Assessment of the levels of resistance ofTropilaelaps mercedesaeto a variety of synthetic miticide

Asses sme n t of t he lev els o f r esis t ance of Tropilaelaps mercedesae to a variety 1 of synthetic miticide. 2 Mag gie C Gill 1 * , Baja r ee Chu t t ong 2 , P aul D avie s 1 , A vril Earl 1 , G eorg e T ong e 1 , D an Etherid g e 1 . 3 1 P HIRA - Sci ence , Lla ndybie, Ammanf o r d, UK. 4 2 Mel iponini a nd Apini R e se ar ch La bor a t o r y , F ac ulty o f Agricu lt u r e, C hia ng Ma i Univer sity , C hia ng 5 Mai, Th ailand . 6 * C o rr es p ond i n g au t hor p hir a .s c i enc e @gmail .c om 7 h ttps:/ / or c id .o r g /0000 - 0002 - 0 683 - 8491 8 9 Abs tr ac t 10 The int roduc tio n of the w e stern honey b ee Api s melli f e r a t o A s ia ha s se en t h e pa r asi t i c mit e s V arroa 11 de s t r uc t or and T r o pilael a ps spp. tr a n s f e r fr om t he ir na t i v e A s i an honey be e ho s ts ( Api s cer a na and 12 Apis dors a ta resp ective ly) t o in f e st the br ood o f A. melli f er a cau sing signifi ca n t damage t o coloni e s 13 and c ol on y los s e s. T . me r c ed e sae w a s recently det ec t ed in Eur ope f or th e fi r st time i n A. mel lif er a 14 c olo nie s an d i s c on side r ed a more dam ag ing par asi t e o f A. melli f e r a th an V a r roa. Be e k eep e r s r e ly 15 hea v ily on t he u se o f s y nthetic mi tic ide s a nd o r ganic c hemic al s t o c ontr ol V ar roa and T r opil aelap s 16 whic h has re s u l t e d in V a r roa d evelop ing r e si s t an ce t o m any s ynthetic miticid e s a n d the s e t rea t me n ts 17 bec o ming le ss eff ec t i v e . L e ss i s kno wn ab ou t c hemic al resi st a nce in T r opi la elap s a s no s tudy ha s b een 18 under t ak e n t h at spec i fic a lly lo ok s at thi s is s u e, b u t the r e i s evi dence to sug g e s t t ha t T r opil a elap s d o 19 ha v e r e si st a nc e t o chemi c al s s uc h a s A mitr az, C o umap ho s, F lumethrin and Flu valina t e . T he u s e of 20 s y n t hetic mit ici de s i s widely r e commen de d f o r s ur v e ill anc e a nd de t ec tion o f T ropila elap s a nd t his 21 r ec omme ndation f o r ms a pa rt o f the con tin g e ncy r e spon se of sev er al gov ern ment age ncie s. The 22 s tudy d ev elo p ed a nov e l c hemical r e si s tan ce te s t f or T ropila elaps and sought t o t e st t h e effi c ac y of 23 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint commer cia lly a vail ab le s y nt h e t i c mitici d es and f ound th a t mites we r e r e s i st a n t t o al l t he s ynthetic 24 ch emic a l treatme n t s t e s t ed a p art fr om A mi t r az whi ch was s how n t o be 64 % e ff ec tiv e. U nd e r st anding 25 and ma naging m it i cide r e si st a nce in thi s s pec i es i s c r i tica l to pr event it s f urth er spread a n d colon y 26 los s e s. 27 28 K ey w or ds – Tropilaelaps , Apis mellifera, m iticide , re s i s tanc e, treatment, hone y be e . 