2287 in Bt Bt Bt Bt B. fusca damaged plants, and Bt maize were studied. Data Bt b thereby eliminating the prob- Bt A random refuge is therefore and non- 9 9 Bt seed, thereby eliminating the need Bt maize, provide useful tools for pest refuges. to Bt Bt Bt maize in Africa, evolved resistance to Bt 8 , 7 protein expressed in the crop. Refuge compliance has Non-compliance to refuge requirements most likely 6 Bt larval mobility. In the laboratory, different scenarios of This susceptible offspring is then controlled by the high 4 Correspondence to: A Erasmus, ARC-GrainPotchefstroom Crops 2520, Institute, South Africa. Private E-mail: Bag [email protected] X1251, ARC – Grain Crops Institute, Potchefstroom, South Africa Unit for EnvironmentalPotchefstroom, Sciences South Africa and Management, North-West University, Another IRM approach is the use of seed mixtures or the ‘refuge fusca ∗ a b . to plant separate non- that mate withcrop. the few resistant adults that survive on the contributed significantly to resistance development of South Africa. dose of been identified as astrategy. possible weakness of the high-dose/refuge in a bag’ strategy. In this case, a singletermined bag of ratio seed of contains a non- prede- included within the cultivated area, lem of low or non-compliancemixture to strategy refuge requirements. simplifies The seed adherence to refuge requirements, B ) 5 Bt Bt 3 ( , 2 and Johnnie Van den Berg Busse- refuge b Bt proteins in , which has is of such a nature that seed mixtures as an IRM strategy may not be effective The non- 4 Bt In South Africa, 1 B. fusca crop. B. fusca seed only). Bt Bt Bacillus thuringiensis Jaco Marais plants, more rapid resistance evolution has been proposed as a possibility with deployment of a* (: ), the target stem borer of maize expressing Cry1Ab protein Bt Bt and Several IRM strategies can be used, of Bt 4 : 2287–2294 www.soci.org © 2016 Society of Chemical Industry 72 Busseola fusca 2016; Cry proteins; genetically modified crops; larval movement; refuge; resistance evolution; stem borers (Lepidoptera: Noctuidae). A stacked event expressing dif- larval movement between single-gene (Cry1Ab) and stacked-trait (Cry1A.105 and Cry2Ab2)

crop cultivation.

Bt As with any pesticide, resistance evolution of target pests is Keywords: to delay resistance evolution. © 2016 Society of Chemical Industry indicating that the movement behaviour of CONCLUSION: Larval movement continued for 5 weeks and resulted in a significant incidence of seed as well as a control treatment (non- on larval survival and mass over timefield indicated study that with Cry the proteins single do not gene kill and larvae the above stacked certain event developmental was stages. conducted A using 2 year seed mixtures containing 5, 10, 15 and 20% non- B. fusca seed mixture strategies. RESULTS: Laboratory and field studies were conducted to study maize expressing Cry1Ab protein inmoving South between Africa. non- Owing to high larval mobility and subsequent sublethal exposure of larvae Cry proteins that control specific target . management. The benefits provided by -resistant plantspest are, however, species. threatened by The the high-dose/refuge evolution insect ofIRM resistance resistance by strategies. management target strategy (IRM) as well as seed mixtures are globally used as a concern. Insectcrucial in resistance delaying resistance evolution management and ensuring sustainability (IRM)of strategies are of predetermined size is planted near the

Pest Manag Sci seed Along with this high-dose expression, a separate non- 1 INTRODUCTION The use of genetically modified (GM) cropserties with insecticidal is prop- a significanttransgenic crops step are modified forward to in express pest management. These BACKGROUND: Products of plant biotechnology, for example genetically modified Abstract Annemie Erasmus,

Movement and survival of plantings with different ratios of non- (wileyonlinelibrary.com) DOI 10.1002/ps.4273 (Lepidoptera: Noctuidae) larvae within maize Research Article Received: 22 January 2016 Revised: 3 March 2016 Accepted article published: 15 March 2016 Published online in Wiley Online Library: 13 April 2016 refuge serves as a source of susceptible adults of the target pest ferent proteins (Cry1A.105 andSouth Africa Cry2Ab2) since 2011 has in an been attempt to deployed control in were planted until recently to control the African stemola borer, fusca become resistant to Cry1Ab produced by single-gene events. only single-gene events of which the high-dose/refuge strategystrategy is relies the on a most crop common. expressing This a high dose of order to kill as many of the target pest population as possible. Bt ,as and Bt 30 m) Bt × non- and non- : 2287–2294 → plants in each 72 Bt Bt1 Bt seed was recorded 2016; and . Bt Bt maize planted adjacent Bt2 and non- and Bt → non- Bt Bt . The five treatments were 5, larvae 4 weeks after seedling → Bt1 5 m area, with five rows planted maize, similar treatments com- , Bt2 Pest Manag Sci × Bt2 Bt1 Bt2 and seed ratios, and a control treatment B. fusca → seeds were marked with a trial marker. and Bt Bt1 Bt and Bt1 plants was planted at the same time as Bt1 to Bt1 → Bt Bt seed only. Each treatment was replicated 4 plant, in the middle row of each treatment, was events, Bt2 larvae each, on the same day that the main trial , Bt Bt Bt Bt2 → C, 50–60% humidity and a 10:14 D:L photoperiod. ∘ B. fusca 2 plants in the experiment. Bt2 ± Bt In order to assess larval development over time without destruc- Maize was grown under field conditions,The and experiment commenced whorl by inoculating whorl and and ear ear tissue The experimental layout consisted of a randomised block design A single non- the main experiment. Plants inneonate this plot were inoculated with ten was inoculated. Fourteen maizefrom plants this plot were and randomly dissectedThe selected number on of surviving a larvae and weekly larval mass basisrecorded. per plant to were then recover larvae. 2.3 Data analysis For the laboratory experiment, larval survivalysed and by mass means were of anal- ANOVA followedon by Fisher the protected incidence tests. of Data stem borerstem-borer-damaged damage, plants expressed per as plot percentage overiment, time in were the analysed field using exper- program the to generate Table curves Curve that 2D describedage the v.5.01 over incidence statistical of time. dam- The incidence of damaged plants in the different For larvae reared on to the experiment. Monitoring of stem borer damage3 commenced days after inoculation, and the incidencerecorded of thereafter damaged at plants weekly was intervals for 9 weeks. tive sampling inside thecontaining experiment, 150 a non- reference plot (7 binations were used, i.e. emergence. Fifty larvaethe per range plant of egg wereinfestation batch used, size in of as both this experiments thisseedling species. were emergence, is The on determined a levels within 6 block of of weeks natural non- after well as tissues were removed as needed for the laboratory study. with first-instar larvae and allowing larvae21 to days feed before for transfer to either feed 3, on 9 tissueof of or the maize. different Larvae treatments were weighed andear placed tips on in the plastic whorl containers tissue10 (100 or mL). times This with was five replicated larvae atreplicate), per least depending container on (container the representing numbersplants a of they were larvae initially that reared on survivedwere before on the then first allowed transfer. Larvae toand feed mass for were 7 determined. days, Theseat after experiments 25 were which maintained larval survival 2.2 Field trial The field(2011/2012 study and was 2012/2013) atcil conducted – the Grain Agricultural over Crops ResearchSouth Institute, two Africa. Coun- Potchefstroom, growing North-West Province, seasons with five treatments planted to different ratios of 10, 15 and 20% non- seeds for both containing non- non- inoculated with 50 neonate A map was created of the positionstreatment of plot in order to distinguish between damage to times, and plots consisted of a 7 at an interrow spacing of 0.9At m planting, and the an position intrarow of spacing eachinside non- of each 17 plot, cm. and non- 12 14 Bt Bt2 iso- Larger events Bt and 10 Bt Bt1 An increased (Lepidoptera: © 2016 Society of Chemical Industry 15 if the www.soci.org A Erasmus, J Marais, J Van den Berg ).Thelarvaeused , as it was one of plants all affect a Bt2 Bt as a control treatment. B. fusca ) and the stacked event E), North-West Province, 11 maize and the B. fusca Bt ′′ Bt1 Bt 22.6 Ostrinia nubilalis plants could be exposed to sub- ′ to non- Bt maize seed mixtures on survival Furthermore, IRM faces challenges 45 Bt ∘ 16 to Bt larvae have not been done before. maize is not practical in these systems. Bt S, 26 Bt could be found in South Africa, and the ′′ maize. and non- Bt proteins, leading to survival of heterozygous A target pest species exhibiting no or limited B. fusca 14.0 Although seed mixture products are very prac- 13 larvae of different ages are effectively controlled Bt ′ Bt2 10 20 B. fusca to ∘ Cry2Ab2) (referred to below as 15 Bt + B. fusca maize events and their near-isogenic, non- . maize, although not at very high levels, which makes it a plants using the seed mixture strategy, and (ii) if there is , non- Bt Bt Bt Bt1 -tolerant population. At the time of this study, no susceptible Studies of the effects of The feeding behaviour and movement of target pest insects that The objectives of the study were to determine (i) to what extent to events. The treatments were designed to simulatetion different scenarios migra- and were as follows: transferring larvae from non- 2.1 Laboratory experiments Two experiments were conducted,and one the with other maize whorl with(completely tissue maize randomised) ear were tissue.experiments, similar The the migration for experimental of larvae these designs ofdays trials. different old) was ages simulated In (3, between non- 9 these and 21 population used wasnumbers the were available most to conduct susceptible this study. of which sufficient lethal dosages of tical from a plantingthere and is compliance monitoring concern pointevolution, regarding especially of for its migrating view, pests. efficacy in managing resistance migration between plants would probablyway. not Apart be affected from