A peer-reviewed version of this preprint was published in PeerJ on 22 February 2016.

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1 A peer-reviewed version of this preprint ws pulished in PeerJ on 22 Ferury View the peer-reviewed version (peerj.om/rtiles/1665), whih is the preferred itle pulition unless you speifilly need to ite this preprint. Khn T, Shhid AA, Khn HAA. (2016) Could iortionl insetiides e used in the mngement of fltoxigeni Aspergillus prsitius nd its inset vetors in stored whet? PeerJ 4:e1665

2 Could iortionl insetiides e used in the mngement of fltoxigeni Aspergillus spp. nd their inset vetors in stored whet? Tiyyh Khn, Ahmd Ali Shhid, Hfiz Azhr Ali Khn Inset pests in stored whet use signifint losses nd ply n importnt role in the dispersl of vile fungl spores of vrious speies inluding fltoxin produing Aspergillus spp. The prolems of insetiide resistne in stored insets nd environmentl hzrds ssoited with fumignts nd onventionl grin protetnts undersore the need to explore redued risk insetiides to ontrol stored insets nd the ultimte effet on fungl infetion inhiition. The purpose of this study ws to investigte the insetiidl potentil of four iortionl insetiides: spinosd, thimethoxm, imidloprid nd indoxr on whet ginst Rhyzoperth domini nd Sitophilus oryze nd the susequent effet of insets mortlity on Aspergillus flvus nd A. prsitius infetion in grins. Spinosd nd thimethoxm were the most effetive insetiides ginst R. domini ompred to S. oryze followed y imidloprid. Spinosd pplied t 0.25, 0.5 nd 1ppm nd thimethoxm t 2 nd 4ppm onentrtions resulted in omplete mortlity of R. domini nd >90% infetion inhiition of A. flvus nd A. prsitius. However, indoxr ws more toxi ginst S. oryze ompred to R. domini. The mortlity of R. domini ws diretly relted to the perent infetion inhiition of A. flvus nd A. prsitius in ll the tretments. Wheres, mortlity of S. oryze ws only relted to the perent infetion inhiition of A. prsitius in ll the tretments. The results show tht lthough oth spinosd nd thimethoxm n provide protetion ginst R. domini nd fungl infetions in stored grins, more potent redued risk insetiides nd/or their omintions would e needed thn either of these to provide rod spetrum protetion of stored grins. In onlusion, the results of the present study provide seline dt for the mngement of fltoxigeni fungi y ontrolling stored insets using iortionl insetiides. PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

3 2 Could iortionl insetiides e used in the mngement of fltoxigeni Aspergillus spp. 3 nd their inset vetors in stored whet? 4 Tiyyh Khn, Ahmd Ali Shhid, Hfiz Azhr Ali khn* 5 Institute of Agriulturl Sienes, University of the Punj, Lhore Emil ddresses: tiyyhkhn@yhoo.om (TK) 9 hmdli.shhid@gmil.om (AAS) 10 zhr_nturlist@yhoo.om (HAAK) *orrespondene: zhr_nturlist@yhoo.om (HAAK) PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

