatum,+and+have+been+documented+moving+engorged+larval+and+nymphal+ ticks+thousands+of+kilometers+during+spring+and+fall+migrations+from+the+ancestral+range+of+

A.#maculatum+in+the+neotropics+north+to+Canada+(Ogden+et+al.,+2008;+Teel+et+al.,+2010;+Scott+ et+al.,+2012;+Florin+et+al.,+2014).+These+birds+may+transport+A.#maculatum#immatures+ southward+during+the+fall,+and+northward+during+the+spring.+Immature+A.#maculatum#were+ collected+during+the+spring,+summer,+and+fall+in+Virginia.+These+ticks+may+move+northward+ during+the+spring,+continuing+the+invasion+into+the+mid%Atlantic,+or+southward+in+the+fall+ back+to+sites+in+areas+where+A.#maculatum#is+already+established.+Once+engorged+immatures+ drop+off+birds+in+an+appropriate+habitat,+they+must+survive+to+molt,+mate,+and+reproduce+in+ order+to+seed+a+new+population.+Because+of+challenges+associated+with+survival+in+the+ environment,+including+predation+and+desiccation+(Needham+and+Teel,+1991),+models+of+ tick+dispersal+have+shown+faster+population+establishment+with+mammalian+dispersers+

(Leighton+et+al.,+2012).+

Genetic+evidence+indicates+that+long+distance+dispersal+must+be+taking+place+in+order+ to+facilitate+gene+flow+between+remote+populations,+and+that+geographically+proximate+ populations+of+A.#maculatum#do+not+have+higher+levels+of+gene+flow+than+distant+ones+

(Nadolny+et+al.,+2015).+This+recent+study+suggests+that+a+combination+of+mammalian+and+ avian+hosts+are+likely+responsible+for+dispersal,+but+that+dispersal+of+immatures+by+ migratory+birds+is+necessary+to+facilitate+the+observed+gene+flow+between+far%flung+ populations.+Combining+genetic+evidence+of+gene+flow+between+populations+with+our+ understanding+of+preferred+hosts+that+are+abundant+in+Virginia+(Table+13),+the+most+likely+ pathway+for+a+new+population+of+these+ticks+to+arise+in+the+Mid%Atlantic+seems+to+be+ through+the+introduction+of+multiple+genotypes+to+an+anthropogenically+disturbed+isolated+ habitat+patch+by+migratory+birds+and+long%distance+dispersing+mammals+(C3+and+C4+hosts).+

The+habitat+patch+must+be+at+an+appropriate+stage+in+ecological+succession+to+be+dominated+ by+large+populations+of+rodent+hosts+in+order+to+provide+sufficient+blood+meals+for+ immature+ticks.+Using+abundant+local+hosts+(C1+and+C2+hosts),+a+population+can+grow+to+ large+numbers+in+a+short+period+of+time.+Once+established,+the+A.#maculatum#population+will+ persist+only+until+the+canopy+closes+and+the+community+of+C1+hosts+declines+(Langley+and+

Shure,+1980).+Ticks+may+then+disperse+to+other+patches,+either+geographically+proximate+

(facilitated+by+C2+and+C3+hosts)+or+distant+(facilitated+by+C3+and+C4+hosts).+

+ Adult+A.#maculatum+were+found+feeding+on+domestic+dogs+and+cats+in+Virginia,+and+ anthropogenic+movement+of+these+ticks+may+be+an+important+factor+in+this+ticks+range+ expansion.+Amblyomma#maculatum+adults+have+been+removed+from+dogs+and+cats+with+no+ travel+history+in+Ontario,+Canada,+where+these+ticks+are+not+established+(Scott+et+al.,+2001).+

A+feeding+preference+for+domesticated+animals+may+enable+A.#maculatum+to+invade+human% dominated+areas+faster+than+otherwise+predicted.++

In+conclusion,+tick+ranges+are+not+fixed+in+time+or+space+and+may+change+rapidly;+the+ geographic+range+of+A.#maculatum#is+much+more+extensive+today+than+it+was+70+years+ago+

(Teel+et+al.,+2010;+Paddock+and+Goddard,+2015).+This+tick+species+is+one+of+the+top+four+tick+ species+parasitizing+humans+in+the+southeastern+US,+and+human+infections+with+R.#parkeri+ seem+to+arise+concurrently+with+the+discovery+of+newly+established+A.#maculatum# populations+(Paddock+and+Goddard,+2015).+Because+of+the+morphological+similarities+ between+A.#maculatum+and+D.#varabilis#at+all+life+stages,+it+may+be+difficult+to+tell+when+A.# maculatum#have+arrived,+so+vigilance+is+required.+Enhanced+surveillance+is+recommended+ in+anthropogenically+disturbed+open+habitats+during+the+summer+months+in+Mid%Atlantic+ states+to+detect+new+populations+of+this+invader.+Once+detected,+medical+and+veterinary+ personnel+should+be+alerted+to+the+increased+threat+of+pathogens+vectored+by+this+tick+ species.+

+ +

ANTHROPOGENIC,DISTURBANCE,AND,SUBSEQUENT,RESTORATION,EFFORTS,

FACILITATE,BIOLOGICAL,INVASIONS,AND,VECTOR5BORNE,DISEASE,

INTRODUCTION,

The+influence+of+ecosystem+disturbance+on+human+health+is+well+documented,+ particularly+with+respect+to+an+increase+in+emerging+vector%borne+and+zoonotic+diseases+

(Patz+et+al.,+2005;+Bradley+and+Altizer,+2007;+Wood+and+Lafferty,+2013;+Keesing+and+Ostfeld,+

2015;+Speldewinde+et+al.,+2015).+Changes+in+land+use+have+resulted+in+an+increased+ abundance+and+diversity+of+arthropod+disease+vectors,+including+mosquitoes+(Norris,+2004)+ and+ticks+(Childs+and+Paddock,+2003;+Brownstein+et+al.,+2005).+Lyme+disease+is+the+best% known+tick%borne+disease+associated+with+landscape+changes,+as+forest+fragmentation+and+ loss+of+biodiversity+have+been+linked+to+increased+human+disease+risk+(Ostfeld+and+Keesing,+

2000;+Allan+et+al.,+2003a;+LoGiudice+et+al.,+2003;+Wood+and+Lafferty,+2013).+Other+tick%borne+ diseases+are+also+intimately+tied+to+their+environments,+such+as+the+increased+abundance+of+ the+lone+star+tick+(Amblyomma#americanum)+and+its+associated+diseases+(including+human+ monocytic+ehrlichiosis+and+human+granulocytic+ehrliciosis)+following+the+suburbanization+ of+the+eastern+United+States+and+the+subsequent+explosion+of+white%tailed+deer+(Odocoileus# virginianus)+populations+(Childs+and+Paddock,+2003).++

Because+the+results+of+anthropogenic+disturbance+can+be+costly+in+terms+of+human+ health+risk+and+in+many+other+ways+(Speldewinde+et+al.,+2015),+ecological+restoration+ projects+are+often+undertaken+to+reestablish+ecosystem+services+that+were+lost.+In+studies+of+

Ixodes#ticks+and+Lyme+disease,+the+removal+of+weedy+species+and+restoration+of+habitat+ decreased+Lyme+disease+risk+(Morlando+et+al.,+2012;+Gilbert,+2013).+Similarly,+the+removal+

92 of+invasive+plant+species+has+resulted+in+a+reduction+in+lone+star+ticks+infected+with+agents+ of+human+ehrlichiosis,+effectively+reducing+disease+risk+(Allan+et+al.,+2010).+While+ restoration+can+increase+biodiversity+of+native+flora+and+fauna,+restored+ecosystems+often+ provide+less+effective+ecosystem+services+than+intact+systems+(Benayas+et+al.,+2009).+

Specifically,+ecosystem+restoration+and+the+corresponding+community+re%assembly+do+not+ necessarily+mitigate+disease+risk,+and+may+even+exacerbate+it+(Speldewinde+et+al.,+2015).+In+ some+cases,+reforestation+may+result+in+an+increase+in+Lyme+disease+risk+(Barbour+and+Fish,+

1993).+Given+the+ongoing+rise+of+tick%borne+disease+incidence+and+the+increasingly+ disturbed+habitat+matrix+in+which+most+humans+live,+it+is+paramount+to+examine+the+effects+ of+habitat+restoration+across+a+spectrum+of+potential+disease+vector+species.++

The+act+of+restoration+can+permanently+change+ecological+communities+by+ temporarily+creating+habitats+that+provide+opportunities+for+invasive+species+presenting+ risks+to+human+health+to+establish+(D’Antonio+and+Meyerson,+2002).+Little+is+known+about+ the+potential+vector%borne+disease+risks+associated+with+these+interim+habitat+states+ created+during+successional+processes.+Most+studies+focus+on+health+risks+after+the+ environment+has+achieved+a+steady+state+and+restoration+activities+are+complete.+There+is+ evidence+that+some+vector+species+can+bounce+back+quickly+after+ecosystem+disturbance;+

Gulf+Coast+tick+Amblyomma#maculatum#densities+were+unchanged+only+a+year+after+a+burn,+ indicating+that+this+species+may+be+adapted+to+take+advantage+of+disturbed+habitats+(Scifres+ et+al.,+1988).+When+an+environment+is+under+heavy+human+use,+such+as+agriculture+or+urban+ development,+managers+often+take+steps+to+minimize+unwanted+species,+including+mowing+ and+the+application+of+pesticides.+Once+restoration+is+complete,+some+of+the+integrity+of+the+ biological+community+is+restored,+along+with+intrinsic+processes+that+can+limit+the+

93 establishment+of+invasive+species+(Benayas+et+al.,+2009).+Disturbed+habitats+left+unmanaged+ may+provide+unique+opportunities+for+invading+vectors.+

+Amblyomma#maculatum#is+one+such+invader+that+has+relevance+to+human+health.+

The+historic+range+of+A.#maculatum+is+throughout+South+and+Central+America+and+across+the+

Gulf+Coast+region+of+the+US,+where+it+is+found+in+xeric+grassland+habitats+(Teel+et+al.,+2010;+

Paddock+and+Goddard,+2015).+This+tick+species+has+only+recently+established+populations+in+

Virginia+and+Delaware,+where+all+populations+have+been+found+in+disturbed+grass% dominated+habitats+(Fornadel+et+al.,+2011;+Wright+et+al.,+2011;+Florin+et+al.,+2014).+Most+ open+habitats+in+the+US+southeast+are+anthropogenically+influenced+through+tree+clearing+or+ mowing+to+create+fields+or+other+open+spaces+for+human+use.+Once+these+open+habitats+are+ created,+successional+processes+often+follow+because+of+seed+banks+or+colonizing+species;+ open+habitats+in+the+eastern+part+of+the+US+typically+only+persist+as+long+as+disturbance+ regimes+are+maintained.++

Amblyomma#maculatum+is+a+large,+aggressive,+human%biting+tick+that+that+can+infect+ hosts+with+Rickettsia#parkeri,+the+bacterial+agent+of+Tidewater+spotted+fever+(Paddock+et+al.,+

2004).+Human+infections+with+R.#parkeri+are+reported+throughout+the+range+of+A.# maculatum+(Sumner+et+al.,+2007;+Jiang+et+al.,+2012;+Paddock+and+Goddard,+2015).+However,+ newly+established+populations+of+these+ticks,+especially+in+Mid%Atlantic+states,+have+tended+ to+exhibit+higher+infection+prevalences+than+in+the+core+of+the+range+(Fornadel+et+al.,+2011;+

Wright+et+al.,+2011).+Recent+evidence+suggests+that+spillover+of+R.#parkeri#to+other+common+ tick+species,+including+the+lone+star+tick+(A.#americanum)+and+the+American+dog+tick+

(Dermacentor#variabilis),+may+occur+in+areas+invaded+by+A.#maculatum#(Gaines+et+al.,+2014;+

Henning+et+al.,+2014,+Wright+et+al.,+2015).+Amblyomma#americanum#is+the+most+abundant+

94 human%biting+tick+in+much+of+the+southeastern+and+Mid%Atlantic+US+(Stromdahl+and+

Hickling,+2012),+and+its+ability+to+transmit+R.#parkeri#after+acquiring+the+pathogen+from+ short%lived+populations+of+A.#maculatum#could+represent+a+significant+public+health+risk+

(Wright+et+al.,+2015).+As+A.#maculatum+continues+to+expand+its+range,+understanding+the+ ecological+mechanisms+by+which+new+populations+arise+and+persist+is+critical+to+protecting+ human+and+animal+health.+

To+determine+the+influence+of+successional+processes+on+A.#maculatum#population+ establishment+and+persistence,+tick+sampling+was+conducted+in+two+successional+ communities.+Sampling+locations+representative+of+both+anthropogenic+and+natural+ disturbance+were+chosen.+Long%term+sampling+was+conducted+on+the+Virginia+mainland+in+ an+early+successional+old+field+community,+and+spatial+sampling+was+conducted+on+a+

Virginia+barrier+island+in+a+naturally+disturbed+community.+Here,+the+results+of+these+ sampling+efforts+are+described,+and+the+importance+of+disturbance+regimes+on+A.# maculatum#population+dynamics+and+the+resulting+implications+for+human+health+are+ discussed.+++

MATERIALS,AND,METHODS,

+ To+understand+the+relationship+between+disturbance+regimes+and+A.#maculatum# populations,+both+temporal+and+spatial+sampling+methods+were+used.+Questing+adult+ticks+ were+collected+using+1+m2+white+denim+flags+attached+to+wooden+dowel+rods+swept+over+the+ ground+and+through+low+vegetation.+All+ticks+found+clinging+to+the+flag+material+were+ removed+from+the+flag+with+forceps+and+placed+in+containers+labeled+with+the+time+and+date+ of+collection,+as+well+as+the+sampling+location,+temperature,+and+weather+data.+Ticks+were+

95 frozen+at+%20+C+until+morphologically+identified+and+then+stored+frozen+at+%80+C+(Keirans+ and+Durden,+1998).+

Temporal#sampling#on#the#Virginia#mainland#

Site+description+

Questing#ticks#were+collected+from+a+restoration+site+owned+by+The+Nature+

Conservancy+(TNC)+adjacent+to+the+Great+Dismal+Swamp+in+Chesapeake,+Virginia,+ previously+referred+to+as+site+CHS+(Fig.+9).+This+site+was+an+agricultural+old+field+last+under+ cultivation+in+2002+and+left+fallow+to+naturally+succeed+back+to+forested+swamp+habitat.+The+ vegetation+on+site+was+representative+of+a+moist,+early%successional+old+field+that+changed+ substantially+over+the+course+of+sampling+(2005%2015).+In+general,+the+site+consisted+of+a+ dense+herbaceous+layer+with+a+mid%story+of+shrubs+and+small+trees+with+little+to+no+canopy+ layer+of+large+trees+until+canopy+closure+occurred+in+2015.+The+tree+layer+was+eventually+ dominated+by+loblolly+pine#(Pinus#taeda),+sycamore+(Platanus#occidentalis),+sweetgum#

(Liquidambar#styraciflua),+red+maple#(Acer#rubrum)+and+several+oak+species+(Quercus+spp.),+ many+of+which+were+planted+as+saplings+by+TNC+in+2003.+The+shrub+layer+was+dominated+by+ various+species+of+blackberries+(Rubus#spp.)+as+well+as+wax+myrtle+(Morella#cerifera)+and+ seedlings+and+saplings+of+the+tree+species.+A+dense+herbaceous+layer+dominated+the+site+and+ was+made+up+of+a+diverse+mixture+of+typical+wetland+grasses,+sedges+and+herbs+including:+

Juncus,#Carex,#Solidago,#Scirpus,#Schoenoplectus,#Panicum,#Eutrochium,#Asclepias+and+Typha# latifolia.++Most+grasses+were+shaded+out+as+the+canopy+closed+in+2015.+

Tick+sampling+

Tick+sampling+took+place+from+2010+to+2015,+while+the+field+progressed+from+early+ successional+habitat+to+closed+canopy+riparian+forest.+Sampling+was+conducted+weekly+ during+the+summer+months+(May+through+August)+and+at+least+monthly+throughout+each+ year+of+collection.+In+order+to+determine+the+area+sampled,+GPS+coordinates+were+taken+ along+the+sampling+transect.+A+1500+m+transect+was+marked+with+surveyors+flags+in+2010+ and+consistently+walked+during+each+sampling+event+throughout+the+years.+ArcGIS+was+used+ to+demarcate+a+2+m+buffer+along+the+transect+for+a+total+of+3000+m2+sampled+during+each+ visit.++

Mammal+trapping+

The+small+mammal+community+was+sampled+at+this+site+from+2005+to+2012.+At+the+ time+of+small+mammal+sampling,+this+site+represented+an+early+successional+habitat+and+ was+dominated+by+grasses+and+forbs+with+mixed+coniferous+and+deciduous+trees+between+5+ and+15+years+old.+A+1+ha+8+x+8+grid+of+modified+Fitch+live+traps+(Rose,+1994)+with+two+traps+ at+each+grid+point+was+established+in+2005;+each+trapping+event+represented+128+traps+ checked.+The+flagging+transect+was+established+along+one+side+of+the+grid,+although+it+ stretched+past+the+trapping+grid+further+into+the+site.+Traps+were+baited+with+birdseed+and+ sunflower+seeds,+were+set+in+the+evening+and+were+checked+for+an+average+of+three+ consecutive+mornings.+During+winter+months,+polyfill+provided+insulation+for+trapped+ animals.+Small+mammals+were+sexed,+weighed,+examined+for+reproductive+status,+tagged+ using+numbered+metal+ear+tags,+and+then+released+after+recording+the+trap+coordinate.+Ticks+ were+not+collected+from+small+mammals+during+this+study.+All+mammals+were+handled+ according+to+the+guidelines+set+forth+by+the+American+Society+of+Mammalogists+(Sikes+and+

Gannon,+2011).+To+determine+mammal+density,+minimum+number+alive+(MNA)+was+ calculated+on+an+annual+basis+by+comparing+tag+numbers+between+each+year.+

Spatial#sampling#on#Virginia#barrier#islands#

Site+description+

Questing#ticks#were+collected+from+Hog+Island,+a+barrier+island+located+on+the+ eastern+shore+of+Virginia+that+is+part+of+the+Virginia+Coastal+Reserve+Long%Term+Ecological+

Research+site.+The+vegetation+at+this+site+is+representative+of+regularly+disturbed+barrier+ island+habitat.+Coastal+dune+habitats,+salt+water+marshes,+fluctuating+fresh+and+brackish+ inland+marshes,+and+shrub+thickets+make+up+a+complex+mosaic+of+habitat.+Each+of+these+ habitats+exists+within+a+small+geographic+area+and+habitats+are+usually+divided+by+narrow+ ecotone+borders.+Ammophila#breviligulata#typically+creates+tall+primary+dunes+that+are+ effective+at+protecting+interior+habitats+from+overwash+disturbances.+Interior+secondary+ dunes+predominantly+include+A.#breviligulata,#Schizachyrium#scoparium,+Panicum#amarum,#

Spartina#patens,#Festuca#rubra,+as+well+as+many+other+other+grasses+and+forbs.#Thick+stands+ of+wax+myrtle+(M.#cerifera)#dominate+areas+with+consistent+access+to+freshwater.+Ecotonal+ regions+between+open+canopy+secondary+dunes+and+M.#cerifera+thickets+are+only+1%3+m+ wide+but+provide+habitat+for+a+diverse+set+of+over+a+dozen+grasses+and+forbs.+

Sampling+methods+

Ticks+were+sampled+in+2014+and+2015+on+five+separate+field+visits.+In+2014,+sampling+ occurred+on+15+July+and+27+September.+The+goal+of+these+initial+two+visits+was+to+survey+tick+ populations+across+as+many+barrier+island+habitats+as+possible.+Marshland,+secondary+

98 dunes,+and+stands+of+M.#cerifera+were+surveyed.+The+presence+of+A.#maculatum#across+the+ island+in+2014+led+to+a+more+formal+assessment+of+Hog+Island’s+tick+population+in+the+ summer+of+2015.+

In+2015,+three+trips+were+conducted+on+14,+16,+and+21+July+during+the+peak+of+A.# maculatum#adult+questing+activity+on+the+mainland.+The+surveys+focused+on+the+habitats+ that+yielded+the+most+ticks+in+2014.+Habitats+were+classified+as+primary+dune,+secondary+ dune,+or+ecotone+regions+based+on+vegetation+characteristics+and+location.+High+elevation+ areas+directly+above+the+intertidal+zone+and+dominated+by#A.#breviligulata#and+S.#patens# were+classified+as+primary+dune+habitat.+Older+dunes+that+did+not+have+direct+contact+with+ the+ocean+were+considered+secondary+dunes.+Ecotone+regions+were+defined+as+the+1%3+m+ wide+areas+between+M.#cerifera#stands+and+secondary+dunes.+Tick+flagging+sessions+were+ conducted+in+1+hr+segments+that+targeted+each+habitat.+In+total,+primary+dunes,+secondary+ dunes,+and+ecotone+regions+were+flagged+for+2+hrs,+3+hrs,+and+3+hrs+20+min,+respectively.+

GPS+points+were+collected+for+the+start+and+stop+point+of+each+collection+hour+in+2015.+

Mammal+trapping+

Small+mammal+live%trapping+was+opportunistically+conducted+on+Smith+Island,+ approximately+20+miles+southeast+of+Hog+Island,+during+the+fall+of+2015+to+collect+immature+ ticks+from+barrier+island+small+mammals.+The+purpose+of+this+trapping+event+was+to+ determine+if+A.#maculatum#immature+ticks+were+present+on+barrier+island+rodents,+ indicating+that+they+could+complete+their+life+cycle+on+barrier+islands+rather+than+simply+ being+dropped+of+onto+the+islands+by+birds.+Smith+Island+is+characterized+by+primary+and+ secondary+dune+communities,+marshes,+wax+myrtle+thickets,+and+ecotonal+zones+similar+to+

99 those+found+on+Hog+Island.+Previous+research+has+also+shown+that+the+rodent+communities+ of+both+Smith+and+Hog+Islands+are+dominated+by+marsh+rice+rats+(Oryzomys#palustris)+

(Forys+and+Dueser,+1993;+Porter+and+Dueser,+2011).+Two+transects+of+64+collapsible+

Sherman+traps+were+set+through+habitats+dominated+by+S.#patens,+S.#alterniflora,+and+M.# cerifera,+and+baited+with+peanut+butter+balls+in+wax+paper.+Traps+were+set+and+checked+for+2+ consecutive+days.+All+mammals+captured+were+tagged+and+processed+as+described+above.+

Trapped+mammals+were+also+examined+for+ticks+around+the+ears+and+snout.+Ticks+were+ removed+using+forceps,+placed+in+vials,+and+returned+to+the+lab+to+be+frozen+at+%20+C+until+ identification+and+processing.+If+the+mammals+were+infested+with+many+ticks,+a+subsample+ of+up+to+50+ticks+was+taken.+Because+engorged+immature+ticks+can+be+difficult+to+identify+ using+morphology+alone,+sequencing+of+the+16S+gene+was+conducted+on+individual+nymphs+ or+pools+of+up+to+10+larvae+to+determine+tick+species,+as+previously+described+(Wright+et+al.,+

2011).+

Trend#Analysis#

To+visualize+data+trends+and+facilitate+exploration+of+population+dynamics,+ polynomial+curves+were+fitted+to+mainland+tick+and+rodent+density+data+using+the+statistical+ software+R+version+3.2.1+(R+Core+Team,+2015).+To+visualize+a+curve+for+tick+density+on+Hog+

Island+where+limited+data+was+collected,+a+cubic+spline+was+fit+to+the+data+points+using+the+R+ package+“splines”.+To+map+the+different+habitats+present+on+Hog+Island,+a+1+m+resolution+

LiDAR%based+bare+earth+Digital+Elevation+Model+(DEM)+was+used,+from+data+collected+on+26+

May+2013+by+USACE%TEC+and+JALBTCX+using+the+CZMIL+system+(USACE%TEC+and+JALBTCX,+

2014).+Polygons+were+visually+corrected+in+some+areas+using+satellite+imagery.+Primary+

100 dunes+were+greater+than+2+m+in+height+and+occurred+closest+to+the+beach.+Secondary+ dunes/grasslands+were+interior+elevations+greater+than+2+m.+Shrub+thickets+were+ elevations+from+1.4%2.0+m,+and+marshes+included+any+area+lower+than+1.4+m+in+elevation.++

RESULTS,

Temporal#sampling#on#the#Virginia#mainland#

Tick+Sampling+

+ Questing+adult+A.#maculatum+were+collected+during+the+first+five+years+of+sampling,+ with+a+peak+of+104+individuals+(1.16+ticks/1,000+m2)+collected+during+the+summer+of+2011+

(Table+14).+Amblyomma#maculatum#densities+were+reduced+in+2012,+and+plummeted+in+

2013+to+0.17+ticks/+1,000+m2.+The+population+was+extirpated+by+2015.+Questing+adult+A.# americanum+were+collected+in+low+densities+throughout+the+summer+of+2010,+but+ populations+peaked+in+2013,+with+densities+>3+ticks/1000+m2+persisting+through+2015+as+ the+canopy+closed+at+the+site.+Other+species+of+ticks,+including+Dermacentor#variabilis,+Ixodes# affinis,+Ixodes#scapularis,+and+Haemophysalis#leporispalustris#were+also+collected+at+the+site,+ as+were+immature+life+stages+of+A.#americanum.++

Mammal+trapping+

Monthly+small+mammal+trapping+at+this+site+from+2005+through+2012+revealed+ robust+populations+of+small+mammals+in+early+succession,+including+meadow+voles+

(Microtus#pennsylvanicus),+hispid+cotton+rats+(Sigmodon#hispidus),+marsh+rice+rats,+and+ eastern+harvest+mice+(Reithrodontomys#humulis)+(Table+15).+Shifts+in+small+mammal+

Table&14.!Amblyomma(maculatum(and!A.(americanum(ticks!collected!from!mainland!successional!site,!201052014.!The!total! number!of!adult!A.(maculatum((AM)!and!A.(americanum((AA)!ticks!collected!of!each!species!is!reported,!with!the!density!of! ticks!per!1000!m2!included!in!parentheses.!The!number!of!sampling!events!along!a!30005m!transect!and!the!total!area!sampled! for!each!year!are!indicated.! ! !! 2010& 2011& 2012& 2013& 2014& 2015& AM!adults!(AM!adults/1000!m2)! 54!(0.67)! 104!(1.16)! 88!(.89)! 17!(.17)! 7!(0.08)! 0!(0)! AA!adults!(AA!adults/1000!m2)! 43!(0.53)! 211!(2.34)! 346!(3.49)! 451!(4.56)! 309!(3.32)! 256!(3.16)! Number!of!sampling!events! 27! 30! 33! 33! 31! 27! Area!sampled!(m2)! 81000! 90000! 99000! 99000! 93000! 81000! ! ! ! Table& 15.! Small! mammal! minimum! number! alive! (MNA)! throughout! succession,! 200552012.! Each! trap! night! included! 128! traps!per!night,!and!all!trapping!was!conducted!on!a!permanent!grid.!The!total!number!of!trap!nights!indicates!all!trap!nights! over!a!calendar!year.!The!total!number!of!mammals!per!trap!night!for!each!year!was!calculated!by!summing!the!MNA!for!all! mammal!species,!then!dividing!by!the!number!of!trap!nights!for!that!year.!Blarina(spp.!shrews!may!have!been!B.(brevicauda(or! B.(carolinensis,!and!were!often!found!dead!in!traps,!facilitating!more!thorough!tick!collections.! ! Scientific&name& Common&Name& 2005& 2006& 2007& 2008& 2009& 2010& 2011& 2012& ((Blarina(spp*( Short5tailed!shrews! 5! 6! 5! 7! 8! 11! 4! 31! ((Microtus(pennsylvanicus( Meadow!vole! 109! 516! 206! 136! 129! 27! 25! 20! ((Mus(musculus( House!mouse! 0! 0! 57! 62! 11! 5! 0! 2! ((Ochrotomys(nuttalli( Golden!mouse! 0! 0! 0! 0! 0! 2! 4! 6! ((Oryzomys(palustris( Marsh!rice!rat! 39! 84! 47! 6! 21! 24! 4! 15! ((Peromyscus(leucopus( White5footed!mouse! 0! 0! 0! 0! 0! 1! 2! 7! ((Reithrodontomys(humulis( Eastern!harvest!mouse! 9! 37! 59! 71! 67! 56! 43! 29! ((Sigmodon(hispidus( Hispid!cotton!rat! 19! 33! 37! 120! 250! 103! 34! 53! Total!mammal!MNA! !! 181! 676! 411! 402! 486! 229! 116! 163! Total!number!trap!nights! 1280! 4480! 5504! 4608! 4736! 4096! 3456! 3584! Total!mammals!per!trap!night! !! 0.14! 0.15! 0.07! 0.09! 0.10! 0.06! 0.03! 0.05! 101 101

102 community)composition)were)evident)as)ecological)succession)progressed.)The)dominant) species)shifted)from)meadow)voles)to)cotton)rats)in)2009,)and)in)2010)the)first)forest) specialist)species)were)trapped)on)the)grid,)including)white=footed)mice)(Peromyscus* leucopus))and)golden)mice)(Ochrotomys*nuttalli).)By)2012,)species)that)had)peaked)in)early) succession)were)in)significant)decline)(Fig.)12).)

))

Spatial*sampling*on*Virginia*barrier*islands*

Tick)sampling)

) Preliminary)sampling)events)on)Hog)Island)over)the)summer)of)2014)revealed)the) presence)of)large)numbers)of)adult)A.*maculatum*(12)ticks)collected)hr=1))and)A.* americanum*(11.5)ticks)hr=1))in)secondary)dune)habitat.)Sampling)in)M.*cerifera)thickets) revealed)no)ticks)of)either)species)in)2014.)More)thorough)sampling)during)the)summer)of)

2015)revealed)that)the)ecotone)region)between)secondary)dune)grass)habitat)and)M.* cerifera*forests)yielded)the)highest)densities)of)A.*maculatum)encountered,)with)34)ticks) collected)per)hour)in)this)habitat)zone)(Table)16).)Amblyomma*maculatum*were)found)in)all) habitats)on)Hog)Island)except)for)closed)canopy)M.*cerifera*forest.)Amblyomma*americanum* were)never)found)in)primary)dune)habitat,)but)adults)were)found)in)secondary)dune)and) ecotonal)habitats.)Amblyomma*americanum*larvae)were)also)collected)in)secondary)dunes)

(870)larvae)hr=1))and)ecotonal)habitats)(1275)larvae)hr=1;)Fig.)13).

103

) ! Figure!12.!Tick)and)mammal)densities)throughout)ecological)succession.)Visualization)of) small)mammal,)A.*maculatum*(AM))and)A.*americanum)(AA))tick)densities)throughout)old) field)ecological)succession,)using)data)collected)at)a)mainland)site)over)10)years.)Tick) density)(adult)ticks)collected)per)m2,)see)Table)14))is)indicated)on)the)left)axis,)and) mammal)density)(minimum)number)alive,)or)MNA,)of)all)species)as)shown)in)Table)15))is) on)the)right)axis.)Open)diamonds)indicate)AM)per)m2)annual)data)points,)with)a)black)solid) line)showing)fit)with)a)third)order)polynomial)(R2)=)0.81).)Solid)black)squares)indicate)AA) per)m2)annual)data)points,)with)a)black)dashed)line)showing)fit)with)a)second)order) polynomial)(R2=0.89).)Solid)black)circles)show)annual)small)mammal)MNA,)with)a)gray)line) indicating)fit)with)a)third)order)polynomial)(R2)=)0.68).)The)photographs)above)the)axis) show)the)site)undergoing)succession)in)2005)(courtey)of)TNC),)2009,)2011,)and)2015.)The) sketch)behind)the)axis)indicates)the)stage)of)succession)throughout)the)years)of)sampling.)) ) ) Table!16.!Hog)Island)collections)of)adult)A.*maculatum)and)A.*americanum.)Numbers)of) adult)A.*maculatum)(AM))and)A.*americanum)(AA))indicate)ticks)collected)per)hour)during) July)2015.)Primary)dunes)are)the)located)directly)above)the)intertidal)zone,)and)secondary) dunes)are)generally)located)further)from)shore,)separated)from)primary)dunes)by)low,) marshy)areas.)Ecotonal)regions)have)over)a)dozen)grasses)and)forbs)present)and)are) located)in)a)narrow)band)between)secondary)dune)systems)and)wax)myrtle)thickets.)See) text)for)more)details)on)vegetation)types.) ) Ticks! per! Primary! Secondary! Ecotonal! hour! dunes! dunes! regions!! AM)hr=1) 7) 6.6) 34) AA)hr=1) 0) 11) 4.8)

104

) Figure! 13.) Hog) Island) map) and) barrier) island) habitat) effects) on) A.* maculatum* density.) Visualization)of)the)effects)of)habitat)on)A.*maculatum*(AM))abundance)on)Hog)Island,)as) observed)during)summer)2015.)Solid)circles)indicate)the)number)of)adult)AM)collected)per) hour)(see)Table)16),)fit)to)a)cubic)spline)curve)for)ease)of)viewing.)The)map)indicates)the) different)habitats)present)on)Hog)Island,)with)colors)indicating)habitat)zones)described)in) the)legend.)These)colors)are)mirrored)along)the)x=axis,)with)the)sketch)behind)the)graph) indicating)the)habitat)zones)where)AM)were)found.)* ! !