in the this concernstrategy about to delay the resistance ability evolution, ofdeploying some a this studies strategy seed suggest is mixture that still better than using no refuge at all. and migration of Suitable IRM strategies arethe needed first for in theprotein expressed world in to develop resistance against the Cry1Ab are highly mobile play an importanttiveness role in of determining the seed effec- mixtures to delay resistance evolution. wileyonlinelibrary.com/journal/ps 2 EXPERIMENTALTwo METHODS are not high dose. seed mixture’s ability to delay resistance evolution. The success rate ofrates high-dose expression, and predispersal the mortality rate of larval dispersal from This may be even more of a problem for with producers not needing to take responsibilityseparate for refuge. planting of a South Africa. This populationon was previously shownBt to survive population of risk of rapidmixtures resistance are deployed evolution to manage may be present when seed Crambidae). larvae that move from non- (RS) individuals and an increased rate of resistance evolution. potential for using this strategy to manageB. resistance evolution fusca by by in African smallholder farmingseparate systems refuges of because non- the planting of migrating line were(Cry1Ab) used event (referred in to below this as study.to These inoculate plants in weregeneration the of diapause laboratory the larvae and collected from field single-gene maizedorp in trials the area Venters- were (26 the F1 (Cry1A.105

2288 2289 Bt1 a maize 0.001 and < (Table 1). , and did Bt2 Bt Bt Bt2 a non- → maize could therefore Bt1 from the start survived after 21 days. No larvae Bt1 0.001 and Bt2 or < maize tissue to Bt2 Bt Bt1 Bt1 maize for the whole period or → wileyonlinelibrary.com/journal/ps Bt and Bt1 whorl tissue, and no evaluation of a to non- Bt Bt2 Bt2 , non- Bt 0.001 0.054 0.015 0.001 < < non- → Bt www.soci.org Many 21-day-old larvae survived the 7 day period in nearly all Larval mass was the greatest for the following movement sce- maize ears was high andfrom in most the cases control did (Table not differ 2). significantly Larval survival was only significantly longer than 9nine-day-old days, larvae and could be therefore conducted. no evaluation of survivaltreatment scenarios, of although survival waslarvae significantly lower that for werefor reared those on that were transferred to 3.1.2 Larval growthTherateofsurvivaloflarvaerearedfor3daysonanyofthetypes and survival on maize ear tissue of maize followed by a 7 day feeding period on non- survived for 21 days on not differ significantly between thesefeeding period treatments (Table after 1). the Larval 7 massfeeding was day period very in low all after treatments thetissue. 7 involving None day exposure of to the larvae reared on migration scenario from be conducted. The lowest larvalwere mass transferred was from non- recorded when larvae narios: non- 0.016 0.001 < < plants control Bt Bt treatments. Bt1 0.001 0.001 and non- < < Bt larvae (3, 9 and 21 days old) 7 days after being transferred between ears of different maize types larvae (3, 9 and 21 days old) 7 days after being transferred between whorl tissue of different maize types Three-day-old larvae Nine-day-old larvae 21-day-old larvae Three-day-old larvae Nine-day-old larvae 21-day-old larvae B. fusca B. fusca 0 a 0 a 20.0 a 1.55 a 78.2 bc 40.10 a 0.001 0.001 0.1a0.02a–––– 7.5 a 0.05 a 80.0 ab 9.35 a 86.0 a 133.5 ab 3.3a0.17a–––– ) (Table 1). Survival of nine-day-old 10.32 12.73 3.05 19.14 2.34 3.11 18.72 11.48 45.31 10.99 15.95 21.10 73.3 c 5.35 c 90.0 ab 61.59 b 96.0 a 151.0 ab 62.0 c78.2 c52.0 c 6.88 cd 5.92 bcd 2.42 ab 80.0 b 92.0 b 78.0 b 21.89 b 30.76 b 21.09 b 87.3 c 50.1 ab 30.2 a 140.30 b 152.80 b 166.40 b 62.0 c 9.67 d 70.9 b 38.79 b 85.5 c 154.90 b 25.0a0.85ab–––– 10.0 a 0.15 a 76.0 a 9.32 a 94.0 a 115.4 a < < 52.0bc0.98ab–––– 50.0 bc 4.27 bc 83.6 ab 53.13 b 96.0 a 151.7 ab 53.3 bc46.7 bc 6.67 c 6.47 c 98.3 b 90.9 ab 68.09 b 68.55 b 100.0 a 100.0 a 173.9 b 181.0 b 50.0bc0.98a–––– 50.0bc3.72abc–––– 22.0 ab 2.35 ab 2.0 a 1.48 a 20.3 a 14.20 a Busseola fusca Bt2 Survival (%) Mass (mg) Survival (%) Mass (mg) Survival (%) Mass (mg) Survival (%) Mass (mg) Survival (%) Mass (mg) Survival (%) Mass (mg) → maize was high (70 and 80% respectively) : 2287–2294 © 2016 Society of Chemical Industry Bt2 72 Bt1 0.05. 0.05. < < Bt Bt and (GenStat, 2014). P P 2016; 17 Bt Bt Bt Bt Survival and mass of Survival and mass of non- Bt1 Bt2 non- Bt2 Bt1 Bt2 → → → → → → → Bt2 non- Bt1 Bt2 non- Bt1 Bt2 non- Bt1 Bt2 non- Bt1 Bt Bt Bt Bt Bt Bt Bt → → → → → → → → → → → → Significance Significance P Bt2 F Bt2 Bt2 Bt1 a non- Table 2. non- non- Bt1 Bt1 a Bt2 non- F Table 1. P non- Bt1 Bt1 Bt1 Bt2 Bt2 non- and did not differ(Table 1). significantly between the different scenarios larvae after feeding for another 7 days on the non- treatment and on Pest Manag Sci per plot was also calculated and compared betweenmeans treatments by of ANOVA. Larval massence and plots survival were on also determined. plants ANOVA in was17th done the edition using refer- GenStat 3.1 Laboratory experiment 3.1.1 Larval growthLarval and survival on survival maize whorl tissue