29 30 T h e i n t r o d u c t i o n o f t h e w e s t e r n h o n e y b e e Apis mellifera t o A s i a h a s s e e n t h e p ar a s i t i c m i t e s Varroa 31 destructor and Tropilaelaps s pp . tr an sf er f r om their native Asia n ho ney bee ho st s ( Apis cerana a n d 32 Apis dorsata r e spec tively) t o in f e st t h e br ood o f A. mellifera cau sing sign i fic a nt dam ag e t o c o l onie s 33 and colon y lo sse s [1 , 2] . Of the four sub - s pec ie s o f Tropilaelaps T . mercedesae i s t h e m o s t 34 wi des p r ea d and wa s rec ently de t ec t ed i n Eur ope f o r the fi rs t time in th e K r a sno dar r e gion o f Ru s s i a 35 and th e Sa meg r elo -Zemo S v a neti r e gion of G e o r g ia in A. mellifera colon ie s [3, 4 ]. T . mercedesae is 36 con s ide r e d a more da maging par a si t e of A. mellifera than Varroa [ 5 ] . H o w e v e r , Varroa i s m o r e 37 wi des p r ea d th an Tropilaelaps and is n ow pr es ent in alm os t every c o untr y global ly wh er e A. mellifera 38 is f ou n d [2 ]. B ee k e ep er s r e ly hea v ily o n the u se o f s y n the tic mitic ide s an d o rg a nic chem ic al s t o 39 cont r ol Varroa a nd Tropilaelaps [ 6] w hi ch h as r es ul t e d i n Varroa dev eloping r e si s t an ce t o man y 40 s yn t hetic mitici d e s a nd the s e t r e a tme n t s bec omi ng le s s eff ective [7, 8] . Le s s i s k n own a bout che mi c al 41 re s i sta n c e i n Tropilaelaps a s no study h as b een und er t ak en t h at spec i fic a lly lo o ks a t thi s is su e, bu t 42 there i s evide nce t o sug g e st that Tropilaelaps do hav e r e s i st anc e t o chemical s such a s Amitr az , 43 Couma pho s, F lumethrin and Fl uv ali nate [1, 6, 9, 10]. The us e of or g a nic c hemica ls a nd biotec hnic a l 44 method s t o c ontr ol Tropilaelaps o ff ers a r esi stance f r e e treatment option , but t her e i s uncer t ai nt y 45 ar ound eff ec tiv e do sa g e ra te and t r e a t men t r egime n s [6 , 11, 12] . The us e o f syn thetic miticid e s i s 46 wi dely r ec ommended f or s u rveilla nc e and de t e ction of Tropilaelaps [ 1 3 , 1 4 ] a n d t h i s 47 r ecomme ndation f orm s a p art of th e c o nt i ngenc y r e sp on s e o f s eve r al governme n t a gencie s [14, 1 5 ] . 48 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint With Tropilaelaps ’ incr e a s ing gl obal spread it is now imp or t a nt to e s tabli sh t he eff ec tiven e ss o f 49 det ec tion and contr ol met hod s. 50 The study sought t o t e st th e e f f i c ac y of c o mmer cia lly a vaila bl e mitic id es and w a s und e rt ak en in July 51 2024 a t C hiang Mai University , T haila nd . All bee s, b r ood ( A. mellifera ) and T. merc ede sae m i t e s w e r e 52 collec t e d fr om c olonie s a t the univ er si ty and coloni e s we r e ma na g ed in L an gs t r oth hiv e s u s ing 53 s t a n d a r d m a n ag e m e n t p r a ct i c e s . T h e ‘ B e l t s v i l l e t es t’ [ 1 6 ] , w h i c h i s c o m m o n l y u s e d b y b e e k e e p e r s t o 54 t e s t f o r p y r e t h r o i d r es i s t a n ce i n Varroa mit e s, and t he t e chniq u e s use d by Bahr eini, et al. (2021 ) [17] 55 w e r e ad apted f or thi s s tudy . F ig 1 s hows the de s i gn of the bi oa ssa y c a ge s us e d . A 13mm ø ven tilat i on 56 hole c ove r e d by 0.0 25mm n yl on me sh cl oth wa s added to 500 ml pla stic pot s to a llow v en til a t ion for 57 