Influence of Zwitterionic Buffer Effects with Thermal Modification Treatments of Wood on Symbiotic Protists in Reticulitermes grassei Clément

insec s A icle In luence o Zwi e ionic Bu e E ec s wi h The mal Modi ica ion T ea men s o Wood on Symbio ic P o is s in Re iculi e mes g assei Clémen Sónia Dua e 1,* , Lina Nunes 2,3 , Da o K žišnik 4, Miha Huma 4and Dennis Jones 5,6   Ci a ion: Dua e, S.; Nunes, L.; K žišnik, D.; Huma , M.; Jones, D. In luence o Zwi e ionic Bu e E ec s wi h The mal Modi ica ion T ea men s o Wood on Symbio ic P o is s in Re iculi e mes g assei Clémen . Insec s 2021,12, 139. h ps://doi.o g/10.3390/ insec s12020139 Academic Edi o s: Ma k E. Mankowski and B ian T. Fo schle Recei ed: 7 Janua y 2021 Accep ed: 3 Feb ua y 2021 Published: 6 Feb ua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1LEAF (Linking Landscape, En i onmen , Ag icul u e and Food) Resea ch Cen e, Ins i u o Supe io de Ag onomia, Uni e sidade de Lisboa. Tapada da Ajuda, 1349-017 Lisboa, Po ugal 2 LNEC, Na ional Labo a o y o Ci il Enginee ing, S uc u es Depa men , A . do B asil, 101, 1700-066 Lisbon, Po ugal; [email p o ec ed] 3 cE3c, Cen e o Ecology, E olu ion and En i onmen al Changes/Azo ean Biodi e si y G oup, Uni e si y o he Azo es, 9700–042 Ang a do He oísmo, Po ugal 4Bio echnical Facul y, Uni e si y o Ljubljana, SI-1000 Ljubljana, Slo enia; da o [email p o ec ed] (D.K.); [email p o ec ed] (M.H.) 5Depa men Wood Science and Enginee ing, Luleå Uni e si y o Technology, Fo ska ga an 1, S-93197 Skelle eå, Sweden; [email p o ec ed] 6 Depa men o Wood P ocessing and Bioma e ials, Facul y o Fo es y and Wood Sciences, Czech Uni e si y o Li e Sciences P ague, Kamýcká1176, 16521 P aha 6–Suchdol, Czech Republic *Co espondence: [email p o ec ed] Simple Summa y: O e he pas hi y yea s, he he mal modi ica ion o wood has become a uni e sally ecognised and comme cialised wood modi ica ion p ocess. The mal modi ica ions may a ec wood p ope ies, ei he posi i ely (dimensional s abili y and decay esis ance) o nega i ely (mechanical p ope ies). The combina ion o he imp egna ion o speci ic eagen s wi h he mal modi ica ion may help o o e come he nega i e e ec s on wood p ope ies. In his s udy, we e alua e he e ec o a combina ion o wo zwi e ionic bu e s, bicine and icine, and he mal modi ica ion o wo wood species (beech and sp uce) agains sub e anean e mi es and hei symbio ic auna. Bicine and icine ea men s alone had a clea in luence on wood mass loss and e mi e su i al. The lagella e p o is symbio ic communi y was a ec ed by he ea men s and esponded di e en ly o hem, as a highly adap able communi y. Howe e , he combina ion o bicine wi h he he mal modi ica ion showed a nega i e e ec on e mi es and hei symbion s on bo h wood species. The combina ion o hese di e en ac o s should be u he in es iga ed, as hese esul s seem o be p omising wi h ega d o he enhancemen o he e mi e esis ance o wood. Abs ac : The majo i y o he mal modi ica ion p ocesses a e a empe a u es g ea e han 180 ◦ C, esul ing in a p oduc wi h some p ope ies enhanced and some diminished (e.g., mechanical p ope ies). Howe e , he du abili y o he mally modi ied wood o e mi e a ack is ecognised as low. Recen a emp s a combining he mal modi ica ion wi h chemical modi ica ion, ei he p io o o di ec ly a e he he mal p ocess, a e p omising. Bu e s, al hough no in luencing he eac ion sys ems, may in e ac on exposu e o ce ain condi ions, po en ially ac ing as p omo e s o biological changes. In his s udy, wo zwi e ionic bu e s, bicine and icine, chosen o hei po en ial o o m Mailla d- ype p oduc s wi h agmen ed hemicelluloses/ ola iles, we e assessed wi h and wi hou he mal modi ica ion o wo wood species (sp uce and beech), wi h subsequen e alua ion o hei e ec agains sub e anean e mi es (Re iculi e mes g assei Clémen ) and hei symbio ic p o is s. The e ec o he wood ea men s on e mi es and hei symbion s was isible a e ou weeks, especially o sp uce ea ed wi h icine and bicine and hea ea men (bicine HT), and o beech ea ed wi h bicine and bicine and hea ea men (bicine HT). The chemical beha iou o hese subs ances should be u he in es iga ed when in con ac wi h wood and also a e hea ea men . This is he i s s udy e alua ing he e ec o po en ial Mailla d eac ions wi h zwi e ionic bu e s on sub e anean e mi e symbio ic auna. Insec s 2021,12, 139. h