after(52–92%) the initial in 7 allafter day movement transfer feeding scenarios to period (Tablenot whorl was differ 1). tissue significantly high Larval of betweenSignificantly survival the the lower control respective and survival treatmentswhere larvae did was were transferred recorded to whorl tissue between(non- of the treatments stacked event 3RESULTS treatments was compared using Tukey’s tests obtained from multi- factor ANOVA. The incidence (%) of damaged Movement and survival of Bt2 plant Bt2 : 2287–2294 or maize whorl 72 . The incidence Bt1 Bt1 Bt2 2016; seed mixtures suffered the treatments at the different , showing its current high Bt plants per plot was, however, Bt Bt Pest Manag Sci B. fusca ) was highly effective against this treatments in either season (Table 3). and non- Bt2 Bt1 Bt1 maize expressing Cry1A.105 and Cry2Ab2 maize only. Larval mass was similar between Bt plots with 5% non- Bt plant tissue were similar in size to those that were Bt2 Bt1 The mean percentage larvae recovered per plant from the plants with damage in the different plots was negligible, plants was recorded in season 2. Bt2 (a) (b) Bt2 -resistant population of The stacked event ( Figure 1. reference plot as well as mean larval mass2. over Bars time: (a) represent season standard 1; errors (b) (SE). season between any ofThe the incidence of damaged non- significantly higher in theThe 15 and overall 20% incidence seedtreatments mixture was of low treatments. and damaged ranged between 4.4 plantsseason, and and 12.3% per in between the 2.4 first plot andseasons 5.4% in the in the the second season.lowest In incidence of both damaged plants, whichsignificantly in most from cases differed the otherof treatments with ranging between 0.0 andto 0.6% in season one, while no damage 4 DISCUSSION The level of larval survival when introduced to tissue, larvae that survived the7dayson 21 day period andreared the on additional non- susceptibility to proteins. The fact thatsimilar larval between survival the and damage to plants was assessment times in thelevel laboratory of experiment resistance indicates in ainitially this high significantly population. lower Although when larval larvae mass fed was on tissue increased as larvaelarvae became was also older. higher The on level maize ears of than on survival maize of whorl tissue. Bt , Bt Bt Bt Bt1 76%), maize. move- > non- Bt → fusca . Bt1 seed mixture B © 2016 Society of Chemical Industry maize in grow- larvae off plants www.soci.org A Erasmus, J Marais, J Van den Berg maize under field Bt Bt1 maize ear tips and . Larvae transferred Bt plants in the different Bt1 Bt1 B. fusca seed mixture treatments Bt → and (Table 1), while mean larval Bt1 Bt1 1 Bt maize in the laboratory study − Bt and maize. None of the larvae reared Bt1 maize ear tissue (Table 2). Survival of Bt2 → (40.1 mg) was approximately the same as ear tips was significantly lower than that Bt1 Bt Bt2 maize was also high (86 and 94% respectively) Bt2 ear tips for another 7 days was high ( to and maize to , non- Bt2 to Bt plots until pupation commenced. In general, the Bt maize whorl tissue in the laboratory study ranged Bt2 Bt to Bt Bt1 Bt1 Bt1 Bt1 non- and and survived 7 days after inoculation. Mass of three-day-old and → . The mass of a 21-day-old larva 7 days after being trans- were very small and non-viable after 7 days of feeding on was the same as that of a 14-day-old larva on non- and 1 − Bt Bt Bt Bt Bt2 Bt2 Bt2 Bt2 Recorded larval mass on non- Survival of nine-day-old larvae was high after feeding for another The incidence of plants exhibiting stem borer feeding damage in The percentage of damaged plants in all between 140.3 and 166.4 mg larva ing season 2 (Fig. 2).non- Larval mass of 28-day-old larvae feeding on over time (Fig. 1). Aalso proportion be of ascribed the to reductioncould a could, take however, degree of place predation underlarvae on recovered field young per conditions. larvae plant The4 that in weeks mean the to percentage reference 26 plot andprepupae were 18% decreased observed respectively in over the in samples the takenafter on two day inoculation 35 seasons. (5 (data The weeks) not first shown),recorded and further. larval numbers were not 3.2 Field trials Natural infestation during both seasonsof was plants low, showing with symptoms 5after of and crop larval 3% emergence feeding during damagereduction seasons 8 in larval 1 weeks numbers and onreference 2 a plot illustrates per respectively. the plant The migration basis of over time in the (Table 2). Whilenon- mass of nine-day-oldin other larvae movement scenarios, transferred thelargely mass similar from between of treatments. 21-day-old larvae was was similar to that of the reference block of non- wileyonlinelibrary.com/journal/ps while that of 21-day-old larvae in ato migration scenario from non- lower in migration scenarios wherenon- larvae were transferred from to thelattertissue(Table2). 7 days on non- nine-day-old larvae reared on non- transferred to ferred from non- treatments was higher thanplots, in except for the the non- 20% ratio, 11 weeksThe after inoculation mean (Table overall 3). incidence (%) of damaged plants did not differ that of a 14–16-day-old larva that fed on non- whorls (during vegetative growth stages) and onsue stem (during and ear reproductive tis- stages) inover the the different two treatment seasons plots dence is of shown damaged in plants wasseasons Fig. (Fig. in 2. 