4 18 Astrt 19 Inset pests in stored whet use signifint losses nd ply n importnt role in the dispersl of 20 vile fungl spores of vrious speies inluding fltoxin produing Aspergillus spp. The 21 prolems of insetiide resistne in stored insets nd environmentl hzrds ssoited with 22 fumignts nd onventionl grin protetnts undersore the need to explore redued risk 23 insetiides to ontrol stored insets nd the ultimte effet on fungl infetion inhiition. The 24 purpose of this study ws to investigte the insetiidl potentil of four iortionl insetiides: 25 spinosd, thimethoxm, imidloprid nd indoxr on whet ginst Rhyzoperth domini 26 nd Sitophilus oryze nd the susequent effet of insets mortlity on Aspergillus flvus nd A. 27 prsitius infetion in grins. Spinosd nd thimethoxm were the most effetive insetiides 28 ginst R. domini ompred to S. oryze followed y imidloprid. Spinosd pplied t 0.25, nd 1ppm nd thimethoxm t 2 nd 4ppm onentrtions resulted in omplete mortlity of 30 R. domini nd >90% infetion inhiition of A. flvus nd A. prsitius. However, indoxr 31 ws more toxi ginst S. oryze ompred to R. domini. The mortlity of R. domini ws 32 diretly relted to the perent infetion inhiition of A. flvus nd A. prsitius in ll the 33 tretments. Wheres, mortlity of S. oryze ws only relted to the perent infetion inhiition of 34 A. prsitius in ll the tretments. The results show tht lthough oth spinosd nd 35 thimethoxm n provide protetion ginst R. domini nd fungl infetions in stored grins, 36 more potent redued risk insetiides nd/or their omintions would e needed thn either of 37 these to provide rod spetrum protetion of stored grins. In onlusion, the results of the 38 present study provide seline dt for the mngement of fltoxigeni fungi y ontrolling 39 stored insets using iortionl insetiides. PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

5 40 Introdution 41 Afltoxins re the group of struturlly diverse myotoxins tht re minly produed y 42 Aspergillus flvus nd A. prsitius, oth elonging to setion Flvi [1,2]. These myotoxins 43 re reognized s immunosuppressive, rinogeni, heptotoxi, mutgeni nd tertogeni [3], 44 sine they led to serious humn nd niml helth hzrds, inluding ute nd hroni liver 45 diseses, tumor indution, reprodutive disorders, genotoxiity nd nephrotoxiity [4-6]. These 46 myotoxins re known to ontminte more thn 25% of the world stored grin erel 47 ommodities of whih more thn 300 fungl metolites re reported to use humn nd niml 48 toxiity [7]. 49 Stored grin ontmintion with inset pests nd fungi is serious prolem resulting in more 50 thn 20% losses in overll prodution y deresing seed germintion nd downgrding of grins 51 [8]. Contmintion of stored grins with fungl spores is minly soure of myotoxins whih 52 usully results from stored inset pest s infesttion [9]. Insets disseminte the fungl spores ll 53 over the grin ulk y their onstnt movement, whih re rried on their ody nd/or deposited 54 in inset frss [10]. These insets rek seed ot s nturl rrier to fungus nd provide entry 55 point for fungl infetion [11]. Therefore strtegies to ontrol insets re needed. The most 56 serious inset pests of stored grins tht use >20% posthrvest losses in developing ountries 57 re: rie weevil (Sitophilus oryze), red flour eetle (Triolium stneum), lesser grin orer 58 (Rhyzoperth domini), rusty grin eetle (Cryptolestes ferrugineus) nd khpr eetle 59 (Trogoderm grnrium) [8,12]. 60 Control of these insets is of prime importne s they hve the ility to rry nd trnsmit 61 spores of Aspergillus spp. internlly [13]. Current ontrol mesures for these insets rely on the PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

6 62 extensive use of fumignts nd onventionl insetiides tht hve resulted in inresed inset 63 resistne [14], primry pest resurgene [15], seondry pest outrek [16], nd their use s grin 64 protetnt is eing reonsidered for their effet on helth nd environmentl sfety [17]. 65 Therefore, there is need to explore redued risk or iortionl insetiides with miniml 66 environmentl effet nd mmmlin toxiity for judiious inset pest mngement. 67 Biortionl insetiides re usully trget speifi new insetiides with low mmmlin 68 toxiity. Some of the iortionl insetiides like spinosd, thimethoxm hve een proved s 69 potentil grin protetnts ginst stored inset pests in different prts of the world [17, 18]. 70 However, suh studies re rre t Pkistn level. Therefore, in the present study, our ims were 71 to evlute the toxiity of spinosd, thimethoxm, imidloprid nd indoxr ginst two 72 mjor pests of stored whet R. domini nd S. oryze, nd the susequent effet of insets 73 mortlity on A. flvus nd A. prsitius infetion in whet grins. The results would e helpful 74 in the mngement of insets nd fltoxigeni fungi in stored ommodities. 75 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