ps://doi.o g/10.3390/insec s12020139 h ps://www.mdpi.com/jou nal/insec s Insec s 2021,12, 139 2 o 15 Keywo ds: bicine; symbion s; e mi e gu ; he mal modi ica ion; icine; wood; Re iculi e mes g assei 1. In oduc ion The sub e anean e mi es a e known as majo pes s o applied wood and hei con ol has always been challenging, and e en mo e so in an en i onmen ally iendly way. The e a e se e al me hods and app oaches o ob aining his con ol, which o en need o be combined o gain a deg ee o syne gy o hei e ec s [ 1 ]. Fo example, hea - ea ed wood may exhibi di e en deg ees o esis ance o e mi es, acco ding o he wood p oduc and hea ea men echnique, among o he ac o s (e.g., [ 2 – 4 ]), bu is gene ally ecognised as low [5]. The he mal modi ica ion o wood is accep ed as being en i onmen ally iendly and is ecognised as he mos comme cialised o he wood modi ica ion p ocesses o da e [ 6 , 7 ]. The majo i y o he mal ea men s o wood in ol e empe a u es g ea e han 180 ◦ C, esul ing in a p oduc wi h some p ope ies enhanced (e.g., dimensional s abili y, du abili y, aes he ical colou ) and some diminished (e.g., mechanical p ope ies). A empe a u es be ween high- empe a u e kiln d ying and con en ional he mal modi ica ion, changes in p ope ies a e limi ed [ 8 ]. The loss o mechanical s eng h has been a limi a ion o he use o con en ional he mally modi ied wood in ce ain p oduc s. The mechanical s eng h educ ion is linked in a ying deg ees o he elease o acidic ola ile species, he acid-ca alysed depolyme isa ion o he hemicelluloses p esen , and he plas icisa ion and edis ibu ion o he lignin componen s p esen . Mo e ecen ly, he e ha e been a emp s o combine he mal modi ica ion wi h chemi- cal modi ica ion me hods. The combina ion o he mal modi ica ion a mode a e ea men empe a u es wi h wax imp egna ion has been epo ed [ 9 ] o he opical wood P e o- ca pus mac oca pus Ku z. The educ ion in he modulus o up u e (MOR) esul ing om mild he mal ea men a 150 ◦ C was elimina ed as a esul o wax imp egna ion. This con inued he wo k ou lined by Huma and collabo a o s [ 10 ], whe e Mon an wax was imp egna ed in o No way sp uce (Picea abies (L.) H. Ka s .) p io o he mal modi ica ion a empe a u es be ween 185 and 230 ◦ C. The use o esins has also been epo ed, such as he ea men o eucalyp us wi h melamine–u ea– o maldehyde esin p io o hea ea - men [ 11 ] and he imp egna ion o beech (Fagus syl a ica L.), ash (F axinus excelsio L.), lime (Tilia spp.) and popla (Popla spp.) wi h me hyla ed melamine o maldehyde esin (MMF) a e he mal ea men a 230 ◦ C [ 12 ]. The po en ial o he imp egna ion o compounds such as inylic-polyglyce ol, inylic-glyce ol, and maleic anhyd ide ollowed by he mal modi ica ion has been assessed as a means o achie ing a non-biocidal an i ungal and an i- e mi e wood ea men [13]. The da kening o he wood colou du ing he mal modi ica ion has been sugges ed as a esul o a Mailla d eac ion [ 14 , 15 ]. Mo e commonly associa ed wi hin ood science, his eac ion is he esul o an amine eac ing wi h a educing suga induced by hea . The possibili y o a Mailla d eac ion occu ing in wood modi ica ion was p oposed by Haup mann and collabo a o s [ 16 ] o he eac ion o xylose wi h icine. The Mailla d eac ion may inc ease he wood dimensional s abili y, h ough he bulking o he cell wall, depending on he chemical componen s used o pe o m he eac ion [ 17 ]. T icine was shown o eac wi h hemicellulose, esul ing in an inc ease in wood ha dness [ 16 ]. Addi ional examina ion o he po en ial o icine and he e ia y amine compound bicine [2-(Bis(2-hyd oxye hyl) amino) ace ic acid] as po en ial wood modi ica ion agen s was ecen ly unde aken [ 18 ]. E alua ion o ea ed samples by Fou ie - ans o m in a- ed spec ome y [ 19 ] indica ed he eagen s unde wen eac ion wi h wood a e imp egna ion in o samples, ollowed by he mal modi ica ion a 160 ◦ C, suppo ed by hie a chical analyses o he spec al da a. Insec s 2021,12, 139 3 o 15 Zwi e ionic bu e s a e neu al molecules wi h a posi i e and a nega i e elec ical cha ge, wi h some ad an ages ega ding o he ypes o bu e s, due o hei s abili y and educed memb ane pe meabili y [ 20 , 21 ]. The ad an ages o s udying hese subs ances o wood ea men is hei low human and en i onmen al oxici y, low p ice, and ela i e ease o p epa a ion, as desc ibed in he scien i ic li e a u e (e.g., [ 20 ]). Bicine