2). the The The same increase overall range inthe mean across damage movement of over inci- the larvae time two to is neighbouring indicativedamaged plants. plants of The increased over incidence the of firstafter 6 weeks which after it inoculation, levelled off. There was a high rate of conditions, while mass of ato 21-day-old larva transferred from incidence of damaged plants in(Figs the 2a and b) was similarson to that 1, of but the the control incidence treatment for of sea- damage was lower than in the non- control in season 2. mass of 28-day-oldlarva larvae under field conditions was 130 mg on larvae feeding for another 7was days on highest the in respective maize the tissues following migration scenarios: non- ment in non-

2290 2291 larvae , season 1; Bt1 B. fusca In grain sorghum 20 seed: (a) . 2 Bt has a long larval phase that This dispersal phenomenon and non- Migration does not cease after 18 After hatching underneath the Bt 25 , 23 B. fusca , wileyonlinelibrary.com/journal/ps 24 18 Furthermore, a significant positive 22 , 21 8 18 Approximately 4% of larvae leave the natal neonate larvae ascend to the whorl, where Field studies also indicated that 25 19 , 18 B. fusca www.soci.org Clear patterns in the intraseasonal progression of larval infes- Larvae of this species were previously described to migrate over from a single egg batch couldnatal plant, migrate implying up that to the 3.6 larvae m frompotentially away a infest from single plants egg the over batch an could area of 40 m tations have been described. may range between 31 andthroughout 50 all days, larval with stages. migration taking place long distances. is generally density dependent andhost plant might quality. From also the third be instar influenced onwards,the larvae lower by parts migrate to of the plant wherecan they penetrate the remain stem. Larvae inplants, plant up whorls, to the especially fourth in instar. older (6–8-week-old) leaf sheath, they either commence feeding deep insidevia the ‘ballooning whorl off’. or disperse larvae commence feedinguntil the inside sixth maize instar. Itof stems, has their been but cycle, reported larvae continues that,indicating occurred towards singly their the in end highly plant4–5 active whorls and weeks migration stems, post-hatching. behaviour during the it was observed that upmigration to off seven the natal plants plant. were infested after larval plant immediately after hatching. relationship has been reported between plant stand and the Bt protein, Using the Bt 4 plots during did not suffer can therefore Bt fusca . B. fusca B larvae migrate is evident component of the study are , season 2. plots are therefore only used to Bt2 B. fusca Bt1 Bt1 treatments, which could technically Busseola fusca Bt1 : 2287–2294 © 2016 Society of Chemical Industry , season 1; (d) 72 Bt2 maize ears on each of the respective assessment 2016; Bt -resistant population was still highly susceptible to the Mean incidence of damaged plants over time after inoculation of plots in the field with different ratios of control plots and Bt (c) (d) (a) (b) plants will be exposed to sublethal dosages of , season 2; (c) Bt and non- Bt Bt1 The high levels of migration observed in non- The use of a Cry1Ab-resistant strain of this pest in a study such The long period during which useful in illustrating larval migration and damagemixture plantings. patterns Results in from seed the period of approximatelyIRM 4–5 strategies weeks for provides this ato pest. challenge Older to larvae migrating from non- from the increase inweek incidence period after of infestation damaged (Fig. 2). plants This wasnon- over especially evident the in 5–7 also serve as control plots for the field component of this study. thereby increasing potential risk ofvious resistance studies development. indicated that Pre- seed mixturesIRM were strategy not for the optimum pests with high migratory behaviour. stacked event, results from the any fitness costs. as this would have defeated theexposure purpose of of studying larvae the effect toas of Cry1Ab this maize in seed mixtures. However, illustrate the migratory potential of larvae, similarbe to in what non-GM it would maize. stages, indicating that this resistant strain of (b) Pest Manag Sci Bt1 Figure 2. Movement and survival of strategy to manage resistance evolutionbe in questioned. 