the bee s , w hile pr ev e n ting mit e s fr om escaping . R e s i st a nc e t o t he activ e i ngr e dients Coum apho s , 58 Amitr az, F luv al in at e a nd Flumethri n f ou nd in commer c ially a vaila bl e miticide st r ips w a s t e st e d. A 59 sec tion of mi t i cid e strip equival ent to a 3% dose , whic h is t h e dosa g e u sed f o r t he B e lt s v il l e t es t [ 16] , 60 w a s a d ded t o e a ch cage a nd a tt ac h ed wi t h a s t ee l pin. A s Tropilaelaps ha ve b ee n s how n t o ha ve only 61 limi t e d s ur vi v al withou t th e pres ence o f fr es h br ood [1 ] a s e c tion o f ope n br ood con ta ining 1 6 lar v ae 62 w a s c ut from a br ood fr ame and fix e d within ea ch cag e t o pr ovi de f ood f o r t he m it e s . A sticky t r a p 63 w a s a t t a ched t o t he i nsi de o f the po t l id s t o c at c h d ead mi t e s . As T. mercedesae h av e a s ho rt ph or e tic 64 phas e and a r e typ i c al ly f ound w ithi n s eal ed brood [1] a dult mi t e s we r e colle ct ed f r om c ol onie s . 65 Br ood fr am es w e r e s elec t e d f rom 10 col onie s and s eal ed brood w a s unc a p ped u sing for ce p s . Mi t e s 66 w e r e c oll ec t ed e ith er with an e nt omolog ical a s pirat or or a slig htly moi s t fine ti pp e d paintbru sh . They 67 w e r e v is ua lly e xamined an d confi rmed as T. m ercede sae b e f o r e bei ng pooled i n a sea la ble pla s tic 68 cont ain er wi t h f r e sh 5 th i n s t ar hone y bee lar v ae prior t o being individ ually t r a n s f er r ed t o th e bi oa ssa y 69 c a g e s . T h e l e v e l o f Tropilaelaps inf e s ta tio n w ithin col onie s wa s a sse s sed u sing the t ech niqu es 70 desc ribed by Gill, et al . ( 2 024 ) [15 ] and a colon y with an i n f e s tat i on leve l of <1% was s el e cted. A f t e r 71 t h e q u e e n h a d b e e n l o ca t e d t he ad u l t bee s w e r e s h a k e n f r o m e v e r y b r oo d f r a me in t o a l ar ge b o w l. A 72 ½ cup s c oo p c o nt ai ning on av er a ge 300 a dult bee s w a s add e d t o eac h bioa s s ay ca g e a long w it h 20 T. 73 mercedesae from the p ooled c ont ain er . A cir c le of 3 mm pla stic me s h w a s adde d 10 mm fr om t he t o p 74 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint of t h e bi oa ssa y cage to pr eve n t the b ee s f r om becomi ng s tuc k t o the stick y t r a p. T he lid w a s the n 75 fix ed o n to the bi oa ssa y c ag es b ef ore bein g in v e r ted. 76 77 Fig 1. De sign of bi o a ssa y c a g e . 78 79 Five r epli cates p e r t r e a tm ent gr oup we re us ed with th e add it i o n of a con t r ol g r o u p with no mitic ide 80 tr e a tment (n = 1 00 mite s/treatme nt). T o l imit po s s ibl e sp atial e ff ect s t he bi oa ssa y c ag es w e re 81 r andomly s t ored in a dark c upboar d at 34 o C 60% RH f or 24 hour s bef ore mit e mort ali ty w as as se ssed . 82 The pe r c e nt age of mi t e mo rtali t y w a s cal cu la ted by as se s s i ng the num b er o f de ad mi t e s f ou n d on th e 83 s tic ky t r aps an d liv e mite s f ound wi t h in t he bioa ssa y cage . 