7 76 Mterils nd Methods 77 Commodities, formultions nd fungl ultures 78 Untreted, len nd infesttion free whet grins (vr Seher-06) with 11.1% moisture ontents 79 were used in the present study. The insetiide formultions were spinosd (Trer 24% tive 80 ingredients [IA], Aryst Life Sienes, Pkistn), thimethoxm (Atr 25% [AI], Syngent, 81 Pkistn), imidloprid (Confidor 20% [AI], Byer Crop Sienes, Pkistn) nd indoxr 82 (Stewrd 15%, DuPont, Pkistn). Aspergillus flvus nd A. prsitius ultures were otined 83 from First Fungus Culture Bnk, Pkistn (FFCB). 84 Insets 85 Helthy ultures of Rhyzoperth domini nd S. oryze dults were olleted from grin 86 mrket, Lhore ( N, E), nd rered on whole whet in the lortory t 26±1 87 o C nd 70±5%. The insets were rered for five genertions efore strting experiments. Adults 88 of oth speies used in iossys were mixed sex nd 2-3-weeks-old [19]. 89 Biossys 90 Insetiidl iossys were done y following the methodology of Athnssiou et l. [19] with 91 some modifitions. Briefly, insetiides were pplied s solution diluted with distilled wter. 92 Individul replite lots of 200 g of whet grins were pled in 0.5 litre glss jrs for insetiide 93 tretments: 0 (ontrol; the grins treted with distilled wter), 0.25, 0.5 nd 1 mg/kg for 94 spinosd; 0 (ontrol; the grins treted with distilled wter), 1, 2 nd 4 mg/kg for thimethoxm; 95 0 (ontrol; the grins treted with distilled wter), 1, 2 nd 4 mg/kg for imidloprid; 0 (ontrol; 96 the grins treted with distilled wter), 1, 2 nd 4 mg/kg for indoxr. To hieve ove 97 onentrtions, 2 ml solution of eh tretment ws prepred nd pplied to the 200 g grins. In 98 order to mximize insetiide distriution, the jr ws shken mnully for 5 minutes [19,21]. PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

8 99 The jrs were left in the lortory for 24 h t 25 o C nd omplete drkness to dry. After 24 h, 100 the jrs with insetiide treted whet grins were inoulted with 0.5 ml spore suspension ( spores/ml) of A. flvus (Aren A ) or A. prsitius (Aren B ) seprtely. Twenty dults of 102 R. domini or S. oryze were introdued in eh jr, nd the insets were disinfeted with 0.5% 103 sodium hypohlorite for 2 min nd wshed with sterile distilled wter efore entering into the 104 jrs [20]. To hek the diret effet of insetiides on fungl infetion inhiition, the sme 105 proedure ws repeted ut without introduing insets in the treted jrs. The treted jrs were 106 losed with muslin loth nd mintined in the lortory t 26±1 o C nd 70±5%. After 14 dys, 107 the jrs were opened for mortlity ounts nd perent infetion inhiition of A. flvus or A. 108 prsitius. 109 Perent infetion inhiition ws determined y following the methodology of Krishnmurthy et 110 l. [21]. Briefly, 60 treted nd inset infested whet grins were withdrwn from the individul 111 replite nd pled on three lyers of sterilized moistened lotter diss in sterilized petri pltes 112 (90 mm dimeter) t the rte of 20 grins per lyer. The pltes were inuted for 7 dys in the 113 lortory t 26±1 o C. After inution, the pltes were exmined under the stereoinoulr 114 mirosope for the presene A. flvus or A. prsitius nd the numer of fungl olonies nd 115 helthy seeds (i.e., without olony) were ounted. Infetion inhiition perentge ws lulted 116 s follows: 117 Perent infetion inhiition = (numer of helthy (uninfeted) seeds/totl numer of seeds) * The entire proedure ws replited five times y prepring new lots of treted whet for eh 119 replite, nd exposing, ounting, nd reording dt for dult mortlity nd fungl infetion s 120 desried ove. 121 Dt nlysis PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