26 Bt Bt to Bt plants dispersal or non- plants Bt2 : 2287–2294 Bt a larvae on Bt plants. This is damaged 72 % Helicoverpa zea proteins within Bt2 larvae from Bt (number of plants) plants earlier than This could indicate 2016; O. nubilalis A concern, however, Bt 26 proteins are detected 12 , O. nubilalis 10 Bt stacked event) = The possibility of increased maize and seed mixtures. O. nubilalis proteins, as indicated in this Bt 26 Bt2 Bt 0.001 – Bt Pest Manag Sci < non- Mean % seed. plants that were damaged Bt A study conducted with 5 plots was observed. Movement of Bt 5 single-gene event; = 0.001 0.002 0.182 0.001 < < Mean plants Bt1 plants. overall % damaged maize showed that larvae detected 5 Bt Bt plants was identified as a potential contributing factor to larvae ( Bt A study of dispersal and movement of The presence of larvae inside stems was not determined in this plants B. fusca non- resistance development that could havea adverse seed implications mixture for strategy. behaviour. supported by results from the laboratorymigration study scenarios in which were different damage evaluated. between As 5 and the 20%cantly, incidence seed a 20% mixtures of ratio did might plant not bepressure differ the signifi- better and option to generate reduce selection agronomical disadvantage more of increased susceptible larval survival in adults.mixture 20% seed plantings However, could the be offsetviduals to by mate higher with resistant numbers individuals. of RS indi- the first hour, and that larvae dispersedfrom from non- and non- by feeding larvae, similarly to observations on in all plots suggests that no larvae survived on Numbers of larvae and damage were, however, significantly higher in plantings with only non- with a 20% seedlarvae mixture is to the sublethal potentialstudy, dosages exposure which may of of eventually migrating lead to survival of some individuals. increased movement between plants if dispersal under low larvalence density of conditions the owing toxin to in the pres- (Lepidoptera: Noctuidae) indicated thatdifference there in was the no significant occurrenceears of larvae between on plantings and of damage pure to maize maize. However, the negligible number of damaged study, and no informationlarvae that can successfully completed be their life provided cycles on on the number of Bt (number of plants) damaged . % Bt Bt maize plants (mean number of damaged plants in brackets) in plots planted with Bt 5%), farm- plants that and non- < Bt Bt Bt seed mixtures field plots, the © 2016 Society of Chemical Industry non- www.soci.org A Erasmus, J Marais, J Van den Berg Mean % - and non- Bt2 plants that Season 1 Season 2 Bt2 Bt were damaged plants, did, however, plants within plots was Bt Bt2 and non- 0.001 0.002 0.476 1.21 8.70 0.17 9.66 6.92 1.79 Bt maize seed, 11 weeks after inoculation with 0.349 0.002 0.917 52.81 9.57 0.89 90.98 10.37 – < plants 4.4 a (5.5)6.2 a (8.0) 3.8 a (4.8) 6.2 a (8.0) 0.6 a (0.8) 0.0 a (0) 2.4 a (4.3) 4.2 b (7.8) 2.4 a (4.3) 4.2 b (4.2) – – Bt damaged 29.1 c (36.3) – – 27.5 b (52.2) – – 24.1 a (27.0)15.4 a (19.0)16.3 a (20.3)20.8 a (27.0)19.1 a (27.0) 2.7 a – (3.3) 4.8 ab (6.0) 8.5 b (10.8) 8.7 b (12.3) 12.7 a (15.8) 11.5 a (14.3) 12.3 a (16.3) 19.0 a (33.0) 10.4 a 15.5 – (14.8) a (28.0) 16.3 a (30.8) 16.4 a (29.7) 1.0 a (1.7) 1.2 a (2.2) 29.5 b (53.2) 2.8 ab (5.2) 3.6 b (6.3) 18.0 a (31.3) 14.3 a (25.8) 13.6 – a (25.7) 12.8 a (23.3) – 7.9 ab (11.5) 7.5 ab (11.0) 0.4 a (0.5) 5.3 b (9.3) 5.3 b (9.3) – 12.3 b (19.0) 12.1 b (18.8) 0.2 a (0.3) 5.4 b (9.5) 5.4 b (9.5) – Mean overall % and non- is when 10% of plants in a field show symptoms 0.05. Bt Bt2 Bt2 Bt2 Bt1 Bt1 Bt1 Bt2 Bt1 < Bt Bt P plants was in the treatment with 20% 24 Incidence (%) of stem-borer-damaged B. fusca Bt 20 Significance Results from the field plots planted with Larval movement patterns in maize are affected by plant archi- F a 10% seed ratio – 15% seed ratio – 20% seed ratio – P Bt1 Control – non- 5% seed ratio – Treatment different ratios of 5% seed ratio – 10% seed ratio – 15% seed ratio – 20% seed ratio – F P Bt2 Control – non- Table 3. in this study canmixtures be on used to the assess rate the of potential effect larval of movement seed and exposure to tecture at the timeplace of during the oviposition, vegetative growth i.e. stages whether orstages during infestation of reproductive takes plant growth.may commence In feeding on late-infested silk of ears, maize, panicles or first-instaring in panicles young before emerg- larvae migrating and commencing feeding inside ears or stems. It was reportedinstars that to there feed is on no ears clear ofof preference late-infested for first maize, first and instars that on occurrence earsstage was at the most time likely of oviposition a andand function larvae shelter. searching of for soft plant tissue growth plants. Although the stacked eventing effectively larvae, controlled which migrat- is evidentincidence from the of laboratory damaged results plants and observed lower overall in incidence the ofseason plants was showing high (4.4–12.3%) borer fromaction damage an threshold agronomical in that standard. The the triggerscontrol the first of application of insecticides for of stem borer damage. Although the incidencefor of damaged the plants 5% seed mix ratio in both seasons was low ( wileyonlinelibrary.com/journal/ps number of larvae that migrateplants. successfully and survive on maize reveal that the incidence of damage to negligible. None of theof treatments exhibited damaged a plants higher incidence than the proportion of non- ers may not acceptresults, such separating damage damage to levels. Further analysis of the were present inside the respective plots.damaged The highest incidence of This shows that there was a high larval movement.