)

Mammal)trapping)

) Trapping)conducted)over)two)days)on)neighboring)Smith)Island)during)October)

2015)revealed)a)robust)population)of)73)marsh)rice)rats)and)two)house)mice)(Mus* musculus).)Almost)every)rodent)sampled)was)liberally)covered)in)larval)ticks,)with)13) nymphal)ticks)also)collected)from)7)individual)rice)rats.)Sequencing)three)pools)of)10)larvae) and)12)nymphs)revealed)100%)sequence)identity)confirmed)with)D.*variabilis.*One)nymph) was)found)to)have)100%)sequence)identity)with)A.*maculatum.)

105

) DISCUSSION!

Disturbed*habitats*support*A.*maculatum*invasion*

At)the)mainland)field)site,)the)A.*maculatum*population)reached)its)peak)in)2011) during)secondary)succession,)nine)years)after)the)field)was)left)fallow.)The)A.*maculatum* population)was)well)established)during)the)inaugural)tick)sampling)season)in)2010,)and) was)likely)first)established)several)years)prior)when)rodent)densities)were)still)high.)Prior) to)2010,)rodent)densities)were)over)400)mammals)per)ha)(Table)15),)including)robust) populations)of)hispid)cotton)rats,)harvest)mice,)and)marsh)rice)rats,)all)known)hosts)for) immature)A.*maculatum*(Teel)et)al.,)2010).)The)diverse)mixture)of)grasses)and)forbs) present)in)early)successional)habitat)provides)abundant)forage)that)can)support)high) densities)of)herbivorous)small)mammals)(Schweiger)et)al.,)2000),)and)provides)habitat) conditions)that)are)suitable)for)A.*maculatum*(Teel)et)al.,)2010;)Paddock)and)Goddard,)

2015).)White=tailed)deer,)and)other)large,)wide=ranging)mammals)such)as)black))bears)

(Ursus*americanum),)raccoons)(Procyon*lotor),)and)gray)foxes)(Urocyon*cinereoargenteus)) are)all)hosts)of)adult)A.*maculatum)and)can)be)abundant)in)patchy)landscapes)including) open)and)forested)habitats)(Lotze)and)Anderson,)1979;)Fritzell)and)Haroldson,)1982;)

Smith,)1991;)Sutherland)et)al.,)2000;)Larivière,)2001).)The)life)cycle)of)A.*maculatum)is) generally)completed)in)1=2)years;)engorged)females)lay)an)average)of)8,000)and)up)to)

15,000)eggs)(Drummond)and)Whetstone,)1970;)Wright,)1971;)Teel)et)al.,)2010),) demonstrating)the)ability)of)this)tick)to)undergo)rapid)population)expansion)after)arrival)in) a)suitable)new)habitat)with)high)hosts)densities.)

Numbers)of)A.*maculatum*at)the)mainland)site)began)dwindling)in)2012,)two)years) after)the)numbers)of)rodent)hosts)underwent)a)substantial)reduction)from)>400)mammals)

106 ha=1)to)229)mammals)ha=1.)The)number)of)A.*maculatum)collected)declined)each)year)after) peaking)in)2011,)and)the)A.*maculatum*population)was)extirpated)in)2015)(Table)14).)Our) trapping)efforts)documented)shifts)in)small)mammal)community)composition)as) successional)processes)advanced)(Table)15).)The)arrival)of)forest)specialist)small)mammal) species)such)as)golden)and)white=footed)mice)in)2010)signaled)the)closing)of)the)canopy) and)the)transition)from)open)habitat)to)forest)(Hirth,)1959;)M’Closkey)and)Fieldwick,)

1975).)After)canopy)closure,)habitat)and)host)communities)became)unable)to)sustain) populations)of)A.*maculatum,)possibly)because)of)the)precipitous)drop)in)rodent)density) resulting)in)insufficient)hosts)available)for)larval)and)nymphal)A.*maculatum.)During)this) time)the)number)of)A.*americanum,)a)tick)species)that)will)readily)feed)on)deer)and)other) large)mammals)at)all)life)stages)(Childs)and)Paddock,)2003),*dramatically)increased)(Table)

14).))

Most)open)habitats)in)Virginia)are)anthropogenically)influenced)through)prescribed) burning,)tree)clearing,)or)mowing)to)create)fields)or)other)open)spaces)for)human)use.)If) open)areas)are)allowed)to)return)to)forest)through)natural)succession,)a)temporal)window) is)created)during)which)appropriate)habitat)and)abundant)small)mammal)hosts)exist)for)A.* maculatum)to)invade)and)thrive.)This)ephemeral)nature)may)be)unique)to)A.*maculatum) populations)in)the)eastern)US,)as)open)habitats)and)rangelands)persist)along)the)Gulf)Coast) and)in)the)central)US)where)A.*maculatum*are)well)established)(Paddock)and)Goddard,)

2015).)Habitat)islands)of)anthropogenically)disturbed)land)undergoing)restoration)may)be) the)only)habitat)able)to)facilitate)the)establishment)and)spread)of)A.*maculatum)populations) in)the)Mid=Atlantic)region.)As)A.*maculatum*ticks)move)northward)into)the)Mid=Atlantic,)it) becomes)increasingly)probable)that)ephemeral)tick)populations)will)appear)in)response)to)

107 ecosystem)restoration)projects,)where)short=lived)open)habitats)can)be)used)as)stepping) stones)to)facilitate)the)process)of)expansion)into)otherwise)unsuitable)habitat.))

)

Barrier*islands:*frequent*disturbance*events*support*high*tick*densities*

Barrier)islands)are)dynamic)landscapes)that)rapidly)accrete)and)erode)as)sediment) supplies,)storm)energies,)and)currents)fluctuate.)Succession)begins)with)primary)dune) building)grasses)(A.*breviligulata,*S.*patens,)and)Uniola*paniculata)*that)stabilize)the)sands) with)developing)root)networks,)and)by)reducing)the)capacity)of)wind)to)carry)soil)particles) through)aboveground)structures)(Lancaster)and)Baas,)1998).)Island)geomorphology)is) driven)by)storm)frequencies,)sediment)supply,)and)relative)sea)levels,)resulting)in)a)fluid) ecosystem)with)areas)of)intermediate)disturbance.)Given)enough)time)without)severe) disturbances)that)bury)or)remove)vegetation)from)the)beach)or)dunes,)successional) processes)begin,)with)primary)colonizers)competing)with)grasses)and)forbs)that)were) originally)excluded)by)mobile)sands.)While)successional)habitats)are)only)available)for) colonization)for)brief)windows)of)time)on)the)mainland,)the)rapidly)changing)barrier)island) ecosystems)may)provide)more)consistent)habitat)and)host)conditions)that)are)amenable)to)

A.*maculatum*establishment.)Amblyomma*maculatum)were)encountered)at)a)rate)of)6=7) ticks)hr=1)in)primary)and)secondary)dune)habitats)on)Hog)Island)(Table)16).)Previous)work) from)1989)through)2011)has)also)demonstrated)consistently)high)densities)of)marsh)rice) rats)across)Hog)Island,)with)occasional)captures)of)house)mice)and)Norway)rats)(Rattus* norvegicus))(Porter)and)Dueser,)2011).)These)rodents,)especially)the)abundant)rice)rats,)are) likely)to)provide)a)substantial)portion)of)the)bloodmeals)for)island=dwelling)immature)A.* maculatum.)

108

Barrier)island)succession)proceeds)at)an)extremely)rapid)rate)(Young)et)al.)1995),) and)within)a)matter)of)years,)shrub)species)begin)to)compete)with)grasses)and)forbs.)

Morella*cerifera*is)the)dominant)shrub)that)occurs)on)Virginia)coast)barrier)islands.)As)M.* cerifera*populations)expand,)their)canopies)have)been)reported)to)attenuate)up)to)95%)of) light)(Brantley)and)Young,)2007).)Most)barrier)island)grasses)are)shade)intolerant)and) quickly)become)excluded.)The)establishment)and)development)of)shrub)thickets)is) extremely)important)to)the)barrier)island)system,)as)they)are)responsible)for)a)substantial) proportion)of)carbon)and)nitrogen)inputs)(Brantley)and)Young,)2008).)No)A.*maculatum* were)collected)in)these)shrub)thickets)in)2014,)indicating)that)these)closed=canopy)thickets) are)analogous)to)the)closed=canopy)forest)that)contributed)to)the)2015)extirpation)of)A.* maculatum*in)the)successional)mainland)system)(Table)14).)

On)Hog)Island,)ecotonal)regions)on)the)edges)of)M.*cerifera)shrub)thickets)and) secondary)dune)habitats)yielded)the)highest)A.*maculatum)densities)of)34)ticks)hr=1)(Table)

16).)These)ecotones)are)analogous)to)the)early=mid)successional)habitat)that)dominated)the) mainland)site)during)peak)tick)abundance)in)2011.)Ecotonal)regions)create)the)highest) plant)diversity)of)any)barrier)island)habitat)as)a)result)of)the)overlap)of)two)plant) communities,)and)parallel)the)diversity)of)grasses)and)forbs)seen)in)mid=successional) phases)before)woody)species)begin)to)dominate)(Schweiger)et)al.,)2000).)High)plant) diversity)has)been)shown)to)predict)high)rodent)density)and)diversity)(Hafner,)1977);) barrier)island)ecotonal)regions)and)successional)communities)likely)provide)a)means)for)A.* maculatum*population)explosions)by)increasing)feeding)success)for)juvenile)ticks)by) supporting)high)rice)rat)densities.)))

109

While)no)A.*maculatum*were)collected)in)shrub)thickets,)peak)A.*maculatum*density) may)have)been)found)adjacent)to)the)shrubs)because)M.*cerifera)enriches)the)soil)through) deposition)and)decomposition)of)plant)litter)(Brantley)and)Young,)2008))and)may)input) more)nitrogen)than)would)otherwise)be)available)to)adjacent)grasslands.)Shrub)thickets) reduce)insolation)and)evaporation,)salt)spray)and)wind)velocities)for)plants)growing) beneath)or)within)close)proximity)(Shumway,)2000),)and)they)may)facilitate)survival)of) neighboring)species)through)hydraulic)lifting)(Caldwell)et)al.,)1998).)Plant)species)growing) near)or)beneath)M.*pennsylvanica,)a)similar)and)co=occurring)species,)are)larger,)produce) more)flowers)and)seeds,)have)access)to)more)soil)moisture,)and)have)greater)tissue) nitrogen)concentration)(Shumway,)2000).)Increased)nitrogen)input)and)increased) vegetational)standing)crop)have)been)found)to)have)strong)positive)effects)on)mammal) diversity)and)abundance)(Huntly)and)Inouye,)1987).)Flourishing)plant)communities) adjacent)to)wax)myrtle)thickets)likely)provide)excellent)habitat)for)marsh)rice)rats)and) questing)A.*maculatum.*)

Although)mammalian)diversity)on)islands)is)much)reduced)compared)to)equivalent) mainland)habitats)(Lawlor,)1986),)it)appears)that)sufficient)densities)of)preferred)hosts) exist)to)support)the)entire)life)cycle)of)A.*maculatum.)The)consistently)high)densities)of) marsh)rice)rats*support)immature)tick)life)stages,)while)reliable)populations)of)raccoons,) deer,)and)foxes)support)adult)ticks)(Dueser)et)al.,)1979).)The)peak)density)of)rice)rats*was) documented)at)the)mainland)site)in)2006,)which)corresponds)to)when)A.*maculatum*was) likely)beginning)to)invade.)It)is)possible)that)the)diversity)of)mammals)available)at)the) mainland)site)was)incidental)–)large)populations)of)rice)rats)in)early)succession,)followed) by)large)populations)of)hosts)such)as)cotton)rats)and)harvest)mice)in)later)stages)of)early)to)

110 mid=succession,)were)essential)for)successful)colonization)by)A.*maculatum.)As)barrier) islands)are)intact)ecosystems)that)can)support)large)A.*maculatum)populations,)the) presence)of)competent)hosts)at)sufficient)density)appears)to)be)more)important)than)high) host)diversity)for)establishment)of)this)tick)species.)Because)island)habitats)experience) frequent)disturbance)events)with)consistently)robust)populations)of)preferred)hosts,) populations)of)A.*maculatum*on)barrier)islands)may)serve)as)population)sources,)with)birds) or)deer)moving)ticks)to)sink)ephemeral)mainland)populations.)

Population)sources)on)islands)moving)to)sinks)on)the)mainland)is)in)contrast)to) traditional)island)biogeography)theory)(MacArthur)and)Wilson,)1967).)Because)of)their) preference)for)disturbed)habitat,)A.*maculatum)populations)seem)to)function)as) metapopulations)in)a)patchy)landscape,)with)ticks)dispersing)between)patches)on)hosts) such)as)birds)or)large)mammals.)Interestingly,)the)entire)US)range)of)A.*maculatum* corresponds)with)coastal)habitats)in)proximity)to)barrier)islands,)with)the)exception)of) some)inland)disturbed)habitats)maintained)by)the)cattle)industry)(Paddock)and)Goddard,)

2015))(Fig.)14).)Populations)in)Virginia)are)non=contiguous,)and)throughout)the)eastern)US) populations)exhibit)extremely)high)genetic)diversity)and)low)genetic)connectivity)among) sites)(Nadolny)et)al.,)2015).)An)established)source)population)on)the)continuous)barrier) island)chain)along)the)eastern)seaboard)could)supply)the)genetic)diversity)needed)to) overcome)founder)effects)in)new)populations.)

)

Lasting*health*effects*from*ephemeral*tick*populations*

The)landscape)of)eastern)US)coastal)regions)is)extremely)developed,)so)patches)of) open)habitat)where)A.*maculatum*may)invade)occur)only)by)human)design,)such)as)when)

111 an)agricultural)field)is)left)fallow,)or)an)area)is)cleared)for)construction.)These)sites)are) often)near)areas)with)large)human)populations,)including)patches)in)urban)or)suburban) developments.)By)creating)and)maintaining)these)open)habitats,)especially)in)areas)with) abundant)human)habitation,)A.*maculatum)may)be)more)likely)to)establish)and)generate) new)human)health)risks.)

) )

) Figure! 14.) Range) map) of) A.* maculatum* in) the) United) States,) with) barrier) islands) highlighted.) Inset) map) shows) counties) with) reported) populations) of) A.* maculatum* in) Virginia,)Delaware,)and)North)Carolina)highlighted)in)dark)blue.)Highlighted)in)red)are)the) areas)of)the)eastern)seaboard)with)barrier)islands,)with)chains)of)islands)highlighted)in)the) inset)map.)) )

)

112

) Emerging)A.*maculatum*populations)at)the)leading)edge)of)the)A.*maculatum* invasion)have)been)documented)to)have)some)of)the)highest)numbers)of)ticks)infected)with)

R.*parkeri)on)record)(Fornadel)et)al.,)2011;)Wright)et)al.,)2011;)Nadolny)et)al.,)2014).)Over)

50%)of)ticks)at)the)mainland)study)area)were)infected)(Nadolny)et)al.,)2014),)much)higher) than)in)established)populations)along)the)Gulf)Coast)(Wright)et)al.,)2011).)After)ephemeral) mainland)populations)are)extirpated,)however,)the)disease)risk)does)not)immediately) disappear)with)the)ticks.)Rickettsia*parkeri)can)be)transmitted)by)co=feeding)from)A.* maculatum)to)A.*americanum,)and)can)be)maintained)in)A.*americanum)via)transstadial) transmission)(Gaines)et)al.,)2014;)Wright)et)al.,)2015).)Amblyomma*americanum)are)also) able)to)transmit)this)pathogen)transovarially)(Goddard,)2003),)and)preliminary)findings)of)

R.*parkeri)in)the)salivary)glands)of)A.*americanum)suggest)that)A.*americanum)can)transmit) this)pathogen)(Wright)et)al.,)2015).)If)R.*parkeri)can)be)transmitted)from)A.*americanum*to) wildlife)hosts)and)be)maintained)in)an)enzootic)cycle,)even)at)low)rates,)this)could)pose)a) major)threat)to)human)health)(Gaines)et)al.,)2014;)Wright)et)al.,)2015).)Amblyomma* americanum)are)the)most)abundant)human=biting)ticks)throughout)much)of)the)US) southeast)and)Mid=Atlantic,)and)are)known)to)bite)humans)at)all)life)stages)(Stromdahl)and)

Hickling,)2012).)Field=collected)A.*americanum*have)been)found)infected)with)R.*parkeri*at) low)densities)in)Virginia)counties)where)A.*maculatum*co=exist)(Gaines)et)al.,)2014).)After)A.* maculatum)are)extirpated)and)successional)habitat)is)restored)to)forest,)the)A.*americanum) that)dominate)the)forested)habitat)may)have)acquired)R.*parkeri)from)A.*maculatum)during) the)years)when)both)species)overlapped)and)co=fed.)In)fact,)an)increase)in)human)cases)of) spotted)fever)group)rickettsial)disease)has)been)linked)to)the)expansion)of)A.*americanum* ticks,)which)may)be)vectoring)R.*parkeri)to)humans)and)animals)(Dahlgren)et)al.,)2016).)

113

Speldewinde)et)al.)(2015))described)several)alternative)disease)risk)states)that)can) result)from)ecosystem)restoration,)after)disease)risk)had)presumably)been)increased) following)ecosystem)degradation.)Because)restored)ecosystems)do)not)necessarily)return) to)their)original)state)or)level)of)functioning)(Hobbs)et)al.,)2006),)restored)ecosystems)may) result)in)a)variety)of)risk)types)across)a)spectrum)from)low)to)high)(Speldewinde)et)al.,)

2015).)An)adjusted)set)of)pathways)that)can)lead)to)these)altered)disease)risk)states)may)be) warranted,)taking)into)account)the)potential)unintended)consequences)of)ecological) restoration)suggested)by)these)data.)In)an)old)field)system,)after)an)initial)spike)in)disease) risk)immediately)following)ecosystem)disturbance,)the)disease)risk)on)disturbed) ecosystems)that)are)maintained)by)humans)may)be)lower)than)that)of)the)original)intact) ecosystem)because)of)removal)of)unwanted)species)via)spraying)or)weeding.)However,) once)ecosystem)restoration)begins,)colonizing)species)may)bring)new)threats,)resulting)in)a) higher)disease)risk)than)was)present)in)the)initial)intact)forest)with)intrinsic)biological) controls)of)disease.))

In)conclusion,)the)invasive)tick)species)A.*maculatum*is)able)to)establish)and)become) abundant)in)suitably)disturbed)areas)such)as)barrier)islands)or)anthropogenically)produced) patches)on)the)mainland)in)the)southeastern)and)Mid=Atlantic)US.)Mainland)populations)of)

A.*maculatum)are)seemingly)limited)to)relatively)open)sites)transitioning)to)forest.)These) mid=successional)states)on)the)mainland)provide)a)similar)niche)as)ecotonal)regions)on) barrier)islands,)both)of)which)support)robust)A.*maculatum*populations.)On)the)mainland,)

A.*maculatum)are)extirpated)and)replaced)by)A.*americanum)and)other)forest=dwelling)tick) species)as)forests)return.)The)fact)that)A.*maculatum)can)transmit)R.*parkeri)to)A.* americanum)via)co=feeding)allows)threats)to)human)health)to)persist)even)after)A.*

114 maculatum)populations)die)out.)Ironically,)restoration)projects)often)provide)habitat) islands)susceptible)to)colonization)by)A.*maculatum,)enabling)the)species)to)expand) northward)where)it)poses)a)health)threat)through)infection)with)R.*parkeri.)Mitigation)of) these)risks)may)be)possible,)either)by)transitioning)disturbed)sites)to)forests)more)directly) via)tree=planting)or)by)targeting)vulnerable)early)successional)habitats)with)acaricides.)

Regardless,)one)must)be)vigilant)about)increasing)risks)of)tick=borne)pathogens)associated) with)the)creation)of)favorable)habitats)through)anthropogenic)disturbance)and)subsequent) ecological)restoration)efforts.!

! !

115

COMPARATIVE!POPULATION!GENETICS!OF!

IXODES'AFFINIS'AND!AMBLYOMMA'MACULATUM'

**This)chapter)is)published,)and)is)reprinted)here)with)permission:)) Nadolny,)R.)M.,)H.)Gaff,)J.)Carlsson,)D.)Gauthier.)Comparative)landscape)genetics)of) two)invading)ticks:)evidence)of)the)ecological)mechanisms)underlying)tick)range) expansions.)Infection,*Genetics,*and*Evolution.*35:)153=162.* !

INTRODUCTION!

The)spread)of)parasites)is)a)major)source)of)disease)emergence)for)vertebrate)taxa,) including)humans)(Mack)et)al.,)2000;)George,)2008;)Williams)et)al.,)2013).)Climate)change) and)anthropogenic)landscape)alterations,)such)as)fragmentation)and)suburban)sprawl,)have) been)linked)to)tick)range)expansions)worldwide,)resulting)in)changes)to)tick)community) structure)and)altered)tick=borne)pathogen)dynamics)(Childs)and)Paddock,)2003;)Cumming) and)Van)Vuuren,)2006;)Gage)et)al.,)2008;)George,)2008;)Léger)et)al.,)2013).))

In)North)America,)the)expanded)ranges)of)the)lone)star)tick,)Amblyomma* americanum,*and)the)blacklegged)tick,)Ixodes*scapularis,)have)received)much)attention) because)of)the)importance)of)these)ticks)to)human)health)(Childs)and)Paddock,)2003;)

Ogden)et)al.,)2008b;)Springer)et)al.,)2014).)More)recently,)Ixodes*affinis)and)the)Gulf)coast) tick,)Amblyomma*maculatum,*have)invaded)the)eastern)United)States)(US),)changing)tick) community)and)pathogen)dynamics)in)the)affected)areas)(Harrison)et)al.,)2010;)Fornadel)et) al.,)2011;)Nadolny)et)al.,)2011;)Varela=Stokes,)2011;)Wright)et)al.,)2011;)Paddock)and)

Goddard,)2015).)Because)of)their)small)size)and)vulnerability)to)the)environment)when)off= host,)tick)dispersal)is)intricately)linked)to)movements)of)their)hosts)(Falco)and)Fish,)1991).)

116

The)ecological)processes)driving)tick)invasions)require)study)in)order)to)better)predict)and) mitigate)disease)emergence)and)improve)understanding)of)tick)behavior.)

Coalescent)genetic)analyses)can)reveal)the)ancestry)of)newly)established) populations,)and)offer)clues)to)how)organisms)disperse)over)long)distances.)The)use)of) molecular)methods)to)trace)the)ancestry)of)invading)species)is)well)established)(Templeton) et)al.,)1995;)Ibrahim)et)al.,)1996;)Le)Roux)et)al.,)2009).)For)more)than)30)years,)molecular) methods)have)been)used)to)investigate)tick)population)genetic)structure)(Araya=Anchetta) et)al.,)2015).)Previous)studies)have)documented)the)effects)of)host=mediated)dispersal)on) genetic)structure)in)the)seabird)tick)Ixodes*uriae,)using)microsatellite)markers)to)determine) how)gene)flow)patterns)changed)depending)on)the)host)species)exploited)for)dispersal)

(McCoy)et)al.,)2001;)McCoy)et)al.,)2003).)Other)studies)have)used)phylogenetic)analyses)to) determine)recent)population)expansion,)identify)founder)effects,)and)examine)population) structure)at)the)expansion)fronts)of)A.*americanum*and)I.*scapularis)(Ogden)et)al.,)2011;)

Mechai)et)al.,)2013;)Kelly)et)al.,)2014).)Some)broad)conclusions)reached)by)this)body)of) work)are)that)tick)behaviors)and)life)cycle)strategies)are)as)critical)as)host)mobility)in) understanding)tick)population)genetic)structure,)particularly)as)applied)to)different) families)of)ticks)(Araya=Anchetta)et)al.,)2015).))

The)northward)expansions)of)I.*affinis)and)A.*maculatum)into)the)Mid=Atlantic)region) of)the)US)have)been)documented)over)the)last)decade)(Harrison)et)al.,)2010;)Fornadel)et)al.,)

2011;)Nadolny)et)al.,)2011;)Varela=Stokes,)2011;)Wright)et)al.,)2011;)Florin)et)al.,)2014).)

Both)species)are)recent)additions)to)a)diverse)assemblage)of)ticks)and)have)the)potential)to) significantly)affect)pathogen)dynamics)(Oliver,)1996;)Nadolny)et)al.,)2011;)Nadolny)et)al.,)

2014).)Ixodes*affinis)is)a)competent)sylvatic)vector)for)Borrelia*burgdorferi)sensu)stricto,)the)

117 bacterial)agent)of)Lyme)disease,)and)A.*maculatum)is)the)major)vector)for)Rickettsia*parkeri,) the)agent)of)Tidewater)spotted)fever,)and)several)significant)veterinary)pathogens)(Oliver) et)al.,)2003;)Paddock)et)al.,)2004;)Paddock)and)Goddard,)2015).)Both)species)have) expanded)northward)from)historic)ranges)in)the)southern)US)and)Central)America)and)both) parasitize)avian)and)mammalian)hosts)throughout)their)ranges)(Harrison)et)al.,)2010;)Teel) et)al.,)2010;)Paddock)and)Goddard,)2015).))

Despite)some)overlap)in)the)vertebrates)parasitized)by)these)two)tick)species,)I.* affinis*and*A.*maculatum)are)generally)found)in)different)habitat)types)and)are)expanding) their)ranges)in)different)geographic)patterns.)The)northern)edge)of)the)expansion)front)for)

I.*affinis)is)currently)in)Virginia)(Nadolny)et)al.,)2011),)and)individuals)can)be)consistently) collected)at)low)densities)in)woodland)habitats)(Gaff)and)Nadolny,)unpublished)data).)

Amblyomma*maculatum)appears)to)be)expanding)in)isolated)populations)in)ephemeral) successional)habitats,)with)large)areas)containing)few)to)no)A.*maculatum)separating) established)breeding)groups)(Gaff)and)Nadolny,)unpublished)data).)These)expansion) patterns)reflect)the)distributions)of)these)ticks)in)their)native)ranges)(Harrison)et)al.,)2010;)

Teel)et)al.,)2010;)Varela=Stokes)et)al.,)2011),)but)the)implications)of)these)two)expansion) types)on)population)connectivity)are)largely)unexplored.)

Several)genetic)markers)have)been)historically)used)to)characterize)population) structure)in)tick)populations,)most)commonly)nuclear)microsatellites)and)variation)in) mitochondrial)genes.)Microsatellites)have)been)characterized)for)twelve)tick)species)

(Araya=Anchetta)et)al.,)2015).)Microsatellites,)however,)are)often)unable)to)achieve)cross= species)amplification)(McCoy)and)Tirard,)2000))and)are)also)not)universally)abundant)in)all) tick)genomes)(Fagerberg)et)al.,)2001).)The)use)of)the)16S)mitochondrial)rRNA)gene)is)

118 pervasive)in)studies)of)tick)population)genetics)and)tick)phylogeny)(Black)and)Piesman,)

1994;)Norris)et)al.,)1996;)Qiu)et)al.,)2002;)Mixson)et)al.,)2006;)Kelly)et)al.,)2014).)This)gene) mutates)at)a)rate)that)is)generally)informative)for)species=level)phylogenetics)and)broad) biogeographic)inferences)(Araya=Anchetta)et)al.,)2015).))

In)this)study,)single)nucleotide)polymorphisms)(SNPs))in)the)16S)mitochondrial) rRNA)gene)were)used)to)examine)patterns)of)genetic)structure)in)I.*affinis*and)A.*maculatum) ticks)across)the)eastern)US.)This)method)enabled)direct)comparison)of)results)across)both) tick)species)without)the)need)to)characterize)two)novel)sets)of)species=specific) microsatellites.)Using)a)mitochondrial)gene)was)also)appropriate)for)the)scale)of)this) research,)investigating)broad)biogeographic)patterns)of)connectivity)across)many)US)states)

(Araya=Anchetta)et)al.,)2015).)The)resulting)information)on)haplotype)frequencies,) geographic)and)genetic)distance)between)new)populations,)and)host)and)habitat) associations)of)each)tick)species)was)used)to)describe)likely)routes)by)which)these)ticks)are) expanding)their)ranges.)

Field=dwelling)ticks)and)generalist)ticks)that)parasitize)a)range)of)hosts)are)less) likely)to)exhibit)genetic)structure)than)nidicolous)(nest=dwelling))ticks,)single=host)ticks,)or) ticks)that)specialize)on)hosts)with)small)home)ranges)(Araya=Anchetta)et)al.,)2015).)In)this) chapter,)I)compare)the)genetic)structure)of)two)non=nidicolous)tick)species)that)are) expanding)their)ranges)simultaneously)across)the)eastern)US,)and)that)share)host) preferences)for)avian)and)mammalian)hosts.)I)discuss)how)tick)ecology)and)behavior)may) influence)population)structure)and)use)this)structure)to)infer)likely)invasion)routes)and) important)life)history)characters)that)have)facilitated)range)expansion)for)each)species.)The)

119 original)hypothesis)for)this)work)was)that)both)species)would)exhibit)isolation)by)distance,) with)northern)populations)originating)from)related)southern)populations.)

)

MATERIALS!AND!METHODS!

Study*sites*and*tick*collection*

) Questing)adult)ticks)were)collected)from)populations)in)Virginia,)North)Carolina,)and)

South)Carolina)using)standard)tick)collecting)methods,)as)previously)described)(Nadolny)et) al.,)2011;)Wright)et)al.,)2011).)Ticks)and/or)tick)DNA)extracts)from)additional)populations) in)states)throughout)the)established)US)ranges)of)both)ticks)were)included)to)provide)a) more)complete)analysis)of)population)structure)(Table)17).)After)morphological)species) identification)(Sonenshine,)1979;)Keirans)and)Litwak,)1989),)ticks)were)frozen)at)=80)C) prior)to)DNA)isolation.)

Molecular*methods*

) Prior)to)DNA)extraction,)individual)ticks)were)placed)in)2)mL)microcentrifuge)tubes) containing)1)mm)and)2.5)mm)glass)beads)and)homogenized)in)a)bead=beater)(BioSpec)

Products,)Inc.,)Bartlesville,)OK))for)30)seconds.)DNA)was)then)extracted)from)individual) adult)ticks)using)a)DNeasy)Blood)&)Tissue)Kit)(Qiagen,)Inc.,)Valencia,)CA),)eluted)in)50)µL)of) buffer)AE)and)stored)at)=20)C)until)processing.)

)

! !

120

Table!17.)Ticks)sequenced,)by)year)and)site.)APHC)indicates)Army)Public)Health)Command) ) Amblyomma'maculatum' #) Ticks)submitted) Site) Year)Collected) Ticks) by) City/County,)State) 200 201 201 201 201 8) 0) 1) 2) 4) CHS) ) 1) 49) 40) ) 90) This)study) ) Chesapeake,)VA) ) ) ) Fairfax)Co.) FX1) 18) 20) 38) Health)Dept.) Fairfax)Co.,)VA) ) ) ) Fairfax)Co.) FX2) 32) 32) Health)Dept.) Fairfax)Co.,)VA) HAM) ) ) 2)) ) 2) This)study) Hampton,)VA) ) ) ) ) Melissa)Miller,) KY1) 3) 3) APHC) Hardin)Co.,)KY) ) ) ) ) Melissa)Miller,) KY2) 36) 36) APHC) Greenville,)KY) ) ) ) ) Varela=Stokes) MS2) 13) 13) Lab) Pass)Christian,)MS) ) ) ) ) Varela=Stokes) MS3) 9) 9) Lab) Moss)Point,)MS) ) ) ) ) Varela=Stokes) MS4) 9) 9) Lab) Moss)Point,)MS) ) ) ) ) Varela=Stokes) MS5) 4) 4) Lab) Gautier,)MS) ) ) ) ) Varela=Stokes) MS6) 1) 1) Lab) Bryam,)MS) ) ) ) ) Varela=Stokes) MS7) 7) 7) Lab) Moss)Point,)MS) NCA) ) 10) ) 30) ) ) 40) Apperson)Lab) Raleigh,)NC) PT1) ) 3)) ) 3) This)study) Portsmouth,)VA) ) ) ) ) Melissa)Miller,) TNS) 19) 19) APHC) Clarksville,)TN) VB1) ) 9)) 5)) 36) ) 50) This)study) Virginia)Beach)VA) VB2) ) 1) ) 1) This)study) Virginia)Beach,)VA) YRK) ) ) 1) 12) ) ) 13) This)study) York)Co.,)VA) Total)A.*maculatum) ) ) ) sequenced) 370) ) ) ) ) ) ! !