9 122 Perent mortlity nd infetion inhiition perentge dt were nlyzed seprtely for eh 123 inset nd fungl speies using one-wy nlysis of vrine (ANOVA) with Sttistix softwre [22]. Mens were ompred y Tukey s Honestly Signifint Differene (HSD) test, t proility. 126 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

10 127 Results 128 Mortlity 129 In se of spinosd, mortlity for R. domini ws higher thn for S. oryze. Rhyzoperth 130 domini showed 100% mortlity t the spinosd onentrtions of mg/kg, in oth the 131 ren (F=6273; df=3, 16; p<0.01, for ren A, nd F=2401; df=3, 16; p<0.01, for ren B). 132 Wheres, S. oryze showed the highest mortlity t the onentrtion 1ppm followed y 0.5 nd ppm (F=235; df=3, 16; p<0.01, for ren A, nd F=218; df=3, 16; p<0.01, for ren B) (Fig ). 135 Rhyzoperth domini showed omplete mortlity on whet treted with thimethoxm t 136 onentrtions 2 nd 4ppm (F=858; df=3, 16; p<0.01, for ren A, nd F=262; df=3, 16; p<0.01, 137 for ren B). Wheres, S. oryze showed omplete mortlity only t 4 ppm, in oth ren 138 (F=1421; df=3, 16; p<0.01, for ren A, nd F=498; df=3, 16; p<0.01, for ren B) (Fig. 2). 139 In se of imidloprid, mortlity for R. domini ws higher thn for S. oryze. R. domini 140 showed 92 nd 94% mortlity in ren A nd B respetively, t the highest onentrtion 4ppm 141 (F=234; df=3, 16; p<0.01, ren A, nd F=192; df=3, 16; p<0.01, for ren B) (Fig. 3). Wheres, 142 S. oryze showed 33 nd 39% mortlity in ren A nd B respetively, t the highest 143 onentrtion 4ppm (F=43.6; df=3, 16; p<0.01, for ren A, nd F=50.8; df=3, 16; p<0.01, for 144 ren B). 145 For indoxr, in ontrst, mortlity for R. domini ws lower thn for S. oryze. R. domini 146 showed 94 nd 92.3% mortlity in ren A nd B, respetively t the onentrtion 4ppm 147 (F=140; df=3, 16; p<0.01, for ren A, nd F=150; df=3, 16; p<0.01, for ren B). However, S. PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

11 148 oryze showed omplete mortlity t 2 nd 4ppm (F=403; df=3, 16; p<0.01, for ren A, nd 149 F=661; df=3, 16; p<0.01, for ren B) 150 Fungl infetion inhiition 151 Mortlity of R. domini seems to influene diretly the perent infetion inhiition of A. flvus 152 nd A. prsitius in ll the tretments. In the ontrols, where the survivl perentge ws high, 153 less infetion inhiition of oth fungi ws oserved. In se of spinosd, where mortlity of R. 154 domini ws 100% t the tested onentrtions, more thn 93% infetion inhiition of A. flvus 155 nd A. prsitius ws oserved. Wheres, in ontrols where the survivl perentge of R. 156 domini ws high (>90%), signifintly more inidene of oth fungi ws oserved. The sme 157 trend ws oserved with rest of the insetiides tested (Fig. 1-4). Similrly, perent mortlity of 158 S. oryze seems to influene only the infetion inhiition of A. prsitius (Fig. 1-4). Moreover, 159 highly signifint positive orreltions (p<0.01) were oserved etween mortlity of R. domini 160 nd infetion inhiition of A. flvus or A. prsitius, nd mortlity of S. oryze nd infetion 161 inhiition of A. prsitius (p<0.01; Tle 1). However, in the present study there ws 162 sttistilly no effet of insetiides on fungl infetion inhiition when the insetiides were 163 tested without introduing insets in the treted jrs (dt not shown here). 164 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