2292 2293 Bt Bull Crop (Lepi- crops: maize: Insects Bt Bt Crop Prot :215–218 :183–187 :510–521 :137–142 5 4 31 82 VSN Interna- J Econ Entomol Proc R Soc Lond Nat Biotechnol J Econ Entomol J Econ Entomol J Econ Entomol plants. S Afr J Plant Soil maize and refuge (Lepidoptera: Noc- Entomol Exp Applic Bt Bt Chilo partellus :38–50 (2012). corn. :1–6 (2014). Bt 136 S Afr J Plant Soil 55 (Lepidoptera: Noctuidae) to S Afr J Plant Soil Nat Biotechnol Bull Entomol Res :147–157 (2010). 103 Busseola fusca Crop Prot -transgenic maize. (Fuller) (Lepidoptera: Noctuidae). (Lepidoptera: Noctuidae) on an artificial Bt wileyonlinelibrary.com/journal/ps on an artificial diet. J Appl Entomol and transgenic Busseola fusca maize. Bt Bt ,inSouthAfrica. refuge strategies in the field. :154–160 (2013). J Econ Entomol :1–9 (2001). :585–593 (1998). 9 54 crops: definition, theory, and data. Bt (Fuller), to 91 (Lepidoptera: Noctuidae) and :255–269 (1987). (Lepidoptera: Noctuidae). Bt Busseolafusca Busseola fusca 77 Busseola fusca , Seeds of change: corn seed mixtures for resistance :684–689 (2009). Crop Prot Busseola fusca :165–169 (1992). Afr Entomol et al. 28 :343–352 (2011). :331–339 (2010). :2011–2025 (2009). :147–151 (2007). :937–948 (2000). :139–144 (1994). :266–267 (2000). :74–79 (2012). 250 :539–563 (2014). www.soci.org farmers’ perceptions, refuge compliance and reportsresistance of in South stem Africa. borer CM management and integrated104 pest management. transgenic maize by European corn borer (Lepidoptera: Crambidae). J Econ Entomol 24 tional Ltd, Hemel Hempstead, UK (2014). stalk borer, Entomol Res of the African maizetuidae), stalk with borer, special reference to5 insect–plant interactions. Busseola fusca (2013). the development of stem borercompliance at resistance the to Vaalharts irrigation schemeProt in South Africa. ment strategy for European corninfesting transgenic borers corn expressing (Lepidoptera: Cry1Ab Crambidae) protein. 93 crops: are seed mixtures or refugia the bestB strategy? (1988). Busseola fusca doptera: Pyralidae) on grain sorghum. (1991). stem borer 73 mass-rearing diet. cultivar effects onBusseola yield fusca losses caused by the maize stalk borer, maize: field resistance, contributing factors andAfrica. lessons from South ciated with resistance of genetically modified behaviour ofCrambidae) on neonate non- European corn borer (Lepidoptera: 103 resistance to 102 lessons from the first billion acres. worm (Coleoptera: Chrysomelidae) emergence and root damagea in seed mix refuge. rainfall on seasonal abundance and flightborer, activity of the maize stalk (1987). 18 makes sense for managing insect resistance to 38 GenStat Seventeenth Edition, Version 17.1.0.13780 (64 bit). 8 Kruger M, Van Rensburg JBJ and Van den Berg J, Transgenic 9 Onstad DW, Mitchell PD, Hurley TM, Lundgren JG, Porter RP, Krupke 2 Van den Berg J, Hilbeck H and Bøhn T, Pest resistance to Cry1Ab 7 Kruger M, Van Rensburg JBJ and Van den Berg J, Perspective on 3 Kruger M, Van Rensburg JBJ and Van den Berg J, No fitness costs asso- 4 Gould F, Testing 5 Goldstein JA, Mason CE and Pesek J, Dispersal and movement 6 Tabashnik BE, Brévault T and Carrière Y, Insect resistance to 15 Onstad DW and Gould F, Modelling the dynamics of adoption to 17 18 Van Rensburg JBJ, Walters MC and Giliomee JH, Ecology of the maize 19 Calatayud PA, Le Ru BP, Van den Berg J and Schulthess F, Ecology 16 Van Rensburg JBJ, First report of field resistance by the stem borer, 10 Davis PM and Onstad DW, Seed mixtures as a resistance manage- 11 Mallet J and Porter P, Preventing insect adaptation to insect-resistant 21 Van den Berg J and Van Rensburg JBJ, Comparative injuriousness of 22 Onyango FO and Ochieng’-Odero JPR, Continuous rearing of the maize 23 Ratnadass A, Traore T, Sylla M and Diarra D, Improved techniques for 20 Van Rensburg JBJ, Walters MC and Giliomee JH, Plant population and 13 Tabashnik BE, Van Rensburg JBJ and Carrière Y, Field-evolved insect 12 Murphy AF, Ginzel MD and Krupke CH, Evaluating western corn root- 24 Van Rensburg JBJ, Walters MC and Giliomee JH, The influence of 14 Carroll MW, Head G and Caprio M, When and where a seed mix refuge Bt O. .A Bt1 plant. larvae Bt plant in a B. fusca Bt event is high :1–16 (2011). B. fusca Bt plants and were 140 event to kill nearly plants in such seed plants 9 days there- Bt2 migrates extensively , predispersal feeding Bt Bt or a non- Bt