121

Table!17.!Continued.! ! Ixodes'affinis' #) Ticks)submitted) Site) Year)Collected) Ticks) by) City/County,)State) 200 201 201 201 201 8) 0) 1) 2) 4) ) ) Marcee)Tolliver,)) ) BEA) NC)State)Health) 30) 30) Dept.) Beaufort)Co.,)NC) BRU) ) ) 15) ) ) 15) Lance)Durden) Brunswick)Co.,)NC) ) ) ) ) Marcee)Tolliver,) BUL) NC)State)Health) 5) 5) Dept.) Bulloch)Co.,)GA) ) ) ) ) Marcee)Tolliver,) CAM) NC)State)Health) 15) 15) Dept.) Camden)Co.,)NC) CHA) ) ) ) 7)) 7) This)study) Charleston)Co.,)SC) CHS) ) ) 10) ) 13) ) 23) This)study) Chesapeake,)VA) CRS) ) ) 2)) 2) This)study) Creswell,)NC) HAM) ) ) ) 2)) 2) This)study) Hampton,)VA) ILW) ) ) 1)) ) 1) This)study) Isle)of)Wight)Co.,)VA) KEL) ) ) ) ) 1)) 1) This)study) Kelly,)NC) LYN) 8) 8) This)study) Lynchburg,)SC) PT2) ) ) 6)) 15) ) 21) This)study) Portsmouth,)VA) RAV) ) ) 9)) 9) This)study) Ravenel,)SC)) RID) ) ) ) ) 6) 6) This)study) Ridgeville,)SC) SEV) ) ) ) ) 4) 4) This)study) Seven)Springs,)NC) TOP) ) ) ) 1)) 1) This)study) Topping,)VA) VB3) ) 15) ) 20) ) 19) ) 54) This)study) Virginia)Beach,)VA) WAS) ) 1)) 1) This)study) Washington,)NC) YRK) ) 6)) 12) ) 13) ) 31) This)study) York)Co.,)VA) Total)I.*affinis) )sequenced) )) ) 236) )) )) * *

122

) Standard)PCR)was)used)to)amplify)a)fragment)of)the)tick)mitochondrial)16S)rRNA) gene)using)primers)16S+1)(5′=CTGCTCAATGATTTTTTAAATTGCTGT=3′))and)16S=1)(5′=

GTCTGAACTCAGATCAAGT=3′))(Macaluso)et)al.,)2003;)Nadolny)et)al.,)2011).)PCR)reactions) were)performed)in)15)µL)reaction)volumes,)with)0.05)U/µL)Taq)DNA)Polymerase)

(BioPioneer)Inc.,)San)Diego,)CA),)1)µM)each)primer,)1.5)mM)MgCl2,)1X)PCR)buffer)(Qiagen)

Inc.,)Valencia,)CA))and)2)µL)DNA)template.)The)PCR)protocol)consisted)of)an)initial)3=min) denaturation)step)at)95°C)followed)by)30)cycles)of)95°C)for)30)seconds,)52°C)for)45)seconds) and)72°C)for)1)minute,)with)a)final)extension)at)72°C)for)7)minutes.*PCR)products)were) visualized)on)1.5%)agarose)gels,)and)purified)using)ExoSap=IT)(Affymetrix)Ltd.,)Santa)Clara,)

CA).)Sequencing)reactions)were)performed)using)the)BigDye)Terminator)v.3.1)Cycle)

Sequencing)Kit)(Applied)Biosystems,)Foster)City,)CA).)Amplicons)with)overlapping)peaks,) indicating)heteroplasmy,)were)cloned)into)the)PCR)2.1)TOPO)vector)using)the)TOPO)TA) cloning)kit)(Invitrogen,)Carlsbad,)CA),)and)resulting)plasmid)inserts)were)sequenced,)as) above.)DNA)sequences)were)identified)by)BLAST)search)(Altschul)et)al.,)1990).)Successfully) amplified)DNA)provided)unambiguous)bi=directional)sequence)data)along)the)length)of)the) sequence)for)all)samples.)All)sequences)were)aligned)and)consensus)sequences)were) constructed)using)Geneious)R7)(http://www.geneious.com;)Kearse)et)al.,)2012).)

Quantitative*analysis*

Sample)size)and)haplotype)variation)

To)determine)the)approximate)sample)size)required)to)capture)all)haplotypes)in)a) given)population,)the)“vegan”)package)in)R)(R)Core)Team,)2015))was)used)to)compute) rarefaction)curves,)following)methods)outlined)by)Lindblom)(2009).))Rarefaction)uses)

123 individual=based)resampling)curves)to)quantify)the)number)of)haplotypes)found)in)each) population)(Gotelli)and)Colwell,)2001))by)randomly)subsampling)the)community)and) plotting)the)number)of)observed)haplotypes)against)the)number)of)observations)until)an) asymptote)is)reached.)To)assess)whether)sample)sizes)provided)total)coverage)of)the) haplotypes)present)in)the)populations)sampled,)the)software)EstimateS)(Colwell)et)al.,)

2004))was)used)to)apply)two)non=parametric)methods,)a)Chao)1)richness)estimator)and)an) abundance=based)coverage)estimator)(ACE))(Lindblom,)2009).)EstimateS)and)the)“vegan”) package)in)R)were)also)used)to)create)haplotype)accumulation)curves)to)visualize)the) number)of)haplotypes)detected)in)relation)to)the)number)of)sites)sampled)(Lindblom,)

2009).)These)methods)enabled)an)estimation)of)how)many)haplotypes)were)likely)missed) by)the)sampling)efforts)as)described.*

)

Phylogenetic)tree)construction)

The)software)MEGA)v)6.06)(Tamura)et)al.,)2007))was)used)to)select)the)best=fitting) molecular)evolutionary)models,)and)to)create)neighbor=joining)and)maximum)likelihood) phylogenies)with)1000)bootstrap)replicates.)JModelTest)(Guindon)and)Gascuel,)2003;)

Darriba)et)al.,)2012))was)used)to)validate)the)model)selections)made)by)MEGA,)and)the)

MrBayes)plugin)in)Geneious)was)used)to)create)Bayesian)trees)(Huelsenbeck)and)Ronquist,)

2001;)Ronquist)and)Huelsenbeck,)2003).*

)

Population)genetic)structure)

Analysis)of)molecular)variance)(AMOVA))was)implemented)in)Arlequin)v)3.5)

(Excoffier)and)Lischer,)2010))to)explore)patterns)of)population)genetic)structure)within)

124

and)among)groups.)Population)structure)was)assessed)by)generating)pairwise)ΦST)values) among)sites.)False)discovery)rate)q)values)based)on)the)ΦST)values)were)used)to)minimize) the)probability)of)Type)I)error)in)multiple)pairwise)comparisons)(Pike,)2011).)Minimum) spanning)trees)(MSTs))of)confirmed)haplotypes)were)created)using)output)from)Arlequin) visualized)using)HapStar)(Teacher)and)Griffiths,)2011).*

A)spatial)analysis)of)molecular)variance)based)on)Φ)statistics)was)calculated)using)

SAMOVA,)v)2.0)(Dupanloup)et)al.,)2002))to)define)groups)of)populations)that)were) geographically)homogeneous)and)maximally)differentiated.)The)method)uses)a)simulated) annealing)procedure)to)maximize)the)proportion)of)total)genetic)variance)as)a)result)of) differences)between)groups)of)populations.)The)analysis)was)run)for)2=11)groups)for)each) tick)species,)with)100)permutations.))

To)determine)if)there)was)significant)genetic)isolation)by)distance)the)“vegan”) package)and)“ade4”)library)were)implemented)in)R)to)perform)Mantel)and)partial)Mantel) tests.)These)tests)used)distance)matrices)based)on)ΦST)and)the)over=land)distance)between) the)latitude)and)longitude)coordinates)of)each)collection)site.)If)clustering)of)haplotypes) into)clades)was)observed,)partial)Mantel)tests)were)also)conducted)to)determine)if) populations)exhibited)hierarchical)clustering)rather)than)true)isolation)by)distance)

(Merimans,)2012).)Partial)Mantel)tests)were)conducted)using)a)third)matrix)of)zeroes)and) ones)indicating)the)dominant)clade)of)each)population.)Using)Arlequin,)mismatch) distributions)(τ))were)tested)within)individual)sites)as)well)as)among)all)samples)for) departure)from)models)representing)spatial)and)demographic)expansion.)Departures)from) selective)neutrality)were)detected)using)Tajima’s)D)and)Fu’s)FS)statistics,)both)of)which)

125 assess)the)difference)between)observed)and)expected)numbers)of)alleles)based)on) observed)nucleotide)diversity)(Tajima,)1989;)Fu,)1997).)

)

RESULTS!

Ixodes*affinis*

Sample)size)and)haplotype)variation)

Ixodes*affinis)were)collected)from)19)sites)in)the)US)from)Georgia)to)Virginia.)The) number)of)I.*affinis)collected)and)sequenced)from)each)site)varied)from)1)to)55)over)the) span)of)4)years,)totaling)236)individuals)(Table)17).)No)sequences)exhibited)heteroplasmy.)

Haplotype)rarefaction)curves)were)generated)for)all)sites,)treating)all)years)as)independent) samples,)with)most)curves)approaching)an)asymptote,)indicating)no)new)haplotypes)being) discovered,)at)5)or)fewer)individuals)(Fig.)15A).)A)population)haplotype)accumulation) curve)failed)to)reach)an)asymptote,)indicating)that)haplotype)sampling)was)not)nearing) completeness)when)all)samples)and)sites)were)included,)which)was)supported)by)ACE)and)

Chao)1)richness)estimators)(Fig.)16).*

)

126

) ) Figure!15.)Haplotype)rarefaction)curves)for)I.*affinis)and)A.*maculatum.)Rarefaction)curves) for)A))Ixodes*affinis)and)B))Amblyomma*maculatum)use)haplotypes)instead)of)species.)Each) line)is)representative)of)a)single)geographic)population)from)one)year,)and)indicates)the) number)of)individuals)sampled)and)the)number)of)haplotypes)found.) ) ) ) )

) !

Figure!16.)16S)haplotype)accumulation)curves)for)I.*affinis)and)A.*maculatum.)16S) haplotype)accumulation)curves)(Sobs))showing)the)mean)cumulative)number)of)haplotypes) in)relation)to)the)number)of)populations)sampled.)The)original)sampling)was)permuted) 1000)times.)Filled)diamonds)indicate)Amblyomma*maculatum*haplotypes,)including) heteroplasmic)haplotypes)(n)=)43);)filled)squares)indicate)Ixodes*affinis*haplotypes)(n)=)18).) Pan=population)haplotype)richness)was)estimated)for)I.*affinis*at)32)and)24)by)ACE)and) Chao)1)methods,)respectively,)implying)that)between)58%)and)78%)of)haplotypes)present) in)the)population)were)detected.)Haplotype)richness)for)A.*maculatum*was)estimated)at)60) and)72)by)ACE)and)Chao)1)methods,)respectively,)implying)that)between)73%)and)61%)of) haplotypes)present)in)the)population)was)detected.!

127

Phylogeny)

Eighteen)unique)16S)haplotypes)were)identified)(Genbank)accession)numbers)

KT037632–KT037649),)one)of)which)had)been)previously)reported)in)the)US)by)two) research)teams)(AFF5,)Genbank)accession)numbers)IAU95879)from)Sapelo)Island,)GA)

[Norris)et)al.,)1999])and)AF549834)from)an)unspecified)US)collection)location)[Xu)et)al.,)

2003]).)Ixodes*affinis)haplotypes)grouped)in)two)mitochondrial)clades,)and)are)labeled) numerically)as)AFF1)to)AFF12)(Clade)A))and)AFF13)to)AFF18)(Clade)B,)Fig.)17A).)One) additional)complete)16S)sequence)reported)in)Genbank)(Genbank)accession)number)

AF549861,)from)Colombia)[Xu)et)al.,)2003]))was)not)included)in)these)analyses;)therefore,) all)analyses)focused)on)US)samples.)The)18)haplotypes)detected)in)this)study)were) visualized)on)a)MST,)where)the)two)clades)observed)in)the)generated)phylogenetic)tree) were)clearly)separated)by)six)base)pair)changes)(Fig.)18A).)The)two)clades)showed) evidence)of)star)radiations)from)AFF4)and)AFF17,)with)an)intermediate)grouping)of)AFF9) to)AFF11.)))

)

128

) ! Figure! 17.) Phylogenetic) relationships) among) 16S) rRNA) mitochondrial) haplotypes) for) I.* affinis*and)A.*maculatum.)Haplotypes)are)based)on)Bayesian)(BA))inference)for)I.*affinis*(A)) and)A.*maculatum)(B).)Topology)was)consistent)between)neighbor=joining)(NJ),)maximum= likelihood) (ML)) and) Bayesian) inferences.) Branch) lengths) are) according) to) the) indicated) scale)of)nucleotide)substitutions)per)base)pair.)Node)values)indicate)branch)support,)based) on)1000)replicates.)(A))Ixodes*affinis)specimens)(n)=)236))collected)across)the)southeastern) United)States)resolved)in)18)haplotypes)(454)base)pairs).)The)evolutionary)model)that)best) fit) these) data) was) the) Tamura) 3=parameter) model) with) a) discrete) gamma) distribution) (T93+G).) A) single) Ixodes* scapularis* sample) (Genbank) accession) number) KF146631)) was) used)to)root)the)tree.)(B))Amblyomma*maculatum)specimens)(n)=)357))collected)across)the) eastern)United)States)resolved)in)36)non=heteroplasmic)haplotypes)(416)base)pairs).)The) Tamura) 3=parameter) evolutionary) model) best) fit) these) data.) A) single) Amblyomma* cajennense*sample)(Genbank)accession)number)L34317))was)used)to)root)the)tree.)) )

! !

129

! Figure!18.)Minimum)spanning)trees)for)I.*affinis)and)A.*maculatum.)Minimum)spanning) trees)of)(A))18)I.*affinis)haplotypes)(AFF1)–)AFF18))and)(B))36)non=heteroplasmic)A.* maculatum)haplotypes)detected)in)this)study)(MAC1)–)MAC36).)Each)labeled)circle) represents)a)haplotype)that)differs)by)one)base)pair)from)a)connecting)haplotype;)black) circles)indicate)the)number)of)additional)base)pair)changes)needed)to)connect)two) haplotypes.)Each)haplotype)is)labeled)with)the)number)of)individuals)with)that)haplotype.) Each)haplotype)is)labeled)with)the)number)of)individuals)with)that)haplotype.)Ixodes*affinis* Clade)A)haplotypes)were)mostly)found)in)the)south,)while)Clade)B)haplotypes)were)mostly) found)in)the)north.)There)was)no)strong)evidence)of)clades)in)these)A.*maculatum)samples.) !

Population)structure.))

Sampled)sites)with)≥)5)individuals)were)used)for)AMOVA)and)pairwise)ΦST))analyses.)

Global)structure)within)(accounting)for)55.7%)of)variation))and)among)groups)(44.3%)of) variation))was)detected)among)the)12)resulting)sites)(AMOVA)ΦST)=)0.44,)p<0.001).)

Haplotype)frequency)as)measured)by)ΦST)was)significantly)different)between)many)of)the) described)sampling)sites)(Table)18).)Haplotype)frequencies)were)most)often)significantly) different)between)sites)in)the)north)(North)Carolina)northward))and)in)the)south)(South)

Carolina)southward),)while)sites)within)each)region)showed)little)difference)in)haplotype) frequency.)

130

AFF17)was)the)most)frequently)encountered)haplotype)(115)individuals),)followed) by)AFF4)(50)individuals),)the)latter)of)which)had)been)detected)in)previous)studies)in)

Georgia)(Genbank)accession)numbers)IAU95879)and)AF549834).)A)Mantel)test)comparing) matrices)for)spatial)and)genetic)distance)between)the)12)sites)with)≥)5)ticks)showed) evidence)for)isolation)by)distance)(r)=)0.58,)p)=)0.003,)2000)permutations).)Partial)Mantel) tests)indicated)that)this)initial)result)was)due)to)spatial)clustering)of)I.*affinis)populations,) with)no)significant)isolation)by)distance)observed)when)using)a)matrix)accounting)for) clustering)as)a)covariate)(r)=)0.00,)p)=)0.51),)and)significant)isolation)by)distance)when) using)the)geographic)distance)matrix)as)a)covariate)(r)=)0.41,)p)=)0.006).)Haplotypes)from)

Clade)A)were)predominant)in)southern)sites,)while)haplotypes)from)Clade)B)were) predominant)in)northern)sites)(Fig.)19A).)SAMOVA)analysis)also)supported)that)the) southern)sites)represented)a)separate)population)from)the)northern)sites,)with)the)model) containing)two)groups)yielding)the)best=supported)FCT)value)(Table)19,)FCT)=)0.63).))

Four)sites)were)sampled)in)multiple)years)to)determine)if)there)were)significant) shifts)in)the)dominant)I.*affinis*haplotypes)at)each)site)over)time)(Table)20).)Sites)were) chosen)if)≥)5)individuals)were)collected)for)at)least)two)consecutive)years.)Only)one)site,)

CHS,)had)significantly)different)haplotype)frequencies)between)two)years)(AMOVA)ΦST)=)

0.17,)p)=)0.03),)although)82%)of)variation)was)still)observed)within)years.)

Table&18.!Pairwise!ΦST!and!false!discovery!rate!(FDR)!adjusted!q!values!for!I.#affinis.!Columns!and!rows!are!arranged!from! northernmost!site!to!southernmost!site.!Above!the!grey!cells!with!stars!are!FDR!adjusted!q!values,!those!that!are!significantly! different!are!marked!with!*.!Below!are!pairwise!ΦST!values!for!all!sites!with!≥!5!ticks.! ! YRK& PT2& VB3& CHS& CAM& BEA& BRU& LYN& CHA& RID& RAV& BUL& (n=31)& (n=21)& (n=54)& (n=23)& (n=15)& (n=30)& (n=15)& (n=8)& (n=7)& (n=6)& (n=9)& (n=5)& YRK! & 0.6554! 0.1471! 0*! 0*! 0.1642! 0*! 0*! 0*! 0*! 0*! 0*! PT2& ! Q0.0146! 0.0084*! 0*! 0*! 0.0499*! 0*! 0*! 0*! 0*! 0*! 0*! VB3& 0.0216! ! 0.0774! 0*! 0*! 0.0386*! 0*! 0*! 0*! 0*! 0*! 0*! CHS& 0.4684! 0.5164! ! 0.3944! 0.5341! 0*! 0*! 0*! 0*! 0*! 0*! 0*! CAM& 0.3412! 0.4035! 0.2666! ! Q0.0083! 0*! 0*! 0*! 0*! 0*! 0*! 0*! BEA& 0.0231! 0.0622! 0.0333! 0.3754! ! 0.2484! 0*! 0*! 0*! 0*! 0*! 0*! BRU& 0.6477! 0.7381! 0.5270! 0.3837! 0.4165! ! 0.5029! 0.0653! 0.5139! 0.0084*! 0.0326*! 0.6107! ! 0.0318 LYN& 0.4542! 0.5548! 0.3316! 0.2039! 0.1837! 0.2732! 0.0577! 0.3569! 0.5732! 0.2278! *! CHA& 0.7363! 0.8628! 0.5994! 0.4631! 0.5058! 0.5909! 0.0177! ! 0.1905! 0.0184*! 0.0791! 0.9879! ! 0.0326 RID& 0.5557! 0.6605! 0.4319! 0.2503! 0.2442! 0.3679! 0.2722! 0.0131! 0.4815! 0.1525! *! RAV& 0.5442! 0.6353! 0.4254! 0.2431! 0.2511! 0.3872! 0.0678! Q0.0239! 0.1771! ! 0.1258! 0.2634! BUL& 0.7232! 0.8522! 0.5859! 0.4336! 0.4687! 0.5706! Q0.0233! 0.1304! 0! 0.4128! ! 0.1211! ! ! 131 131

Figure&19.!Maps!of!I.#affinis!and!A.#maculatum!haplotypes!across!the!eastern!United!States.!(A)!Map!of!I.#affinis#haplotypes! collected!across!19!sites.!Red!haplotypes!are!from!Clade!A!(AFF1!–!AFF12)!and!blue!haplotypes!are!from!Clade!B!(AFF13!–! AFF18).!(B)!Map!of!haplotypes!of!A.#maculatum!ticks!collected!across!19!sites.!Although!there!were!significant!differences!in! haplotype!frequencies!between!some!sites,!no!geographic!pattern!was!evident.! 132 132

Table&19.!Ixodes#affinis#mitochondrial!rRNA!SAMOVA!population!groupings.!! & Groups&(K)& Structure&recovered& FCT& 2& [BRU,!BUL,!RAV,!RID,!CHA,!LYN]![BEA,!CAM,!YRK,!PT2,!VB3,!CHS]! 0.637! 3& [RID]![BRU,!BUL,!RAV,!CHA,!LYN]![BEA,!CAM,!YRK,!PT2,!VB3,!CHS]! 0.630! 4& [RID]![LYN]![BRU,!BUL,!RAV,!CHA]![BEA,!CAM,!YRK,!PT2,!VB3,!CHS]! 0.629! 5& [RID]![BUL]![LYN]![BRU,!RAV,!CHA]![BEA,!CAM,!YRK,!PT2,!VB3,!CHS]! 0.614! 6& [RID]![BUL]![RAV]![LYN]![BRU,!CHA]![BEA,!CAM,!YRK,!PT2,!VB3,!CHS]! 0.605! 7& [RID]![BUL]![RAV]![LYN]![CAM]![BRU,!CHA]![BEA,!YRK,!PT2,!VB3,!CHS]! 0.553! 8& [RID]![BUL]![RAV]![LYN]![CAM]![CHS]![BRU,!CHA]![BEA,!YRK,!PT2,!VB3]! 0.506! 9& [RID,!LYN]![BUL,!RAV,!CHA]![CAM]![CHS]![BRU]![BEA]![YRK]![PT2]![VB3]! 0.499! 10& [RID]![LYN]![BUL,!RAV,!CHA]![CAM]![CHS]![BRU]![BEA]![YRK]![PT2]![VB3]! 0.510! 11& [RID]![LYN]![RAV]![BUL,!CHA]![CAM]![CHS]![BRU]![BEA]![YRK]![PT2]![VB3]! 0.539! ! ! ! Table&20.!Changing!haplotype!frequencies!of!I.#affinis#and!A.#maculatum#ticks!sampled!over!time.!The!number!of!haplotypes! present!in!each!year!is!reported,!with!the!number!of!individuals!collected!in!that!year!in!parentheses.!AMOVA!analysis!over! multiple!years!resulted!in!ΦST!and!p!values!for!each!site,!and!indicated!the!amount!of!haplotype!variation!accounted!for!within! and!between!years!at!each!site.!Sites!marked!with!*!showed!significant!changes!in!haplotype!frequency!across!years.! ! Tick!Species! Site! 2010! 2011! 2012! ΦST! p! Variation!within!years! I.#affinis# CHS*! 0! 5!(10)! 3!(13)! 0.17! 0.03! 82%! I.#affinis# PT1! 0! 2!(6)! 2!(15)! Q0.06! 0.50! 100%! I.#affinis# VB3! 4!(15)! 3!(20)! 4!(19)! Q0.03! 0.70! 100%! I.#affinis# YRK! 2!(6)! 2!(13)! 4!(13)! Q0.06! 0.50! 100%! A.#maculatum# CHS! 1!(1)! (10)!44! 9!(40)! Q0.02! 0.88! 100%! A.#maculatum# FX2! 0! 7!(18)! 14!(50)! 0.03! 0.07! 96%! A.#maculatum# NCA*! 0! 6!(10)! 9!(28)! 0.09! 0.04! 90%! A.#maculatum# VB1! 3!(9)! 3!(5)! 6!(34)! Q0.04! 0.73! 100%! 133 133

134

Mismatch)distributions)revealed)no)evidence)of)spatial)(τ!=)7.79,)p)=)0.19))or) demographic)expansion)(τ!=)9.4,)p)=)0.08))for)the)population)when)analyzed)as)a)whole.)

Neutrality)tests)(D)=)0.50,)p)=)0.72;)FS)=)I0.12,)p)=)0.55))also)revealed)no)significant) evidence)for)departures)from)neutrality)when)all)samples)were)included.)There)were)also) no)significant)indicators)for)spatial)expansion)or)departures)from)neutrality)when)we) analyzed)separately)the)northern)(τ)=)8.22,)p)=)0.35;)FS)=)0.23,)p)=)0.57;)D)=)0.23,)p)=)0.64)) and)southern)(τ)=)2.04,)p)=)0.42;)FS)=)I1.47)p)=)0.19)D)=)I1.37,)p)=)0.06))populations)that) were)supported)by)SAMOVA)analysis.)Both)northern)and)southern)populations,)however,) showed)evidence)of)recent)demographic)expansion)(τ)=)0,)p)<)0.001).)

)

Amblyomma!maculatum!

Sample)size)and)haplotype)variation)

Amblyomma!maculatum)were)collected)from)18)sites)across)the)eastern)US.)The) number)of)A.!maculatum)collected)and)sequenced)from)each)site)varied)from)one)to)90) across)the)four)years,)totaling)370)individuals)(Table)17).)Rarefaction)curves)were) generated)for)all)sites,)treating)all)years)as)independent)samples,)and)curves)started) approaching)asymptote)at)around)10)individuals)(Fig.)15B).)The)haplotype)accumulation) curve)failed)to)reach)an)asymptote,)indicating)that)haplotype)sampling)was)not)nearing) completeness)when)all)sites)were)included,)which)was)supported)by)ACE)and)Chao)1) richness)estimators)(Fig.)16).!

)

135

Heteroplasmy!

Of)the)A.!maculatum)analyzed,)13)out)of)370)ticks)exhibited)heteroplasmy)(3.5%)of) individuals))at)one)or)more)sites)in)the)mitochondrial)16S)rRNA)gene.)Most)heteroplasmic) sites)were)within)a)short)stretch)of)base)pairs)on)a)hairpin)loop)region)of)the)rRNA) molecule.)Heteroplasmic)sites)were)confirmed)using)TA)cloning)on)a)representative)sample) of)individuals)exhibiting)multiple)peaks)at)a)given)base)pair)site.)Cloned)plasmids) containing)rRNA)from)one)individual)exhibited)one)phase)when)sequenced.)The)ticks)that) exhibited)heteroplasmy)were)from)six)sites)that)were)geographically)distant)from)one) another,)and)included)five)ticks)from)CHS,)two)ticks)from)VB1,)two)from)NCA,)two)from)

FX2,)one)from)KY2)and)one)from)MS5.)The)ticks)that)exhibited)heteroplasmy)were)not) included)in)these)analyses,)due)to)the)inability)to)resolve)the)phase)of)the)heteroplasmic) sites.!

)

Phylogeny)

ThirtyIsix)unique)haplotypes)were)identified)that)were)not)heteroplasmic)(Genbank) accession)numbers)KT037650–KT037685),)several)of)which)had)been)partially)sequenced) by)other)researchers)(Black)and)Piesman,)1994;)Ketchum)et)al.,)2009;)Ferrari)et)al.,)2013).)

To)construct)phylogenic)trees,)only)complete)416)bp)sequences)were)used,)thus)conserving) all)variable)sites)within)analyses)(Fig.)17B).)Although)there)was)abundant)haplotype) diversity)in)the)A.!maculatum!samples)sequenced,)no)evidence)of)clades)emerged)from)the) generated)trees.)By)visualizing)the)36)haplotypes)using)an)MST,)it)was)evident)that)the)lack) of)clades)was)due)to)very)few)base)pair)changes)between)haplotypes)(Fig.)18B).)There)was) evidence)for)star)radiations)from)haplotypes)MAC16,)MAC8,)and)MAC9,)and)haplotypes)

136

MAC1)and)MAC2)were)distinct)from)the)rest)of)the)haplotypes)by)seven)base)pair)changes.)

Population)structure)

Sites)with)≥)10)and)5)individual)A.!maculatum)were)used)for)AMOVA)and)pairwise)

ΦST)analyses,)respectively.)Global)structure)within)(85.44%)of)variation))and)among)groups)

(14.56%)of)variation))was)detected)among)the)9)resulting)populations)with)≥)10) individuals)(ΦST)=)0.15,)df)=)318,)p)<)0.001).)Haplotype)frequency)as)measured)by)ΦST)was) significantly)different)between)almost)all)of)the)sampling)sites,)indicating)little)connectivity) between)sites)with)≥)5)individuals)(Table)21).)Sites)VB1)and)FX2)(ΦST)=)0.04,)q)=)0.12))and)

MS2)and)TNS)(ΦST)=)0.05,)q)=)0.08),)had)haplotype)frequencies)that)were)not)significantly) different.)Those)sites)with)the)smallest)sample)sizes)(MS3,)MS4,)and)MS7,)all)from)

Mississippi))exhibited)more)statistically)similar)haplotype)frequencies)to)other)sites)in)the) pairwise)ΦST)analysis)(Table)21).!

A)Mantel)test)comparing)matrices)for)physical)and)genetic)distance)among)the)12) sites)with)≥)5)ticks)showed)no)evidence)for)isolation)by)distance)(r)=)10.01,)p)=)0.44,)2000) permutations).)Although)there)were)significant)differences)in)haplotype)frequencies) between)most)sites,)no)geographic)pattern)was)evident)(Fig.)19B).)Sites)in)geographic) proximity)to)each)other)were)not)more)likely)to)have)similar)haplotype)frequencies.)MAC8) was)the)most)frequently)encountered)haplotype)(60)individuals),)followed)closely)by)

MAC16)(52)individuals),)and)both)of)these)were)centers)of)star)radiations)(Fig.)18B))and) found)at)multiple)sites)(Fig.)19B).)SAMOVA)analysis)supported)that)each)of)the)sampling) sites)represented)a)genetically)isolated)population)(Table)22).)

137

Mismatch)distributions)revealed)no)evidence)of)spatial)(τ!=)2.26,)p)=)0.20))or) demographic)expansion)(τ)=)3.09,)p)=)0.27))when)all)individuals)were)analyzed)as)one) population.)Tajima’s)D)statistic)revealed)no)evidence)for)departures)from)neutrality)for)the) whole)population)(D)=)I0.45,)p)=)0.38),)but)Fu’s)FS)statistic)suggested)evidence)of)recent) expansion)(FS)=)I13.88,)p)=)0.001).)There)were)few)significant)indicators)for)departures) from)neutrality)or)expansion)when)examining)individual)sites,)with)only)populations)YRK)

(FS)=)I1.41,)p)=)0.03))and)MS3)(FS)=)I2.95,)p)=)0.02))departing)from)neutrality)by)Fu’s)F) statistic,)and)only)MS3)indicating)spatial)expansion)(τ!=)1.16,)p)=)0.04).)

Four)sites)were)sampled)in)multiple)years)to)determine)if)there)were)significant) shifts)in)the)dominant)I.!affinis!haplotypes)at)each)site)over)time)(Table)20).)Sites)were) chosen)if)≥)5)individuals)were)collected)for)at)least)two)consecutive)years.)Only)one)site,)

NCA,)had)significantly)different)haplotype)frequencies)between)two)years)(AMOVA)ΦST)=)

0.09,)p)=)0.04),)although)90%)of)variation)was)still)observed)within)years.)

Table&21.!Pairwise!ΦST!and!false!discovery!rate!(FDR)!adjusted!q!values!for!A.#maculatum.!Above!the!grey!cells!with!stars!are! FDR!adjusted!q!values,!those!that!are!significantly!different!are!marked!with!*.!Below!are!pairwise!ΦST!values!for!all!sites!with! ≥!5!ticks.! ! VB1& YRK& TNS& FX1& FX2& MS2& MS3& MS4& MS7& NCA& CHS& KY2& Site&& (n=48)& (n=13)& (n=19)& (n=38)& (n=30)& (n=13)& (n=9)& (n=9)& (n=7)& (n=38)& (n=85)& (n=35)& VB1& 0*! 0.0234*! 0.0234*! 0.1167! 0*! 0.4836! 0.0234*! 0.4675! 0*! 0*! 0*! ! YRK& 0.4369! 0*! 0*! 0*! 0*! 0*! 0*! 0.0118*! 0*! 0*! 0*! ! TNS& 0.0705! 0.2293! 0.0118*! 0.0438*! 0.0840! 0.1285! 0.1277! 0.8918! 0*! 0.0118*! 0.0334*! ! FX1& 0.0432! 0.3497! 0.0854! 0*! 0*! 0.1277! 0.0626! 0.2557! 0*! 0*! 0*! ! FX2& 0.0374! 0.3563! 0.0540! 0.0954! 0*! 0.3134! 0.0334*! 0.4675! 0.0371*! 0*! 0*! ! MS2& 0.2294! 0.1085! 0.0500! 0.1526! 0.1714! 0*! 0.7781! 0.0969! 0*! 0.0334*! 0*! ! MS3& S0.0235! 0.5769! 0.0360! 0.0298! 0.0150! 0.2092! 0.0118*! 0.6166! 0.2779! 0.0334*! 0.0371*! ! MS4& 0.1496! 0.2251! 0.0497! 0.0739! 0.1206! S0.0600! 0.1436! 0.2140! 0*! 0.3960! 0.0277*! ! MS7& S0.0407! 0.4707! S0.0719! 0.0165! S0.0233! 0.0722! S0.0513! 0.0331! 0.3576! 0.2291! 0.1703! ! NCA& 0.0648! 0.4334! 0.0798! 0.1059! 0.0355! 0.2498! 0.0194! 0.2096! 0.0048! 0*! 0*! ! CHS& 0.0894! 0.2020! 0.0772! 0.0870! 0.0960! 0.0632! 0.1280! S0.0024! 0.0215! 0.1644! 0*! ! KY2& 0.2085! 0.3360! 0.0990! 0.1715! 0.1846! 0.1166! 0.1363! 0.1186! 0.0936! 0.2033! 0.2251! ! ! !