12 165 Disussion 166 The results of the present study reveled vrying level of toxiities of insetiide ginst R. 167 domini nd S. oryze. The results lso suggest tht ontrol of these insets my help limit the 168 infetion of fltoxigeni fungi in stored whet. 169 Overll, spinosd ws the most effetive insetiide ginst R. domini ompred to S. oryze. 170 Spinosd pplied t 0.25, 0.5 nd 1ppm onentrtions resulted in omplete mortlity of R. 171 domini. In ontrst, S. oryze showed 94% mortlity t the highest onentrtion of spinosd 172 tested. Spinosd is reltively new insetiide of low mmmlin toxiity nd hs rod 173 spetrum of trget speies, inluding stored inset pests [23], nd hs een registered for use in 174 the United Sttes [19]. The findings on spinosd toxiity ginst the tested inset speies re in 175 greement with numer of previous reports [19,23-26]. It seems tht if R. domini is the sole 176 speies in stored ommodities, spinosd my e s n effetive grin protetnt. 177 In se of thimethoxm, R. domini showed omplete mortlity t onentrtions 2 nd 4ppm, 178 wheres S. oryze showed omplete mortlity t only 4 ppm. Thimethoxm is neoniotinoid 179 nd reltively new insetiide of low mmmlin toxiity nd does not produe mutgeni 180 nd/or tertogeni effets [18]. It hs een extensively used s seed ot tretment with 181 stisftory results ginst wide rnge of pests [27]. Thimethoxm ws the first time evluted 182 s grin protetnt y Arthur et l. [18] nd found it effetive ginst R. domini nd S. 183 oryze. It ws further reported tht the mortlity of S. oryze nd R. domini inresed with 184 time nd it hd rehed pproximtely 100% fter 6 d of exposure to thimethoxm. But in our 185 study, S. oryze, showed less thn 100% mortlity t 2ppm fter 14 d of exposure. This 186 differene ould e due to the different origin of S. oryze. PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

13 187 Imidloprid is lso neoniotinoid with low mmmlin toxiity nd hs een used s seed 188 tretment for the mngement of numer of suking inset pests [27]. Reently, Dglish et l. 189 [28] investigted the potentil of imidloprid ginst different eetle pests of stored grins. 190 They reported tht the toxiity of imidloprid ws speies nd dose dependent, nd tht R. 191 domini ws more suseptile to imidloprid thn S. oryze. The results of the present study 192 with respet to imidloprid lso reveled tht the mortlity for R. domini ws higher thn for 193 S. oryze. Rhyzoperth domini showed up to 94% mortlity t the highest onentrtion 4 ppm 194 s ompred to 39% mortlity for S. oryze. 195 Indoxr elongs to n oxdizine insetiide group nd hs strong potentil ginst different 196 inset pests [29]. In the present study mortlity for R. domini ws lower thn for S. oryze, 197 whih is in greement with Dglish et l. [28] who evluted indoxr potentil s grin 198 protetnt first time. 199 Conerning the infetion inhiition of tested fungi, mortlity of R. domini ws diretly relted 200 to the perent infetion inhiition of A. flvus nd A. prsitius in ll the tretments. In the 201 ontrols, where survivl perentge ws high, less inhiition of oth fungi ws oserved. 202 Moreover, highly signifint positive orreltion ws oserved etween mortlity of R. 203 domini nd infetion inhiition of A. flvus or A. prsitius, nd mortlity of S. oryze nd 204 infetion inhiition of A. prsitius. There ws n indiret effet of insetiides on the 205 inhiition of oth fungi. Insetiides used mortlity of the inset speies whih ultimtely 206 resulted in infetion inhiition of fungi. One most prole reson ould e tht inresed 207 mortlity of insets resulted in redued movement nd seed ot dmge whih ultimtely led to 208 redued infetion of the fungi. After the hrvest of rops, temperture nd humidity ply n 209 importnt role in the growth of toxigeni fungi nd insets in the storge eosystem [30]. Stored PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

