the h. zea susceptible colony was a laboratory colony maintained at the mississippi state university department of entomology , mississippi state , ms insect rearing facility . this colony originated from non - bt corn in 2006 and wild individuals collected from non - bt corn were incorporated into the colony on a yearly basis . prior to the initiation of this experiment the susceptible colony was not known to be exposed to insecticides . field - derived populations for this study were composed of 15 h. zea colonies collected during 2013 and 2014 ( table 1 ) . larvae were placed in 36-ml solo cups ( bio - serv , frenchtown , nj ) containing stonefly heliothis diet ( product no . 38 - 0600 , ward s natural science , rochester , ny ) with matching lids . at pupation , 50 pupae were placed in 3.79-liter cardboard containers with matching lids with the corresponding colony and generation information labeled on the outside of each bucket . water solution that was changed daily . for the purpose of egg collection for bioassays , the cardboard containers were lined with reynolds cut - rite wax paper ( reynolds consumer products , lake forest , il ) . cotton cloth was placed over each bucket and kept in place by the lid to serve as an oviposition substrate . eggs were collected daily and new cloths and wax paper were applied to every bucket . collected egg sheets and wax paper from each colony were kept in 3.79-liter ziploc ( s.c . johnson & johnson , inc . , racine , wi ) bags until larvae hatched for use in bioassays . the laboratory susceptible colonies and field derived populations of each species were reared at the mississippi state university insect rearing facility under the following conditions : 25  c , 80% relative humidity ( rh ) , and a photoperiod of 16:8 ( l : d ) h. all assays were conducted on first- and/or second - generation progeny of field - collected colonies . table 1.description of h. 
 zea field - derived populations and laboratory colonies by identification code , collection host , month collected , and collection locationcolonyspeciescollection hostmonthlocationlabh . description of h. 
 zea field - derived populations and laboratory colonies by identification code , collection host , month collected , and collection location   all collections from z. mays were from non - bt . mortality bioassays were conducted to determine the susceptibility of h. zea to commercial formulations of flubendiamide ( belt ; bayer cropscience , raleigh , nc ) and chlorantraniliprole ( prevathon ; dupont crop protection , newark , de ) . dilutions of flubendiamide and chlorantraniliprole in distilled water were made from a stock solution with a concentration of 1,000  ng / ml and 500  ng / ml , respectively , to yield eight concentrations ranging from 0 to 35  ng / ml for flubendiamide and 0 to 6.8  ng / ml for chlorantraniliprole . aliquots from these solutions were combined with stonefly heliothis diet to yield 400  g of insecticide - treated diet for each concentration . insecticide - treated diet for each concentration was dispensed into 16 wells of a 128-well bioassay tray ( product no . each well was infested with one neonate ( < 24  h after hatching ) . infested assay trays were labeled and placed in a rearing chamber maintained at 25  c , 80% rh , and a photoperiod of 16:8 ( l : d ) h. all bioassays were replicated at least four times over different days based on date of oviposition . typically the ability of larvae to right themselves after being flipped onto their dorsal surface is considered an appropriate criterion for determining mortality with intoxicated larvae ( temple et al . zea colonies ( data not presented ) , it was observed that intoxicated larvae , though severely stunted , could still right themselves when flipped onto their dorsal surface . given the mobility of intoxicated larvae , therefore , the criteria for mortality was defined as larvae that had not molted to the second instar and weighed < 10  mg after 7 d ( siegfried et al . corrected data were analyzed with probit analysis to calculate slope , lc50 , lc90 , and confidence intervals ( proc probit , sas institute 2012 ) . goodness of fit tests ( p  > the h. zea susceptible colony was a laboratory colony maintained at the mississippi state university department of entomology , mississippi state , ms insect rearing facility . this colony originated from non - bt corn in 2006 and wild individuals collected from non - bt corn were incorporated into the colony on a yearly basis . prior to the initiation of this experiment the susceptible colony was not known to be exposed to insecticides . field - derived populations for this study were composed of 15 h. zea colonies collected during 2013 and 2014 ( table 1 ) . larvae were placed in 36-ml solo cups ( bio - serv , frenchtown , nj ) containing stonefly heliothis diet ( product no . 38 - 0600 , ward s natural science , rochester , ny ) with matching lids . at pupation , 50 pupae were placed in 3.79-liter cardboard containers with matching lids with the corresponding colony and generation information labeled on the outside of each bucket . water solution that was changed daily . for the purpose of egg collection for bioassays , the cardboard containers were lined with reynolds cut - rite wax paper ( reynolds consumer products , lake forest , il ) . cotton cloth was placed over each bucket and kept in place by the lid to serve as an oviposition substrate . eggs were collected daily and new cloths and wax paper were applied to every bucket . collected egg sheets and wax paper from each colony were kept in 3.79-liter ziploc ( s.c . johnson & johnson , inc . , racine , wi ) bags until larvae hatched for use in bioassays . the laboratory susceptible colonies and field derived populations of each species were reared at the mississippi state university insect rearing facility under the following conditions : 25  c , 80% relative humidity ( rh ) , and a photoperiod of 16:8 ( l : d ) h. all assays were conducted on first- and/or second - generation progeny of field - collected colonies . table 1.description of h. 