84 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint One -w ay ANOV A ana lysi s w a s under t ak e n in R S t u dio a nd show e d a sligh t s tati s ti c a lly s ig nifi cant 85 diff erenc e between the t r e a tm ent g r o ups (F = 2.5, p 4i±W=4i±W0. 078 ) (F ig. 2). D u nn ett t e sts f or multipl e 86 compari s o n s r ev e al ed th a t Amitr az h ad a sig ni fic a nt eff e ct on the mean mo rtali ty of T. m ercede sae 87 ( p 4i±W= 0 .035) w hen comp ared to the control and Amitr a z w as s how n t o be 64% e f f e ctiv e . 88 89 Fi g 2. Mea n (±SE ) mort a li ty ( %) o f T . mercedesae e xpo s ed to d i ff e r ent tr e atm ents (n=2 5). 90 91 The se r e sult s demon st rat e t h at t hi s T. mercedesae pop ul a ti on is r e si s tant to a ll the s yn t hetic 92 c he m i c al t r ea t m e n ts t es t e d apa r t fr o m Am i t r a z . W h i l e tr ea t m en t e f f ic a cy o f < 5 0% i n d i ca t es c h em i c a l 93 r e si s t an ce a n effic a cy o f only 64 % indi c ates a de v el opi ng c hemical resi s tanc e, a nd the level o f contr o l 94 after u sing a n y s ynthetic ch emi c a l s shoul d pr ef er ably be >95 % [18] . 95 The r ang e o f s u rv iv al variabili ty ob ser ved in thi s s tud y p r im arily indi cate s po ten ti a l di ff e r e nce s i n 96 su s c epti bility within the mi t e popul ation . Futur e w ork s ho ul d e xpl or e how genet ic or envir onmen tal 97 f ac tor s may co n t r i bu t e to mitic ide r e si st anc e in di ff e r e nt Tropilaelaps popul ation s a s i t sp r ead s 98 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint glo bally . This me thod al s o o ff e r s a novel t e s t for chemi cal re si s t a nce in Tropilaelaps spp . whic h ha s 99 not p r e viou sly bee n a v a ilabl e . Unde rs ta nding and ma nagi ng m it i cide resi s tan c e i n thi s spec ie s is 100 critical t o pr e vent fu rth er spr e ad a nd c ol on y lo ss e s . It i s impo rtant th a t g overnme n t a g enc ie s, 101 r e sea r che r s and beekeep ers a ppr e cia t e the level s of c hemical r e s i st anc e w hen c a r ryi ng out 102 surveill a nce f or ne w incur sion s of Tropilaelaps as in eff ec tiv e monitoring t echn i que s could lead to 103 new inc ur sion s remain ing unde t e c t e d a n d allow Tropilaelaps spp . t o b e c om e e s t a bl is h e d. 104 105 Re f e re n c e s . 106 1. de G u z man , Li lia I ., et al. "Ecology , li f e hi st o ry , and manage ment o f Tropilaelaps 107 mit e s. " Jou r na l o f ec onomic ent omo log y 110 .2 (2017): 319 -332. 108 2. T r aynor , Ki r st en S ., et al . " Varroa destructor : A c omplex par a si te, c ripp ling h oney bee s 109 w o r ld wide. " T r en d s in P ar a si t ol ogy 36.7 (2 020): 592-6 06 . 110 3. Br andor f , Ann a , et al . "Fi rs t r e por t o f e stabli s h ed mi t e popul at i o ns, Tropilaelaps mercedesae , 111 in E ur ope. " Jou r na l o f Apic ul tur al R e s earc h (2024): 1 -3. 112 4. Jana shia , Ir akli , e t al. "Sc iendo ." J ournal of Apicul tur al Sci enc e. (2024 ). 113 5. Anderson , Den i s L. , an d M a thew J. Mo rg an. " G e ne tic and morphol ogi c al va riati on of be e-114 par a sitic Tropilaelaps mit e s (Aca ri: Lael a pidae ): new and r e -d efined s p eci es ." E xperime n ta l 115 and Applied Acarology 43 (200 7): 1 -24. 