& & 138 138

Table&22.!Amblyomma#maculatum#mitochondrial!rRNA!SAMOVA!population!groupings.& & Groups&(K)& Structure&recovered& FCT& 2& [VB1,!TNS,!FX1,!FX2,!MS2,!MS3,!MS4,!MS7,!NCA,!CHS,!KY2]![YRK]! 0.174! 3& [VB1,!TNS,!FX1,!FX2,!MS2,!MS3,!MS4,!MS7,!NCA,!CHS]![KY2]![YRK]! 0.176! 4& [MS2]![VB1,!TNS,!FX1,!FX2,!MS3,!MS4,!MS7,!NCA,!CHS]![KY2]![YRK]! 0.164! 5& [MS2]![MS4]![VB1,!TNS,!FX1,!FX2,!MS3,!MS7,!NCA,!CHS]![KY2]![YRK]! 0.150! 6& [MS2]![MS4,!CHS]![FX1]![VB1,!TNS,!FX2,!MS3,!MS7,!NCA]![KY2]![YRK]! 0.134! 7& [FX1]![KY2]![VB1,!FX2,!MS3,!MS7]![TNS]![NCA]![MS4,!CHS]![YRK,!MS2]!! 0.139! 8& [MS2,!MS4]![YRK]![FX1]![NCA]![VB1,!FX2,!MS3,!MS7]![TNS]![CHS]![KY2]! 0.151! 9& [TNS,!MS7]![YRK]![FX2]![KY2]![VB1,!MS3]![MS2,!MS4]![NCA]![CHS]![FX1]! 0.177! 10& [MS3]![VB1]![FX1]![KY2]![YRK]![NCA]![MS2,!MS4]![FX2]![TNS,!MS7]![CHS]! 0.184! 11& [VB1]![YRK]![TNS!MS7]![FX1]![FX2]![MS2]![MS3]![MS4]![NCA]![CHS]![KY2]! 0.192! ! 139 139

140

DISCUSSION(

Because'of'the'recent'reports'documenting'the'range'expansions'of'I.#affinis'

(Harrison'et'al.,'2010;'Nadolny'et'al.,'2011)'and'A.#maculatum#(reviewed'by'Paddock'and'

Goddard,'2015),'evidence'of'northward'population'expansion'was'expected'in'both' species.'Instead,'the'genetic'population'structure'of'each'species'was'unique,'and'neither' pattern'supported'the'original'simple'hypothesis'of'populations'spreading'north'from' closely'related'southern'populations.'Populations'of'both'species'from'sites'in'northern' and'southeastern'Virginia'are'<10'years'old,'and'are'unlikely'to'contain'significant'onKsite' mutation.'Populations'of'both'I.#affinis'and'A.#maculatum'sampled'over'multiple'years' tended'to'have'relatively'stable'haplotype'frequencies'(Table'20),'suggesting'that' haplotypes'introdued'in'previous'years'tended'to'persist'over'time.'

Ixodes#affinis#

This'is'the'first'study'of'the'population'genetics'of'I.#affinis.'Ixodes#affinis'was' characterized'by'two'genetic'clades'with'a'clear'geographic'break,'with'Clade'A'dominant' in'South'Carolina'and'Georgia'and'Clade'B'dominating'in'North'Carolina'and'Virginia'(Fig.'

19A,'Table'18,'Table'21).'Clade'A'was'more'diverse'than'Clade'B,'indicating'that'I.#affinis' has'been'established'longer'at'the'southern'sites'than'the'northern'sites.'This'pattern'was' expected,'but'was'especially'striking'since'82%'of'individual'I.#affinis#in'this'study'were' collected'from'northern'sites,'but'66%'of'haplotypes'were'from'southern'sites.'The'star' radiations'evident'in'each'clade,'however,'indicate'that'both'clades'are'expanding' independently'of'each'other'(Fig.'18A).'Mismatch'analyses'further'support'that'these' clades'are'both'undergoing'significant'demographic'expansion.'Results'from'SAMOVA'and'

141 partial'Mantel'tests'suggest'that'each'of'these'populations'is'independent.'These'results' indicate'that'I.#affinis'are'newly'arrived'in'the'north'(North'Carolina'and'Virginia)'and'that' this'population'is'expanding.'The'source'of'northern'populations'is,'however,'not'southern' populations,'but'rather'neighboring'northern'populations.'

So'how'did'the'northern'population'become'established?'There'are'a'number'of' possible'explanations'for'the'establishment'of'and'subsequent'genetic'barrier'between'the' two'I.#affinis'populations.'One'possibility'is'that'this'population'is'the'result'of'a'founder' event,'facilitated'by'the'longKdistance'dispersal'of'an'engorged'I.#affinis'female'north'of'

South'Carolina.'Those'initial'offspring'from'a'female'of'haplotype'AFF17'may'have'since' colonized'the'rest'of'the'northern'portion'of'the'I.#affinis'range,'radiating'into'closely' related'haplotypes'in'Clade'B'(Fig.'18A,'Fig.'19A).'Ixodes#affinis'feed'on'a'variety'of' mammalian'and'avian'hosts,'but'are'most'commonly'found'on'cervids,'canids,'and'felids'as' adults'(Harrison'et'al.,'2010).'These'large'mammals'could'transport'adult'I.#affinis'over' long'distances;'however,'such'longKdistance'dispersal'events'are'relatively'uncommon'for' hosts'of'I.#affinis,'which'would'account'for'the'lack'of'genetic'exchange'between'the'two' populations'after'the'initial'establishment.'Anthropogenic'movement'of'I.#affinis'on'whiteK tailed'deer'(Odocoileus#virginianus)'is'unlikely,'as'adult'I.#affinis'are'not'active'during'the' fall'and'winter'seasons'when'deer'are'killed'and'transported'by'hunters'(Oliver'et'al.,'

1987).'

Other'possible'explanations'for'the'genetic'segregation'of'the'two'I.#affinis' populations'include'haplotypeKcorrelated'behavioral'or'physiological'differences'that'allow' increased'survival'in'their'respective'regions.'As'I.#affinis#has'moved'northward'along'a' latitudinal'and'climatic'gradient,'adaptations'corresponding'with'haplotype'clade'may'

142 have'resulted'in'differential'survival.'For'example,'the'Northern'and'American'clades'of'I.# scapularis'exhibit'different'hostKseeking'behaviors'and'survival'ability'dependent'upon' their'respective'regional'clades'(Goddard'and'Piesman,'2006;'Ginsberg'et'al.,'2014).'A'third' explanation'could'be'the'presence'of'a'geographic'break'in'the'range'of'a'critical'host'that' dictates'the'presence'or'absence'of'particular'haplotypes.'For'example,'the'ranges'of'the' northern'shortKtailed'shrew'(Blarina#brevicauda)'and'the'southern'shortKtailed'shrew'(B.# carolinensis)'overlap'only'in'the'area'where'northern'clade'I.#affinis'are'found'(George'et' al.,'1986;'McCay,'2001).'Field'studies'on'shrews'and'the'genetic'makeup'of'their'associated' ticks'would'be'needed'to'test'this'hypothesis.'

Ixodes#affinis'haplotype'diversity'was'generally'low'with'few'haplotypes'present'at' each'site,'indicating'that'only'a'few'individual'ticks'would'be'needed'to'initiate'a' population.'Adult'I.#affinis'likely'play'an'important'role'in'forming'these'populations'via' shortKdistance'founder'events,'where'one'or'two'engorged'adult'females'might'drop'off'a' deer'or'other'large'mammal.'Few'of'the'sampled'populations'were'monohaplotypic,'and' gene'flow'was'documented'between'nearby'sampling'sites,'indicating'that'multiple'shortK distance'dispersal'events'are'needed'to'establish'and'maintain'populations.'Ixodes#affinis' are'predominantly'dispersed'by'mammals'at'all'stages'of'their'lives'(Harrison'et'al.,'2010).'

The'small'mammals'that'play'an'important'role'as'hosts'for'juvenile'I.#affinis'have'limited' vagility,'thereby'limiting'the'dispersal'potential'of'immature'I.#affinis.'Long'distance' dispersal'events'as'discussed'above'would'also'be'relatively'uncommon'for'most'hosts'of' adult'I.#affinis;'studies'of'the'role'of'whiteKtailed'deer'in'seed'dispersal'found'that'most' dispersal'events'were'only'within'hundreds'or'thousands'of'meters'(Myers'et'al.,'2004).'

Each'of'the'two'I.#affinis'clades'is'likely'to'be'undergoing'demographic'expansion'through'

143 these'shortKdistance'dispersal'events,'assisted'by'mammals'and'perhaps'nonKmigratory' birds.'These'shortKdistance'dispersal'events'would'be'facilitated'by'the'riparian'woodland' habitats'that'I.#affinis'favor,'which'are'abundant'in'the'eastern'US'and'have'relatively'high' connectivity'between'habitat'patches'(Griffith'et'al.,'2003).''

At'present,'all'populations'of'I.#affinis'in'the'US'are'in'Atlantic'coastal'plain' ecosystems'(Oliver'et'al.,'2003;'Harrison'et'al.,'2010;'Nadolny'et'al.,'2011).'Ixodes#affinis'are' found'in'secondary'successional'woodland'habitats,'and'the'adults'are'relatively'easy'to' collect'using'sampling'flags'where'they'are'found'(Nadolny'et'al.,'2011).'It'is'possible'that' coastal'plains'ecosystems'provide'all'the'essential'resources'I.#affinis'need'to'survive,' including'common'flooding'events'and'abundant'woodland'habitat,'but'these'ticks'are' unable'to'establish'outside'of'these'environmental'constraints.'Ixodes#affinis'may'yet' establish'in'areas'north'of'Virginia'considered'part'of'the'Atlantic'coastal'plain'(Auch,'

2014).''

Amblyomma#maculatum#

In'contrast'to'I.#affinis,'the'genetic'structure'of'A.#maculatum'indicated'that'there'is' low'gene'flow'among'sites,'including'those'that'are'geographically'proximate'(Fig.'19B,'

Table'21,'Table'22).'Little'evidence'of'genetic'clades'was'uncovered,'and'most'haplotypes' were'separated'by'only'1K3'base'pairs'(Fig.'18B).'Fu’s'FS'statistic'indicates'recent' population'growth'of'the'population'as'a'whole,'with'star'radiations'(Fig.'18B)'supporting' recent'expansion'of'A.#maculatum'throughout'the'eastern'US.'Populations'at'individual' sample'sites,'however,'were'generally'not'undergoing'expansion,'with'only'a'few' exceptions.'This'was'contrary'to'expectations,'as'A.#maculatum'has'been'documented'

144 spreading'into'the'MidKAtlantic,'presumably'from'established'populations'further'south'

(Fornadel'et'al.,'2011;'Wright'et'al.,'2011),'so'evidence'of'northward'population'expansion' was'expected.'''

Previous'studies'of'A.#maculatum#population'genetics'have'generally'documented' low'genetic'diversity'and'high'levels'of'gene'flow'(ArayaKAnchetta'et'al.,'2015).'Ferrari'et' al.'(2013)'described'four'16S'mitochondrial'rRNA'haplotypes'in'Mississippi'and'three'in'

North'Carolina#A.#maculatum,'with'little'genetic'variation'between'all'sampled'populations'

(FST'='0.02).'However,'they'found'strong'support'for'genetic'differences'between'individual' sites'within'regions.'A'separate'study'on'A.#maculatum#ticks'from'Texas,'Oklahoma'and'

Kansas'(Ketchum'et'al.,'2009)'reported'seven'16S'mitochondrial'rRNA'haplotypes,'with' low'genetic'variation'among'populations'that'had'been'established'for'20'or'more'years.'

This'study'was'performed'on'a'larger'scale'on'younger'populations,'which'could'explain' the'moderate'genetic'variation'observed'across'the'eastern'US'(ΦST'='0.15),'no'isolation'by' distance,'and'significant'genetic'differences'between'sites.'

Each'population'of'A.#maculatum'comprised'multiple'haplotypes,'indicating'that' multiple'females'were'involved'in'their'formation.'Interestingly,'the'same'haplotypes'were' found'widely'dispersed'throughout'the'eastern'US,'indicating'that'there'must'be'some' limited'gene'flow'between'even'widely'separated'populations'(Fig.'19B).'In'contrast,' populations'geographically'close'to'one'another'were'not'more'likely'to'share'haplotypes'

(Table'21).'Only'sites'with'very'few'individuals'sampled,'all'from'Mississippi,'exhibited' evidence'of'significant'gene'flow'among'populations'(Table'21),'which'may'be'a'statistical' artifact'of'small'sample'size.'Previous'studies'of'A.#maculatum#populations'have'shown' polyphyletic'haplotypes'and'implicated'multiple'introductions'in'the'genetic'diversity'

145 observed'(Ketchum'et'al.,'2009;'Ferrari'et'al.,'2013).'Ferrari'et'al.'(2013)'have'suggested' that'observed'A.#maculatum#interKpopulation'gene'flow'could'be'facilitated'by'longK distance'anthropogenic'movements'of'cattle'or'by'migratory'birds.''

In'order'for'gene'flow'to'occur'between'geographically'distant'populations,'long' distance'dispersal'must'be'taking'place.#Immature'A.#maculatum'parasitize'many'species'of' birds,'which'could'provide'opportunities'for'longKdistance'dispersal'(Teel'et'al.,'2010).'In' one'study,'up'to'2.4%'of'ticks'collected'from'mistKnetted'songbirds'over'2'years'were'A.# maculatum'larvae'and'nymphs'(Florin'et'al.,'2014).'Like'I.#affinis,'the'adults'of'A.# maculatum'largely'parasitize'deer'and'other'large'mammals'–'none'of'which'regularly' migrate'between'northern'Virginia'and'Mississippi.'Unlike'I.#affinis,'A.#maculatum#nymphs' and'larvae'have'been'reported'from'more'than'35'species'of'birds,'including'migratory' species'that'regularly'travel'long'distances'(Ogden'et'al.,'2008;'Teel'et'al.,'2010;'Florin'et' al.,'2014).'Immature'A.#maculatum'have'been'collected'from'migratory'birds'in'Canada,' well'outside'this'species’'range'(Ogden'et'al.,'2008).'Larvae'or'nymphs'arriving'at'a'new' area'after'feeding'on'birds'are'more'likely'to'initiate'populations'with'multiple'haplotypes' than'a'single'adult.'This'is'especially'likely'considering'mixed'flocks'of'birds'can'support' nymphs'or'larvae'that'are'unlikely'to'represent'identical'haplotypes,'possibly'founding'a' population'with'more'than'one'A.#maculatum'haplotype'during'a'migration'stop.'Even'new' populations'in'northern'Virginia'(<2'years'old)'generally'exhibited'multiple'haplotypes'in' the'adult'ticks'collected,'indicating'population'establishment'by'more'than'one'female.''

Migratory'birds'are'important'agents'of'longKdistance'dispersal'of'other'tick'species,' especially'I.#scapularis'as'it'expands'its'range'northward'into'Canada'(Smith'et'al.,'1996;'

Ogden'et'al.,'2008).'However,'models'of'I.#scapularis#range'expansion'into'Canada'suggest'

146 that'migratory'birds'are'not'the'sole'source'of'new'ticks,'with'mammalian'dispersal' resulting'in'faster'population'establishment'(Leighton'et'al.,'2012).'One'challenge'in' understanding'how'immature'birdKborne'ticks'could'initiate'new'populations'is'the'low' likelihood'of'survival'to'adulthood'and'successful'reproduction.'Ticks'spend'up'to'90%'of' their'lives'offKhost,'surviving'environmental'stressors'and'predation'(Needham'and'Teel,'

1991).'Risk'of'desiccation'is'a'particularly'important'consideration'for'ticks'of'all'life' stages,'and'A.#maculatum'is'sensitive'to'low'humidity'levels'(Fleetwood,'1985).'As'such,'the' range'of'A.#maculatum'is'restricted'to'warm,'moist'microhabitats'in'coastal'environments' with'temperate'climates'and'high'humidity'(Cooley'and'Kohls,'1944;'Goddard'and'

Norment,'1983;'Yoder'et'al.,'2008).''

Amblyomma#maculatum'adults'mate'onKhost'with'males'using'pheromones'to' attract'females,'which'eliminates'some'reproductive'challenges'when'ticks'are'at'low' densities'(Gladney,'1971;'Gladney'et'al.,'1974).'Adults'are'generally'considered'more'likely' to'establish'populations,'as'one'female'A.#maculatum'can'lay'15,000'eggs'(Drummond'and'

Whetstone,'1970;'Wright,'1971).'One'or'more'deer'arriving'at'a'successional'site'carrying' engorged'A.#maculatum#females'of'several'haplotypes'could'be'responsible'for'founding'a' genetically'diverse'new'population.'However,'deer'do'not'disperse'far'enough'to'account' for'the'presence'of'identical'haplotypes'in'geographically'distance'populations,'such'as'the' sampling'sites'in'southern'Mississippi'and'Northern'Virginia.'No'evidence'of'isolation'by' distance'was'found,'which'should'be'evident'if'there'was'significant'migration'between' sites'that'are'close'to'one'another.'It'is'likely'that'some'combination'of'avian'and' mammalian'hosts'is'responsible'for'the'patterns'observed,'and'additional'research'will'be'

147 required'to'conclusively'answer'questions'of'what'hosts'are'important'in'A.#maculatum# range'expansion.'

A'preference'for'grassy,'often'xeric'habitat'helps'to'explain'why'A.#maculatum' populations'are'so'genetically'isolated'from'nearby'populations.'Amblyomma#maculatum' are'often'found'in'grassy,'successional'habitats'(Wright'et'al.,'2011;'Paddock'and'Goddard,'

2015),'which'are'common'but'often'ephemeral'in'the'southeastern'US.'Due'to'a'history'of' fire'suppression'in'the'eastern'US,'anthropogenic'actions'such'as'clearKcutting'and' prescribed'burns'are'the'major'mechanism'by'which'open'habitats'are'created'and' preserved.'In'particular,'prescribed'burns'to'recreate'lost'grassland'habitat'have'been' increasing'in'recent'decades,'with'the'unintended'result'of'creating'ideal'patches'of'A.# maculatum#habitat'(Paddock'and'Goddard,'2015).'Burned'areas'are'recolonized'quickly'by'

A.#maculatum,'and'have'even'resulted'in'increased'tick'populations'within'a'year'of'a' prescribed'burn'(Scifres'et'al.,'1988).'Human'manipulation'may'create'patches'of'ideal' habitat'in'which'a'particular'haplotype'may'establish'and'thrive'for'a'sufficient'amount'of' time'to'be'picked'up'by'a'traveling'host'and'redistributed'across'the'landscape'to'another' suitable'spot.'

Populations'of'A.#maculatum'are'patchy,'and'are'likely'mediated'by'local' extirpations'as'these'open'habitats'complete'the'successional'process'and'become' unsuitable'for'A.#maculatum'and'its'preferred'host'community.'Small'mammals'are'likely'to' be'important'for'A.#maculatum#population'maintenance'in'successional'habitats,'but'are' not'likely'to'contribute'to'dispersal'due'to'canopy'closure'extirpating'local'rodent' populations'(Langley'and'Shure,'1980).'To'facilitate'formation'of'genetically'diverse' populations,'multiple'founders'must'arrive'at'an'appropriate'site'at'the'correct'time'in'the'

148 successional'process'when'hosts'are'abundant.'However,'appropriate'mixes'of'hosts'and' successional'habitat'are'only'available'until'succession'completes,'which'might'not'provide' sufficient'time'for'subsequent'generations'of'ticks'to'disperse'locally'like'I.#affinis.'Unless' open'habitat'is'maintained'by'prescribed'burning'or'other'intervention,'the'successional' process'may'swiftly'extirpate'populations'of'A.#maculatum,'so'the'nearest'neighbor' populations'remain'genetically'and'geographically'isolated,'seeded'only'by'longKdistance' dispersers.'As'a'result'of'the'ephemeral'and'geographically'isolated'nature'of'these' populations,'one'limitation'of'this'study'is'the'high'likelihood'of'missing'many'such' existing'populations'in'the'described'sampling.''

Heteroplasmy'was'discovered'in'the'16S'rRNA'gene'of'3.5%'of'sampled'A.# maculatum'ticks.'In'the'resolved'haplotypes,'heteroplasmy'was'distributed'across'the' eastern'US'and'was'not'limited'to'any'site'or'geographic'region.'Heteroplasmy'has'been' reported'previously'in'Amblyomma'ticks;'Xiong'et'al.'(2013)'described'166'heteroplasmic' sites'in'A.#cajennense,'including'22'sites'in'the'rRNA'genes.'Heteroplasmy'may'therefore'be' common'in'ticks'of'the'genus'Amblyomma;'recognizing'the'possibility'of'heteroplasmy'in' these'ticks'is'important'for'accurate'population'genetics'studies.''

In'conclusion,'the'present'study'is'unique'because'it'compares'patterns'of'genetic' structure'across'two'tick'species'that'share'many'of'the'same'vertebrate'hosts,'but'inhabit' different'habitats'across'the'eastern'US.'Marked'differences'were'found'in'population' structure,'connectivity'and'isolation,'as'well'as'differing'levels'of'genetic'diversity.'This' evidence'suggests'that'diverse'populations'of'A.#maculatum#are'founded'via'multiple' founder'events,'including'longKdistance'dispersal'events,'and'are'maintained'in'relative' isolation'by'ephemeral'assemblages'of'hosts'in'successional'habitats.'The'northern'I.#affinis'

149 population'was'likely'founded'by'a'rare'longKdistance'founder'event,'but'has'since'been' maintained'and'expanded'via'shortKdistance'dispersals'by'mammalian'hosts'with'small' home'ranges.'While'host'choice'is'a'key'ecological'factor'in'tick'range'expansions,'equally' important'are'habitat'stability,'habitat'connectivity'and'the'life'stages'that'are'responsible' for'dispersal.'Although'there'is'considerable'host'overlap'at'all'life'stages'for'I.#affinis'and'A.# maculatum,'these'other'ecological'differences'result'in'strikingly'different'geographic'and' genetic'patterns.''

' '

150

MODELING(THE(EFFECTS(OF(HABITAT(AND(HOST(PARAMETERS((

ON(TICK(INVASIONS(

INTRODUCTION(

Ticks'are'bloodKfeeding'ectoparasites'that'parasitize'humans'and'animals'and'are' second'only'to'mosquitoes'in'spreading'vectorKborne'diseases'worldwide'(Goodman'et'al.,'

2005).'Many'tick'species'are'expanding'their'ranges'as'a'result'of'anthropogenic'changes'in' the'landscape,'shifting'climatic'variables,'and'increasing'populations'of'suitable'host' species'and'suitable'tick'habitat'(Childs'and'Paddock,'2003;'Ogden'et'al.,'2006;'Ogden'et'al.,'

2008a).'Climate'change'has'been'forecasted'to'lead'to'an'overall'increase'in'tick'habitat'in' the'coming'years,'and'is'already'facilitating'tick'range'expansions'worldwide,'leading'to' increasing'disease'risks'(Cumming'and'Van'Vuuren,'2006;'George,'2008;'Léger'et'al.,'

2013).'It'is'essential'to'understand'factors'that'limit'tick'distribution'in'order'to'predict' disease'emergence,'as'eradication'of'ticks'and'their'associated'pathogens,'once'established,' may'be'impossible'(Cumming'and'Van'Vuuren,'2006;'Léger'et'al.,'2013).'

Tick'invasions'are'different'from'invasions'by'other'nonKhitchhiking'taxa'because' tick'life'history'is'sharply'demarcated'between'periods'of'movement'onKhost'and'longer' periods'offKhost.'In'order'to'understand'the'movement'patterns'of'ticks'across'a'landscape,' the'suitability'of'both'abiotic'and'biotic'factors'must'be'considered.'Ticks'have'a'complex' life'history'involving'differing'host'preferences'throughout'ontogeny,'and'thus'they'must' find'suitable'hosts'to'survive'in'the'environment.'Ticks'depend'on'the'largeKscale' movements'of'their'hosts'to'transport'them'across'a'landscape,'and'are'particularly' vulnerable'to'environmental'pressures,'such'as'desiccation,'when'they'are'freeKliving'offK

151 host'(Léger'et'al.,'2013).'Host'specificity'is'key'to'any'parasite’s'ability'to'disperse'across'a' landscape'and'invade'new'areas'(Kruse'et'al.,'2012).'Because'many'humanKbiting'species' of'ticks'are'generalists'and'can'feed'on'a'variety'of'avian,'mammalian,'and'reptilian'species' throughout'ontogeny,'tick'range'expansions'are'likely'limited'only'by'climatic'variables'

(Cumming'and'Van'Vuuren,'2006).'Ticks'are'strongly'dependent'on'both'host'availability' and'environmental'factors'for'their'survival'and'reproduction'in'any'habitat'(Léger'et'al.,'

2013),'but'the'relative'importance'of'hosts'and'habitats'in'tick'range'expansions'has'never' been'fully'explored.''

Models'have'been'used'to'elucidate'the'complex'life'history'of'ticks'and'to'mitigate' tickKborne'disease'risk.'Differential'equationK'based,'ageKstructured'difference,'and'matrixK based'models'have'provided'insight'into'the'population'dynamics'of'ticks'and'the' dynamics'of'tickKborne'disease'(Haile'and'Mount,'1987;'Sandberg'et'al.,'1992;'Mount'et'al.,'

1997;'Rosà'and'Pugliese,'2007;'Gaff'et'al.,'2009).'Spatially'explicit'components'have'been' added'by'using'remote'sensing,'GIS,'and'partial'differential'equation'models'(Radcliffe'and'

Rass,'1984;'Bunnel'et'al.,'2003;'DiukKWasser'et'al.,'2010).'While'helpful,'most'of'these' models'focus'primarily'on'proportional'interactions'between'ticks'and'hosts'that'inform' our'understanding'of'tick'populations'and'pathogens,'but'not'individual'movement.'

SpatiallyKexplicit'agentKbased'models'simulate'the'actions'of'individual'ticks'and'hosts'and' can'be'used'to'capture'the'mechanistic'phenomena'underlying'individual'episodes'of'range' expansion'(Madhav'et'al.,'2004;'Gaff,'2011;'Wang'et'al.,'2012;'Gaff'and'Nadolny,'2013;'

Wang'et'al.,'2015)'.'

Here,'a'spatiallyKexplicit'agentKbased,'stochastic'model'was'developed'to'simulate' the'spatioKtemporal'dynamics'of'tick'populations'in'the'years'immediately'following'

152 introduction'of'ticks'to'a'novel'environment.'Using'this'model,'derived'from'the'TICKSIM' model'(Gaff,'2011;'Gaff'and'Nadolny,'2013),'it'was'possible'to'determine'the'relative' strength'of'influence'that'host'and'habitatKbased'parameters'have'on'invasion'rate,' population'density,'geographic'pattern'of'tick'invasion,'and'the'genetic'diversity'in' resulting'tick'populations.'In'addition'to'addressing'broad'questions,'the'model'was'used' to'investigate'the'case'study'of'two'recent'tick'species'invasions'in'Virginia.'The'recent' simultaneous'range'expansions'of'the'two'ixodid'tick'species,'Ixodes#affinis#and'

Amblyomma#maculatum,#into'the'MidKAtlantic'region'of'the'US'provided'an'opportunity'to' compare'the'relative'influences'of'host'and'habitat'choice'on'invasion'dynamics'and' genetic'connectivity'(Nadolny'et'al.,'2015).'Information'gleaned'from'literature'values,' field'studies'on'tick'ecology,'and'lab'studies'on'tick'genetic'connectivity'was'used'to' parameterize'the'model,'and'the'emergent'properties'were'compared'to'determine'if'the' invasion'patterns'seen'in'the'field'could'be'replicated'in#silico'on'a'small'scale.''

In'the'sections'that'follow,'I'will'provide'some'background'information'and' modeling'considerations,'and'describe'the'model'following'the'protocol'recommended'for' agentKbased'models'by'Grimm'et'al.'(2010).'The'general'performance'of'the'model'will'be' evaluated,'as'will'the'sensitivity'of'simulated'tick'dynamics'to'changes'in'habitat'and'hostK related'parameters.'Finally,'the'applications'of'the'model'will'be'demonstrated'through' determining'the'relative'influence'of'habitat'suitability'and'host'density'on'simulated' invasions'by'a'generalized'tick,'and'the'influence'of'habitat'connectivity'on'the'genetic' signatures'of'newly'established'populations'of'simulated'I.#affinis#and'A.#maculatum.'

(

153

BACKGROUND(INFORMATION(AND(MODELING(CONSIDERATIONS'

Two'tick'species,'I.#affinis'and'A.#maculatum,'are'concurrently'expanding'their' ranges'into'the'MidKAtlantic'region'of'the'US'and'have'been'observed'invading'in'different' geographic'patterns,'and'with'different'genetic'signatures'(Nadolny'et'al.,'2015).'Ixodes# affinis'is'generally'found'in'disturbed'forested'habitat'and'is'a'generalist'tick'species'that' feeds'on'small'mammals'and'birds'during'immature'stages,'and'medium'and'large' mammals'during'the'adult'stage.'This'tick'species'exhibits'genetically'wellKmixed' populations'that'are'likely'created'and'maintained'through'shortKdistance'dispersal'events' throughout'the'contiguous'forested'habitat'that'is'abundant'in'the'MidKAtlantic'(Nadolny'et' al.,'2015).'Amblyomma#maculatum#is'another'generalist'tick'species'that'feeds'on'birds'and' mammals,'but'is'found'only'in'disturbed'open'habitats,'which'are'patchily'distributed' throughout'the'MidKAtlantic'(Harrison'et'al.,'2010;'Wright'et'al.,'2011).'Populations'of'this' tick'species'are'genetically'isolated'from'other'nearby'populations,'and'each'population'is' likely'founded'by'multiple'longKdistance'founding'events,'and'then'maintained'by'the'high' densities'of'rodent'hosts'that'are'present'in'grassKdominated'habitats'(Nadolny'et'al.,'

2015).'Both'I.#affinis'and'A.#maculatum#generally'complete'their'life'cycle'in'one'year'and' have'significant'overlap'in'the'hosts'parasitized'at'all'life'stages'(Harrison'et'al.,'2010;'Teel' et'al.,'2010).'Their'different'range'expansion'patterns'and'differences'in'genetic' connectivity'can'likely'be'explained'by'the'disparate'habitat'needs'of'these'tick'species' rather'than'differences'in'host'preferences.'I'hypothesize'that'through'the'inclusion'of' heterogeneous,'patchy'habitats'and'differential'survival'in'different'habitat'types,'it'is' possible'to'simulate'the'genetic'and'spatial'spread'patterns'exhibited'in#situ#by'these'two' tick'species.''

154

While'genetic'connectivity'has'long'been'used'as'a'proxy'for'measuring'species' dispersalamong'habitat'patches,'there'has'been'no'inclusion'of'genetic'parameters'in' models'describing'ticks.'It'has'been'suggested'that'incorporating'population'genetics'into' agentKbased'models'would'be'useful'for'describing'many'evolutionary'processes'

(DeAngelis'and'Mooij,'2005),'but'inclusion'of'genetic'components'in'agentKbased'models'of' range'expansion'and'invasion'of'any'species'is'rare'(Pertoldi'and'Topping,'2004;'Bialozyt' et'al.,'2006;'Kekkonen'et'al.,'2012).'By'including'genetics'in'agentKbased'models'of'species' undergoing'range'expansions,'it'is'possible'to'validate'models'using'the'genetic'diversity' and'connectivity'observed'in'the'field.'

The'agentKbased'model'described'here'is'derived'from'previous'TICKSIM'models,' which'modeled'tickKhost'interactions'and'emergent'patterns'of'disease'prevalence'(Gaff,'

2011;'Gaff'and'Nadolny,'2013).'The'current'model'has'been'altered'in'some'significant' ways'from'these'previous'iterations.'First,'the'presence'of'a'pathogen'passed'between'ticks' and'hosts'has'been'removed'in'order'to'generalize'the'model'beyond'a'specific'tickK pathogen'system,'and'to'focus'specifically'on'tick'range'expansions.'Pathogen'dynamics' can'be'reintroduced'in'later,'more'complex'models.'Second,'while'the'initial'TICKSIM'only' tracked'ticks'and'hosts,'my'version'of'the'model'also'tracks'spatially'explicit'tick' populations,'and'their'appearance'in'space'and'time,'by'colorKcoding'habitat'patches'based' on'the'presence'or'absence'of'ticks.'This'allows'measurement'of'invasion'rate,'spatial' pattern'of'invasion,'and'tick'population'densities'overall'and'in'specific'habitats.'Third,'this' model'includes'heterogeneous'habitats,'and'includes'a'desiccation'parameter'that'affects' ticks'directly'to'induce'mortality'in'poorKquality'habitat'patches.'Finally,'this'model'

155 includes'maternally'inherited'genetic'“haplotypes”'to'simulate'patterns'of'gene'flow'among' tick'populations.''

Other'recent'agentKbased'models'have'included'heterogeneous'habitats,'as'well'as' multiple'hosts'which'which'ticks'can'interact.'Recent'models'of'lone'star'tick'(Amblyomma# americanum)'populations'in'Texas'used'tickKhostKclimateKlandscape'interactions'to' simulate'field'conditions'and'determine'the'influence'of'climate'change'and'seasonality'on' tick'populations'(Wang'et'al.,'2012;'Wang'et'al.,'2015).'These'models'predicted'tick'density' increases'with'the'addition'of'a'greenbelt'to'a'Texas'city,'and'changes'in'tick'densities'with' the'effects'of'climate'change'on'the'seasonal'activities'of'tick'hosts.'While'both'these' models'and'the'present'model'include'heterogeneous'habitat,'multiple'host'types,'host' home'ranges,'and'climate'variables;'the'present'model'differs'in'several'important'ways.'

The'focus'of'the'present'model'is'on'tick'invasions,'not'on'established'populations'of'ticks.'