14 210 inset pests in the grnry eosystem move for reeding nd feeding purposes from one point 211 nother nd ontriute the dispersl of vile spores of fungi of vrious speies, inluding A. 212 flvus nd A. prsitius, whih re rried on the inset ody surfe or deposited in their frss 213 [30]. During feeding in stored ommodities, insets rek the seed ot of grins, whih is 214 nturl rrier to fungl growth, nd ultimtely promote esy spred of fungl spores [11]. 215 Hene, in the present study fungi infetion ws more in those tretments where the survivl rte 216 of insets ws high, most proly due to high rte of feeding nd movement tivities. 217 Dispersl of fungl spores in storge ommodities usully depends on the type of inset speies 218 present in those ommodities [31]. During storge, the invsion y vriety of insets uses 219 losses to storge grins nd t s mehnil vetors of vile fungl spores [20]. If storge 220 grins left untreted, insets within the grnry eosystem n rete lol onditions of 221 humidity nd temperture tht fvor the rpid growth of fungi, resulting in deteriortion of the 222 grin mss nd prodution of myotoxins [32]. The results of the orreltion in the present study 223 re in ordne with those of Lmoni nd Hell [31] who reported signifint nd positive 224 ssoition etween the tivity of Sitophilus sp. nd A. prsitius. Similrly, Nesi et l. [30] 225 reported wek involvement of Sitophilus sp. in the distriution of A. flvus spores in stored 226 mize wheres R. domini ws more suseptile to fungl ontmintion nd spred. From the 227 results it is ssumed tht if the storge ommodities left untreted nd hve vile spores of A. 228 flvus nd A. prsitius, nd R. domini nd S. oryze infesttion, there would e more 229 hnes of spred of the sid fungi. 230 Sine the insetiides tested in the present study re not reommended yet s grin protetnts in 231 Pkistn, further studies should e onduted to ssess their long term effiy or persistene 232 ginst different stored pests ross different stored grin ommodities. Spinosd nd PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

15 233 thimethoxm proved very effetive ginst R. domini nd the susequent infetion inhiition 234 of oth fltoxin produing fungi thn the rest of the insetiide tested. In grnry systems where 235 R. domini is the sole prolem, spinosd nd thimethoxm my provide dequte protetion. 236 However, tested insetiides exept indoxr did not provide dequte ontrol of S. oryze. 237 Therefore, in storge ins where oth inset speies oexist, there is need to evlute other 238 insetiides or insetiide omintions whih n provide dequte protetion ginst inset 239 pests nd susequent fungl infetion. In onlusion, the results of the present study provide 240 seline dt for the mngement of fltoxigeni fungi y ontrolling R. domini nd S. oryze 241 using iortionl insetiides. Whether this is prtil solution in grnry systems for long 242 time needs to e further hrterizing in future studies PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