and icine, bo h conside ed wi hin his s udy, a e examples o zwi e ionic bu e s; hese subs ances a e commonly used as non- e men able bu e ing agen s o some eac ions, including he manu ac u e o p ima y cells (bicine) and animal issue cul u e ( icine) [22,23]. The pH ange o bicine is 7.6–9.0, and ha o icine is 7.4–8.8 [ 23 ]. Howe e , hese al- ues may dec ease wi h a empe a u e inc ease, hough his does no in e e e signi ican ly wi h hei ionic s eng hs ( o a empe a u e ange be ween 25 and 50 ◦C [22]). T icine has been desc ibed as an e icien sca enge o -OH adicals [ 24 ], hough la e esea ch sugges ed lowe ac i i y o bicine and icine o hyd oxyl adicals compa ed o o he Good’s bu e s [25]. Bu e s a e designed o ha e li le in luence he eac ions in which hey a e used, so i is impo an o in es iga e he compa ibili y o he bu e wi h he eac ion sys em no only ega ding he bu e ing ange bu also wi h ega d o o he possible bu e in e ac ions. Such examples o his may be he possible impac s o he bu e on cell g ow h and su i al, he in e ac ions o bu e s wi h mac omolecules ( o example, icine may o m complexes wi h DNA, and zwi e ionic bu e s in luence mRNA exp ession o some eac ion sys ems), and he impac o bu e s on edox s udies [ 21 ]. Bu e s, al hough selec ed so as no o in luence he eac ion sys ems, may ha e some in e ac ions when exposed o some condi ions and ac as p omo e s o biological changes on hose sys ems. T icine has been p oposed o o m a xylosamine a empe a u es a ound 103 ◦ C (as ypical o labo a o y d ying condi ions) [ 13 ]. Fu he ea angemen s we e sugges ed, whe eby he xylosamine was con e ed o a ke osamine. Haup mann and collabo a o s [ 16 ] ep esen ed one o he i s documen ed s udies in o he Mailla d eac ion in wood, e en hough he concep has long been conside ed a p obabili y. By be e unde s anding he depolyme isa ion/deg ada ion p ocess wi hin he hemicellulose, i may be possible o apply Mailla d- ype eac ions and eac he suga moie ies wi hin he cell wall ma ix, ins ead o being ola ilised. The Mailla d eac ion is dependen upon he in e ac ion be ween he amine and he suga , wi h he ease o eac ion ypically being g ea e o p ima y amines compa ed o seconda y amines ( ep esen ing he eac i i y o he N-H bond). Typically, e ia y amines a e no expec ed o eac in a simila way, since he e a e no N-H bonds p esen . Howe e , o compounds such as bicine, he e may be an oppo uni y o he mal deg ada ion eac i a ing he ni ogen g oup p esen . On he o he hand, i is known ha N, N-bis(2- hyd oxye hyl) glycine (bicine) and N-(2-hyd oxy-1,1-bis(hyd oxyme hyl) e hyl) glycine ( icine) a e impo an hyd ogen ion bu e s o biological media, and hei use has been no ed in s udies in o he bu e ing o e mi e gu p o is s (e.g., [ 21 ]). The ad an age o he applica ion o Mailla d eac ion o wood elies on i s simplici y, as i in ol es an aqueous p ocess, and i is igni ed by hea [17]. The bulking o cell wall and he wood ha dening p ocess, e e ed o as e ec s o he Mailla d eac ion, may con ibu e posi i ely o an inc ease in ea ed wood e mi e esis ance, as hese p ope ies di icul he ask o a acking and p ocessing wood. Mo eo e , he lowe e mi es’ hindgu , which ha bou s hei symbio ic auna (p oka y- o es and lagella e p o is s), is na u ally designed o p o ide a ich and complex en i onmen able o ul il he speci ic equi emen s, ega ding pH, oxygen, and hyd ogen, o he se e al di e en species o symbion s li ing inside e mi es [ 26 ]. The e o e, a ious g adien s o pH, oxygen and hyd ogen exis , uelled by a combina ion o hos and symbion ac i i ies inside he e mi e hindgu [ 27 ]. The bulk o he cellulose diges ion p ocess is pe o med by lagella ed p o is s, which belong o wo sepa a e lineages o he unicellula euka yo es: he o de Oxymonadida (Phylum P eaxos yla) and he phylum Pa abasalia [ 28 – 30 ]. The e is g owing in e es in he lignocelluloly ic deg ada ion p ocess pe o med by he e mi e and Insec s 2021,12, 139 4 o 15 symbio ic auna holobion [ 31 ]. Flagella e p o is s seem o be sensi i e o he e mi e die , which may lead o changes in he lagella e p o is species composi ion and communi y s uc u e [32–34]. The analysis o e mi e su i al and consump ion o wood (ei he by measu ing wood mass loss and/o g ading he a ack) a e common me hods o e alua e wood ea men s’ e icacy agains e mi es [ 35 ]. The e ec o hese wood ea men s on