Although'the'landscape'in'the'present'model'is'markedly'less'complex'than'the'landscapes' modeled'by'Wang'et'al.'(2012;'2015),'each'individual'tick'is'tracked,'each'host'movement' is'tracked,'and'there'is'higher'temporal'resolution'than'in'the'Wang'et'al.'(2012;'2015)' models.'#

One'other'recent'model'that'examines'tick'range'expansions'focused'on'the'role'of' different'host'types'on'the'range'expansion'of'the'blacklegged'tick'(Ixodes#scapularis)'into'

Canada'(Madhav'et'al.,'2004).'Madhav'et'al.'(2004)'found'that'longKrange'hosts'(e.g.'deer)' increase'invasion'rate,'high'densities'of'shortKdistance'hosts'(e.g.'mice)'can'slow'invasions,' and'migratory'birds'play'an'important'role'in'the'movement'of'these'ticks'across' landscapes.'In'this'cellular'automata'model,'the'authors'measured'only'the'effects'of' varying'host'parameters'on'area'colonized,'using'a'simplified,'spatiallyKexplicit'landscape.'

156

While'my'model'shares'many'commonalities'with'the'I.#scapularis'model,'Madhav'et'al.'

(2004)'did'not'test'the'influence'of'different'habitat'types'on'invasions,'nor'did'they' monitor'invasion'rate,'tick'population'connectivity,'or'geographic'patterns'of'tick' populations.'The'I.#scapularis'model'was'also'deterministic,'rather'than'stochastic,'which' does'not'take'into'account'individual'tick'and'host'interactions,'and'modeled'the'influence' of'tick'burden'on'invasion,'which'was'not'tested'in'the'model'presented'here.'A'final' difference'is'that'the'I.#scapularis'model'operated'at'a'large'spatial'scale,'which'is' inappropriate'for'testing'hypotheses'about'local'invasion'patterns.''

This'model'is'based'on'the'premise'that'interactions'between'individual'ticks,'their' hosts,'and'their'habitats'generate'the'patterns'observed'in'tick'range'expansions.'By' varying'host'and'habitat'parameters'and'including'stochastic'effects,'it'is'possible'to' determine'the'relative'influence'of'host'and'habitat'parameters'on'tick'invasions'at'the' local'scale.'By'measuring'invasion'rate,'tick'population'density,'geographic'patterns'of'tick' population'establishment,'and'genetic'diversity'and'connectivity'of'tick'populations,'it'is' possible'to'answer'the'following'questions:'1)'Does'host'density'or'habitat'quality'have'the' greater'influence'on'tick'invasions'and'2)'How'does'habitat'connectivity'influence'the' genetic'connectivity'and'genetic'diversity'of'invading'ticks?''

MODEL(DESCRIPTION(

This'model'description'follows'the'Overview,'Design'concept'and'Details'(ODD)' protocol'for'describing'agentKbased'models'developed'by'Grimm'et'al.'(2006)'and'consists' of'six'elements.'The'first'three'elements'provide'an'overview,'the'fourth'element'explains' general'concepts'underlying'the'model'design,'and'the'last'two'elements'provide'details.'

157

1.! Purpose#

The'purpose'of'this'model'is'to'simulate'the'spatioKtemporal'dynamics'and'

genetic'diversity'of'new'tick'populations'after'an'initial'introduction'event'to'a'

novel'area'in'response'to'varying'host'and'habitat'parameters'and'to'better'

understand'the'underlying'mechanisms'leading'to'the'establishment'and'dispersal'

of'tick'populations'across'a'landscape.'The'results'of'these'simulations'will'help'

determine'the'relative'importance'of'host'density,'host'dispersal'distance,'and'

habitat'suitability'in'shaping'the'spatial'patterns,'invasion'rate,'population'density,'

and'genetic'diversity'and'connectivity'of'newly'establishing'tick'populations.'The'

ability'to'reproduce'the'spatial'and'genetic'connectivity'patterns'observed'in#situ'of'

invading'tick'species'I.#affinis'and'A.#maculatum#is'of'particular'interest.'

2.! Entities,#state#variables,#and#scales#

Agents/individuals''

This'model'considered'the'interactions'among'three'populations'of'agents:'

longKdistance'dispersing'hosts'(e.g.'deer),'shortKdistance'dispersing'hosts'(e.g.'

mice),'and'ticks.'Hosts'were'characterized'by'the'state'variables:'identification'

number,'home'base,'home'range'size,'mortality'rate,'number'of'ticks'currently'

feeding'on'the'host,'and'the'maximum'number'of'ticks'able'to'attach'to'the'host'at'

one'time.'To'keep'host'populations'constant,'if'a'host'died'it'was'immediately'

replaced'by'another'host,'which'was'created'on'a'random'patch.'The'home'base'of'

each'host'was'the'X,'Y'coordinate'of'the'patch'it'was'created'on.'Each'host'had'a'

158 specific'home'range'and'was'only'able'to'move'within'a'certain'subset'of'patches' away'from'their'home'base.'Host'categories'varied'in'the'distance'they'could'travel' per'time'step,'and'the'size'of'their'home'range.'Hosts'moved'ticks'that'were' attached'across'the'landscape,'and'ticks'could'only'move'when'on'a'host.'A'host' could'carry'up'to'the'specified'maximum'number'of'ticks,'and'if'a'host'died,'all'ticks' on'that'host'also'died.'

Ticks'were'characterized'by'the'state'variables:'identification'number,'sex,' life'stage,'activity,'identity'number'of'current'host,'and'a'maternally'inherited' genetic'haplotype.'Ticks'were'assigned'a'sex'(male'or'female)'at'birth,'and'moved' through'the'following'four'life'stages'throughout'ontogeny:'egg,'larvae,'nymph,'and' adult.'Tick'host'preferences'changed'depending'on'life'stage'and'were'reflected'by' probabilities'of'successful'attachment'to'each'host'category.'Ticks'moved'through' three'activities'during'each'life'stage:'resting,'questing,'and'feeding.'Adult'female' ticks'completed'a'final'activity,'laying'eggs,'after'feeding.'The'tick'population'did'not' remain'constant.'Mating'was'not'explicitly'included'in'this'model,'but'ticks'were' assumed'to'mate'onKhost,'so'female'ticks'were'able'to'lay'eggs'after'a'successful' bloodmeal.'There'was'a'set'number'of'haplotypes'divided'equally'between'the' initial'ticks'at'the'start'of'each'simulation'(e.g.'if'there'were'eight'initial'haplotypes' and'32'initial'ticks,'there'would'be'four'ticks'of'each'haplotype);'and'each'new'tick'

“hatched”'throughout'the'course'of'the'simulation'inherited'its'haplotype'from'its' mother.'Parameter'values'for'hosts'and'ticks'can'be'found'in'Table'23.'

159

Spatial'units'

New'tick'populations'were'observed'through'the'color'variables'of'patches,' with'color'reflecting'patch'occupation'by'ticks.'A'green'color'variable'indicated'the' background'environment'where'no'ticks'are'present.'Once'a'tick'was'hatched'or' moved'on'to'a'patch,'that'color'variable'turned'to'yellow'to'indicate'that'ticks'were' present.'If'six'or'more'adult'ticks'occupied'a'patch'simultaneously,'that'color' variable'changed'to'red'to'indicate'that'a'population'of'ticks'was'present.'Once'the' color'variable'for'a'patch'had'changed'color,'it'would'remain'changed'until'the'first' day'of'the'next'year,'when'all'color'variables'were'reset'to'green.'This'enabled' output'at'the'end'of'the'simulation'to'reflect'only'the'most'recent'year’s'tick' occupancy'patterns'across'the'simulated'landscape.''

The'presence'or'absence'of'ticks'of'each'haplotype'across'space'was'also' monitored'by'a'patch'variable.'The'haplotype'variable'recorded'if'there'were'any' ticks'of'each'haplotype'as'a'simple'presence/absence'variable'for'each'patch.'Like' the'color'variable,'once'a'haplotype'had'been'recorded'in'a'patch,'that'record' remained'until'the'first'day'of'the'next'year,'when'all'haplotype'variables'were'reset' to'enable'only'the'most'recent'year’s'distribution'of'haplotypes'across'the' landscape'to'be'recorded'at'the'end'of'the'simulation.'

Environment'

The'environment'was'set'up'as'a'grid'of'51x51'patches,'with'each'patch' roughly'representing'0.06'ha'for'a'total'simulated'area'of'roughly'165'ha,'with'hard'

(reflective)'boundaries.'The'highest'hierarchical'level'in'the'model'was'the'abiotic'

160

environment'and'its'fluctuations.'Type'of'habitat'was'determined'by'a'desiccation'

parameter'that'influenced'tick'survival'when'the'ticks'were'offKhost.'Desiccation'

could'be'increased'to'increase'tick'mortality'(indicating'habitat'of'poorer'quality),'

or'decreased'to'not'influence'tick'mortality'(indicating'good'habitat'where'ticks'

were'easily'able'to'survive'offKhost).'Once'habitats'were'created,'they'were'constant'

and'the'desiccation'parameter'did'not'change'during'the'simulation.'The'model'

could'be'run'with'one'habitat'(one'desiccation'parameter'for'the'whole'

environment),'or'with'multiple'habitats'in'different'parts'of'the'environment,'each'

with'its'own'desiccation'parameter.'#

Time'of'year'also'influenced'tick'mortality.'For'simplicity,'each'year'was'

divided'into'360'days,'and'each'month'(30'days)'had'a'parameter'that'influenced'

tick'mortality.'Ticks'were'more'likely'to'die'during'summer'and'winter'months,'

when'cold'or'heat'are'significant'stressors'(Table'23).'

3.! Process#overview#and#scheduling#

The'model'proceeds'in'daily'time'steps.'Within'each'day'or'time'step,'six'

modules'happen'in'the'following'order:'set'day'of'year,'tick'changes,'host'changes,'

calculate'new'populations,'calculate'haplotype'distributions,'and'reset'(Fig.'20).'

Within'each'module,'individuals'are'processed'in'random'order.'

( (

161

Table(23.'Baseline'parameter'values'used'in'model.'LD'indicates'longKdistance'dispersing' hosts'(e.g.'deer),'SD'indicates'shortKdistance'dispersing'hosts'(e.g.'mice).' ' Entities( Parameter( Category(or(value/unit( Environment( Simulation'size'(ha)' 165' Number'of'patches' 2601' ' ha/patch' 0.06' ' 0.1'in'Jan,'Feb,'Mar,'Jul,'Oct,'Nov'and'Dec;'' Tick'mortality' 0.01'in'Apr,'May,'Jun,'Aug,'Sep' ' Desiccation'parameter'(D)# 1' ' Occupied'(yellow)'patch'' 1'tick'of'any'life'stage' Patches( (#'ticks'needed)' Population'(red)'patch'' At'least'6'adults' (#'ticks'needed)' LD(hosts' ( Initial'deer'population' 50' Deer'rate' 1'patch'per'time'step,'random'walk' ' 13'patches'in'any'direction'from'home' Deer'movement' base,'total'of'729'patches'(50'ha)' ' Deer'mortality' 0.02' ' Max'ticks'per'deer' 30' SD(hosts' ( Initial'mouse'population' 800' Mouse'rate' 0.5'patches'per'time'step,'random'walk' ' 1'patch'in'any'direction'from'home'base,' Mouse'movement' total'of'9'patches'(.56'ha)' ' Mouse'mortality' 0.02' ' Max'ticks'per'mouse' 30' ' Prob.'larva'attachment'on' 0.01' Ticks( deer' Prob.'larva'attachment'on' 0.9' mouse' ' Prob.'nymph'attachment'on' 0.01' deer' ' Prob.'nymph'attachment'on' 0.75' mouse' ' Prob.'adult'attachment'on' 0.75' deer' ' Prob.'adult'attachment'on' 0.01' mouse' ' Initial'tick'population'' 32'nymphs' ' Eggs'laid'per'female' 1500' ' Time'from'egg'to'hatching' 120'days' ' Molt'time'larva'to'nymph' 90'days' ' Molt'time'nymph'to'adult' 90'days' ' Maximum'questing'time' 120'days' ' Length'of'blood'meal' 6'days'(for'adults,'nymphs,'and'larvae)' '' Initial'number'of'haplotypes' 8' ' '

162

' ' Figure(20.'Flow'diagram'for'processes'for'each'time'step'of'agentKbased'model.'Boxes'and' solid'lines'indicate'model'processes'and'transitions.'Inset'shows'subKprocesses'within' changing'tick'activities.'Reset'tick'populations'and'haplotypes'reverted'back'to'initial' conditions,'which'were'updated'to'reflect'current'occupancy'at'the'next'time'step.' '

4.! Design#concepts#

Basic'principles#

The'underlying'principle'of'this'model'is'that'independent'agents'interact'

with'one'another'and'simulate'the'interactions'that'ticks'would'have'with'hosts'in'

the'field.'Ticks'interacted'with'hosts'by'using'them'as'blood'meals,'and'ticks'had'a'

probability'of'attaching'to'each'host'type'that'changed'throughout'ontogeny,'with'

163 higher'probibilities'indicating'increased'preference'for'that'host.'Each'tick'was' assigned'a'genetic'haplotype'that'was'passed'on'to'offspring.'Hosts'moved'across' the'landscape'according'to'their'type'(i.e.'species),'and'were'constrained'by'home' ranges.'Tick'mortality'was'influenced'by'habitat'(desiccation),'time'of'year,'and'host' availability.'Through'these'interactions,'ticks'were'transported'across'the' environment'by'hosts,'and'established'new'populations'with'specific'genetic' signatures.'

Emergence'

The'emergent'property'being'modeled'is'the'establishment'of'new' populations'of'genetically'distinct'ticks'that'then'either'sustain'themselves'or'die' out.'Population'establishment'was'measured'in'four'ways:'invasion'rate,'tick' population'density,'genetic'diversity'and'connectivity,'and'patterns'of'spatial' spread.''

Sensing'

Ticks'sensed'hosts'only'within'their'own'patch,'and'had'a'given'probability' of'successful'attachment'and'feeding'on'that'host.'Ticks'could'not'attach'to'hosts' that'were'already'carrying'the'maximum'number'of'ticks'for'that'host.'Ticks'also' interacted'with'the'environment,'as'both'desiccation'and'time'of'year'were'factored' into'calculating'tick'mortality'during'each'time'step.'

Hosts'did'not'sense'or'interact'with'other'hosts'and'moved'around'the' environment'independently'of'one'another'in'this'simple'model.'Ticks'did'not'sense'

164

or'interact'with'other'ticks,'even'to'mate;'any'adult'female'that'had'successfully'

completed'a'bloodmeal'would'reproduce,'as'ticks'mate'onKhost'and'probability'of'

predation'on'fed'females'was'not'included'in'this'model.'

Interaction'

Ticks'sensed'hosts'on'their'patch'and'attached'to'a'host'to'attempt'to'

successfully'obtain'a'blood'meal.'Once'a'tick'had'successfully'attached'to'a'host,'it'

switched'from'“questing”'activity'to'“feeding”.'A'tick'would'feed'on'a'host'for'a'

specific'number'of'days.'Once'the'tick'was'engorged,'it'would'detach'from'the'host'

and'drop'off'on'whatever'patch'that'host'had'moved'to'during'the'interim'time'

steps.'The'tick'would'then'transition'to'“resting”,'while'it'transitioned'from'one'life'

stage'to'the'next.'After'a'given'number'of'time'steps,'the'tick'would'again'begin'

questing'and'would'attempt'to'attach'to'its'next'host.'Adult'female'ticks'laid'a'set'

number'of'eggs'after'completing'their'final'bloodmeal.'Through'the'interactions'of'

ticks'with'hosts,'ticks'could'be'transported'across'the'landscape'and'seed'new'

populations'with'specific'genetic'signatures.''

Stochasticity'

Stochasticity'was'included'in'calculating'host'movement,'host'mortality,'tick'

mortality,'and'successful'tick'attachment'to'hosts'while'questing.'Probabilities'for'

stochastic'parameters'are'described'in'Table'23.'The'processes'were'stochastic'for'

each'run'and'each'agent'has'equivalent'fitness.'

165

Observation'

The'following'metrics'were'monitored'throughout'each'time'step:'the'

number'of'ticks'in'each'life'stage,'the'number'of'occupied'(yellow)'and'populated'

(red)'patches,'the'location'of'each'occupied'patch,'the'location'of'each'populated'

patch,'the'number'of'ticks'of'each'haplotype,'and'the'presence'or'absence'of'ticks'of'

each'haplotype'in'each'patch.'

5.! Initialization#

Each'simulation'began'with'a'landscape'with'a'set'number'of'randomly'

distributed'hosts'of'both'breeds,'and'an'initial'number'of'ticks'created'in'the'center'

of'the'simulation.'Each'simulation'ran'for'1080'time'steps,'or'3'years,'to'give'tick'

populations'sufficient'time'to'establish'and'spread.'The'initial'state'of'the'

simulation'used'base'parameters'outlined'in'Table'23,'which'were'derived'from'

values'in'the'literature,'and'in'previous'versions'of'this'model'(Gaff,'2011;'Gaff'and'

Nadolny,'2013).'

6.! Input#data#

This'model'did'not'use'input'data'to'represent'timeKvarying'processes.'

The'model'was'programmed'using'NetLogo'version'5.0.'This'software'was' programmed'by'Uri'Wilensky'in'1999'and'is'freely'available'

(http://ccl.northwestern.edu/netlogo/).'The'model'was'run'on'the'Old'Dominion'

University'high'performance'computing'cluster'using'BehaviorSpace'to'run'experiments'

166

“headless”,'from'the'command'line.'All'statistical'analyses'on'model'results'were' completed'in'R'and'MATLAB'(Mathworks,'2015;'R'Core'Team,'2015).'

MODEL(EVALUATION(

General#model#performance#

To'evaluate'general'model'performance,'the'model'was'initialized'with'32'nymphal' ticks'of'eight'haplotypes,'four'ticks'of'each'haplotype,'dropped'at'the'center'of' homogenous'habitat'with'a'desiccation'parameter'of'1.0,'which'does'not'influence'tick' mortality.'Tick'mortality'was'therefore'influenced'only'by'season'and'stochastic'effects.''

The'simulation'was'started'on'01'March,'to'begin'in'the'spring'when'nymphs'of'I.#affinis# are'active.'All'parameter'values'were'set'at'base'values'as'indicated'in'Table'23.'The' resulting'phenologies'of'each'tick'life'stage'(Fig.'21)'show'nymphs'peaking'in'the'spring,' adults'peaking'in'the'summer,'and'larvae'peaking'in'the'winter'each'year.'Growing'annual' numbers'of'nymphs'and'adults'indicated'that'the'tick'population'was'increasing'every' year,'and'that'the'model'was'coded'properly.''

Sensitivity#analyses#

To'identify'the'factors'with'the'greatest'influence'on'tick'invasion,'sensitivity' analyses'were'performed'on'nine'parameters'(Table'24).'Each'of'the'parameters'was' varied'one'at'a'time'as'shown'in'Table'24,'while'keeping'all'other'parameters'constant'as' shown'in'Table'23.'Each'variant'of'each'parameter'was'run'25'times'until'1080'time'steps'

(3'years),'or'until'the'tick'population'was'extirpated.'A#priori'power'analyses'conducted''

167

( ( Figure(21.(Seasonal'phenology'of'A)'larvae,'B)'nymphs,'and'C)'adults'ticks'in'a'generalized' model.'' '

' with'G*Power'version'3.0.10'indicated'that'25'runs'per'parameter'set'would'be'sufficient' to'provide'statistical'significance'(Faul'et'al.,'2007).'For'each'parameter'variation,'the' following'was'recorded'at'the'last'time'step'of'the'simulation:'tick'population'density'

(measured'by'number'of'red'patches'and'number'of'yellow'patches),'invasion'rate''

168

Table(24.(Parameters'varied'in'sensitivity'analyses.'

' Parameter( Min( Max( Increment( Initial'ticks' 1' 121' 30' Initial'deer' 10' 170' 40' Initial'mice' 200' 2000' 600' Deer'home'range'size' 1' 16' 5' Mouse'home'range' 0.25' 3.25' 0.75' size' Deer'rate' 1' 11' 5' Mouse'rate' 0.25' 1.75' 0.5' Max'ticks/deer' 30' 310' 70' Max'ticks/mouse' 5' 130' 25' Desiccation'(D)# 1' 41' 10' Initial'#'of'haplotypes' 2' 16' 2,'4,'8,'16' '

(measured'by'distance'from'origin'to'furthest'yellow'patch,'distance'from'origin'to'furthest' red'patch,'mean'distance'from'origin'to'yellow'patches,'mean'distance'from'origin'to'red' patches),'genetic'diversity'(measured'by'the'number'of'surviving'haplotypes),'and'spatial' pattern'of'invasion'(measured'by'the'number'of'clusters'of'red'and'yellow'patches).'A' cluster'was'defined'as'being'three'or'more'patches'away'from'the'nearest'red'or'yellow' patch,'and'made'up'of'two'or'more'red'or'yellow'colored'patches.'Cluster'analysis'was' computed'using'the'DBSCAN'package'for'MATLAB'(Mathworks,'2015).'Spearman'rank' correlation'coefficients'were'calculated'to'determine'the'relative'influence'each'parameter' exerted'on'the'measured'variables'(Table'25).'

Some'patterns'that'emerged'from'the'sensitivity'analyses'were'quite'striking,' though'not'wholly'unexpected.'Mouse'density'and'mouse'home'range'size'exerted'the' largest'influences'on'tick'population'density'at'the'end'of'the'third'year,'with'more'mice' with'larger'ranges'resulting'in'higher'tick'densities.'Deer'home'range'size'had'the'greatest'

169 influence'on'invasion'rate'and'on'the'spatial'patterns'of'tick'invasions'in'heterogeneous' habitat.'Larger'home'ranges'of'long'distance'dispersers'resulted'in'faster'spread'and'more' clustering.'The'maximum'number'of'ticks'attached'to'a'deer'was'not'significantly' correlated'with'any'invasion'metric,'although'increasing'the'number'of'ticks'that'could'be' attached'to'a'mouse'positively'influenced'the'number'of'populations'established.'The' initial'number'of'haplotypes'had'the'greatest'influence'on'the'percentage'of'haplotypes' surviving'at'the'end'of'three'years.'More'initial'haplotypes'resulted'in'more'dieKoff'of' haplotypes'through'stochastic'effects'and'a'smaller'percentage'surviving'through'the'end' of'the'third'year.'However,'increasing'the'number'of'initial'haplotypes'significantly' increased'the'mean'number'of'haplotypes'surviving'at'the'end'of'year'three'(Spearman' correlation'coefficient'='0.96,'p'<0.01).'

Table&25.&Spearman(rank(correlation(table(from(sensitivity(analyses.(Correlation(coefficients(with(a(strong(direct(correlation( are(close(to(1,(and(those(with(a(strong(inverse(correlation(are(close(to(<1.(Spearman(rank(correlation(coefficients(are(presented( here,(with(stars(indicating(a(significance(level(of(p(<(0.01.(Bolded(correlation(coefficients(indicate(the(parameter(that(resulted( in(the(largest(effect(size(for(each(metric.( & Spearman&Rank&Correlation&Coefficients& Deer& Mouse& Max& Max& Initial& Mouse& Deer& Deer& Mouse& Initial&No.& Metric& Home& Home& Ticks/ Ticks/ D" Ticks& Density& Density& Rate& Rate& Haplotypes& Range& Range& Deer& Mouse& Distance(to(furthest( <0.13( 0.23( 0.57*( 0.87*& 0.28( 0.41*( 0.07( <0.06( 0.33*( <0.45*( <0.05( red(patch( Distance(to(furthest( 0.14( 0.21( 0.71*( 0.9*& 0.54*( 0.63*( 0.12( <0.01( <0.06( <0.51*( 0.18( yellow(patch( Mean(distance(to( <0.28( <0.31( 0.66*( 0.85*& <0.21( 0.34( 0.04( <0.02( 0.31*( <0.21( 0.09( red(patches( Mean(distance(to( <0.23( <0.35( 0.78*( 0.9*& 0.2( 0.53*( <0.09( <0.03( <0.14( <0.2( 0.04( yellow(patches( Number(of(red( <0.01( 0.81*& <0.31*( 0.12( 0.53*( <0.18( 0.15( <0.11( 0.58*( <0.62*( <0.04( patches( Number(of(yellow( 0.58*( 0.89*( 0.88*( 0.82*( 0.92*& 0.14( 0.38*( 0.05( <0.09( <0.89*( 0.21( patches( Number(of(clusters( <0.02( <0.32( 0.14( 0.65*& <0.24( 0.23( <0.23( <0.09( 0.03( <0.05( 0.18( Surviving(%( 0.68*( 0.16( 0.49*( 0.54*( 0.24( 0.06( <0.03( 0.08( 0.19( <0.63*( I0.84*& haplotypes( 170 170

171

MODEL&APPLICATION:&HOSTS&DENSITY&VS.&HABITAT&QUALITY&

! One!essential!question!when!dealing!with!tick!range!expansion!is!the!relative! influence!of!habitat!and!hosts!in!determining!dispersal!and!establishment.!Because!ticks! spend!much!of!their!lives!off

To!test!whether!host!density!or!habitat!quality!had!the!largest!influence!on!tick! invasions,!a!set!of!four!models!was!run!100!times!per!parameter!set.!The!models!varied! habitat!quality!between!good!and!poor,!and!host!density!between!high!and!low!(Fig.!22).!

Changing!the!number!of!available!mice!and!deer!varied!host!density,!and!changing!the! desiccation!parameter!varied!habitat!quality.!High!host!levels!were!set!at!1600!mice!and!

120!deer,!and!low!host!levels!were!set!at!200!mice!and!15!deer.!Good!quality!habitat!was! created!by!a!low!desiccation!parameter!that!did!not!influence!tick!mortality,!while! introducing!a!desiccation!parameter!of!30!created!poor!quality!habitat.!Each!model!was! run!on!a!landscape!of!homogenous!habitat!that!did!not!change!over!time,!and!all!metrics! other!than!host!density!and!habitat!were!kept!at!baseline!levels!(Table!23).!Invasion! metrics!were!measured!at!the!last!time!step!after!3!years!for!each!run.!Partial!rank! correlation!coefficients!(PRCC)!were!computed!to!determine!the!relative!influence!of!hosts! and!habitats!on!invasion!metrics!(Table!26),!and!boxplots!were!generated!to!display!the! differences!between!means!between!the!four!models!(Fig.!23).!

172

! & Figure&22.!Models!testing!the!influence!of!habitat!quality!and!host!density!on!tick! invasions.!These!images!represent!example!runs!of!the!tick!populations!exposed!to!these! conditions!across!homogeneous!landscapes!after!three!years.!Green!cells!are!habitat,!red! cells!indicate!cells!with!>6!adult!ticks,!and!yellow!cells!are!occupied!by!at!least!one!tick!of! any!life!stage.!! !

& &

173

Table&26.&Partial!rank!correlation!coefficients!(PRCC)!measuring!effects!of!host!density!and! habitat!quality!on!tick!invasions.!PRCCs!are!between!input!parameters!of!the!models!(host! density!vs.!habitat!quality)!and!the!output!metrics!measuring!density,!clustering,!genetic! diversity,!and!invasion!rate.!All!PRCCs!except!those!indicated!with!*!were!significantly! different,!with!p!values!

174

Figure&23.&Boxplots!comparing!means!between!four!treatments!groups!testing!influence!of! host!density!and!habitat!quality!on!invasion!metrics.!Metrics!are!A)!invasion!rate,! measured!by!adding!together!all!rate!metrics,!B)!invasion!rate,!measured!by!multiplying! together!all!rate!metrics!C)!tick!density,!measured!by!adding!together!all!density!metrics,! D)!tick!density,!measured!by!multiplying!together!all!density!metrics,!E)!genetic!diversity,! and!F)!spatial!spread,!measure!by!number!of!clusters.!Rate!metrics!included!maximum!and! average!distances!to!yellow!and!red!patches,!and!density!metrics!included!number!of!red! and!yellow!patches.!In!lieu!of!presenting!all!box!plots!produced!for!rate!and!density! metrics,!only!the!additive!and!multiplicative!plots!are!shown!to!give!an!overview!of!the! trends!seen.!Lines!through!the!middle!of!each!box!represent!the!median,!and!whiskers! extend!to!lower!and!upper!quartiles!(25th!and!75th!percentile).!Outliers!are!represented! with!clear!circles.!! !

175

In!homogenous!habitat,!host!density!was!found!to!have!a!relatively!greater!influence! on!almost!all!invasion!metrics,!although!both!host!density!and!habitat!quality!showed! significant!effects!on!all!invasion!metrics!(Table!26).!Higher!host!densities!resulted!in! higher!tick!densities,!faster!invasion!rate,!more!clusters,!and!more!genetic!diversity!of!ticks! at!the!end!of!three!years.!Higher!desiccation,!or!poor!habitat,!resulted!in!lower!tick!density,! slower!invasion!rate,!fewer!population!clusters,!and!less!genetic!diversity.!

MODEL&APPLICATION:&INFLUENCE&OF&HABITAT&CONNECTIVITY&ON&GENETICS&

! While!the!above!results!suggested!that!host!density!is!more!important!than!habitat! quality,!field!data!shows!otherwise.!Ticks!must!move!between!suitable!habitat!patches!to! disperse!across!a!landscape,!which!is!especially!evident!in!the!range!expansion!of!A.! maculatum,!a!tick!species!that!thrives!only!in!patchy!disturbed!environments.!Amblyomma! maculatum!tick!populations!found!in!these!habitat!patches!have!shown!genetic! dissimilarity!to!other!populations,!with!each!population!being!genetically!distinct!from!its! neighbors.!Conversely,!I.!affinis!disperses!throughout!well

24).!!

176

! & Figure&24.!Models!for!testing!the!influence!of!habitat!connectivity!on!genetic!connectivity,! and!reproducing!patterns!exhibited!by!I.!affinis!(left)!and!A.!maculatum!(right).!In!the!I.! affinis!model,!initial!ticks!are!dropped!in!the!center!of!the!completely!connected!“forest”! habitat,!which!has!a!desiccation!parameter!(D)!of!1,!indicating!good!tick!habitat.!The!two! “field”!patches!(red!and!yellow)!both!are!poor!habitat!quality!for!I.!affinis!ticks.!The!A.! maculatum!model!has!the!reverse!setup,!with!initial!ticks!dropped!in!forest!habitat!that! poor!quality,!with!nearby!field!patches!of!higher!quality.!In!both!simulations,!ticks!were! initialized!at!the!0,0!coordinate,!in!the!center!of!the!forest!habitat.! !

! The!two!models!were!each!run!100!times,!with!one!modeling!I.!affinis!moving!through! well

177 habitat!at!the!end!of!three!years.!To!replicate!what!was!seen!in!the!field,!the!I.!affinis!model! should!result!in!similar!genetic!makeup!throughout!the!simulation,!while!the!A.!maculatum! model!should!result!in!genetically!distinct!populations!in!each!habitat.!!

! To!analyze!the!similarity!between!each!habitat,!a!binary!similarity!coefficient!was! used.!These!are!simple!similarity!measures!based!on!presence

Sorensen’s!index!was!used!to!compute!a!similarity!coefficient!between!each!pair!of!habitat! types!(Krebs,!2014).!Because!there!were!three!habitats!(one!forest!and!two!field!patches),! the!results!of!all!three!pairwise!comparisons!were!summed!to!create!a!general!similarity! index!that!was!used!for!statistical!analysis.!Because!many!patches!did!not!have!haplotypes! present,!the!data!was!zero!inflated!and!non

! To!answer!the!question!of!which!tick!species!exhibits!populations!that!are!more! genetically!similar,!the!general!similarity!index!was!compared!between!the!I.!affinis!model! and!the!A.!maculatum!model.!The!I.!affinis!populations!were!more!similar!among!habitats,! while!populations!in!each!habitat!in!the!A.!maculatum!models!tended!to!be!more! genetically!distinct.!Habitat!connectivity!also!influenced!the!number!of!habitats!that! persisted!until!the!end!of!the!simulation,!with!I.!affinis!models!resulting!in!more!haplotypes! surviving!than!A.!maculatum!models.!In!I.!affinis!models,!there!was!also!a!greater!diversity! of!haplotypes!in!forested!habitat,!while!A.!maculatum!models!resulted!in!proportionally! more!haplotypes!present!in!field!habitats.!!

178

! & & Figure&25.&Boxplots!depicting!differences!between!means!of!four!different!invasion!metrics! for!I.!affinis!and!A.!maculatum!models.!Whiskers!indicate!lower!and!upper!quartiles,!and! circles!indicate!outliers.!Results!and!significance!levels!of!Wilcoxon!rank!sum!test!are! indicated!below!each!set!of!plots.&Significant!differences!were!observed!between!I.!affinis! and!A.!maculatum!A)!general!similarity!indices,!B)!overall!genetic!diversity!(number!of! haplotypes!surviving)!at!the!end!of!three!years,!C)!the!proportion!of!ticks!(haplotypes)! surviving!in!forested!habitat!at!the!end!of!three!years,!and!D)!the!proportion!of!ticks! (haplotypes)!surviving!in!field!habitats!at!the!end!of!three!years.! & &

179

DISCUSSION&

No!model!is!a!perfect!representation!of!the!agents!and!environment!that!it! represents,!and!the!present!model!has!several!limitations!that!are!fully!acknowledged.!