116 6. R obert s , J o hn MK, et al . "E ff ec t i v ene s s o f co n trol s t rat eg ie s for Varroa jacobsoni and 117 Tropilaelaps mercedesae in P a pua New G ui nea." Exp erim ent al a nd Applie d Ac a r o lo gy 80 118 (2020 ) : 399 -407. 119 7. Mitt on, Giul ia A., e t al. " Mo r e t ha n six ty y e ars liv ing with Varroa destructor : a r evi ew o f 120 acarici d e r e s i s t anc e. " Intern a tio nal journ al of pe s t ma nageme n t (2022 ): 1 -18. 121 8. Le s ter , P hilip J. " Int egrat ed r e si s tan c e man ageme nt for acarici d e u se on Varroa 122 destructor . " Fr o n ti ers i n Be e S cie nce 1 (2 023): 12 97326. 123 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint 9. K ong pit ak, Picha i, et al . " The ef fi c a cy of Ba yvar ol® a nd C hec k Mite® i n the con tr ol of 124 Tropilaelaps mercedesae i n t h e E u r o p e a n h o n e y b e e (A p is m e l l i f e r a ) i n T h a i l a n d . " A p i a c t a 4 3 125 (2008 ) : 12 -16. 126 10. P et t is , Je f f e r y S ., R obyn R ose, and V eerana n Ch aiman ee . "Ch emical and c ultur a l c on trol of 127 Tropilaelaps mercedesae m i t e s i n h o n e y b e e ( Apis mellifera ) c o lo n i es i n N o r t he r n 128 Tha iland. " PloS on e 1 2.11 (201 7 ): e0188 0 63. 129 11. Mahmood , R a s h id, et al . " I nt e g ra te d co n t ro l o f Tropilaelaps clareae a n d Varroa destructor i n 130 Apis mellifera L. c o lon ie s. " S chola r ly Jour nal of Ag r ic ul tur al S cien c e 4. 1 (2014 ): 3 2- 37. 131 12. Sa nk ov it z, Madi son , et al . " E valuat i on of ef fi c a cy of formic aci d and th ermal rem edi ation f o r 132 mana g eme nt o f Tropilaelaps a nd Varroa mi t e s i n cen t r al Thail and. " bioRxi v (2024 ): 2024-03. 133 13. Anderson , De ni s L ., and J ohn MK R o bert s. "S t and a r d meth od s f or Tropilaelaps mi t e s 134 r e sea r ch. " Jou rnal o f A p icult ur al R e se ar c h 52. 4 (2013): 1 - 1 6. 135 14. EFSA P ane l on Animal H e alth and W el f a r e (A HA W ). " S cie nt i fic Opinion o n t h e r i s k o f e ntr y o f 136 Aethina tumida an d Tropilaelaps spp . in t he EU . " EFSA J ournal 11 .3 (201 3) : 3128. 137 15. Gill, Mag g ie C ., et al . "A sse s s me n t o f the effic ac y of fie ld a n d l aborat o ry methods f or t h e 138 det ec tion o f Tropilaelaps spp. " Pl oS one 19.9 (2024): e 0301880. 139 16. APH A. “‘Be l t s v ille’ a nd pyr e thr oi d r e si st a nce t e s ting . ” A v aila ble fr om 140 h ttps:/ /www .n a tion a lbe euni t.com / a sset s/P DFs/3 _R e sou r ce s_ f or _be ek eep ers/F a ct_S hee t s/F141 ac t_07_ Bel ts vil l e_and _ Pyr et hr oid _R e si st a nce_ T e s ti ng.pd f . (2019 ). 142 17. Bahrein i, Ra s s o l, et al. "N ew bioa ssa y c age metho do logy f or in vi t r o s tudi e s on Varroa 143 destructor and Apis mellifera ." Plo s one 1 6.4 (202 1): e0250 594. 144 18. Gumula, E w a . "G ui d eline on ve t e r i na ry med icina l produc ts contr olli ng Varroa destructor 145 par a sito si s i n bee s. " (2011 ). 146 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted December 16, 2024. ; https://doi.org/10.1101/2024.12.10.627757doi: bioRxiv preprint