symbio ic lagella e p o is communi y may also shed a ligh on he ea men mode o ac ion and he ex en o e ec s on e mi es. The ad an ages o his mode o ac ion, ocused on he e ec s on he e mi e symbio ic lagella e p o is communi y, will be he speci ici y o he ea men s, which mean ha i will p obably ha e ewe side e ec s on non- a ge o ganisms. This wo k objec i es a e o e alua e he e ec o zwi e ionic bu e s and hea ea - men on 1) sub e anean e mi e heal h and 2) he sub e anean e mi e symbio ic lagella e p o is communi y. 2. Ma e ials and Me hods 2.1. Wood T ea men Wood samples o sp uce (P. abies) and beech (Fagus syl a ica L.) o dimensions 30 × 10 × 10 mm (LxRxT, Go enjska, Slo enia) we e ea ed wi h solu ions o icine and bicine (ob ained om Fishe Scien i ic, Hamp on, NH, USA), using a acuum imp eg- na ion me hod: he samples we e placed in con aine s and weighed down, 1 M solu ions o bicine o icine in dis illed wa e we e espec i ely added; he imp egna ion sched- ule ollowed he ull cell p ocess, 20 min o acuum o 10 kPa ollowed by wo hou s o o e p essu e a 900 kPa and a he end o 10 min o acuum o 10 kPa. Be o e and a e he imp egna ion, he samples we e d ied and weighed o de e mine up ake. The ea ed samples we e allowed o d y unde low hea ing condi ions (50 ◦C). Once d y condi ions had been achie ed, samples we e hen he mally ea ed using a modi ied p ocess o he one desc ibed by Rep and collabo a o s [ 36 ], whe eby a educed maximum empe a u e o 160 ◦ C was used (ins ead o he con en ional 180–230 ◦ C em- ployed o he mal modi ica ion o wood) unde acuum. This was o minimise he he mal deg ada ion o he bicine and icine espec i ely a empe a u es a ound 180 ◦ C. Sp uce and beech samples we e also subjec o he same hea ea men p ocess o 160 ◦ C wi hou any imp egna ion. The ollowing combina ions o ea men s we e e alua ed agains sub e anean e - mi es and hei p o is symbion s o bo h wood species: (1) un ea ed; (2) hea - ea ed (HT); (3) bicine; (4) bicine and hea ea men (bicine HT); (5) icine; (6) icine and hea ea men ( icine HT). 2.2. E ec s on Sub e anean Te mi es Sub e anean e mi es belonging o he species Re iculi e mes g assei Clémen , and o h ee di e en colonies, we e cap u ed in a pine o es (Pinus pinas e Ai on), in Sesimb a, Se úbal dis ic o Po ugal. Each colony was cap u ed on a dead wood log (app oxima ely 1 m long and 0.08 m diame e ) mo e han 100 m apa . Logs we e b ough o he labo a o y and kep in a condi ioned oom a 24 ± 1 ◦ C and 80% ± 5% ela i e humidi y. G oups o 150 e mi e wo ke s we e cap u ed manually and es ablished in 200 mL glass ja s wi h mois ened sand (Fon ainebleau sand and wa e ; 4:1 / ) as subs a e. P e iously ea ed sp uce and beech wood (as explained in he p e ious poin ) was o e ed o he e mi es as ood, and also pine con ols we e es ablished. Th ee eplica es pe ea men we e hen placed in con ac wi h he e mi es and he es was un o ou weeks in he desc ibed condi ions. Ma i ime pine es specimens wi h he same dimensions we e also included as in e nal i ulence con ols [37,38]. Insec s 2021,12, 139 5 o 15 The ini ial mois u e con en o he blocks was measu ed in se s o h ee addi ional eplica es pe ea men and hese alues we e used o de e mine he heo e ical ini ial d y mass (IDM) o he exposed specimens. A he end o he ial, he inal mois u e con en was eco ded, and he mass loss was ob ained acco ding o Equa ion (1). % mass loss = (FDM −IDM)/IDM ×100 (1) whe e FDM is he d y mass o he block a he end o he es . The su i al a e (%) o he e mi es was also eco ded. All wood blocks we e g aded acco ding o e mi e a ack using he scale: 0 = no damage; 1 = a emp ed a ack; 2 = sligh damage; 3 = supe icial and inne damage; 4 = hea y inne damage [37]. 