 zea field - derived populations and laboratory colonies by identification code , collection host , month collected , and collection locationcolonyspeciescollection hostmonthlocationlabh . description of h. 
 zea field - derived populations and laboratory colonies by identification code , collection host , month collected , and collection location   all collections from z. mays were from non - bt . concentration  mortality bioassays were conducted to determine the susceptibility of h. zea to commercial formulations of flubendiamide ( belt ; bayer cropscience , raleigh , nc ) and chlorantraniliprole ( prevathon ; dupont crop protection , newark , de ) . dilutions of flubendiamide and chlorantraniliprole in distilled water were made from a stock solution with a concentration of 1,000  ng / ml and 500  ng / ml , respectively , to yield eight concentrations ranging from 0 to 35  ng / ml for flubendiamide and 0 to 6.8  ng / ml for chlorantraniliprole . aliquots from these solutions were combined with stonefly heliothis diet to yield 400  g of insecticide - treated diet for each concentration . insecticide - treated diet for each concentration was dispensed into 16 wells of a 128-well bioassay tray ( product no . each well was infested with one neonate ( < 24  h after hatching ) . infested assay trays were labeled and placed in a rearing chamber maintained at 25  c , 80% rh , and a photoperiod of 16:8 ( l : d ) h. all bioassays were replicated at least four times over different days based on date of oviposition . ingestion of the diamides results in feeding cessation ( nauen et al . 2007 , hannig et al . 2009 ) . typically the ability of larvae to right themselves after being flipped onto their dorsal surface is considered an appropriate criterion for determining mortality with intoxicated larvae ( temple et al . ( data not presented ) , it was observed that intoxicated larvae , though severely stunted , could still right themselves when flipped onto their dorsal surface . given the mobility of intoxicated larvae , severe growth inhibition was a more reliable indication of mortality . therefore , the criteria for mortality was defined as larvae that had not molted to the second instar and weighed < 10  mg after 7 d ( siegfried et al . corrected data were analyzed with probit analysis to calculate slope , lc50 , lc90 , and confidence intervals ( proc probit , sas institute 2012 ) . goodness of fit tests ( p  > significant differences in lc50 and lc90 values were observed among populations of h. zea for both chlorantraniliprole ( table 2 ) and flubendiamide ( table 3 ) . overall , mean lc50 and lc90 data suggest that h. zea were 6.4-fold ( 23.43 vs. 3.65  ng / ml ) to 5.4-fold ( 30.51 vs. 5.65  ng / ml ) more tolerant to flubendiamide than chlorantraniliprole , respectively . a similar study suggested that s. frugiperda was 13.67-fold more tolerant to flubendiamide compared with chlorantraniliprole ( hardke et al . lepidopteran larvae appear to be more sensitive to chlorantraniliprole than flubendiamide at lower concentrations . table 2.comparative susceptibility of h. 
 zea neonates to chlorantraniliprole in dose  mortality curves generated with concentration  mortality bioassays with insecticide - treated dietcolonyspeciesnslope ( se)lc50 ( 95% cl ) ( ng / ml)lc90 ( 95% cl ) ( ng / ml) ( df)plabh . zea9913.35 ( 0.24)3.58 ( 3.413.73)5.25 ( 5.015.55)6.13 ( 5)0.2935ar14h . zea2562.83 ( 0.48)3.38 ( 2.853.76)5.32 ( 4.796.23)3.86 ( 5)0.5700ga14h . zea5124.02 ( 0.64)3.62 ( 3.423.84)4.98 ( 4.545.86)1.56 ( 5)0.9059msstone13 - 1h . zea8813.13 ( 0.40)2.94 ( 2.753.11)4.43 ( 4.075.03)4.93 ( 4)0.2947msstone13 - 2h . zea16432.76 ( 0.23)3.21 ( 3.053.36)5.10 ( 4.745.61)4.44 ( 5)0.4882msstone13 - 3h . zea11664.51 ( 0.64)3.52 ( 3.363.68)4.68 ( 4.355.27)6.09 ( 5)0.2974msyaz 14h . zea1282.12 ( 0.43)4.09 ( 3.254.84)7.47 ( 6.1510.72)8.43 ( 5)0.1340nc14h . zea2565.54 ( 1.01)4.22 ( 3.864.49)5.32 ( 4.985.91)6.38 ( 5)0.2702sc14h . zea3845.72 ( 0.93)4.05 ( 3.764.26)5.06 ( 4.85.49)9.07 ( 5)0.1064tn14 - 1h . zea2565.21 ( 0.96)3.72 ( 3.373.98)4.76 ( 4.445.32)0.43 ( 5)0.9946tn 14 - 2h . zea2562.49 ( 0.61)3.15 ( 2.193.67)5.26 ( 4.656.53)6.83 ( 5)0.2336 total number of insects tested . confidence limits . table 3.comparative susceptibility of h. 