Because!of!the!stochasticity!inherent!in!agent

(Wang!et!al.,!2012;!Wang!et!al.,!2015).!Also,!in!this!simple!model,!hosts!do!not!interact!with! habitat!or!other!hosts,!and!the!host!population!is!kept!constant,!so!host!demography!is!not! included.!Also,!the!edges!of!the!model!simulation!are!reflective,!which!would!have!strong! influences!after!more!than!a!few!years!of!simulated!time.!For!that!reason,!simulations!were! kept!to!a!maximum!time!of!three!years,!which!enabled!tick!populations!sufficient!time!to! establish!and!grow,!but!not!“outgrow”!the!small!area!of!the!simulation.!

This!simple!model!yielded!several!interesting!inferences!regarding!the!effects!of! hosts!and!habitats!on!tick!range!expansions.!The!results!of!sensitivity!analyses!indicated! that!in!homogeneous!habitat,!long

180 results!from!a!spatially

Results!from!comparing!the!relative!influences!of!host!density!with!habitat!quality! suggested!that!both!significantly!influence!tick!invasion!rate,!pattern,!density,!and!genetic! diversity.!Interestingly,!in!homogenous!habitat,!host!density!had!the!largest!influence!on!all! invasion!parameters!measured.!However,!once!heterogeneous!habitat!was!introduced,! connectivity!between!high!quality!habitat!patches!significantly!influenced!tick!invasions,! especially!the!genetic!connectivity!between!tick!populations,!even!at!a!very!small!spatial! scale.!Greater!habitat!connectivity!resulted!in!more!genetic!similarity!between!populations,! and!more!overall!genetic!diversity!surviving!at!the!end!of!three!years.!Isolated!habitat! patches!resulted!in!more!genetically!distinct!populations,!with!less&overall!genetic!diversity! surviving!after!three!years.!!

These!changes!in!genetic!connectivity!and!diversity!mimic!the!genetic!patterns! exhibited!by!I.!affinis!and!A.!maculatum!in!the!field,!where!habitat!preference!resulted!in! significant!differences!between!the!two!species!(Nadolny!et!al.,!2015).!In!the!model,!the!I.! affinis!simulation!resulted!in!one!genetically!heterogeneous!population!concentrated!in!the! forest,!and!the!A.!maculatum!simulation!resulted!in!several!genetically!distinct!populations! concentrated!in!the!field!patches.!Simply!changing!the!connectivity!between!preferred! habitat!patches!for!each!tick!species!simulated!these!genetic!and!spatial!differences.!

181

Knowledge!of!the!genetic!makeup!of!these!populations!in!situ!provides!a!measure!of! validation!for!this!model,!and!the!generation!of!such!close!patterns!suggests!that!the!agent< based!model!was!robust,!even!at!such!a!small!spatial!scale.!

FUTURE&DIRECTIONS&

! As!written,!the!model!presented!here!is!a!useful!tool!for!learning!about!the!relative! influence!of!habitat!and!host!parameters!on!tick!range!expansions!and!genetic!connectivity.!

For!future!incarnations!of!this!model,!it!will!be!useful!to!measure!the!spatial!patterns!of! spread!more!directly,!including!measuring!the!size!of!clusters!and!distance!between! clusters.!Monitoring!the!persistence!of!clusters!of!tick!populations!would!also!provide! useful!information!on!the!stability!of!tick!populations!throughout!the!years!immediately! following!a!range!expansion.!Including!more!complex!host!dynamics,!such!as!herding! behavior,!territory!defense,!and!interaction!of!hosts!with!the!landscape!will!also!be! important!for!more!realistic!models!of!tick

! The!effects!of!changing!habitat!over!time!are!important!for!tick!population! establishment!and!persistence,!so!for!future!models!it!would!be!interesting!to!change!the! desiccation!parameter!over!time!to!model!the!influence!of!succession!or!deforestation!on! tick!populations.!Also,!the!simulations!presented!here!were!at!a!very!small!spatial!scale,! where!edge!effects!may!influence!invasion!rate!after!only!a!few!years.!Increasing!the!size!of! the!model,!perhaps!through!the!creation!of!a!mixed!deterministic/stochastic!model,!would! allow!for!more!realistic!model!of!tick!invasions!over!regional!spatial!scales.!A!larger!spatial! scale!could!include!more!host!categories,!such!as!migratory!birds,!and!allow!a!more!

182 detailed!investigation!of!the!influence!of!different!host!categories!on!the!ability!of!tick! populations!to!establish!and!persist!outside!their!native!range.!!

! !

183

GENERAL&DISCUSSION&

! Ticks!and!other!blood

Parasite!range!expansions!introduce!pathogens!to!new!areas!where!healthcare!providers! may!be!ill

There!has!been!much!speculation!on!the!underlying!causes!of!these!range!expansions,!and! blame!has!been!cast!on!increasing!global!temperatures,!changing!patterns!of!human! landscape!use,!and!other!large

Vuuren,!2006;!Léger!et!al.,!2013).!However,!there!is!a!paucity!of!data!for!parasites! expanding!their!ranges,!and!even!less!hypothesis!testing!on!determining!the!most! important!ecological!mechanisms!that!underlie!range!expansions.!

! Climate!change!is!perhaps!the!single!most!notorious!factor!in!parasite!range! expansions,!with!rising!temperatures!expanding!the!geographic!ranges!of!many!parasites.!

Recent!studies!have!predicted!significant!increases!in!the!geographic!areas!at!risk!of! vector

(Gage!et!al.,!2008).!In!addition,!recent!surveillance!has!documented!increasing!cases!of! vector

Florida!(Ogden!et!al.,!2015;!Seppa,!2015).!Many!studies!have!implicated!climate!change!in! tick!range!expansions,!because!of!direct!effects!on!temperature,!rainfall!and!humidity,!as! well!as!indirect!effects!on!host!and!plant!communities!important!for!tick!survival!(see!

Léger!et!al.,!2013!for!review).!Interestingly,!although!many!studies!report!tick!range! expansions!associated!with!climate!change,!there!are!few!studies!documenting!the!

184 disappearance!of!ticks!from!areas!that!have!become!climatically!unsuitable.!Although!long! periods!of!drought!have!been!found!to!decrease!tick!abundance!(Jones!and!Kitron,!2000),! colonization!and!extinction!probabilities!may!not!balance!each!other,!resulting!in!a!global! net!gain!of!areas!with!increased!risks!of!tick

! Changes!in!the!host!community!have!also!been!associated!with!tick!invasions!and! range!expansions.!The!direct!introduction!and!transportation!of!livestock!by!humans!has! facilitated!numerous!tick!introductions,!including!A.!maculatum!into!the!central!United!

States!(Semtner!and!Hair,!1973),!as!well!as!the!Asian!cattle!tick!(Rhipicephalus!microplus)! to!Africa!(Adakal!et!al.,!2013)!and!the!African!tropical!bont!tick!(Amblyomma!variegatum)! to!the!Caribbean!(Beati!et!al.,!2012).!While!these!introduced!livestock!ticks!can!cause!huge! economic!losses!(Estrada

(Childs!and!Paddock,!2003).!Migratory!birds!have!been!implicated!in!the!expansion!of!the! blacklegged!tick!(I.!scapularis)!into!Canada;!changing!migration!patterns!may!increase!the! number!of!ticks!introduced,!while!increased!populations!of!terrestrial!hosts!will!facilitate! faster!establishment!and!local!dispersal!(Madhav!et!al.,!2004;!Ogden!et!al.,!2008b;!Leighton! et!al.,!2012).!!

! A!final!factor!that!is!associated!with!tick!invasions!is!anthropogenic!habitat! disturbance,!or!landscape!changes.!Deforestation,!reforestation,!and!habitat!fragmentation! all!have!influences!on!microclimates!that!influence!tick!survival!off

185 influencing!the!host!communities!available!to!ticks!for!bloodmeals!and!dispersal.!

Microclimate!influences!tick!behavior,!with!dry!conditions!reducing!questing!height!or! inducing!quiescence!in!order!for!ticks!to!avoid!desiccation,!which!could!affect!the!fitness!or! survival!of!populations!if!dry!conditions!persist!(Randolph!and!Storey,!1999).!Reforestation! of!the!northeastern!United!States!is!thought!to!have!facilitated!the!expansion!of!I.!scapularis! and!the!subsequent!outbreak!of!Lyme!disease,!and!similar!changes!are!likely!driving! expansions!of!Ixodes!ricinus!ticks!and!Lyme!in!Europe!(Ogden!et!al.,!2013).!Habitat! fragmentation!has!been!shown!to!increase!tick!densities,!and!may!increase!invasion!rate!of!

I.!scapularis!into!Canada!(Barbour!and!Fish,!1993;!Simon!et!al.,!2014).!The!biodiversity!loss! resulting!from!deforestation!and!fragmentation!has!also!been!implicated!in!increased! pathogen!transmission!between!ticks!and!hosts,!and!may!increase!risks!to!humans!

(Logiudice!et!al.,!2003;!Brownstein!et!al.,!2005).!Recent!work!has!suggested!that!knowledge! of!tick!habitat!is!insufficient!to!predict!tick!densities!(Trout!Fryxell!et!al.,!2015).!The!work! presented!in!this!dissertation!provides!evidence!for!the!importance!of!landscape!use,! specifically!the!availability,!connectivity,!and!stability!of!preferred!habitats,!in!facilitating! tick!range!expansions.!

! The!aim!of!this!research!was!to!understand!the!ecological!mechanisms!underlying! the!differences!in!the!patterns!of!biological!invasion!exhibited!by!hitchhiking!parasites.!The! opportunity!to!test!hypotheses!pertaining!to!parasite!range!expansion!was!provided!by! two!tick!species,!I.!affinis!and!A.!maculatum,!as!they!simultaneously!invaded!Virginia.!Field! studies!conducted!throughout!southeastern!Virginia!revealed!that!these!two!species!were! expanding!in!different!geographic!patterns.!Ixodes!affinis!populations!were!found!primarily! in!disturbed!forests!habitat,!which!is!abundant!and!well

186 expansion!patterns!exhibited!by!A.!maculatum!populations!were!strikingly!different,!with! populations!found!only!in!isolated!patches!of!grassy,!open!habitat.!These!open!habitat! patches!are!dependent!on!disturbance!to!be!maintained,!and!are!short

(Heller!et!al.,!2015).!It!is!therefore!unlikely!that!differences!in!host!preference!between! these!two!ticks!resulted!in!the!different!geographic!patterns!of!expansion.!

The!genetic!connectivity!and!structure!of!populations!of!each!of!these!two!tick! species!were!also!strikingly!different.!By!using!FST!and!isolation!by!distance!as!proxies!for! dispersal,!it!was!determined!that!I.!affinis!ticks!in!Virginia!and!North!Carolina!were!one! large,!interconnected!population,!likely!spread!via!short

Because!the!habitats!favored!by!A.!maculatum!are!transient!and!maintained!by!disturbance! events,!it!is!likely!that!long

187 host!mobility,!but!that!data!presented!here!indicates!that!habitat!connectivity!may!be!an! equally!important!predictor!for!tick!genetic!structure,!and!perhaps!more!important!in!some! generalist!species!(Araya

Field!and!genetic!studies!yielded!abundant!evidence!for!the!importance!of!habitat! availability,!connectivity,!and!stability!in!tick!invasions.!An!agent

Other!models!of!tick!range!expansions!have!focused!on!the!relative!importance!of! host!diversity!(Madhav!et!al.,!2004)!or!the!predicted!effects!of!climate!change!(Eisen!et!al.,!

2006;!Leighton!et!al.,!2012)!on!tick!invasions!at!large!scales.!Previous!tick!models!including! habitat!heterogeneity!have!predicted!different!habitats!as!sources!and!sinks,!or!predicted! increased!disease!risk!based!on!habitat!changes!(Hoch!et!al.,!2010;!Li!et!al.,!2012;!Wang!et! al.,!2012).!However,!inclusion!of!habitat!heterogeneity!in!models!of!tick!range!expansion! may!allow!more!accurate!predictions!of!invasion!patterns!at!regional!scales.!Inclusion!of! genetic!connectivity!into!models!also!allows!for!models!to!be!more!easily!validated!through! population!genetics!studies.!The!simulations!presented!here!indicated!that!for!generalist! parasites,!habitat!connectivity!is!a!better!indicator!than!host!mobility!for!spatial!and! genetic!patterns!of!parasite!range!expansion.!Knowledge!of!preferred!tick!habitats!paired!

188 with!simple!models!could!provide!healthcare!providers!with!improved!local!scale! information!on!risks!of!emerging!tick

! !

189

CONCLUSIONS&

! This!dissertation!presents!a!case!study!of!two!invading!parasites.!I!have!used!field< derived!knowledge!of!the!ecology!of!the!organisms,!genetic!connectivity!data,!and!agent< based!models!to!better!understand!the!ecological!mechanisms!that!influence!range! expansion!patterns!in!these!two!ticks.!Here!I!present!the!following!general!conclusions! derived!from!this!work!that!can!be!used!as!guidelines!to!better!understand!and!predict! hitchhiker!invasions!across!taxa!with!similar!ecological!constraints.!Each!bolded,! numbered!statement!is!a!general!conclusion,!with!the!following!brief!summary!explaining! its!relevance!for!hitchhiker!invasion!ecology.!

1)! Generalist&hitchhikers&that&disperse&using&many&types&of&host&make&successful&

invaders.&

By!utilizing!dispersers!that!travel!over!short,!medium,!and!long!distances,!generalist!

hitchhikers!maximize!their!opportunities!for!introduction!to!new!environments.!!

2)! LongUdistance&dispersers&are&critical&for&facilitating&hitchhiker&expansion&across&a&

landscape.&&

Hitchhikers!cannot!invade!new!territory!without!being!moved!away!form!the!source!

population!by!a!dispersal!agent.!By!regularly!dispersing!on!organisms!that!move!

over!long!distances,!hitchhikers!are!able!to!move!farther!from!source!populations!

than!would!be!possible!if!only!short!distance!dispersing!organisms!were!used.!

190

3)! The&farther&dispersers&move,&the&faster&hitchhikers&can&invade.&

Hitchhiking!on!organisms!that!disperse!over!long!distances!not!only!enables!farther!

dispersal!from!source!populations,!but!increases!the!rate!of!the!invasion!process!as!

well!by!increasing!the!number!of!opportunities!for!population!establishment!far!

from!the!source!population.!

4)! High&densities&of&shortUdistance&dispersers&are&critical&for&facilitating&population&

establishment&once&an&invader&arrives.&

Once!a!hitchhiker!arrives!to!a!new!environment,!there!must!be!abundant!short<

distance!dispersers!in!order!for!the!hitchhikers!to!establish!a!population.!Without!

sufficient!short

die!out!or!be!translocated!to!another!habitat!patch!by!another!long

disperser.!

5)! One&cannot&infer&invasion&patterns&through&knowledge&of&dispersers&alone&–&

understanding&limiting&environmental&factors,&such&as&habitat,&is&critical&as&well.&

For!generalist!hitchhikers!that!disperse!using!a!number!of!different!organisms,!

identifying!patches!of!suitable!habitat!may!be!a!better!method!of!predicting!range!

expansions!than!through!tracking!disperser!movement!patterns.!

6)! Anthropogenically&disturbed&habitat&can&facilitate&hitchhiker&invasions.&

Habitats!that!are!anthropogenically!disturbed!have!fewer!barriers!to!establishment!

than!intact!ecosystems,!such!as!an!abundance!of!temporary!niches!available!for!

invading!organisms!to!exploit,!or!suites!of!disturbance

disperse!hitchhikers!among!disturbed!habitat!patches.!Anthropogenically!disturbed!

habitats!are!also!abundant!and!well

191

7)! Availability,&connectivity,&and&permanence&of&suitable&habitat&can&be&more&

important&than&dispersers&for&generalist&hitchhiker&invasions.&

For!a!hitchhiker!to!invade!a!new!area,!three!things!must!be!in!place:!suitable!habitat!

must!exist,!dispersers!must!move!between!source!populations!and!new!suitable!

habitats,!and!the!habitats!must!be!permanent!enough!to!sustain!hitchhikers!until!a!

population!can!establish.!

8)! Inclusion&of&genetic&variables&in&agentUbased&models&of&hitchhiker&invasions&

allows&for&validation&of&dispersal&predictions&using&population&genetics&

techniques.&

By!including!genetic!metrics!in!agent

range!expansion,!it!is!possible!to!validate!model!conclusions!with!genetic!datasets.!

Hitchhiker!dispersal!is!challenging!to!measure!directly,!and!using!genetic!datasets!

as!a!proxy!for!dispersal!in!models!and!in!field!studies!enables!stronger!conclusions!

to!be!drawn.

192

REFERENCES&!

! ! 1.! Adakal,!H.,!Biguezoton,!A.,!Zoungrana,!S.,!Courtin,!F.,!De!Clercq,!E.M.!and!Madder,!M.,!

2013.!Alarming!spread!of!the!Asian!cattle!tick!Rhipicephalus!microplus!in!West!

Africa!

Acarol.!61,!383–386.!

2.! Allan,!B.F.,!Dutra,!H.P.,!Goessling,!L.S.,!Barnett,!K.,!Chase,!J.M.,!Marquis,!R.J.,!Pang,!G.,!

Storch,!G.A.,!Thach,!R.E.,!Orrock,!J.L.,!2010.!Invasive!honeysuckle!eradication!reduces!

tick

18527.!doi:10.1073/pnas.1008362107!

3.! Allan,!B.F.,!Keesing,!F.,!Ostfeld,!R.S.,!2003.!Effect!of!Forest!Fragmentation!on!Lyme!

Disease!Risk.!Conserv.!Biol.!17,!267–272.!doi:10.1046/j.1523<1739.2003.01260.x!

4.! Altschul,!S.F.,!Gish,!W.,!Miller,!W.,!Myers,!E.!W.,!Lipman,!D.J.,!1990.!Basic!local!

alignment!search!tool.!J.!Mol.!Bio.!215,!403<410.!

5.! Ammon,!M.E.,!1995.!Lincoln's!Sparrow!(Melospiza!lincolnii),!The!Birds!of!North!

America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!from!

the!Birds!of!North!America!

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/191!

6.! Apperson,!C.S.,!Levine,!J.F.,!Evans,!T.L.,!Braswell,!A.,!Heller,!J.,!1993.!Relative!

utilization!of!reptiles!and!rodents!as!hosts!by!immature!Ixodes!scapularis!(Acari:!

Ixodidae)!in!the!coastal!plain!of!North!Carolina,!USA.!Exp.!Appl.!Acarol.!17,!719–731.!

doi:10.1007/BF00051830!

7.! Araya

193

population!genetics:!a!comprehensive!review.!Infect.!Genet.!Evol.!29,!164–79.!

doi:10.1016/j.meegid.2014.11.008!

8.! As,!S.,!1984.!To!Fly!or!Not!to!Fly?!Colonization!of!Baltic!Islands!by!Winged!and!

Wingless!Carabid!Beetles.!J.!Biogeogr.!11,!413.!doi:10.2307/2844805!

9.! Auch,!R.F.,!2014.!Middle!Atlantic!Coastal!Plain.!SAIC!TSSC,!work!performed!under!U.!

S.!Geological!Survey!contract!03CRN001!at!USGS!Center!for!EROS.!Available!at:!

http://landcovertrends.usgs.gov/east/eco63Report.html!

10.!Baneth,!G.,!2011.!Perspectives!on!canine!and!feline!hepatozoonosis.!Vet.!Parasitol.!

181,!3–11.!doi:10.1016/j.vetpar.2011.04.015!

11.!Barbour,!A.,!Fish,!D.,!1993.!The!biological!and!social!phenomenon!of!Lyme!disease.!

Science!260,!1610–1616.!doi:10.1126/science.8503006!

12.!Barker!S.C.,!Murrell!A.,!2004.!Systematics!and!evolution!of!ticks!with!a!list!of!valid!

genus!and!species!names.!Parasitology,!129:15<36!

13.!Barney,!J.N.,!Whitlow,!T.H.,!2008.!A!unifying!framework!for!biological!invasions:!The!

state!factor!model.!Biol.!Invasions!10,!259–272.!doi:10.1007/s10530<007<9127<8!

14.!Bart,!J.,!1995.!Acceptance!Criteria!for!Using!Individual

Management!Decisions.!Ecol.!Appl.!5,!411.!doi:10.2307/1942032!

15.!Beati,!L.,!Patel,!J.,!Lucas

Krecek,!R.,!Mertins,!J.W.,!Alfred,!J.T.,!Kelly,!S.,!Kelly,!P.,!2012.!Phylogeography!and!

Demographic!History!of!Amblyomma!variegatum!(Fabricius)!(Acari:!Ixodidae),!the!

Tropical!Bont!Tick.!Vector

doi:10.1089/vbz.2011.0859!

16.!Bekoff,!M.,!1977.!Canis!latrans.!Mamm.!Species!1–9.!

194

doi:10.1177/03063127067078012!

17.!Benayas,!J.M.R.,!Newton,!A.C.,!Diaz,!A.,!Bullock,!J.M.,!2009.!Enhancement!of!

Biodiversity!and!Ecosystem!Services!by!Ecological!Restoration:!A!Meta

Science!325,!1121–1124.!doi:10.1126/science.1172460!

18.!Bendel,!P.R.,!Gates,!J.E.,!1987.!Home!range!and!microhabitat!partitioning!of!the!

southern!flying!squirrel!(Glaucomys!volans).!J.!Mammal.!68,!243<255.!

19.!Best,!L.B.,!1977.!Nestling!biology!of!the!Field!Sparrow.!The!Auk!308<319.!

20.!Bialozyt,!R.,!Ziegenhagen,!B.,!Petit,!R.J.,!2006.!Contrasting!effects!of!long!distance!

seed!dispersal!on!genetic!diversity!during!range!expansion.!J.!Evol.!Biol.!19,!12–20.!

doi:10.1111/j.1420<9101.2005.00995.x!

21.!Black,!W.C.,!Piesman,!J.,!1994.!Phylogeny!of!hard

based!on!mitochondrial!16S!rDNA!sequences.!Proc.!Natl.!Acad.!Sci.!U.!S.!A.!91,!

10034–10038.!doi:10.1073/pnas.91.21.10034!

22.!Blackburn,!T.M.,!Pyšek,!P.,!Bacher,!S.,!Carlton,!J.T.,!Duncan,!R.P.,!Jarošík,!V.,!Wilson,!

J.R.U.,!Richardson,!D.M.,!2011.!A!proposed!unified!framework!for!biological!

invasions.!Trends!Ecol.!Evol.!26,!333–339.!doi:10.1016/j.tree.2011.03.023!

23.!Blair,!W.F.,!1936.!The!Florida!marsh!rabbit.!J.!Mammal.!17,!197<207.!

24.!Bradley,!C.A.,!Altizer,!S.,!2007.!Urbanization!and!the!ecology!of!wildlife!diseases.!

Trends!Ecol.!Evol.!22,!95–102.!doi:10.1016/j.tree.2006.11.001!

25.!Brantley,!S.T.,!Young,!D.R.,!2007.!Leaf

expanding!shrub!thickets.!Ecology!88,!524<530.!

26.!Brantley,!S.T.,!Young,!D.R.,!2008.!Shifts!in!litterfall!and!dominant!nitrogen!sources!

after!expansion!of!shrub!thickets.!Oecologia!155,!337<345.!

195

27.!Brennan,!L.A.,!Hernandez,!F.,!Williford,!D.,!2014.!Northern!Bobwhite!(Colinus!

virginianus),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!

Ornithology;!Retrieved!from!the!Birds!of!North!America!

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/397!

28.!Brinkerhoff,!R.J.,!Folsom

affect!Lyme!disease!risk?!Range!expansion!of!the!vector

burgdorferi.!Front.!Ecol.!Environ.!9,!103–110.!doi:10.1890/090062!

29.!Brown,!R.Z.,!1953.!Social!behavior,!reproduction,!and!population!changes!in!the!

house!mouse!(Mus!musculus!L.).!Ecol.!Monogr.!23,!218<240.!

30.!Brownstein,!J.S.,!Skelly,!D.K.,!Holford,!T.R.,!Fish,!D.,!2005.!Forest!fragmentation!

predicts!local!scale!heterogeneity!of!Lyme!disease!risk.!Oecologia!146,!469–475.!

doi:10.1007/s00442<005<0251<9!

31.!Bull,!C.M.,!Burzacott,!D.,!Sharrad,!R.D.,!1989.!No!competition!for!resources!between!

two!tick!species!at!their!parapatric!boundary.!Oecologia!79,!558<562.!

32.!Bunnell,!F.L.,!Campbell,!R.W.!Squires,!K.A.,!2004.!Conservation!priorities!for!peripheral!

species:!the!example!of!British!Columbia.!Can.!J.!Forest!Rest.!34,!2240<2247.!!

33.!Caldwell,!M.M.,!Dawson,!T.E.,!Richards,!J.H.,!1998.!Hydraulic!lift:!consequences!of!

water!efflux!from!the!roots!of!plants.!Oecologia!113,!151<161.!

34.!Cameron,!G.N.,!Kincaid,!W.B.,!1982.!Species!removal!effects!on!movements!of!

Sigmodon!hispidus!and!Reithrodontomys!fulvescens.!Am.!Midl.!Nat.!1,!60–67.!

35.!Cameron,!G.N.,!Spencer,!S.R.,!1981.!Sigmodon!hispidus.!Mamm.!Species!158,!1–9.!

doi:10.1177/03063127067078012!

196

36.!Carey,!M.,!Carey,!M.,!Burhans,!D.E.,!Nelson,!D.A.,!2008.!Field!Sparrow!

(Spizella!pusilla),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!

Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/103!

doi:10.2173/bna.103!

37.!Carlton,!J.T.,!2001.!Introduced!Species!in!U.S.!Coastal!Waters:!Pew!Oceans!

Commissions!Report.!Pew!Oceans!Commissions:!Washington,!DC.!

38.!Cavitt,!J.F.,!Haas,!C.A.!2014.!Brown!Thrasher!(Toxostoma!rufum),!The!Birds!of!North!

America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!from!

the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/557!

39.!Chapman,!J.A.,!Hockman,!J.G.,!Ojeda,!M.M.,!1980.!Sylvilagus!floridanus.!Mamm.!

Species!136,!1–8.!

40.!Chapman,!J.A.,!Willner,!G.R.,!1981.!Sylvilagus!palustris.!Mamm.!Species!153,!1<3.!

41.!Childs,!J.E.,!Paddock,!C.D.,!2003.!The!ascendancy!of!Amblyomma!americanum!as!a!

vector!of!pathogens!affecting!humans!in!the!United!States.!Annu.!Rev.!Entomol.!48,!

307–337.!doi:10.1146/annurev.ento.48.091801.112728!

42.!Clark,!K.,!2004.!Borrelia!species!in!host

northern!Florida.!J.!Clin.!Microbiol.!42,!5076–5086.!doi:10.1128/JCM.42.11.5076<

5086.2004!

197

43.!Clark,!K.L.,!Oliver!Jr,!J.H.,!McKechnie,!D.B.,!Williams,!D.C.,!1998.!Distribution,!

abundance,!and!seasonal!activities!of!ticks!collected!from!rodents!and!vegetation!in!

South!Carolina.!J.!Vector!Ecol.!23,!89<105.!

44.!Clark,!K.L.,!Oliver,!J.H.,!Grego,!J.M.,!James,!!a!M.,!Durden,!L.!a,!Banks,!C.W.,!2001.!Host!

associations!of!ticks!parasitizing!rodents!at!Borrelia!burgdorferi!enzootic!sites!in!

South!Carolina.!J.!Parasitol.!87,!1379–1386.!!

45.!Clifford,!C.M.,!Anastos,!G.,!Elbl,!A.,!1961.!The!larval!ixodid!ticks!of!the!eastern!United!

States!(Acarina

46.!Colwell,!R.K.,!Mao,!C.!X.,!Chang,!J.!2004.!Interpolating,!extrapolating,!and!comparing!

incidence

47.!Cooley,!R.A.,!Kohls,!G.M.!1944.!The!genus!Amblyomma!(Ixodidae)!in!the!United!

States.!J.!Parasit.!30,!77<111.!

48.!Cumming,!G.S.,!Van!Vuuren,!D.P.,!2006.!Will!climate!change!affect!ectoparasite!

species!ranges?!Glob.!Ecol.!Biogeogr.!15,!486–497.!doi:10.1111/j.1466<

822X.2006.00241.x!

49.!D’Antonio,!C.M.,!Meyerson,!L.A.,!2002.!Exotic!plant!species!as!problems!and!

solutions!in!ecological!restoration.!Restor.!Ecol.!10,!703–713.!doi:10.1046/j.1526<

100X.2002.01051.x!

50.!Dahlgren,!F.S.,!Paddock,!C.D.,!Springer,!Y.P.,!Eisen,!R.J.,!Behravesh,!C.B.,!2016.!

Expanding!range!of!Amblyomma!americanum!and!simultaneous!changes!in!the!

epidemiology!of!spotted!fever!group!rickettsiosis!in!the!United!States.!Am.!J.!Trop.!

Med.!Hyg.!94,!35<42.!

51.!Dale,!V.,!Joyce,!L.,!McNulty,!S.,!Neilson,!R.,!Ayres,!M.,!Flannigan,!M.,!Hanson,!P.,!Irland,!

198

L.,!Lugo,!A.,!Peterson,!C.,!Simberloff,!D.,!Swanson,!F.,!Stocks,!B.,!Wotton,!B.,!2001.!

Climate!Change!and!Forest!Disturbances.!BioScience!51,!723–734.!

doi:10.1641/0006<3568(2001)051[0723:CCAFD]2.0.CO;2.!

52.!Daniels,!T.!J.,!Falco,!R.C.,!Curran,!K.L.,!Fish,!D.!1996.!Timing!of!Ixodes!scapularis!

(Acari:!Ixodidae)!oviposition!and!larval!activity!in!southern!New!York.!J.!Med.!

Ent.!33,!140<147.!

53.!Darriba,!D.,!Taboada,!G.L.,!Doallo,!R.,!Posada,!D.,!2012.!jModelTest!2:!more!models,!

new!heuristics!and!parallel!computing.!Nat.!Methods!9,!772<772.!

54.!Davis,!D.E.,!Emlen,!J.T.,!1948.!The!placental!scar!as!a!measure!of!fertility!in!rats.!J.!

Wildlife!Manage.!12,!162<166.!

55.!DeAngelis,!D.L.,!Mooij,!W.M.,!2005.!Individual

Evolutionary!Processes.!Annu.!Rev.!Ecol.!Evol.!Syst.!36,!147–168.!

doi:10.1146/annurev.ecolsys.36.102003.152644!

56.!Diuk

M.,!Cortinas,!R.,!Hickling,!G.J.,!Tsao,!J.I.,!Barbour,!A.G.,!Kitron,!U.,!Piesman,!J.,!Fish,!D.,!

2010.!Field!and!climate

nymphal!Ixodes!scapularis,!an!important!vector!of!tick

eastern!United!States.!Glob.!Ecol.!Biogeogr.!19,!504–514.!doi:10.1111/j.1466<

8238.2010.00526.x!

57.!Do,!R.,!Shonfield,!J.,!McAdam,!A.G.,!2013.!Reducing!accidental!shrew!mortality!

associated!with!small

supplementation.!J.!Mammal.!94,!754<760.!

58.!Dolan,!P.G.,!Carter,!D.C.,!1977.!Glaucomys!volans.!Mamm.!Species!78,!1<6.!

199

59.!Drake,!J.M.,!Lodge,!D.M.,!2006.!Allee!effects,!propagule!pressure!and!the!probability!

of!establishment:!Risk!analysis!for!biological!invasions.!Biol.!Invasions!8,!365–375.!

doi:10.1007/s10530<004<8122<6!

60.!Drummond,!R.O.,!Whetstone,!T.M.,!1970.!Oviposition!of!the!Gulf!Coast!Tick.!J.!Econ.!

Entomol.!63,!1547–1551.!doi:10.1093/jee/63.5.1547!

61.!Dueser,!R.D.,!Brown,!W.C.,!Hogue,!G.S.,!McCaffrey,!C.,!McCuskey,!S.,!T,!H.G.,!1979.!

Mammals!on!the!Virginia!Barrier!Islands.!J.!Mammal.!60,!425–429.!

62.!Dupanloup,!I.,!Schneider,!S.,!Excoffier,!L.,!2002.!A!simulated!annealing!approach!to!

define!the!genetic!structure!of!populations.!Mol.!Ecol.!11,!2571<2581.!

63.!Durden,!L.A.,!Keirans,!J.E.,!1996.!Nymphs!of!the!genus!Ixodes!(Acari:!Ixodidae)!of!the!

United!States:!Taxonomy,!identification!key,!distribution,!hosts,!and!

medical/veterinary!importance.!Entomological!Society!of!America.!

64.!Durden,!L.A.,!Oliver!Jr,!J.H.,!1999.!Ecology!of!Ixodes!scapularis!and!Lyme!disease!in!

coastal!Georgia,!USA.!Acarology!IX!2,!379<383.!

65.!Eisen,!L.,!Eisen,!R.J.,!Lane,!R.S.,!2006.!Geographical!distribution!patterns!and!habitat!

suitability!models!for!presence!of!host

Mendocino!County,!California.!J.!Med.!Entomol.!43,!415–427.!doi:10.1603/0022<

2585(2006)043[0415:GDPAHS]2.0.CO;2!

66.!Estrada

ticks!that!affect!livestock:!A!review.!Agriculture,!3,!221<235.!

67.!!Estrada

The!Amblyomma!maculatum!Koch,!1844!(Acari:!Ixodidae:!Amblyomminae)!tick!

group:!diagnostic!characters,!description!of!the!larva!of!A.!parvitarsum!Neumann,!