2.3. Scanning Elec on Mic oscopy (SEM) Te mi es o one o he colonies used in 2.2 we e used o mic oscopy. Hindgu s o 50 wo ke s we e dis ibu ed on 250 µ L o T age U medium [ 39 ], cu open and sligh ly homogenised o elease hei con en s. The con en s we e pu on a 0.1% poly-L-lysine coa ed su ace, and hen we e ixed in 1:1 / glu a aldehyde 2.5%/ o maldehyde 2.5% o e nigh . A e cen i uga ion, and h ee bu e washes, o 15 min each, samples we e pos - ixed in 1% osmium e oxide o 30 min. The samples we e dehyd a ed using a g aded e hanol se ies, 15 min in each concen a ion: 30%, 50%, 70%, 80%, 90%, and h ee imes in 100% e hanol, and hen pu on o he d ie agen hexame hyldisilazane. D ied samples we e moun ed on s ubs and spu e coa ed wi h gold be o e obse ing wi h a scanning elec on mic oscope JEOL JSM-5200LV (JEOL, C oissy-Su -Seine, F ance) a Facul y o Sciences o Lisbon Uni e si y. 2.4. E ec s on Symbio ic P o is s The e mi es used belonged o he same colonies e e ed on Sec ion 2.2. Te mi e wo ke s in he 4 h ins a o de elopmen we e selec ed. G oups o 50 e mi e wo ke s o each colony, andomly chosen, we e exposed o he di e en wood samples, and i e e mi es pe ype o wood ea men we e e alua ed o hei symbio ic lagella e p o is communi y di e si y and abundance a e : 24 h, one week, wo weeks, h ee weeks, and ou weeks (end o he ials). Flagella e p o is di e si y and abundance we e e alua ed by di ec obse a ion unde a mic oscope. The quan i ica ion o lagella e p o is s was pe o med wi h a haemocy ome e , in acco dance wi h a p e iously desc ibed me hod [ 40 ]. The h ee di e en colonies’ symbio ic auna was compa ed using e mi es eeding on he o iginal wood whe e hey we e cap u ed. Flagella e p o is s we e dis inguished and iden i ied acco ding o species o majo axa desc ip ions [ 41 – 44 ], based on mo phological cha ac e is ics, sepa a ed in o nine mo pho ypes, and quan i ied. 2.5. S a is ical Analysis In o de o de ec signi ican (p< 0.05) a ia ion in e mi e su i al and wood mass loss caused by di e en wood ea men s, he da a ob ained we e submi ed o an ANOVA, and when he esul was conside ed o be signi ican ( o p< 0.05), Tukey’s hones ly signi ican di e ence es (HSD) was done. The g ade o e mi e a ack was also analysed wi h a non-pa ame ic K uskal–Wallis analysis (and a pos hoc pai wise Wilcoxon es ), as well as da a on symbio ic lagella e p o is s, as he assump ions o da a no mali y we e no espec ed. All hese analyses we e pe o med wi h RS udio (RS udio Team 2015) 0.99.467 and R-3.1.2 (R Founda ion o S a is ical Compu ing, Vienna, Aus ia). Insec s 2021,12, 139 6 o 15 3. Resul s 3.1. E icacy agains Sub e anean Te mi es Rega ding beech, no signi ican di e ence was iden i ied in e mi e su i al (F = 1.83; dF = 5; p= 0.181), despi e he lowe alues o e mi e su i al o e mi es ed wi h bicine and bicine and hea ea men (bicine HT) beech (Table 1). This lack o s a is ical signi icance may be explained by s ong a ia ion wi hin he esul s o di e en ials belonging o he same ca ego y. Howe e , he esul s o wood mass loss (F = 14.40; dF = 5; p< 0.001) and e mi e a ack g ade (Table 2) co obo a e he di e ences obse ed in e mi e su i al a e, and wo g oups a e isible: 1) un ea ed beech and hea - ea ed beech (HT), and 2) bicine and bicine and hea ea men (bicine HT), also including icine, which had signi ican ly lowe mass loss compa ed o g oup 1. Table 1. A e age esul s o e mi e su i al a es, wood mass loss (%; and s anda d de ia ion) o di e en wood species (pine, sp uce and beech) and he ea men s: un ea ed, hea - ea ed (HT), bicine, bicine and hea ea men (bicine HT), icine, icine and hea ea men ( icine HT), a e ou weeks o exposu e. Values ollowed by di e en le e s wi hin he same column we e conside ed o be signi ican ly di e en (p< 0.05; Tukey ´ s hones ly signi ican di e ence es ). The compa isons we e made o di e en ea men s wi hin he same wood species. Wood Species T ea men Su i al (%) Mass Loss (%) Pine Un ea ed 84.4 ±7.4 - 18.3 ±2.9 - Sp uce Un ea ed 88.4 ±6.3 a 19.5 ±0.8 a HT 90.8 ±4.5 a 19.0 ±1.9 a Bicine 64.2 ±38.9 ab 11.7 ±1.0 b Bicine HT 21.1 ±18.7 b 10.8 ±1.5 b T icine 11.3 ±17.4 b 11.5 ±1.1 ab T icine HT 62.3 ±31.2 ab 17.5 ±0.8 ab Beech Un ea ed 82.0 ±9.7 a 15.9 ±0.8 a HT 87.2 ±2.7 a 14.8 ±0.8 a Bicine 38.0 ±22.0 a 12.3 ±0.3 b Bicine HT 57.6 ±47.0 a 11.4 ±1.3 b T icine 71.4 ±17.2 a 14.2 ±0.3 b T icine HT 79.9 ±16.8 a 13.0 ±0.0 ab Table 2. G ade o e mi e a ack o di e en wood species (pine, sp uce, and beech) and he ea men s: un ea ed, hea ea ed (HT), bicine, bicine and hea ea men (Bicine HT), icine, icine and hea ea men (T icine HT), a e ou weeks o exposu e, o each eplica e. The wood blocks we e g aded using he scale: 0 = no damage; 1 = a emp ed a ack; 2 = sligh damage; 3 = supe icial and inne damage; 4 = hea y inne damage [37]. Wood Species T ea men Un ea ed HT Bicine Bicine HT T icine T icine HT Sp uce 4 4 3 4 4 4 4 4 3 3 3 4 4 4 3 1 2 4 Beech 4 4 3 4 4 4 4 4 3 3 4 4 4 4 3 2 3 4 Pine 4 - - - - - 4 - - - - - 4 - - - - - Insec