16 245 Referenes Bennett JW, Klih M (2003) Myotoxins. Clinil Myoiol Rev 16: Horn BW (2007) Biodiversity of Aspergillus setion Flvi in the United sttes; A review. 248 Food dd Contm 24: Ptten RC (1981) Afltoxins nd disese. Am J Trop Med Hyg 30: Cho TC, Mxwell SM, Wong SY (1991) An outrek of fltoxiosis nd ori id 251 poisoning in Mlysi: liniopthologil study. J Pthol 164: Ley J (1988) The miroiology of erel grins from res of Irn with high 253 inidene of oesophgel ner. J Stored Prod Res 24: Desjrdins AE, Mnndhr G, Plttner RD, Mrgos CM, Shresth K, MCormik SP 255 (2000) Ourrene of Fusrium speies nd myotoxins in Neplese mize nd whet 256 nd the effet trditionl proessing method on myotoxin levels. J Agri Food Chem 48: Glvno F, Piv A, Ritieni A, Glvno G (2001) Dietry strtegies to ountert the 259 effet of myotoxins: review. J Food Prote 64: Philip TW, Throne JE (2010) Biortionl pprohes for mnging stored-produt inset. 261 Annu Rev Entomol 55: Birk NMM, Lorini I, Sussel VM (2006) Fungus nd myotoxins in whet grin t post 263 hrvest. In: 9 th Interntionl Working Conferene on Stored Produt Protetion: Sntos JP, Mntovni EC (1997) Perds de grãos do milho pré-olheit, olheit, 265 trnsporte e rmzenmento. Sete Lgos, Emrp/CNPMS, 6p Setmu M, Crdwell KF, Shulthess F, Hell K (1998) Effet of inset dmge to mize 267 ers, with speil refrene to Mussidi nigrivenell (Lepidopter: Pyrlyde) on 268 Aspergillus flvus (Deuteromyetes: Monoliles) infetion nd fltoxin prodution in 269 mize efore hrvest in the Repupli of Benin. J Eon Entomol 91: Pimentel D World resourses nd food losses to pests 37, pp Brr P, Nesi A, Etheverry M (2013) In vitro omptiility of nturl nd food grde 272 fungiide nd insetiide sustnes with Purpureoillium lilinum nd their effet 273 ginst Aspergillus flvus. J Stored Prod Res 54: Gurusurmnin G, Rhmn A, Srmh M, Roy S, Bor S (2008) Pestiide usge 275 pttern in te eosystem, their retrospets nd lterntive mesures. J Environ Biol 29: Hzrik LK, Bhuyn M, Hrrik BN (2009) Inset pest of te nd their mngement. 278 Annu Rev Entomol 54: Crnhm JE (1996) Te pest nd their ontrol. Annu Rev Entomol 11: Vyis BJ, Athnssiou CG, Milons DN, Mvrots C (2010) Persistene nd effiy of 281 spinosd on whet, mize nd rley grins ginst four mjor stored produt pests. Crop 282 Protet 29: Arthur FH, Yue B, Wilde GE (2004) Suseptiility of stored-produt eetles on whet 284 nd mize treted with thimethoxm: effets of onentrtion, exposure intervl, nd 285 temperture. J Stored Prod Res 40: PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

17 Athnssiou CG, Arthur FH, Kvlliertos NG, Throne JE (2011) Effiy of spinosd 287 nd methoprene, pplied lone or in omintion, ginst six stored-produt inset 288 speies. J Pest Si 84: Ferreir-Cstro FL, Potenz MR, Roh LO, Corre B (2012) Intertion etween 290 toxigeni fungi nd weevils in orn grin smples. Food Control 26: Krishnmurthy YL, Shshikl JB, Nik S (2008) Antifungl potentil of some nturl 292 produts ginst Aspergillus flvus in soyen seeds during storge. J Stored Prod Res : Anlytil Softwre (2005). Sttistix Version 8: 1: User's mnul. Anlytil Softwre, 295 Tllhssee, Florid Athnssiou CG, Kvlliertos NG, Chintzoglou GJ (2008) Effetiveness of spinosd 297 dust ginst different Europen popultions of the onfused flour eetle, Triolium 298 onfusum Jquelin du Vl. J Stored Prod Res 44: Athnssiou CG, Kvlliertos NG, Yitilis AE, Vyis BJ, Mvrots CS, Tomnovi Z 300 (2008) Influene of temperture nd humidity on the effiy of spinosd ginst four 301 stored grin eetle speies. J Inset Si 8: Fng L, Surmnym BH, Arthur FH (2002). Effetiveness of spinosd on four lsses 303 of whet ginst five stored produt insets. J Eon Entomol 95: Nyk MK, Dglish GJ, Byrne VS (2005) Effetiveness of spinosd s grin protetnt 305 ginst resistnt eetle nd psoid pests of stored grin in Austrli. J Stored Prod Res : Zhng L, Greenerg SM, Zhng Y, Liu TX (2011) Effetiveness of thimethoxm nd 308 imidloprid seed tretments ginst Bemisi ti (Hemipter: Aleyrodide) on otton. 309 Pest Mng Si 67: Dglish GJ, Nyk MK (2012) Potentil of the neoniotinoid imidloprid nd the 311 oxdizine indoxr for ontrolling five oleoptern pests of stored grin. Inset Si : Khn HAA, Shd SA, Akrm W (2013) Resistne to new hemil insetiides in the 314 house fly, Mus domesti L., from diries in Punj, Pkistn. Prsitol Res 112: doi: / s PuMed: Nesi A, Brr P, Etheverry M (2011) Integrted mngement of inset vetors of 317 Aspergillus flvus in stored mize, using syntheti ntioxidnts nd nturl 318 phytohemils. J Stored Prod Res 47: Lmoni Y, Hell K (2009) Propgtion of myotoxigeni fungi in mize stores y post- 320 hrvest insets. Intl J Trop Inset Si 29: Sinh KK, Sinh AK (1992). Effet of Sitophilus oryze infesttion on Aspergillus flvus 322 infetion nd fltoxin ontmintion in stored whet. J Stored Prod Res 28: PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