200

1901,!16S!rDNA!sequences,!distribution!and!hosts.!Syst.!Parasitol.!60,!99–112.!

doi:10.1007/s11230<004<1382<9!

68.!Evans,!M.,!Gow,!E.,!Roth,!R.R.,!Johnson,!M.S.,!Underwood,!T.J.,!2011.!Wood!Thrush!

(Hylocichla!mustelina),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!

Cornell!Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/246!

69.!Excoffier,!L.,!Lischer,!H.E.,!2010.!Arlequin!suite!ver!3.5:!a!new!series!of!programs!to!

perform!population!genetics!analyses!under!Linux!and!Windows.!Mol.!Ecol.!

Resour.!10,!564<567.!

70.!Falco,!R.!C.,!Fish,!D.,!1991.!Horizontal!movement!of!adult!Ixodes!dammini!(Acari:!

Ixodidae)!attracted!to!CO2

71.!Falls,!J.!B.,!Kopachena,!J.G.,!2010.!White

The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!

Ornithology;!Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu/bna/species/128!!

72.!Faul,!F.,!Erdfelder,!E.,!Lang,!A.

power!analysis!program!for!the!social,!behavioral,!and!biomedical!sciences.!Behav.!

Res.!Methods!39,!175–91.!

73.!Ferrari,!F.A.G.,!Goddard,!J.,!Caprio,!M.,!Paddock,!C.D.,!Mixson

Stokes,!A.S.,!2013.!Population!analyses!of!Amblyomma!maculatum!ticks!and!

Rickettsia!parkeri!using!single

Borne.!Dis.!4,!439–444.!doi:10.1016/j.ttbdis.2013.04.010!

74.!Fish,!D.,!Howard,!C.,!1999.!Methods!used!for!creating!a!national!Lyme!disease!risk!

201

map.!Mor!Mortal!Wkly!Rep!CDC!Surveill!Summ!48,!21–24.!

75.!Fleetwood,!S.C.,!1985.!The!environmental!influences!in!selected!vegetation!

microhabitats!on!the!various!life!stages!of!Amblyomma!maculatum!Koch!(Acari,!

Ixodidae).!Dissertation.!Texas!A!and!M!University.!

76.!Florin,!D.A.,!Brinkerhoff,!R.J.,!Gaff,!H.,!Jiang,!J.,!Robbins,!R.G.,!Eickmeyer,!W.,!Butler,!J.,!

Nielsen,!D.,!Wright,!C.,!White,!A.,!Gimpel,!M.E.,!Richards,!A.L.,!2014.!Additional!U.S.!

collections!of!the!Gulf!Coast!tick,!Amblyomma!maculatum!(Acari:!Ixodidae),!from!the!

State!of!Delaware,!the!first!reported!field!collections!of!adult!specimens!from!the!

State!of!Maryland,!and!data!regarding!this!tick!from!surveilla.!Syst.!Appl.!Acarol.!19,!

257.!doi:10.11158/saa.19.3.1!

77.!Fornadel,!C.M.,!Zhang,!X.,!Smith,!J.D.,!Paddock,!C.D.,!Arias,!J.R.,!Norris,!D.E.,!2011.!High!

Rates!of!Rickettsia!parkeri!Infection!in!Gulf!Coast!Ticks!(Amblyomma!maculatum)!

and!Identification!of!“!Candidatus!Rickettsia!Andeanae”!from!Fairfax!County,!

Virginia.!Vector

78.!Forys,!E.A.,!Dueser,!R.D.,!1993.!Inter

palustris).!Am.!Midl.!Nat.!130,!408.!doi:10.2307/2426141!

79.!Fritzell,!E.K.,!Haroldson,!K.J.,!1982.!Urocyon!cinereoargenteus.!Mamm.!Species!1–8.!

doi:10.2307/3503957!

80.!Fu,!Y.X.!1997.!Statistical!tests!of!neutrality!of!mutations!against!population!growth,!

hitchhiking!and!background!selection.!Genetics!147,!915<925.!

81.!Gaff,!H.,!2011.!Preliminary!analysis!of!an!agent

Math.!Biosci.!Eng.!8,!463–473.!doi:10.3934/mbe.2011.8.463!

82.!Gaff,!H.,!Gross,!L.,!Schaefer,!E.,!2009.!Results!from!a!mathematical!model!for!human!

202

monocytic!ehrlichiosis.!Clin.!Microbiol.!Infect.!15,!15–16.!doi:10.1111/j.1469<

0691.2008.02131.x!

83.!Gaff,!H.,!Nadolny,!R.,!2013.!Identifying!requirements!for!the!invasion!of!a!tick!species!

and!tick

doi:10.3934/mbe.2013.10.625!

84.!Gage,!K.L.,!Burkot,!T.R.,!Eisen,!R.J.,!Hayes,!E.B.,!2008.!Climate!and!Vectorborne!

Diseases.!Am.!J.!Prev.!Med.!35,!436–450.!doi:10.1016/j.amepre.2008.08.030!

85.!Gaines,!D.N.,!Operario,!D.J.,!Stroup,!S.,!Stromdahl,!E.,!Wright,!C.,!Gaff,!H.,!Broyhill,!J.,!

Smith,!J.,!Norris,!D.E.,!Henning,!T.,!Lucas,!A.,!Houpt,!E.,!2014.!Ehrlichia!and!Spotted!

Fever!Group!Rickettsiae!Surveillance!in!Amblyomma!americanum!in!Virginia!

Through!Use!of!a!Novel!Six

14,!307–316.!doi:10.1089/vbz.2013.1509!

86.!Garcia

peripheral!populations.!Evolution!51,!21<28.!!

87.!Genoways,!H.,!Choate,!J.,!1998.!Natural!History!of!the!Southern!Short

Blarina!carolinensis.!Mammalogy!Papers:!U.!Nebraska!State!Museum!244,!1–43.!

88.!George,!J.E.,!2008.!The!effects!of!global!change!on!the!threat!of!exotic!arthropods!and!

arthropod

1149,!249–254.!doi:10.1196/annals.1428.084!

89.!George,!S.B.,!Choate,!J.R.,!Genoways,!H.H.,!1986.!Blarina!brevicauda.!Mamm.!Species!

261,!1<9.!

90.!Gilbert,!L.,!2013.!Can!restoration!of!afforested!peatland!regulate!pests!and!disease?!J.!

Appl.!Ecol.!50,!1226<1233.!doi:10.1111/1365<2664.12141!

203

91.!Ginsberg,!H.S.,!Ewing,!C.P.,!1989.!Comparison!of!flagging,!walking,!trapping,!and!

collecting!from!hosts!as!sampling!methods!for!northern!deer!ticks,!Ixodes!dammini,!

and!lone

313–322.!doi:10.1007/BF01197925!

92.!Ginsberg,!H.S.,!Rulison,!E.L.,!Azevedo,!A.,!Pang,!G.C.,!Kuczaj,!I.M.,!Tsao,!J.I.,!LeBrun,!

R.A.,!2014.!Comparison!of!survival!patterns!of!northern!and!southern!genotypes!of!

the!North!American!tick!Ixodes!scapularis!(Acari:!Ixodidae)!under!northern!and!

southern!conditions.!Parasites!and!Vectors!7,!394.!

93.!Gladney,!W.J.,!1971.!Mate

Ixodidae)!on!a!bovine.!Nature!232,!401<402.!

94.!Gladney,W.J.,!Grabbe,!R.R.,!Ernst,!S.E.,!Oehler,!D.D.,!1974.!The!Gulf!Coast!tick:!

evidence!of!a!pheromone!produced!by!males.!J.!Med.!Ent.!11,!303<306.!

95.!Goddard,!J.,!1992.!Ecological!studies!of!adult!Ixodes!scapularis!in!central!Mississippi:!

questing!activity!in!relation!to!time!of!year,!vegetation!type,!and!meteorologic!

conditions.!J.!Med.!Ent.!29,!501<506.!

96.!Goddard,!J.,!2003.!Experimental!infection!of!lone!star!ticks,!Amblyomma!americanum!

(L.),!with!Rickettsia!parkeri!and!exposure!of!guinea!pigs!to!the!agent.!J.!Med.!

Entomol.!40,!686–689.!doi:10.1603/0022<2585<40.5.686!

97.!Goddard,!J.,!Norment,!B.R.,!1983.!Notes!on!the!geographical!distribution!of!the!Gulf!

Coast!tick,!Amblyomma!maculatum!(Koch)![Acari:!Ixodidae].!Entomological!News!

USA!94,!103<104!

98.!Goddard,!J.,!Paddock,!C.D.,!2005.!Observations!on!distribution!and!seasonal!activity!

of!the!Gulf!Coast!tick!in!Mississippi.!J.!Med.!Entomol.!42,!176–179.!

204

99.!Goddard,!J.,!Piesman,!J.,!2006.!New!records!of!immature!Ixodes!scapularis!from!

Mississippi.!J.!Vector!Ecol.!31,!421<422.!

100.! Goodman,!J.L.,!Dennis,!D.T.,!Sonenshine,!D.E.,!2005.!Tick

humans.!American!Society!of!Microbiology.!

101.! !Gorrochotegui

Beaty,!B.J.,!Black!IV,!W.C.,!2000.!Genetic!isolation!by!distance!among!Aedes!aegypti!

populations!along!the!northeastern!coast!of!Mexico.!Am.!J.!Trop.!Med.!Hyg.!62,!200–

209.!

102.! Gotelli,!N.J.,!Colwell,!R.K.,!2001.!Quantifying!biodiversity:!procedures!and!

pitfalls!in!the!measurment!and!comparison!of!species!richness!.!Ecol.!Lett.!4,!379–

391.!doi:10.1046/j.1461<0248.2001.00230.x!

103.! Gowaty,!P.A.,!Plissner,!J.H.,!2015.!Eastern!Bluebird!(Sialia!sialis),!The!Birds!of!

North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!

from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/381!

doi:10.2173/bna.381!

104.! Gray,!J.S.,!1998.!The!ecology!of!ticks!transmitting!Lyme!borreliosis.!Exp.!Appl.!

Acarol.!22,!249<258.!

105.! Greenlaw,!J.S.,!2015.!Eastern!Towhee!(Pipilo!erythrophthalmus),!The!Birds!of!

North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!

from!the!Birds!of!North!America!

205

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/262!

doi:10.2173/bna.262!

106.! Griffith,!J.A.,!Stehman,!S.V.,!Sohl,!T.L.,!Loveland,!T.R.,!2003.!Detecting!trends!in!

landscape!pattern!metrics!over!a!20

programme.!Int.!J.!Remote!Sens.!24,!175<181.!

107.! Grimm,!V.,!Berger,!U.,!Bastiansen,!F.,!Eliassen,!S.,!Ginot,!V.,!Giske,!J.,!Goss<

Custard,!J.,!Grand,!T.,!Heinz,!S.K.,!Huse,!G.,!Huth,!A.,!Jepsen,!J.U.,!Jørgensen,!C.,!Mooij,!

W.M.,!Müller,!B.,!Pe’er,!G.,!Piou,!C.,!Railsback,!S.F.,!Robbins,!A.M.,!Robbins,!M.M.,!

Rossmanith,!E.,!Rüger,!N.,!Strand,!E.,!Souissi,!S.,!Stillman,!R.A.,!Vabø,!R.,!Visser,!U.,!

DeAngelis,!D.L.,!2006.!A!standard!protocol!for!describing!individual

agent

doi:10.1016/j.ecolmodel.2006.04.023!

108.! Grimm,!V.,!Berger,!U.,!DeAngelis,!D.L.,!Polhill,!J.G.,!Giske,!J.,!Railsback,!S.F.,!

2010.!The!ODD!protocol:!A!review!and!first!update.!Ecol.!Modell.!221,!2760–2768.!

doi:10.1016/j.ecolmodel.2010.08.019!

109.! Grimm,!V.,!Railsback,!S.F.,!2005.!Individual

Princeton!Series!in!Theoretical!and!Computational!Biology.!

110.! Grimm,!V.V.,!1999.!Ten!years!of!individual

have!we!learned!and!what!could!we!learn!in!the!future?!Ecol.!Modell.!115,!129–148.!

111.! Guindon,!S.,!Gascuel,!O.,!2003.!A!simple,!fast,!and!accurate!algorithm!to!

estimate!large!phylogenies!by!maximum!likelihood.!Systematic!biology!52,!696<704.!

112.! Guzy,!M.J.,!Ritchison,!G.,!1999.!Common!Yellowthroat!(Geothlypis!trichas),!The!

Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!

206

Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/448!

113.! Hafner,!M.S.,!1977.!Density!and!diversity!in!Mojave!Desert!rodent!and!shrub!

communities.!J.!Anim.!Ecol.!46,!925<938.!

114.! Haggerty,!T.M.,!Morton,!E.S.,!2014.!Carolina!Wren!(Thryothorus!ludovicianus),!

The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!

Ornithology;!Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/188!

115.! Haile,!D.G.,!Mount,!G.A.,!1987.!Computer!simulation!of!population!dynamics!

of!the!lone!star!tick,!Amblyomma!americanum!(Acari:!Ixodidae).!J.!Med.!Ent.!24,!356<

369.!

116.! Halkin,!S.L.,!Linville,!S.U.,!1999.!Northern!Cardinal!(Cardinalis!cardinalis),!The!

Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!

Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/440!

117.! Harrison,!B.!A,!Rayburn,!W.H.,!Toliver,!M.,!Powell,!E.E.,!Engber,!B.R.,!Durden,!

L.!a,!Robbins,!R.G.,!Prendergast,!B.F.,!Whitt,!P.B.,!2010.!Recent!discovery!of!

widespread!Ixodes!affinis!(Acari:!Ixodidae)!distribution!in!North!Carolina!with!

implications!for!Lyme!disease!studies.!J.!Vector!Ecol.!35,!174–179.!

doi:10.1111/j.1948<7134.2010.00074.x!

118.! Hartl,!D.L.,!1988.!A!primer!of!population!genetics.!Sinauer!Associates,!Inc.!

119.! Heger,!T.,!Trepl,!L.,!2003.!Predicting!biological!invasions.!Biol.!Invasions!5,!

207

301–309.!doi:10.1023/B:BINV.0000005568.44154.12!

120.! Heller,!E.!L.,!Wright,!C.!L.,!Nadolny,!R.!M.,!Hynes,!W.!L.,!Gaff,!H.!D.,!Walters,!E.!

L.,!2015.!New!Records!of!Ixodes!affinis!(Acari:!Ixodidae)!parasitizing!avian!hosts!in!

southeastern!Virginia.!J.!Med.!Ent.!doi:10.1093/jme/tjv175!

121.! Hengeveld,!R.,!1988.!Mechanisms!of!biological!invasions.!J.!Biogeogr.!15,!819–

828.!

122.! Henning,!T.C.,!Orr,!J.M.,!Smith,!J.D.,!Arias,!J.R.,!Norris,!D.E.,!2014.!Spotted!fever!

group!Rickettsiae!in!multiple!hard!tick!species!from!Fairfax!County,!Virginia.!Vector<

Borne!Zoonotic!Dis.!14,!482–485.!doi:10.1089/vbz.2013.1534!

123.! Herkert,!J.R.,!Kroodsma,!D.E.,!Gibbs,!J.P.,!2001.!Sedge!Wren!

(Cistothorusplatensis),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!

Cornell!Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!America!

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/582!

124.! Hirth,!H.F.,!1959.!Small!mammals!in!old!field!succession.!Ecology!40,!417.!

doi:10.2307/1929758!

125.! Hobbs,!R.J.,!Arico,!S.,!Aronson,!J.,!Baron,!J.S.,!Bridgewater,!P.,!Cramer,!V.A.,!

Epstein,!P.R.,!Ewel,!J.J.,!Klink,!C.A.,!Lugo,!A.E.!Norton,!D.,!2006.!Novel!ecosystems:!

theoretical!and!management!aspects!of!the!new!ecological!world!order.!Global!Ecol.!

Biogeog.15,!1<7.!

126.! Hoch,!T.,!Monnet,!Y.,!Agoulon,!A.,!2010.!Influence!of!host!migration!between!

woodland!and!pasture!on!the!population!dynamics!of!the!tick!Ixodes!ricinus:!A!

modelling!approach.!Ecol.!Modell.!221,!1798–1806.!

208

doi:10.1016/j.ecolmodel.2010.04.008!

127.! Huelsenbeck,!J.!P.,!Ronquist,!F.,!2001.!MRBAYES:!Bayesian!inference!of!

phylogenetic!trees.!Bioinformatics,!17,!754<755.!

128.! Huntly,!N.,!Inouye,!R.S.,!1987.!Small!mammal!populations!of!an!old

chronosequence:!successional!patterns!and!associations!with!vegetation.!J.!Mammal.!

68,!739–745.!doi:10.2307/1381550!

129.! Ibrahim,!K.M.,!Nichols,!R.A,!Hewitt,!G.M.,!1996.!Spatial!patterns!of!genetic!

variation!generated!by!different!forms!of!dispersal!during!range!expansion.!

Heredity!77,!282–291.!doi:10.1038/hdy.1996.142!

130.! Jackson,!B.J.,!Jackson,!J.A.,!2000.!Killdeer!(Charadrius!vociferus),!The!Birds!of!

North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!

from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/517!

131.! Jaster,!L.A.,!Jensen,!W.E.,!Lanyon,!W.E.,!2012.!Eastern!Meadowlark!(Sturnella!

magna),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!

Ornithology;!Retrieved!from!the!Birds!of!North!America!

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/160!

doi:10.2173/bna.160!

132.! Jiang,!J.,!Stromdahl,!E.Y.,!Richards,!A.L.,!2012.!Detection!of!Rickettsia!parkeri!

and!Candidatus!Rickettsia!andeanae!in!Amblyomma!maculatum!Gulf!Coast!ticks!

collected!from!humans!in!the!United!States.!Vector

182.!doi:10.1089/vbz.2011.0614!

209

133.! Johnson,!B.J.,!1990.!Seasonal!distribution!and!population!dynamics!of!

Amblyomma!maculatum!Koch:!Ixodidae!in!Oklahoma.!M.S.!Thesis,!Oklahoma!State!

University,!Stillwater.!

134.! Johnson,!L.S.,!2014.!House!Wren!(Troglodytes!aedon),!The!Birds!of!North!

America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!from!

the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/380!

135.! Jones,!C.J.,!Kitron,!U.D.,!2000.!Populations!of!Ixodes!scapularis!(Acari:!

Ixodidae)!are!modulated!by!drought!at!a!Lyme!disease!focus!in!Illinois.!J.!Med!

Ent.!37,!408<415.!

136.! Judson,!O.P.,!1994.!The!rise!of!the!individual

Evol.!9,!9<14.!!

137.! Kearse,!M.,!Moir,!R.,!Wilson,!A.,!Stones

Drummond,!A.,!2012.!Geneious!Basic:!an!integrated!and!extendable!desktop!

software!platform!for!the!organization!and!analysis!of!sequence!data.!

Bioinformatics,!28,!1647<1649.!

138.! Keesing,!F.,!Ostfeld,!R.S.,!2015.!Is!biodiversity!good!for!your!health?!Science!

349,!235–236.!doi:10.1126/science.aac7892!

139.! Keirans,!G.,!Needham,!R.,!Oliver!Jr.,!J.H.,!1999.!The!Ixodes!ricinus!complex!

worldwide:!Diagnosis!of!the!species!in!the!complex,!hosts!and!distribution.!In!

Acarology!IX:!Symposia,!G.!R.!Needham,!R.!Michell,!D.!J.!Horn,!and!W.!C.Welbourn!

(eds.).!Ohio!Biological!Survey,!Columbus,!Ohio.!

210

140.! Keirans,!J.E.,!Clifford,!C.M.,!1978.!The!genus!Ixodes!in!the!United!States:!a!

scanning!electron!microscope!study!and!key!to!the!adults.!J.!Med.!Entomol.!Suppl.!2,!

1–149.!

141.! Keirans,!J.E.,!Durden,!L.A.,!1998.!Illustrated!key!to!nymphs!of!the!tick!genus!

Amblyomma!(Acari:!Ixodidae)!found!in!the!United!States.!J.!Med.!Entomol.!35,!489–

495.!

142.! Keirans,!J.E.,!Litwak,!T.R.,!1989.!Pictorial!key!to!the!adults!of!hard!ticks,!family!

Ixodidae!(Ixodida:!Ixodoidea),!east!of!the!Mississippi!River.!J.!Med.!Entomol.!26,!

435–448.!

143.! Kekkonen,!J.,!Wikström,!M.,!Brommer,!J.E.,!2012.!Heterozygosity!in!an!

isolated!population!of!a!large!mammal!founded!by!four!individuals!is!predicted!by!

an!individual

doi:10.1371/journal.pone.0043482!

144.! Kelly,!R.R.,!Gaines,!D.,!Gilliam,!W.F.,!Brinkerhoff,!R.J.,!2014.!Population!genetic!

structure!of!the!Lyme!disease!vector!Ixodes!scapularis!at!an!apparent!spatial!

expansion!front.!Infect.!Genet.!Evol.!27,!543–550.!doi:10.1016/j.meegid.2014.05.022!

145.! Kenis,!M.,!Auger

M.J.W.,!Settele,!J.,!Augustin,!S.,!Lopez

invasive!alien!insects.!Biol.!Invasions!11,!21–45.!doi:10.1007/s10530<008<9318

146.! Kershner,!E.L.,!Walk,!J.W.,!Warner,!R.E.,!2004.!Postfledging!movements!and!

survival!of!juvenile!Eastern!Meadowlarks!(Sturnella!magna)!in!Illinois.!Auk!121,!

1146<1154.!

147.! Ketchum,!H.R.,!Teel,!P.D.,!Coates,!C.J.,!Strey,!O.F.,!Longnecker,!M.T.,!2009.!

211

Genetic!variation!in!12S!and!16S!mitochondrial!rDNA!genes!of!four!geographically!

isolated!populations!of!Gulf!Coast!ticks!(Acari:!Ixodidae).!J.!Med.!Entomol.!46,!482–

489.!doi:10.1603/033.046.0311!

148.! Kilpatrick,!A.M,!Randolph,!S.E.,!2012.!Drivers,!dynamics,!and!control!of!

emerging!vector

doi:10.1016/j.micinf.2011.07.011.Innate!

149.! Kohls,!G.M.,!Rogers,!A.J.,!1953.!Note!on!the!occurrence!of!the!tick!Ixodes!affinis!

Neumann!in!the!United!States.!J.!Parasitol.!39,!44–45.!

150.! Koprowski,!J.L.,!1994.!Sciurus!carolinensis.!Mamm.!Species!480,!1–9.!

151.! Krebs,!C.J.,!2014.!Estimating!Community!Parameters.!In:!Ecological!Methodology,!2nd!

Ed.!Addison

152.! Krohne,!D.T.,!Dubbs,!B.A.,!Baccus,!R.,!1984.!An!analysis!of!dispersal!in!an!

unmanipulated!population!of!Peromyscus!leucopus.!Am.!Midl.!Nat.!112,!146<156.!

153.! Kruse,!I.,!Hare,!M.P.,!Hines,!A.H.,!2012.!Genetic!relationships!of!the!marine!

invasive!crab!parasite!Loxothylacus!panopaei:!an!analysis!of!DNA!sequence!

variation,!host!specificity,!and!distributional!range.!Biol.!Invasions!14,!701–715.!

doi:10.1007/s10530<011<0111

154.! Kuhnert,!F.,!Diehl,!P.!A.,!&!Guerin,!P.!M.!1995.!The!life

Amblyomma!hebraeum,!in!vitro.!Int.!J.!Parasitol.!25,!887<896.!

155.! Lackey,!J.A.,!Huckaby,!D.G.,!Ormiston,!B.G.,!1985.!Peromyscus!leucopus.!Mamm.!

Species!247,!1–10.!

156.! Lampo,!M.,!Rangel,!Y.,!Mata,!A.,!1998.!Population!genetic!structure!of!a!three<

host!tick,!Amblyomma!dissimile,!in!eastern!Venezuela.!J.!Parasitol.!84,!1137<1142.!

212

157.! Lancaster,!N.,!Baas,!A.,!1998.!Influence!of!vegetation!cover!on!sand!transport!

by!wind:!field!studies!at!Owens!Lake,!California.!Earth!Surf.!Proc.!Land.!23,!69<82.!

158.! !Langley!Jr,!A.K.,!Shure,!D.J.,!1980.!The!effects!of!loblolly!pine!plantations!on!

small!mammal!populations.!Am.!Mid.!Nat.!103,!59<65.!

159.! Larivière,!S.,!2001.!Ursus!americanus.!Mamm.!Species!647,!1–11.!!

160.! Larivière,!S.,!Walton,!L.R.,!1997.!Lynx!rufus.!Mamm.!Species!1–8.!

161.! Lawlor,!T.E.,!1986.!Comparative!biogeography!of!mammals!on!islands.!Biol.!J.!

Linn.!Soc.!28,!99<125.!

162.! Le!Roux,!J.,!Wieczorek,!!a.!M.,!2009.!Molecular!systematics!and!population!

genetics!of!biological!invasions:!towards!a!better!understanding!of!invasive!species!

management.!Ann.!Appl.!Biol.!154,!1–17.!doi:10.1111/j.1744<7348.2008.00280.x!

163.! Léger,!E.,!Vourc’h,!G.,!Vial,!L.,!Chevillon,!C.,!McCoy,!K.D.,!2013.!Changing!

distributions!of!ticks:!causes!and!consequences.!Exp.!Appl.!Acarol.!59,!219–44.!

doi:10.1007/s10493<012<9615<0!

164.! Leighton,!P.!a.,!Koffi,!J.K.,!Pelcat,!Y.,!Lindsay,!L.R.,!Ogden,!N.H.,!2012.!Predicting!

the!speed!of!tick!invasion:!an!empirical!model!of!range!expansion!for!the!Lyme!

disease!vector!Ixodes!scapularis!in!Canada.!J.!Appl.!Ecol.!49,!457–464.!

doi:10.1111/j.1365<2664.2012.02112.x!

165.! Lesica,!P.,!Allendorf,!F.W.,!1995.!When!are!peripheral!populations!valuable!for!

conservation?!Conserv.!Biol.!9,!753<760.!

166.! Li,!S.,!Hartemink,!N.,!Speybroeck,!N.,!Vanwambeke,!S.O.,!2012.!Consequences!

of!landscape!fragmentation!on!Lyme!disease!risk:!a!cellular!automata!approach.!

PLoS!One!7,!e39612.!doi:10.1371/journal.pone.0039612!

213

167.! Lindblom,!L.,!2009.!Sample!size!and!haplotype!richness!in!population!

samples!of!the!lichen

doi:10.1017/S0024282909008743!

168.! Lodge,!D.M.,!1993.!Biological!invasions:!lessons!for!ecology.!Trends!Ecol.!

Evol.!8,!133–137.!doi:10.1016/0169<5347(93)90025

169.! LoGiudice,!K.,!Ostfeld,!R.S.,!Schmidt,!K.A,!Keesing,!F.,!2003.!The!ecology!of!

infectious!disease:!effects!of!host!diversity!and!community!composition!on!Lyme!

disease!risk.!Proc.!Natl.!Acad.!Sci.!U.!S.!A.!100,!567–571.!

doi:10.1073/pnas.0233733100!

170.! Lotze,!J.H.,!Anderson,!S.,!1979.!Procyon!lotor.!Mamm.!Species!1–8.!

171.! Lounibos,!L.P.,!2002.!Invasions!by!insect!vectors!of!human!disease.!Annu.!

Rev.!Entomol.!47,!233–266.!

172.! Lowther,!P.E.,!1993.!Brown

North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!

from!the!Birds!of!North!America!

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/047!

173.! Ludsin,!S.!A.,!Wolfe,!A.D.,!2001.!Biological!invasion!theory:!Darwin’s!

contributions!from!The!Origin!of!Species.!Bioscience!51,!780.!doi:10.1641/0006<

3568(2001)051[0780:BITDSC]2.0.CO;2!

174.! M’Closkey,!R.T.,!Fieldwick,!B.,!1975.!Ecological!Separation!of!Sympatric!

Rodents!(Peromyscus!and!Microtus).!J.!Mammal.!56,!119–129.!doi:10.2307/1379611!

214

175.! Macaluso,!K.R.,!Mulenga,!A.,!Simser,!J.A.,!Azad,!A.F.,!2003.!Differential!

expression!of!genes!in!uninfected!and!Rickettsia

ticks!as!assessed!by!differential

176.! MacArthur,!R.H.,!Wilson,!E.O.,!1967.!The!theory!of!island!biogeography!(Vol.!

1).!Princeton!University!Press.!

177.! Mack,!D.E.,!Yong,!W.,!2000.!Swainson's!Thrush!(Catharus!ustulatus),!The!

Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!

Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/540!

178.! Mack,!R.N.,!Simberloff,!D.,!Lonsdale,!W.M.,!Evans,!H.,!Clout,!M.,!Bazzaz,!F.,!

2000.!Biotic!invasions:!causes,!epidemiology,!global!consequences,!and!control.!Ecol.!

Appl.!10,!689–710.!

179.! Madhav,!N.K.,!Brownstein,!J.S.,!Tsao,!J.I.,!Fish,!D.,!2004.!A!dispersal!model!for!

the!range!expansion!of!blacklegged!tick!(Acari:!Ixodidae).!J.!Med.!Entomol.!41,!842–

852.!doi:10.1603/0022<2585<41.5.842!

180.! Maggi,!R.G.,!Reichelt,!S.,!Toliver,!M.,!Engber,!B.,!2010.!Borrelia!species!in!

Ixodes!affinis!and!Ixodes!scapularis!ticks!collected!from!the!coastal!plain!of!North!

Carolina.!Ticks!Tick.!Borne.!Dis.!1,!168–171.!doi:10.1016/j.ttbdis.2010.08.003!

181.! MathWorks,!Inc.,!2015.!MATLAB:!the!language!of!technical!computing.!

Desktop!tools!and!development!environment.!MathWorks.!

182.! McCay,!T.S.,!2001.!Blarina!carolinensis.!Mamm.!Species!673,!1–7.!

183.! Mccoy,!K.D.!and!Tirard,!C.,!2000.!Isolation!and!characterization!of!

microsatellites!in!the!seabird!ectoparasite!Ixodes!uriae.!Mol.!Ecol.!9,!2212–2213.!

215

184.! McCoy,!K.D.,!Boulinier,!T.,!Tirard,!C.,!Michalakis,!Y.,!2001.!Host!specificity!of!a!

generalist!parasite:!Genetic!evidence!of!sympatric!host!races!in!the!seabird!tick!

Ixodes!uriae.!J.!Evol.!Biol.!14,!395–405.!doi:10.1046/j.1420<9101.2001.00290.x!

185.! McCoy,!K.D.,!Boulinier,!T.,!Tirard,!C.,!Michalakis,!Y.,!2003.!Host

genetic!structure!of!parasite!populations:!differential!dispersal!of!seabird!tick!host!

races.!Evolution!57,!288–296.!doi:10.1111/j.0014<3820.2003.tb00263.x!

186.! McDiarmid,!R.W.,!Foster,!M.S.,!Guyer,!C.,!Gibbons,!J.W.,!Chernoff,!N.,!

2011.!Reptile!biodiversity:!standard!methods!for!inventory!and!monitoring.!

University!of!California!Press.!

187.! McGuire,!B.,!Pizzuto,!T.,!Bemis,!W.E.,!Getz,!L.L.,!2006.!General!ecology!of!a!

rural!population!of!Norway!rats!(Rattus!norvegicus)!based!on!intensive!live!

trapping.!Am.!Midl.!Nat.!155,!221<236.!

188.! McManus,!J.J.,!1974.!Didelphis!virginiana.!Mamm.!Species!1–6.!

189.! Mechai,!S.,!Feil,!E.J.,!Gariepy,!T.D.,!Gregory,!T.R.,!Lindsay,!L.R.,!Millien,!V.,!

Ogden,!N.H.,!2013.!Investigation!of!the!population!structure!of!the!tick!vector!of!

Lyme!disease!Ixodes!scapularis!(Acari:!Ixodidae)!in!Canada!using!mitochondrial!

cytochrome!C!oxidase!subunit!I!gene!sequences.!J.!Med.!Entomol.!50,!560–570.!

190.! Merimans,!P.G.,!2012.!The!trouble!with!isolation!by!distance.!Mol.!Ecol.!21,!

2839<2846.!

191.! Mertins,!J.W.,!Moorhouse,!A.S.,!Alfred,!J.T.,!Hutcheson,!H.J.,!2010.!Amblyomma!

triste!(Acari:!Ixodidae):!new!North!American!collection!records,!including!the!first!

from!the!United!States.!J.!Med.!Entomol.!47,!536–542.!doi:10.1603/ME09165!

192.! Mixson,!T.R.,!Lydy,!S.L.,!Dasch,!G.A.,!Real,!L.A.,!2006.!Inferring!the!population!

216

structure!and!demographic!history!of!the!tick,!Amblyomma!americanum!Linnaeus.!J.!

Vector!Ecol.!31,!181–192.!!

193.! Morlando,!S.,!Schmidt,!S.J.,!LoGiudice,!K.,!2012.!Reduction!in!Lyme!disease!

risk!as!an!economic!benefit!of!habitat!restoration.!Restor.!Ecol.!20,!498–504.!

doi:10.1111/j.1526<100X.2011.00796.x!

194.! Mount,!G.A.,!Haile,!D.G.!and!Daniels,!E.,!1997.!Simulation!of!blacklegged!tick!