s 2021,12, 139 7 o 15 Rega ding e mi e su i al a e, he esul s show a clea di e ence (F = 6.28; dF = 5; p= 0.004) be ween sp uce submi ed o 1) un ea ed sp uce and hea ea ed sp uce and 2) bicine and hea ea men and icine, which caused signi ican ly lowe e mi e su i al a e. Fo mass loss esul s, he di e ences a e signi ican (F = 26.96; dF = 5; p< 0.001) be ween 1) un ea ed and HT sp uce and 2) bicine and bicine HT sp uce, wi h he la e ha ing lowe mass losses. Te mi e a ack g ade showed ha bicine HT and icine had lowe a ack g ading, al hough no signi ican di e ences among hem we e de ec ed (sp uce: χ2 = 10.50; d = 5; p= 0.062; beech: χ2 = 10.88; d = 5; p= 0.054). 3.2. E ec s on Symbio ic P o is s The h ee colonies exhibi ed no signi ican di e ences ega ding hei symbio ic p o- is s ( χ 2 = 0.73; d = 2; p= 0.692). Nine mo pho ypes we e ound and iden i ied based on mo phological cha ac e is ics (Table 3). Va ia ions in p o is di e si y and abundance we e e alua ed o all ea men s es ed (Table 4-sp uce; Table 5-beech). Table 3. Flagella e p o is iden i ica ion o mo pho ypes (n1 o n9) based on mo phological cha ac e s. Phylum Class O de Family Genus Species n1 Pa abasalia T ychonymphea T ichonymphida T ichonymphidae T ichonympha n9 Spi o ichonymphea Spi o ichonymphida Holomas igo oididae Spi o ichonympha S. lagella a n4 Holomas igo es H. elonga um n8 Mic ojoenia M. hexami oides n6 Hypo ichomonadea Hypo ichomonadida Hypo ichomonadidae n2 P eaxos yla Oxymonadida Py sonympha sp.1 n7 Py sonympha sp.2 n3 Dinenympha D. g acilis n5 D. imb ia a Some mo pho ypes we e shown o be suscep ible o some ea men s. Fo example, n1 (T ichonympha sp.) was suscep ible o all ea men s o bo h wood species a e 28 days o exposu e. All wood ea men s showed a clea nega i e impac o e ime on e mi es, ega ding his mo pho ype. Fo sp uce, icine (p= 0.004) and icine HT (0.042) exe nega i e in luences by day 1 ( χ 2 = 20.58; d = 6; p= 0.002), while bicine (p= 0.012) and bicine HT (p= 0.031) exe nega i e in luences by day 7 ( χ 2 = 25.26; d = 6; p< 0.001), and hea - ea ed sp uce- ed e mi es (p= 0.003) exhibi ed a lowe numbe o n1 p o is s by day 14 ( χ 2 = 51.25; d = 6; p< 0.001). Fo beech, he nega i e e ec s o di e en wood ea men s, ela i ely o ini ial pine ed e mi es was isible on day 7 ( χ 2 = 21.29; d = 6; p= 0.002) o icine (p= 0.029) and icine HT (p= 0.011), which was also di e en om e mi es ed on sp uce con ols (p= 0.026), al hough la e icine- ea ed beech- ed e mi es showed a eco e y in hese p o is s’ abundance. Fo bicine (p= 0.018) and bicine HT (p= 0.012), e ec s we e isible a e day 14 ( χ 2 = 25.01; d = 6; p< 0.001) ela i e o pine, and hea - ea ed beech (p< 0.001, p= 0.033, espec i ely) showed a s onge nega i e e ec on T ichonympha sp. abundance on day 28 (χ2 = 95.30; d = 6; p< 0.001). The abundances o mo pho ype n2 (Py sonympha sp1; Figu e 1) luc ua ed, bu we e gene ally signi ican ly lowe han pine con ol ed e mi es, o all ea men s and bo h wood species (sp uce: χ 2 = 75.97; d = 6; p< 0.001; beech: χ 2 = 25.26; d = 6; p< 0.001). Fo sp uce, he nega i e e ec on his mo pho ype abundance was obse ed a he end o he ials (day 28) wi h wood ea ed wi h bicine- (p= 0.023) and bicine HT- ed e mi es (p< 0.001), which exhibi ed a lowe abundance han un ea ed sp uce ed e mi es. Fo beech, his e ec was s onge a he end o he ials on e mi es ed wi h icine (p= 0.008) and bicine HT (p= 0.004) wood ela i e o e mi es ed wi h un ea ed beech. Insec s 2021,12, 139 8 o 15 Table 4. A e age numbe (and s anda d de ia ion) o lagella e p o is s belonging o di e en mo pho ypes (n1 o n9) a e 1, 7, 14, 21, and 28 days o exposu e; e mi es we e exposed o un ea ed pine (Pine), un ea ed sp uce (Con ol), hea - ea ed sp uce (HT), sp uce ea ed wi h: bicine (B), bicine and hea ea men (BHT), icine (T), icine and hea ea men (THT). Di e en capi al le e s wi hin he same column indica e signi ican di e ences (p< 0.05) be ween pine and he es o he ea men s o each day o obse a ion; di e en small le e s wi hin he same column indica e signi ican di e ences (p< 0.05) among sp uce ea men s o each day o obse a ion. T ea men n1 n2 n3 n4 n5 n6 n7 n8 n9 Pine 0.86 ±0.78 A 12.06 ±3.28 A 2.62 ±1.84 A 0.24 ±0.48 A 0.30 ±0.65 A 0.06 ±0.24 A 0.22 ±0.58 A 4.56 ±2.84 A 1.42 ±1.05 A Day 1 Con ol 0.64 ±0.76 Aab 8.24 ±3.53 Ba 2.16 ±1.34 Aa 0.08 ±0.28 Aa 0.24 ±0.60 