18 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

19 Figure 1. Effet of different onentrtions of spinosd on insets mortlity nd fungl infetion 337 inhiition. Dt rs of speifi olor with different letters re signifintly different; in ll ses 338 df=3,16, Tukey s HSD test t p< PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

20 Figure 2. Effet of different onentrtions of thimethoxm on insets mortlity nd fungl 342 infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in 343 ll ses df=3, 16, Tukey s HSD test t p<0.05 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

21 Figure 3. Effet of different onentrtions of imidloprid on insets mortlity nd fungl 347 infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in 348 ll ses df=3, 16, Tukey s HSD test t p<0.05 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

22 Figure 4. Effet of different onentrtions of indoxr on insets mortlity nd fungl 352 infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in 353 ll ses df = 3, 16, Tukey s HSD test t p< Tle 1. orreltion nlysis etween insets mortlity nd fungl infetion inhiition 356 Aspergillus flvus Aspergillus prsitius Ryzoperth domini 0.93** 0.87** Sitophilus oryze 0.18 ns 0.96** 357 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

23 Figure 1(on next pge) figure of insetiide fungus pper1 Figure 1 (Spinosd). Effet of different onentrtions of spinosd on insets mortlity nd fungl infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in ll ses df=3,16, Tukey s HSD test t p<0.05 Figure 2 (Thimethoxm). Effet of different onentrtions of thimethoxm on insets mortlity nd fungl infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in ll ses df=3, 16, Tukey s HSD test t p<0.05 Figure 3 (Imidloprid). Effet of different onentrtions of imidloprid on insets mortlity nd fungl infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in ll ses df=3, 16, Tukey s HSD test t p<0.05 Figure 4 (Indoxr). Effet of different onentrtions of indoxr on insets mortlity nd fungl infetion inhiition. Dt rs of speifi olor with different letters re signifintly different; in ll ses df = 3, 16, Tukey s HSD test t p<0.05 PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

24 Perentge A C R. domini mortlity A. flvus inhiition Control Spinosd R. domini mortlity A. prsitius inhiition Conentrtion B Control D S. oryze mortlity A. flvus inhiition d S. oryze mortlity A. prsitius inhiition Control d d PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

25 Perentge Thimethoxm A R. domini mortlity A. flvus inhiition B S. oryze mortlity A. flvus inhiition C Control R. domini mortlity A. prsitius inhiition Control D S. oryze mortlity A. prsitius inhiition Control Control Conentrtion PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

26 Perentge A C R. domini mortlity A. flvus inhiition Control d d R. domini mortlity A. prsitius inhiition Control d Imidloprid B Conentrtion Control D S. oryze mortlity A. flvus inhiition S. oryze mortlity A. prsitius inhiition Control PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015

27 Perentge Indoxr A C R. domini mortlity A. flvus inhiition Control d R. domini mortlity A. prsitius inhiition Control d d d B Control D Conentrtion S. oryze mortlity A. flvus Inhiition S. oryze mortlity A. prsitius inhiition Control d PeerJ CC-BY 4.0 Open Aess re: 24 My 2015, pul: 24 My 2015