(Acari:!Ixodidae)!population!dynamics!and!transmission!of!Borrelia!burgdorferi.!J.!

Med.!Ent.!34,!461<484.!

195.! Mowbray,!T.B.,!1997.!Swamp!Sparrow!(Melospiza!georgiana),!The!Birds!of!

North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!

from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/279!

196.! Myers,!J.A.,!Vellend,!M.,!Gardescu,!S.!and!Marks,!P.L.,!2004.!Seed!dispersal!by!

white

of!plants!in!eastern!North!America.!Oecologia!139,!35<44.!

197.! Nadolny,!R.,!Gaff,!H.,!Carlsson,!J.,!Gauthier,!D.,!2015.!Comparative!population!

genetics!of!two!invading!ticks:!evidence!of!the!ecological!mechanisms!underlying!

tick!range!expansions.!Infect.!Genet.!Evol.!35,!153–162.!

doi:10.1016/j.meegid.2015.08.009!

198.! Nadolny,!R.M.,!Wright,!C.L.,!Hynes,!W.L.,!Sonenshine,!D.E.,!Gaff,!H.D.,!2011.!

Ixodes!affinis!(Acari:!Ixodidae)!in!southeastern!Virginia!and!implications!for!the!

spread!of!Borrelia!burgdorferi,!the!agent!of!Lyme!disease.!J.!Vector!Ecol.!36,!464–

217

467.!doi:10.1111/j.1948<7134.2011.00191.x!

199.! Nadolny,!R.M.,!Wright,!C.L.,!Sonenshine,!D.E.,!Hynes,!W.L.,!Gaff,!H.D.,!2014.!

Ticks!and!spotted!fever!group!rickettsiae!of!southeastern!Virginia.!Ticks!Tick.!Borne.!

Dis.!5,!53–57.!doi:10.1016/j.ttbdis.2013.09.001!

200.! Needham,!G.R.,!Teel,!P.D.,!1991.!Off

Annual!Rev.!Ent.!36,!659<681.!

201.! Nelder,!M.P.,!Reeves,!W.K.,!2005.!Ectoparasites!of!road

northwestern!South!Carolina,!USA.!Vet.!Parasitol.!129,!313–322.!

doi:10.1016/j.vetpar.2004.02.029!

202.! Norris,!D.,!Klompen,!J.,!Black!IV,!W.,!1999.!Comparison!of!the!mitochondrial!

12S!and!16S!ribosomal!DNA!genes!in!resolving!phylogenetic!relationships!among!

hard!ticks!(Acari:!Ixodidae).!Ann.!Entomol.!Soc.!Am.!92,!117–129.!

doi:10.1093/aesa/92.1.117!

203.! Norris,!D.E.,!2004.!Mosquito

change.!Ecohealth!1,!19–24.!doi:10.1007/s10393<004<0008<7!

204.! Norris,!D.E.,!Klompen,!J.S.,!Keirans,!J.E.,!Black,!W.C.,!1996.!Population!genetics!

of!Ixodes!scapularis!(Acari:!Ixodidae)!based!on!mitochondrial!16S!and!12S!genes.!J.!

Med.!Entomol.!33,!78–89.!

205.! Ogden,!N.H.,!Bigras

Kurtenbach,!K.,!2008a.!Projected!effects!of!climate!change!on!tick!phenology!and!

fitness!of!pathogens!transmitted!by!the!North!American!tick!Ixodes!scapularis.!J.!

Theor.!Biol.!254,!621–632.!doi:10.1016/j.jtbi.2008.06.020!

206.! Ogden,!N.H.,!Lindsay,!L.R.,!Hanincová,!K.,!Barker,!I.K.,!Bigras

218

Charron,!D.F.,!Heagy,!A.,!Francis,!C.M.,!O’Callaghan,!C.J.,!Schwartz,!I.,!Thompson,!R.A.,!

2008b.!Role!of!migratory!birds!in!introduction!and!range!expansion!of!Ixodes!

scapularis!ticks!and!of!Borrelia!burgdorferi!and!Anaplasma!phagocytophilum!in!

Canada.!Appl.!Environ.!Microbiol.!74,!1780–1790.!doi:10.1128/AEM.01982<07!

207.! Ogden,!N.H.,!Maarouf,!A.,!Barker,!I.K.,!Bigras

Morshed,!M.G.,!O’Callaghan,!C.J.,!Ramay,!F.,!Waltner

Climate!change!and!the!potential!for!range!expansion!of!the!Lyme!disease!vector!

Ixodes!scapularis!in!Canada.!Int.!J.!Parasitol.!36,!63–70.!

doi:10.1016/j.ijpara.2005.08.016!

208.! Ogden,!N.H.,!Margos,!G.,!Aanensen,!D.M.,!Drebot,!M.A.,!Feil,!E.J.,!Hanincova,!K.,!

Schwartz,!I.,!Tyler,!S.!Lindsay,!L.R.,!2011.!Investigation!of!genotypes!of!Borrelia!

burgdorferi!in!Ixodes!scapularis!ticks!collected!during!surveillance!in!Canada.!Appl.!

Environ.!Microb.!77,!3244<3254.!

209.! Ogden,!N.H.,!Mechai,!S.!Margos,!G.,!2015.!Changing!geographic!ranges!of!ticks!

and!tick

diversity.!Front.!Cell.!Inf.!Microb.!3,!7Q17.!

210.! Ogden,!N.H.,!Mechai,!S.,!Margos,!G.,!2013.!Changing!geographic!ranges!of!ticks!

and!tick

diversity.!Front.!Cell.!Infect.!Microbiol.!3,!46.!doi:10.3389/fcimb.2013.00046!

211.! Oliver,!J.H.,!1996.!Lyme!borreliosis!in!the!southern!United!States:!a!review.!J.!

Parasitol.!82,!926–935.!

212.! Oliver,!J.H.,!Keirans,!J.E.,!Lavender,!D.R.,!Hutcheson,!H.J.,!1987.!Ixodes!affinis!

Neumann!(Acari:!Ixodidae):!new!host!and!distribution!records,!description!of!

219

immatures,!seasonal!activities!in!Georgia,!and!laboratory!rearing.!J.!Parasitol.!73,!

646–652.!

213.! Oliver,!J.H.,!Lin,!T.,!Gao,!L.,!Clark,!K.L.,!Banks,!C.W.,!Durden,!L.A.,!James,!A.M.,!

Chandler,!F.W.,!2003.!An!enzootic!transmission!cycle!of!Lyme!borreliosis!

spirochetes!in!the!southeastern!United!States.!Proc.!Natl.!Acad.!Sci.!U.!S.!A.!100,!

11642–11645.!doi:10.1073/pnas.1434553100!

214.! Ostfeld,!R.S.,!Keesing,!F.,!2000.!Biodiversity!and!disease!risk:!the!case!of!Lyme!

disease.!Conserv.!Biol.!14,!722–728.!doi:10.1046/j.1523<1739.2000.99014.x!

215.! Ostfeld,!R.S.,!Keesing,!F.,!2012.!Effects!of!host!diversity!on!infectious!disease.!

Annu.!Rev.!Ecol.!Evol.!Syst.!43,!157–182.!doi:10.1146/annurev

145022!

216.! Paddock,!C.D.,!Finley,!R.W.,!Wright,!C.S.,!Robinson,!H.N.,!Schrodt,!B.J.,!Lane,!

C.C.,!Ekenna,!O.,!Blass,!M.A.,!Tamminga,!C.L.,!Ohl,!C.A.,!McLellan,!S.L.F.,!Goddard,!J.,!

Holman,!R.C.,!Openshaw,!J.J.,!Sumner,!J.W.,!Zaki,!S.R.,!Eremeeva,!M.E.,!2008.!Rickettsia!

parkeri!Rickettsiosis!and!its!clinical!distinction!from!Rocky!Mountain!spotted!fever.!

Clin.!Infect.!Dis.!47,!1188–1196.!doi:10.1086/592254!

217.! Paddock,!C.D.,!Fournier,!P.E.,!Sumner,!J.W.,!Goddard,!J.,!Elshenawy,!Y.,!

Metcalfe,!M.G.,!Loftis,!A.D.,!Varela

identification!of!a!novel!spotted!fever!group!Rickettsia!sp.!from!Gulf!Coast!ticks!

(Amblyomma!maculatum)!in!the!United!States.!Appl.!Environ.!Microbiol.!76,!2689–

2696.!doi:10.1128/AEM.02737<09!

218.! Paddock,!C.D.,!Goddard,!J.,!2015.!The!evolving!medical!and!veterinary!

importance!of!the!Gulf!Coast!tick!(Acari:!Ixodidae).!J.!Med.!Entomol.!52,!230–252.!

220

doi:10.1093/jme/tju022!

219.! Paddock,!C.D.,!Sumner,!J.W.,!Comer,!J.A,!Zaki,!S.R.,!Goldsmith,!C.S.,!Goddard,!J.,!

McLellan,!S.L.F.,!Tamminga,!C.L.,!Ohl,!C.A.,!2004.!Rickettsia!parkeri:!a!newly!

recognized!cause!of!spotted!fever!rickettsiosis!in!the!United!States.!Clin.!Infect.!Dis.!

38,!805–811.!doi:10.1086/381894!

220.! Pardieck,!K.L.,!Ziolkowski!Jr.,!D.J.,!Hudson,!M.A.R.,!2015.!North!American!

Breeding!Bird!Survey!Dataset!1966!

Patuxent!Wildlife!Research!Center!.!

221.! Parker,!I.M.,!Simberloff,!D.,!Lonsdale,!W.M.,!Goodell,!K.,!Wonham,!M.,!Kareiva,!

P.M.,!Williamson,!M.H.,!Von!Holle,!B.,!Moyle,!P.B.,!Byers,!J.E.,!Goldwasser,!L.,!1999.!

Impact:!toward!a!framework!for!understanding!the!ecological!effects!of!invader.!

Biol.!Invasions!1,!3–19.!doi:10.1023/A:1010034312781!

222.! Patz,!J.A.,!Campbell

regional!climate!change!on!human!health.!Nature!438,!310–317.!

doi:10.1038/nature04188!

223.! Payne,!N.F.,!1975.!Range!extension!of!the!marsh!rabbit!in!Virginia.!

Chesapeake!Science!16,!77<78.!

224.! !Peer,!B.D.,!Bollinger,!E.K.,!1997.!Common!Grackle!(Quiscalus!quiscula),!The!

Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!

Retrieved!from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/271!

doi:10.2173/bna.271!

221

225.! Pertoldi,!C.,!Topping,!C.,!2004.!The!use!of!agent

in!conservation!genetics!studies.!J.!Nat.!Conserv.!12,!111–120.!

doi:10.1016/j.jnc.2003.12.001!

226.! Pfäffle,!M.,!Petney,!T.,!Skuballa,!J.,!Taraschewski,!H.,!2011.!Comparative!

population!dynamics!of!a!generalist!(Ixodes!ricinus)!and!specialist!tick!(I.!hexagonus)!

species!from!European!hedgehogs.!Exp.!Appl.!Acarol.!54,!151–164.!

doi:10.1007/s10493<011<9432

227.! Pike,!N.,!2011.!Using!false!discovery!rates!for!multiple!comparisons!in!

ecology!and!evolution.!Methods!in!Ecology!and!Evolution,!2,!278<282.!

228.! Piper,!W.!H.,!Wiley,!R.!H.,!1990.!Correlates!of!range!size!in!wintering!White<

throated!Sparrows,!Zonotrichia!albicollis.!Anim.!Behav.!40,!545<552.!

229.! Porter,!J.,!Dueser,!R.,!2011.!Hog!Island!VA!Small

2011.!Virginia!Coast!Reserve!Long

Publication!knb

lter

230.! !Qiu,!W.G.,!Dykhuizen,!D.E.,!Acosta,!M.S.,!Luft,!B.J.,!2002.!Geographic!

uniformity!of!the!lyme!disease!spirochete!(Borrelia!burgdorferi)!and!its!shared!

history!with!tick!vector!(Ixodes!scapularis)!in!the!Northeastern!United!States.!

Genetics!160,!833–849.!

231.! R!Core!Team,!2015.!R:!a!language!and!environment!for!statistical!computing.!

Vienna,!Austria.!Available!at!http://www.R

232.! Radcliffe,!J.,!Rass,!L.,!1984.!The!spatial!spread!and!final!size!of!models!for!the!

deterministic!host

222

233.! Randolph,!S.E.,!Storey,!K.,!1999.!Impact!of!microclimate!on!immature!tick<

rodent!host!interactions!(Acari:!Ixodidae):!implications!for!parasite!transmission.!J.!

Med.!Ent.!36,!741<748.!

234.! Recht,!M.A.,!1988.!The!biology!of!domestic!rats:!telemetry!yields!insight!for!

pest!control.!Proceedings!of!the!13th!Vertebrate!Pest!Conference.!21,!98<100.!

235.! Ronquist,!F.,!Huelsenbeck,!J.P.,!2003.!MrBayes!3:!Bayesian!phylogenetic!

inference!under!mixed!models.!Bioinformatics!19,!1572<1574.!

236.! Rosà,!R.,!Pugliese,!A.,!2007.!Effects!of!tick!population!dynamics!and!host!

densities!on!the!persistence!of!tick

doi:10.1016/j.mbs.2006.10.002!

237.! Rose,!R.K.,!1994.!Instructions!for!building!two!live!traps!for!small!mammals.!

Va.!J.!Sci.!45,!151–158.!

238.! Rudenko,!N.,!Golovchenko,!M.,!Honig,!V.,!Mallatova,!N.,!Krbkova,!L.,!Mikulasek,!

P.,!Fedorova,!N.,!Belfiore,!N.M.,!Grubhoffer,!L.,!Lane,!R.S.,!Oliver,!J.H.,!2013.!Detection!

of!Borrelia!burgdorferi!sensu!stricto!ospC!alleles!associated!with!human!lyme!

borreliosis!worldwide!in!non

southeastern!United!States.!Appl.!Environ.!Microbiol.!79,!1444–1453.!

doi:10.1128/AEM.02749<12!

239.! Sandberg,!S.,!Awerbuch,!T.!E.,!Spielman,!A.,!1992.!A!comprehensive!multiple!

matrix!model!representing!the!life!cycle!of!the!tick!that!transmits!agent!of!Lyme!

disease.!J.!Theor.!Biol.!157,!203<220.!

240.! Schmidly,!D.J.,!2004.!The!mammals!of!Texas:!revised!edition!(No.!59).!

University!of!Texas!Press.!

223

241.! Schmitz,!O.J.,!2000.!Combining!field!experiments!and!individual

identify!the!dynamically!relevant!organizational!scale!in!a!field!system.!Oikos!89,!471<484.!!

242.! Schweiger,!E.W.,!Diffendorfer,!J.E.,!Holt,!R.D.,!Pierotti,!R.,!Gaines,!M.S.,!2000.!

The!interaction!of!habitat!fragmentation,!plant,!and!small!mammal!succession!in!an!

old!field.!Ecol.!Monogr.!70,!383<400.!

243.! Scifres,!C.J.,!Oldham,!T.W.,!Teel,!P.D.,!Drawe,!D.L.,!1988.!Gulf!Coast!tick!

(Amblyomma!maculatum!)!Populations!and!responses!to!burning!of!coastal!prairie!

habitats.!southwest.!Assoc.!Nat.!33,!55–64.!

244.! Scott,!J.D.,!Anderson,!J.F.,!Durden,!L.A.,!2012.!Widespread!dispersal!of!Borrelia!

burgdorferi–infected!ticks!collected!from!songbirds!across!Canada.!J.!Parasitol.!98,!

49–59.!doi:10.1645/GE<2874.1!

245.! Scott,!J.D.,!Fernando,!K.,!Banerjee,!S.N.,!Durden,!L.A.,!Byrne,!S.K.,!Banerjee,!M.,!

Mann,!R.B.,!Morshed,!M.G.,!2001.!Birds!disperse!Ixodid!(Acari:!Ixodidae)!and!

Borrelia!burgdorferi

246.! Semtner,!P.J.,!Hair,!J.A.,!1973.!Distribution,!seasonal!abundance,!and!hosts!of!

the!Gulf!Coast!tick!in!Oklahoma.!Ann.!Entomol.!Soc.!Am.!66,!1264<1268.!

247.! Seppa,!N.,!2015.!Chikungunya:!Is!on!the!Move:!The!virus!has!found!a!new!

hemisphere!and!might!get!a!new!latitude.!Science!News,!187:!16–20.!

doi:!10.1002/scin.2015.187012017!

248.! Shackelford,!C.E.,!Brown,!R.E.,!Conner,!R.N.,!2000.!Red

(Melanerpes!carolinus),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!

Cornell!Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!America!

Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/500!

224

249.! Shumway,!S.W.,!2000.!Facilitative!effects!of!a!sand!dune!shrub!on!species!

growing!beneath!the!shrub!canopy.!Oecologia!124,!138<148.!

250.! Sikes,!R.S.,!Gannon,!W.L.,!2011.!Guidelines!of!the!American!Society!of!

Mammalogists!for!the!use!of!wild!mammals!in!research.!J.!Mammal.!92,!235–253.!

doi:10.1644/10

251.! Simon,!J.A.,!Marrotte,!R.R.,!Desrosiers,!N.,!Fiset,!J.,!Gaitan,!J.,!Gonzalez,!A.,!Koffi,!

J.K.,!Lapointe,!F.

Rogic,!A.,!Roy

habitat!fragmentation!drive!the!occurrence!of!Borrelia!burgdorferi,!the!agent!of!

Lyme!disease,!at!the!northeastern!limit!of!its!distribution.!Evol.!Appl.!7,!750–64.!

doi:10.1111/eva.12165!

252.! Smith,!K.G.,!Tarvin,!K.A.,!Woolfenden,!G.E.,!2013.!Blue!Jay!

(Cyanocitta!cristata),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!

Cornell!Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/469!

253.! Smith,!R.!P.,!Rand,!P.!W.,!Lacombe,!E.!H.,!Morris,!S.!R.,!Holmes,!D.!W.,!Caporale,!

D.!A.,!1996.!Role!of!bird!migration!in!the!long

the!vector!of!Lyme!disease.!J.!Infect.!Dis.!174,!221<224.!

254.! Smith,!Robert!J.,!Margret!I.!Hatch,!David!A.!Cimprich!and!Frank!R.!Moore.!

2011.!Gray!Catbird!(Dumetella!carolinensis),!The!Birds!of!North!America!Online!(A.!

Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!

225

America!Online:http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/167!

doi:10.2173/bna.167!

255.! Smith,!W.P.,!1991.!Odocoileus!virginianus.!Mamm.!Species!388,!1–13.!

256.! Sonenshine,!D.E.,!1979.!Ticks!of!Virginia!(Acari,!Metastigmata).!Virginia!

Polytechnic!Institute!and!State!University.!

257.! Sonenshine,!D.E.,!1991.!Biology!of!Ticks,!Vol.!1.!Oxford!University!Press,!New!

York.!!

258.! Speldewinde,!P.C.,!Slaney,!D.,!Weinstein,!P.,!2015.!Is!restoring!an!ecosystem!

good!for!your!health?!Sci.!Total!Environ.!502,!276–279.!

doi:10.1016/j.scitotenv.2014.09.028!

259.! Springer,!Y.P.,!Eisen,!L.,!Beati,!L.,!James,!A.M.,!Eisen,!R.J.,!2014.!Spatial!

Distribution!of!counties!in!the!continental!united!states!with!records!of!occurrence!

of!Amblyomma!americanum!(Ixodida:!Ixodidae).!J.!Med.!Entomol.!51,!342–351.!

doi:10.1603/ME13115!

260.! Stalling,!D.T.,!1997.!Reithrodontomys!humulis.!Mamm.!Species!565,!1–6.!

261.! Stromdahl,!E.Y.,!Hickling,!G.J.,!2012.!Beyond!Lyme:!Aetiology!of!tick

human!diseases!with!emphasis!on!the!southeastern!United!States.!Zoonoses!Public!

Hlth.!59,!48–64.!doi:10.1111/j.1863<2378.2012.01475.x!

262.! Sumner,!J.W.,!Durden,!L.!a.,!Goddard,!J.,!Stromdahl,!E.Y.,!Clark,!K.L.,!Reeves,!

W.K.,!Paddock,!C.D.,!2007.!Gulf!coast!ticks!(Amblyomma!maculatum)!and!Rickettsia!

parkeri,!United!States.!Emerg.!Infect.!Dis.!13,!751–753.!doi:10.3201/eid1305.061468!

263.! Sutherland,!G.D.,!Harestad,!A.S.,!Price,!K.,!Lertzman,!K.P.,!2000.!Scaling!of!

226

natal!dispersal!distances!in!terrestrial!birds!and!mammals.!Conserv.!Ecol.!4,!16.!

doi:16\nArtn!16!

264.! Sutherst,!R.W.,!Bourne,!!A.!S.,!2009.!Modelling!non

of!invasive!species:!A!tale!of!two!modelling!paradigms.!Biol.!Invasions!11,!1231–

1237.!doi:10.1007/s10530<008<9335

265.! Tajima,!F.,!1989.!Statistical!method!for!testing!the!neutral!mutation!

hypothesis!by!DNA!polymorphism.!Genetics,!123,!585<595.!

266.! Tamura,!K.,!Dudley,!J.,!Nei,!M.,!Kumar,!S.,!2007.!MEGA4:!molecular!

evolutionary!genetics!analysis!(MEGA)!software!version!4.0.!Mol.!Biol.!Evol.!24,!

1596<1599.!

267.! Taylor,!P.D.,!Fahrig,!L.,!Henein,!K.,!Merriam,!G.,!1993.!Connectivity!is!a!vital!

element!of!landscape!structure.!Oikos!68,!571–573.!

268.! Teacher,!A.!G.!F.,!Griffiths,!D.!J.,!2011.!HapStar:!automated!haplotype!network!

layout!and!visualization.!Mol.!Ecol.!Resour,!11,!151<153.!

269.! Teel,!P.D.,!Hopkins,!S.W.,!Donahue,!W.A.,!Strey,!O.F.!1998.!Population!

dynamics!of!immature!Amblyomma!maculatum!(Acari:!Ixodidae)!and!other!

ectoparasites!on!meadowlarks!and!northern!bobwhite!quail!resident!to!the!coastal!

prairie!of!Texas.!J.!Med.!Ent.!35,!483<488.!

270.! Teel,!P.D.,!Ketchum,!H.R.,!Mock,!D.E.,!Wright,!R.E.,!Strey,!O.F.,!2010.!The!Gulf!

Coast!tick:!a!review!of!the!life!history,!ecology,!distribution,!and!emergence!as!an!

arthropod!of!medical!and!veterinary!importance.!J.!Med.!Entomol.!47,!707–722.!

doi:10.1603/ME10029!

271.! Templeton,!A.R.,!Routman,!E.,!Phillips,!C.A.,!1995.!Separating!population!

227

structure!from!population!history:!a!cladistic!analysis!of!the!geographical!

distribution!of!mitochondrial!DNA!haplotypes!in!the!tiger!salamander,!Ambystoma!

tigrinum.!Genetics!140,!767–82.!

272.! Trout!Fryxell,!R.T.,!Moore,!J.E.,!Collins,!M.D.,!Kwon,!Y.,!Jean

Schaeffer,!S.M.,!Odoi,!A.,!Kennedy,!M.,!Houston,!A.E.,!2015.!Habitat!and!vegetation!

variables!are!not!enough!when!predicting!tick!populations!in!the!southeastern!

United!States.!PLoS!One!10,!e0144092.!doi:10.1371/journal.pone.0144092!

273.! Trout,!R.T.,!Steelman,!C.D.,!Szalanski,!A.L.,!Loftin,!K.,!2010.!Establishment!of!

Amblyomma!maculatum!(Gulf!Coast!Tick)!in!Arkansas,!U.S.A.!Florida!Entomol.!93,!

120–122.!doi:10.1653/024.093.0117!

274.! USACE

http://shoals.sam.usace.army.mil/!

275.! Vanderhoff,!N.,!Sallabanks,!R.,!James,!F.C.,!2014.!American!Robin!(Turdus!

migratorius),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!

of!Ornithology;!Retrieved!from!the!Birds!of!North!America!Online:!

http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/462!!

276.! Varela

parkeri!in!Amblyomma!maculatum!Ticks,!North!Carolina,!USA,!2009–2010.!Emerg.!

Infect.!Dis.!17,!2350–2353.!doi:10.3201/eid1712.110789!

277.! Vermeij,!G.!J.,!1991.!When!biotas!meet:!understanding!biotic!interchange.!

Science!253,!1099<1104.!

228

278.! Vickery,!P.D.,!1996.!Grasshopper!Sparrow!(Ammodramus!savannarum),!The!

Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!

Retrieved!from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/239!

279.! Wade

280.! Wang,!H.,!Grant,!W.E.,!Teel,!P.D.,!Hamer,!S.A.,!2015.!Simulation!of!climate

host

fluctuations!on!tick!densities.!J.!Vector!Ecol.!40,!247–255.!doi:10.1111/jvec.12161!

281.! Wang,!H.H.,!Grant,!W.E.,!Teel,!P.D.,!2012.!Simulation!of!climate

landscape!interactions:!A!spatially!explicit!model!for!ticks!(Acari:!Ixodidae).!Ecol.!

Modell.!243,!42–62.!doi:10.1016/j.ecolmodel.2012.06.007!

282.! Whisson,!D.A.,!Quinn,!J.H.,!Collins,!K.C.,!2007.!Home!range!and!movements!of!

roof!rats!(Rattus!rattus)!in!an!old

589<594.!

283.! Whitman,!T.J.,!Richards,!A.L.,!Paddock,!C.D.,!Tamminga,!C.L.,!Sniezek,!P.J.,!

Jiang,!J.,!Byers,!D.K.,!Sanders,!J.W.,!2007.!Rickettsia!parkeri!infection!after!tick!bite,!

Virginia.!Emerg.!Infect.!Dis.!13,!334–336.!doi:10.3201/eid1302.061295!

284.! Wiley,!R.W.,!1980.!Neotoma!floridana.!Mamm.!Species,!139,!1<7.!

285.! Williams,!C.F.,!Britton,!J.R.,!Turnbull,!J.F.,!2013.!A!risk!assessment!for!

managing!non

doi:10.1007/s10530<012<0364<0!

286.! Williams,!R.!E.,!Hair,!J.!A.,!1976.!Influence!of!Gulf!Coast!ticks!on!blood!

229

composition!and!weights!of!eastern!meadowlarks!in!Oklahoma.!Ann.!Entomol.!Soc.!

Am.!69,!403<404.!

287.! Wilson!Jr.,!W.H.,!2013.!Palm!Warbler!(Setophaga!palmarum),!The!Birds!of!

North!America!Online!(A.!Poole,!Ed.).!Ithaca:!Cornell!Lab!of!Ornithology;!Retrieved!

from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/238!

288.! !Wilson,!W.G.,!1998.!Resolving!discrepancies!between!deterministic!population!

models!and!individual

289.! Wolfe,!J.L.,!1982.!Oryzomys!palustris.!Mamm.!Species!176,!1–5.!

290.! Wolfe,!J.L.,!Linzey,!A.V.,!1977.!Peromyscus!gossypinus.!Mamm.!Species!70,!1<5.!!

291.! Wood,!C.L.,!Lafferty,!K.D.,!2013.!Biodiversity!and!disease:!a!synthesis!of!

ecological!perspectives!on!Lyme!disease!transmission.!Trends!Ecol.!Evol.!28,!239<

247.!

292.! Wood,!Gene!W.,!Barrett,!R.H.,!1979.!Status!of!wild!pigs!in!the!United!States.!

Wildlife!Soc.!B.!7,!237<246.!

293.! Wright,!C.L.,!Hynes,!W.L.,!White,!B.T.,!Marshall,!M.N.,!Gaff,!H.D.,!Gauthier,!D.T.,!

2014.!Single

scapularis.!Ticks!Tick.!Borne.!Dis.!5,!48–52.!doi:10.1016/j.ttbdis.2013.08.003!

294.! Wright,!C.L.,!Nadolny,!R.M.,!Jiang,!J.,!Richards,!A.L.,!Sonenshine,!D.E.,!Gaff,!H.D.,!

Hynes,!W.L.,!2011.!Rickettsia!parkeri!in!Gulf!Coast!Ticks,!Southeastern!Virginia,!USA.!

Emerg.!Infect.!Dis.!17,!896–898.!doi:10.3201/eid1705.101836!

295.! Wright,!C.L.,!Sonenshine,!D.E.,!Gaff,!H.D.,!Hynes,!W.L.,!2015.!Rickettsia!parkeri!

transmission!to!Amblyomma!americanum!by!cofeeding!with!Amblyomma!maculatum!

230

(Acari:!Ixodidae)!and!potential!for!spillover.!J.!Med.!Entomol.!52,!1090–1095.!

doi:10.1093/jme/tjv086!

296.! Wright,!J.E.,!1971.!Response!of!ovipositing!Amblyomma!maculatum!Koch!

(Acarina:!Ixodidae)!to!photoperiod.!J.!Med.!Entomol.!8,!529–531.!

doi:10.1093/jmedent/8.5.529!

297.! Xiong,!H.,!Barker,!S.C.,!Burger,!T.D.,!Raoult,!D.,!Shao,!R.,!2013.!Heteroplasmy!in!

the!mitochondrial!genomes!of!human!lice!and!ticks!revealed!by!high!throughput!

sequencing.!PLoS!One!8,!e73329.!doi:10.1371/journal.pone.0073329!

298.! Xu,!G.,!Fang,!Q.Q.,!Keirans,!J.E.,!Durden,!L.A.,!2003.!Molecular!phylogenetic!

analyses!indicate!that!the!Ixodes!ricinus!complex!is!a!paraphyletic!group.!J.!Parasitol.!

89,!452–457.!doi:10.1645/0022<3395(2003)089[0452:MPAITT]2.0.CO;2!

299.! Yasukawa,!K.,!Searcy,!W.A.,!1995.!Red

(Agelaius!phoeniceus),!The!Birds!of!North!America!Online!(A.!Poole,!Ed.).!Ithaca:!

Cornell!Lab!of!Ornithology;!Retrieved!from!the!Birds!of!North!America!

Online:!http://bna.birds.cornell.edu.proxy.lib.odu.edu/bna/species/184!

300.! Yoder,!J.A.,!Christensen,!B.S.,!Croxall,!T.J.,!Schumaker,!L.K.,!Tank,!J.L.,!2008.!

Moisture!requirements!for!activity!and!survival!of!the!Gulf!Coast!tick,!Amblyomma!

maculatum!Koch!(Acari:!Ixodidae),!based!on!a!water!balance!study!of!all!life!cycle!

stages.!Int.!J.!Acarol.!34,!285<292.!

301.! Young,!D.R.,!Shao,!G.,!Porter,!J.H.,!1995.!Spatial!and!temporal!growth!

dynamics!of!barrier!island!shrub!thickets.!Am.!J.!Bot.!82,!638<645.!

& &

231

TABLE&OF&IACUC&PROTOCOL&APPROVAL&NUMBERS&

Permit& Animals& IACUC&approval& Permit&name& holder& specified& number& Hampton!Roads!regional!tick!survey! Holly!Gaff! Small!mammals! 11<102! Hampton!Roads!regional!tick!survey,! Part!B! Holly!Gaff! Reptiles! 13<008! Bird

232

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VITA& & Robyn&M.&Nadolny& Department!of!Biological!Sciences! Old!Dominion!University,!Norfolk!VA!23529! & EDUCATION& Old!Dominion!University! Ecological!Sciences! ! ! PhD,!May!2016!! Old!Dominion!University! Biology! ! ! MS,!2011! ! ! Beloit!College! Ecology,!Evolution!and!Behavioral!Biology! BS,!2008! ! PROFESSIONAL&EXPERIENCE& 2011!–!present! SMART!summer!internships!with!the!Human!Tick!Test!Kit!Program,!Laboratory! Sciences,!Army!Public!Health!Center,!Aberdeen!Proving!Ground,!MD.! & SELECTED&GRANTS&RECEIVED& Nadolny!R.!M.!2014.!The!anatomy!of!an!invasion:!understanding!and!predicting!tick!range! expansions.!Jayne!Koskinas!Ted!Giovanis!Foundation!for!Health!and!Policy.!$14,763.!! Nadolny,!R.!M.!Funded!2011!–!2016.!Science,!Mathematics!And!Research!for!Transformation! (SMART)!Award.!U.S.!Department!of!Defense.!Tuition,!fees,!and!stipend!for!5!years!of!PhD! ! SELECTED&PUBLICATIONS&& Heller,!E.!L.,!C.!L.!Wright,!R.!M.!Nadolny,!W.!Hynes,!H.!D.!Gaff,!E.!L.!Walters.!2015.!New!records!of! Ixodes!affinis!parasitizing!avian!hosts!in!southeastern!Virginia.!Journal!of!Medical!Entomology.! doi:10.1093/jme/tjv175!! Nadolny,!R.!M.,!H.D.!Gaff,!J.!Carlsson,!D.!Gauthier.!Year.!Comparative!landscape!genetics!of!two! invading!ticks:!evidence!of!the!ecological!mechanisms!underlying!tick!range!expansions.! Infection,!Genetics,!and!Evolution.!35:!153<162.! Nadolny,!R.!M.,!K.!Feldman,!B.!Pagac,!E.!Y.!Stromdahl,!H.!Rutz,!S.!B.!Wee,!A.!L.!Richards,!J.!Smith,!M.! Armolt,!H.D.!Gaff.!Year.!Review!of!the!Mid