Aa 0.20 ±0.41 Aa 0.20 ±0.41 Aa 1.88 ±1.05 Ba 1.28 ±1.21 Aab HT 0.76 ±0.66 Ab 10.84 ±4.04 Aa 3.92 ±1.53 Bb 0.32 ±0.48 Aa 0.44 ±0.58 Aa 0.04 ±0.20 Aa 0.28 ±0.46 Aa 2.92 ±1.68 Aa 1.52 ±1.19 Ab B 0.44 ±0.58 Aab 9.40 ±3.86 Ba 2.36 ±1.47 Aac 0.28 ±0.46 Aa 0.16 ±0.37 Aa 0.12 ±0.33 Aa 0.08 ±0.28 Aa 3.40 ±2.45 Aa 0.88 ±0.97 Aab BHT 0.64 ±0.81 Aab 9.68 ±3.58 Ba 2.92 ±1.53 Aab 0.28 ±0.46 Aa 0.04 ±0.20 Aa 0.00 ±0.00 Aa 0.08 ±0.28 Aa 3.12 ±1.90 Aa 0.68 ±0.69 Ba T 0.20 ±0.41 Ba 9.76 ±4.56 Aa 1.72 ±1.14 Aa 0.12 ±0.33 Aa 0.20 ±0.41 Aa 0.08 ±0.28 Aa 0.20 ±0.41 Aa 2.40 ±1.29 Ba 0.64 ±0.76 Ba THT 0.36 ±0.57 Bab 10.32 ±3.16 Aa 3.56 ±1.87 Abc 0.24 ±0.44 Aa 0.20 ±0.41 Aa 0.12 ±0.33 Aa 0.08 ±0.28 Aa 2.80 ±1.80 Ba 1.24 ±1.05 Aab Day 7 Con ol 0.60 ±0.76 Aab 8.00 ±3.35 Ba 1.85 ±1.19 Aa 0.15 ±0.37 Aa 0.10 ±0.28 Aa 0.10 ±0.33 Aa 0.25 ±0.41 Aa 2.90 ±2.27 Ba 1.30 ±1.19 Aa HT 0.72 ±0.68 Ab 8.92 ±2.43 Ba 4.20 ±2.18 Bb 0.12 ±0.33 Aa 0.12 ±0.33 Aa 0.12 ±0.33 Aa 0.12 ±0.33 Aa 4.60 ±3.44 Ba 1.32 ±1.07 Aa B 0.28 ±0.54 Bab 7.68 ±2.70 Ba 1.24 ±1.13 Bac 0.20 ±0.41 Aa 0.00 ±0.00 Ba 0.12 ±0.33 Aa 0.04 ±0.20 Aa 2.64 ±1.91 Ba 0.56 ±0.77 Bb BHT 0.36 ±0.57 Bab 7.48 ±3.92 Ba 0.96 ±0.89 Bc 0.08 ±0.28 Aa 0.00 ±0.00 Ba 0.24 ±0.44 Aa 0.12 ±0.33 Aa 1.44 ±1.56 Ba 0.36 ±0.57 Bab T 0.20 ±0.41 Ba 7.32 ±3.20 Ba 2.32 ±1.25 Aa 0.08 ±0.28 Aa 0.04 ±0.20 Aa 0.20 ±0.41 Aa 0.04 ±0.20 Aa 2.88 ±2.49 Aa 0.84 ±0.80 Bab THT 0.44 ±0.58 Aab 8.64 ±3.65 Ba 2.16 ±1.46 Aa 0.08 ±0.28 Aa 0.00 ±0.00 Ba 0.04 ±0.20 Ba 0.08 ±0.28 Aa 3.12 ±2.01 Aa 0.72 ±0.94 Bb Day 14 Con ol 0.92 ±0.76 Aa 9.88 ±3.31 Bac 4.20 ±1.91 Ba 0.32 ±0.56 Aa 0.08 ±0.28 Aa 0.24 ±0.52 Aa 0.32 ±0.56 Aa 3.40 ±1.80 Aa 1.84 ±1.18 Aa HT 0.28 ±0.46 Bb 10.2 ±2.36 Bac 3.36 ±1.58 Aa 0.08 ±0.28 Aa 0.04 ±0.20 Aa 0.12 ±0.33 Aa 0.28 ±0.54 Aa 6.20 ±3.44 Ab 1.20 ±1.12 Aab B 0.24 ±0.44 Bb 6.32 ±3.67 Bbc 2.12 ±1.62 Ab 0.12 ±0.33 Aa 0.00 ±0.00 Ba 0.12 ±0.33 Aa 0.00 ±0.00 Aa 3.00 ±2.45 Aa 0.52 ±0.59 Bb BHT 0.08 ±0.28 Bb 7.84 ±3.59 Bc 2.08 ±1.53 Ab 0.00 ±0.00 Aa 0.00 ±0.00 Ba 0.08 ±0.28 Aa 0.12 ±0.33 Aa 2.84 ±2.37 Ba 0.76 ±0.88 Bb T 0.16 ±0.37 Bb 11.48 ±3.72 Aa 2.24 ±1.13 Ab 0.12 ±0.33 Aa 0.00 ±0.00 Ba 0.16 ±0.47 Aa 0.08 ±0.28 Aa 3.44 ±2.81 Aa 0.64 ±0.70 Bb THT 0.20 ±0.41 Bb 9.20 ±3.77 Bac 2.72 ±1.70 Ab 0.04 ±0.20 Aa 0.00 ±0.00 Ba 0.28 ±0.54 Aa 0.24 ±0.52 Aa 3.44 ±1.53 Aa 0.72 ±1.02 Bb Day 21 Con ol 0.68 ±0.75 Aa 9.40 ±4.70 Bac 2.00 ±0.96 Aab 0.20 ±0.41 Aa 0.00 ±0.00 Ba 0.16 ±0.37 Aa 0.36 ±0.49 Aa 3.84 ±2.29 Aa 1.08 ±1.00 Aa HT 0.48 ±0.65 Aab 11.20 ±4.32 Aa 2.84 ±2.15 Aa 0.24 ±0.44 Aa 0.00 ±0.00 Ba 0.16 ±0.47 Aa 0.20 ±0.41 Aa 3.44 ±1.87 Aac 1.92 ±1.47 Aa B 0.40 ±0.58 Bab 6.56 ±3.18 Bb 1.40 ±1,41 Bbc 0.04 ±0.20 Aa 0.00 ±0.00 Ba 0.04 ±0.20 Aa 0.04 ±0.20 Ab 2.80 ±1.32 Bab 0.16 ±0.37 Bb BHT 0.16 ±0.37 Bb 7.28 ±3.29 Bc 1.24 ±1.05 Bc 0.00 ±0.00 Aa 0.00 ±0.00 Ba 0.16 ±0.37 Aa 0.04 ±0.20 Ab 2.32 ±2.17 Bb 0.28 ±0.54 Bb T 0.32 ±0.48 Bab 10.36 ±3.39 Ab 1.88 ± 1.13 Aabc 0.24 ±0.52 Aa 0.00 ±0.00 Ba 0.32 ±0.48 Aa 0.08 ±0.28 Aa 2.36 ±1.93 Bbc 0.40 ±0.58 Bb THT 0.20 ±0.50 Bb 5.84 ±2.73 Bc 1.44 ±1.16 Babc 0.08 ±0.28 Aa 0.00 ±0.00 Ba 0.20 ±0.41 Aa 0.16 ±0.37 Aa 2.52 ±2.06 Bab 0.44 ±0.65 Bb Day 28 Con ol 0.92 ±1.04 Aa 6.84 ±2.79 Ba 3.24 ±1.39 Aa 0.28 ±0.46 Aa 0.08 ±0.28 Aa 0.24 ±0.52 Aa 0.28 ±0.54 Aa 2.48 ±1.48 Ba 0.76 ±0.66 Bab HT 0.44 ±0.58 Ba 12.68 ±5.43 Ab 2.56 ±1.61 Aa 0.04 ±0.20 Aa 0.04 ±0.20 Aa 0.12 ±0.33 Aa 0.24 ±0.44 Aa 3.08 ±2.20 Ba 1.48 ±1.48 Ab B 0.08 ±0.28 Bb 5.00 ±1.71 Bc 1.20 ±1.08 Bb 0.04 ±0.20 Aa 0.00 ±0.00 Ba 0.00 ±0.00 Aa 0.04 ±0.20 Aa 0.48 ±0.51 Bb 0.08 ±0.28 Bc BHT 0.08 ±0.28 Bb 3.44 ±2.18 Bd 1.04 ±0.89 Bb 0.00 ±0.00 Ab 0.00 ±0.00 Ba 0.08 ±0.28 Aa 0.00 ±0.00 Aa 0.24 ±0.52 Bb 0.80 ±1.22 Bab T 0.36 ±0.57 Bab 6.32 ±3.21 Bac 0.88 ±0.73 Bb 0.12 ±0.33 Aa 0.00 ±0.00 Ba 0.04 ±0.20 Aa 0.08 ±0.28 Aa 1.20 ±0.96 Bb 0.80 ±0.91 Bab THT 0.36 ±0.49 Ba 8.32 ±4.98 Ba 1.44 ±1.42 Bb 0.00 ±0.00 Ab 0.00 ±0.00 Ba 0.16 ±0.37 Aa 0.12 ±0.44 Aa 2.00 ±1.35 Ba 0.52 ±0.65 Ba Insec s 2021,12, 139 9 o 15 Table 5. A e age numbe (and s anda d de ia ion) o lagella e p o is s belonging o di e en mo pho ypes (n1 o n9) a e 1, 7, 14, 21 and 28 days o exposu e; e mi es we e exposed o un ea ed pine (Pine), un ea ed beech (Con ol), hea - ea ed beech (HT), beech ea ed wi h: Bicine (B), Bicine and hea ea men (BHT), T icine (T), T icine and hea ea men (THT). Di e en capi al le e s wi hin he same column indica e signi ican di