Bt 28 , plants would most likely not 13 . B. fusca B. fusca Bt2 For biology has, however, not been events controlling third-instar 29 Entomol Exp Applic Bt B. fusca event could be regarded as ultra-high plants, and this could influence the effi- Busseola fusca to other dispersal was significantly greater from Bt B. fusca Bt Bt : 2287–2294 © 2016 Society of Chemical Industry 72 maize, it could be concluded that this event is maize could also play a role in the efficacy of seed Bt2 plants, movement and whether the O. nubilalis Bt 2016; Bt use in North America. plants for a short period before migration to a larvae could be considered high dose, and, if fourth-instar Bt If this principle is applied to 21-day-old Bt high-dose/refuge resistance management strategyof after 15 years 27 plants than from non- The 5% seed mixtures seem to be the most efficient in controlling Whether initial infestation occurs on a It has been suggested that 1 Haung F, Andow DA and Buschman LL, Success of the 5% mixture could, however, increaseproteins the on selection larvae while pressure potentially of exposingresistant a adults greater to number each of other during theprocess. mate-finding and mating dose. This study hasover indicated its that whole larval life cycle, overcant long larval distances, and survival that is signifi- possible ifon larvae non- get the opportunity to feed Whether or not lay their eggsmixtures on will non- also affect the likelihood of sublethal exposure. migrating larvae. While this may bestrategy, acceptable this in terms may of not an IRM farmers be use practical a under 10% field action conditions threshold where for the control of behaviour on studied before. mixtures. This aspect of dose. nubilalis larvae are controlled, the seed mixture planting, premigration feedingence behaviour will larval influ- growth, survivalwere not and addressed in migration. this study, While results from theindicated these laboratory that, assays aspects if oviposition were to occur on after, the likelihood ofmigration from the non- survival would be high. Any secondary result in a sufficiently highefficacy level of of mortality a necessary seedlarly to mixture ensure true IRM for late strategy.feeding This infestations, on where would ear neonate be and larvaelow. particu- silk Neonate commence tissue where toxinBt expression levels are transferred to also low dose for this species. Therefore, it was concluded that was a low-dose event against Pest Manag Sci REFERENCES ACKNOWLEDGEMENTS This study was08–001) funded and byacknowledgment. The Biosafety The Maize South technical Trust, AfricaUrsula assistance du and of (Project Plessis and Stefaans Mabel we BS du Grobler, Toit accordingly is highly appreciated. give due 5 CONCLUSIONS The efficacy of ahigh-dose/refuge strategies with seed the production mixture of sufficient num- strategybers of depends SS on individuals structured and the ability of the all the heterozygote RS individuals.mixture Survival plantings of will larvae depend largely in onon the the the seed initial non- establishment Movement and survival of to be followed by primary migration to non- cacy of a seed mixture strategy. . : 2287–2294 Busseola fusca J Econ Entomol 72 corn. 2016; corn in Bt Bt Pest Manag Sci :1276–1285 (2000). 93 (7):e69675 (2013). 8 J Econ Entomol :1214–1223 (2012). PLoS ONE corn borer (Lepidoptera:105 Crambidae) on (Lepidoptera: Crambidae) tunneling and survival inhybrids. transgenic corn inheritance of field-evolved resistance to 29 Razze JM and Mason CE, Dispersal behaviour of neonate European 27 Walker KA, Hellmich RL and Lewis LC, Late-instar European corn borer 28 Campagne P, Kruger M, Pasquet R and Van den Berg J, Dominant :259–272 18 (Noctuidae), (Lepidoptera: (Lepidoptera: corn containing © 2016 Society of Chemical Industry Bt www.soci.org A Erasmus, J Marais, J Van den Berg and :127–132 (2014). Bt 55 Busseola fusca Helicoverpa zea Eldana saccharina J Ga Entomol Soc and Crop Prot traits. TM Sesamia calamistis SmartStax ® (1983). rence, distribution, and ear damage of Noctuidae) in mixed plantingsGenuity of non- in Ibadan, Nigeria, West Africa. three species of maize stemborers: Pyralidae), 26 Yang F, Kerns DL, Head GP, Leonard BR, Niu Y and Haung F, Occur- 25 Kaufmann T, Behavioural biology, feeding habits and ecology of wileyonlinelibrary.com/journal/ps

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