e ences (p< 0.05) be ween pine and he es o he ea men s o each day o obse a ion; di e en small le e s wi hin he same column indica e signi ican di e ences (p< 0.05) among sp uce ea men s o each day o obse a ion. T ea men n1 n2 n3 n4 n5 n6 n7 n8 n9 Pine 0.86 ±0.78 A 12.06 ±3.28 A 2.62 ±1.84 A 0.24 ±0.48 A 0.30 ±0.65 A 0.06 ±0.24 A 0.22 ±0.58 A 4.56 ±2.84 A 1.42 ±1.05 A Day 1 Con ol 0.52 ±0.65 Aa 9.32 ±2.91 Ba 4.08 ±2.33 Ba 0.24 ±0.44 Aa 0.12 ±0.33 Aa 0.12 ±0.33 Aa 0.16 ±0.37 Aa 2.44 ±1.73 Ba 1.36 ±1.04 Aa HT 0.72 ±0.68 Aa 8.88 ±2.91 Bab 3.04 ±1.43 Aa 0.20 ±0.50 Aa 0.28 ±0.46 Aa 0.12 ±0.33 Aa 0.28 ±0.46 Aa 3.28 ±2.21 Aa 1.44 ±1.26 Aa B 0.64 ±0.70 Aa 7.20 ±3.37 Bab 3.12 ±1.81 Aa 0.20 ±0.41 Aa 0.16 ±0.47 Aa 0.12 ±0.33 Aa 0.16 ±0.37 Aa 2.52 ±2.33 Ba 0.64 ±0.86 Bb BHT 0.64 ±0.86 Aa 7.32 ±4.41 Bb 2.80 ±1.66 Aab 0.32 ±0.56 Aa 0.24 ±0.44 Aa 0.16 ±0.37 Aa 0.08 ±0.28 Aa 2.12 ±1.42 Ba 0.72 ±0.79 Bab T 0.40 ±0.50 Aa 7.48 ±2.87 Bab 3.48 ±1.83 Aa 0.08 ±0.28 Aa 0.08 ±0.28 Aa 0.04 ±0.20 Aa 0.16 ±0.37 Aa 1.96 ±1,37 Ba 0.88 ±0.88 Aab THT 0.48 ±0.65 Aa 4.72 ±1.40 Bc 1.76 ±1.16 Ab 0.04 ±0.20 Aa 0.04 ±0.20 Aa 0.04 ±0.20 Aa 0.08 ±0.28 Aa 1.96 ±1,49 Ba 1.28 ±0.89 Aa Day 7 Con ol 0.72 ±0.84 Aa 10.32 ±3.41 Ba 2.80 ±1.71 Aa 0.16 ±0.37 Aa 0.00 ±0.00 Aa 0.12 ±0.33 Aa 0.32 ±0.56 Aa 3.72 ±3.70 Aa 1.28 ±0.98 Aac HT 0.48 ±0.51 Aa 10.2 ±3.50 Bac 2.48 ±1.56 Aab 0.32 ±0.63 Aa 0.00 ±0.00 Aa 0.04 ±0.20 Aa 0.16 ±0.47 Aa 2.76 ±1.20 Aa 1.20 ±0.87 Aac B 0.64 ±0.70 Aa 8.28 ±2.64 Bb 1.60 ±1.26 Abc 0.16 ±0.37 Aa 0.00 ±0.00 Aa 0.12 ±0.33 Aa 0.20 ±0.41 Aa 3.60 ±3.23 Aa 0.84 ±0.75 Bb BHT 0.64 ±0.76 Aa 9.48 ±4.45 Bbc 2.52 ±1.73 Aa 0.12 ±0.44 Aa 0.08 ±0.28 Aa 0.20 ±0.50 Aa 0.20 ±0.50 Aa 3.16 ±1.97 Aa 1.16 ±0.99 Bab T 0.36 ±0.57 Bab 10.6 ±5.02 Bab 3.04 ±1.81 Aa 0.08 ±0.28 Aa 0.04 ±0.20 Aa 0.08 ±0.28 Aa 0.24 ±0.44 Aa 6.28 ±2.70 Ba 0.88 ±0.73 Bbc THT 0.16 ±0.47 Bb 6.96 ±2.37 Bc 1.52 ±0.96 Bc 0.08 ±0.28 Aa 0.00 ±0.00 Ba 0.16 ±0.37 Aa 0.12 ±0.33 Aa 4.60 ±2.50 Ba 0.36 ±0.70 Bb Day 14 Con ol 0.72 ±0.74 Aa 12.40 ±3.52 Aa 3.44 ±1.87 Aa 0.24 ±0.44 Aa 0.00 ±0.00 Ba 0.28 ±0.46 Aa 0.20 ±0.50 Aa 3.84 ±2.15 Aa 1.88 ±1.30 Aa HT 0.60 ±0.71 Aab 8.64 ±3.38 Bbc 2.84 ±1.68 Aa 0.16 ±0.37 Aa 0.04 ±0.20 Aa 0.08 ±0.28 Aa 0.16 ±0.37 Aa 4.56 ±2.57 Aa 1.56 ±1.33 Aa B 0.32 ±0.48 Bab 8.40 ±3.55 Bbc 2.36 ±1.55 Aa 0.12 ±0.33 Aa 0.00 ±0.00 Ba 0.20 ±0.50 Aa 0.12 ±0.33 Aa 3.56 ±2.72 Aa 0.40 ±0.58 Bb BHT 0.28 ±0.54 Bab 12.4 ±3.99 Aa 3.64 ±2.27 Aa 0.20 ±0.41 Aa 0.00 ±0.00 Ba 0.08 ±0.28 Aa 0.24 ±0.44 Aa 3.96 ±2.84 Aa 0.72 ±0.68 Bb T 0.52 ±0.71 Aab 9.16 ±2.84 Bb 2.32 ±1.49 Aa 0.12 ±0.33 Aa 0.00 ±0.00 Ba 0.24 ±0.52 Aa 0.24 ±0.52 Aa 3.96 ±2.79 Aa 0.64 ±0.76 Bb THT 0.20 ±0.41 Bb 7.24 ±2.59 Bc 2.92 ±2.06 Aa 0.08 ±0.28 Aa 0.00 ±0.00 Ba 0.12 ±0.33 Aa 0.28 ±0.61 Aa 4.32 ±2.85 Aa 0.84 ±0.90 Ba Day 21 Con ol 0.64 ±0.70 Aa 9.80 ±3.32 Ba 3.48 ±1.83 Ba 0.24 ±0.52 Aa 0.00 ±0.00 Ba 0.20 ±0.41 Aa 0.20 ±0.41 Aa 3.00 ±1.76 Bab 1.40 ±1.00 Aa HT 0.48 ±0.59 Aa 8.84 ±3.30 Bab 3.68 ±2.17 Bab 0.12 ±0.33 Aa 0.04 ±0.20 Aa 0.12 ±0.33 Aa 0.24 ±0.44 Aa 3.56 ±2.26 Bb 1.32 ±1.38 Aa B 0.20 ±0.41 Ba 10.28 ±4.77 Ba 1.48 ±0.87 Abc 0.04 ±0.20 Aa 0.00 ±0.00 Ba 0.24 ±0.52 Aa 0.12 ±0.33 Aa 2.08 ±1.35 Ba 0.48 ±0.71 Bb BHT 0.36 ±0.64 Aa 8.60 ±2.92 Bb 1.76 ±1.16 Abc 0.08 ±0.28 Aa 0.00 ±0.00 Ba 0.08 ±0.28 Aa 0.04 ±0.20 Aa 1.84 ±1.72 Ba 0.84 ±1.52 Bb T 0.48 ±0.65 Aa 8.56 ±3.81 Bb 2.16 ±1.97 Ab 0.12 ±0.33 Aa 0.00 ±0.00 Ba 0.08 ±0.28 Aa 0.12 ±0.33 Aa 2.04 ±2.41 Ba 1.36 ±1.82 Aab THT 0.16 ±0.37 Ba 7.80 ±2.00 Bc 1.72 ±1.49 Bc 0.16 ±0.37 Aa 0.00 ±0.00 Ba 0.12 ±0.33 Aa 0.04 ±0.20 Aa 3.00 ±2.31 Bab 0.80 ±1.12 Aab Day 28 Con ol 0.76 ±0.72 Aa 8.72 ±3.47 Bab 2.52 ±1.42 Aa 0.40 ±0.87 Aa 0.00 ±0.00 Ba 0.36 ±1.04 Aa 0.16 ±0.37 Aa 2.12 ±1.13 Ba 0.92 ±0.91 Ba HT 0.28 ±0.46 Ba 9.28 ±2.69 Bb 2.84 ±1.86 Aa 0.04 ±0.20 Aab 0.00 ±0.00 Ba 0.16 ±0.37 Aa 0.08 ±0.28 Aa 1.04 ±0.93 Bbd 1.16 ±0.94 Aa B 0.12 ±0.33 Ba 7.32 ±4.49 Bac 1.32 ±1.25 Bb 0.00 ±0.00 Ab 0.00 ±0.00 Ba 0.20 ±0.50 Aa 0.24 ±0.44 Aa 1.12 ±1.09 Bbd 0.16 ±0.47 Bb BHT 0.24 ±0.44 Ba 5.40 ±2.52 Bc 1.76 ±1.64 Ab 0.20 ±0.41 Aab 0.00 ±0.00 Ba 0.08 ±0.28 Aa 0.08 ±0.28 Aa 0.88 ±0.97 Bbc 0.08 ±0.40 Bb T 0.52 ±0.51 Aa 5.84 ±2.27 Bc 1.48 ±1.16 Bb 0.00 ±0.00 Ab 0.00 ±0.00 Ba 0.04 ±0.20 Aa 0.12 ±0.44 Aa 0.40 ±0.71 Bc 0.16 ±0.37 Bb THT 0.08 ±0.28 Ba 6.80 ±2.25 Bac 1.40 ±1.08 Bb 0.04 ±0.20 Aab 0.00 ±0.00 Ba 0.12 ±0.33 Aa 0.08 ±0.28 Aa 1.68 ±1.25 Bad 0.08 ±0.28 Bb