 zea neonates to flubendiamide in dose  mortality curves generated with concentration  mortality bioassays with insecticide - treated dietcolonyspeciesnslope ( se)lc50 ( 95% cl ) ( ng / ml)lc90 ( 95% cl ) ( ng / ml) ( df)plabh . zea4474.54 zea1925.22 ( 1.16)24.04 ( 19.9426.28)30.76 ( 28.5733.74)2.68 ( 3)0.4439msstark13 - 1h . zea4065.23 ( 0.61)17.02 ( 16.1917.91)21.75 ( 20.3723.88)1.25 ( 3)0.5354msstark14 - 1h . zea2565.04 ( 1.03)16.45 ( 15.2517.77)21.22 ( 19.2925.73)1.56 ( 5)0.9060msstone13 - 1h . zea9472.58 ( 0.20)19.72 ( 18.5420.83)32.38 ( 30.2835.21)3.28 ( 3)0.1938msstone13 - 2h . zea6722.75 ( 0.76)21.23 ( 15.3023.73)33.82 ( 30.7543.90)4.60 ( 3)0.2035msvic14h . zea5606.46 ( 0.72)30.74 ( 29.9931.52)37.49 ( 35.9539.87)1.03 ( 3)0.7948nc14h . zea2889.07 ( 1.46)24.47 ( 23.2225.36)28.19 ( 27.2329.60)2.60 ( 3)0.4572sc14h . zea8967.37 ( 0.60)25.70 ( 25.1426.22)30.58 ( 29.7931.61)9.23 ( 5)0.1002tn14 - 1h . zea2563.72 ( 0.63)22.82 ( 20.3724.64)32.20 ( 29.6636.70)3.85 ( 5)0.5718tn14 - 2h . zea2565.19 ( 0.90)22.30 ( 20.2423.80)28.55 ( 26.7431.49)4.34 ( 5)0.5009 total number of insects tested . confidence limits . comparative susceptibility of h. 
 zea neonates to chlorantraniliprole in dose  mortality curves generated with concentration  mortality bioassays with insecticide - treated diet   total number of insects tested . . comparative susceptibility of h. 
 zea neonates to flubendiamide in dose  mortality curves generated with concentration  mortality bioassays with insecticide - treated diet   total number of insects tested . the lc50 values for chlorantraniliprole ranged from 2.94 to 4.22  ng / ml ( 1.44-fold ) , with a mean of 3.66  ng / ml ( table 2 ) . the lc90 values for chlorantraniliprole ranged from 4.52 to 9.17  ng / ml ( 2.02 fold ) , with a mean of 5.68  ng / ml . the lc50 values for flubendiamide ranged from 16.45 to 30.74  ng / ml ( 1.86 fold ) , with a mean of 23.53  ng / ml ( table 3 ) . the lc90 values ranged from 21.22 to 35.33  ng / ml ( 1.66 fold ) , with a mean of 30.59  ng / ml . overall , three field populations had lc50 values that were significantly lower than the laboratory colony and three field populations had lc50 values that were significantly greater than the laboratory colony for chlorantraniliprole ( table 2 ) . similarly , two field populations had lc50 values lower than the laboratory colony and five field populations had lc50 values that were greater than the laboratory colony for flubendiamide ( table 3 ) . statistical differences among populations were minimal and most likely represent natural variation among field populations . although it can not be ruled out because these insecticides have been used commercially since 2008 , this does not appear to represent a major shift in h. zea susceptibility to this class of insecticides . flubendiamide and chlorantraniliprole have been used extensively in soybean and grain sorghum to manage h. zea across the southern united states ( catchot et al . 2015 ) because of their high level of efficacy and long residual control ( tohnishi et al . , h. zea populations in the southern united states have likely been exposed to some level of selection pressure to these active ingredients prior to being tested . concentration mortality values of chlorantraniliprole for h. zea neonates in the current studies are considerably lower than those previously reported . ( 2009 ) reported mean lc50 values that were 15-fold greater than the results reported here ( 56 vs 3.6  ng / ml ) . the susceptibility of multiple lepidopteran species to several insecticides has been shown to increase at later instars ( yu 1983 ) . although direct comparisons can not be made to previous research , the overall purpose of this study was to develop a more rapid method for testing h. zea susceptibility to diamide insecticides . in many cases , growers and consultants need to respond to a control failure and need an answer as quickly as possible to determine if resistance is the cause of poor control . rearing larvae to the third instar increases the time needed to confirm resistance and will negatively impact the ability of growers to respond in a timely manner . baseline susceptibility to the diamide insecticide class was generated in louisiana by hardke et al . however , data have not been produced for these species in mississippi or other states included in this study . furthermore , differences observed between this study and previous studies can be attributed to differences in growth stages of larvae tested . the long residual efficacy of the diamide insecticides may potentially expose multiple generations of the same species to the insecticide . adams ( 2016 ) observed up to 90% and 60% control of h. zea 32 d after treatment in soybean field studies with both chlorantraniliprole and flubendiamide , respectively . this falls well within the generation time frame for h. zea during the warm summer months . therefore , neonates were used in this study to account for subsequent populations that could potentially be exposed to the diamide insecticides as the growing season progresses and to develop a more rapid method for confirming resistance . nevertheless , it is critical to document the variability in the response of field populations prior to the occurrence of control failures in the field . resistance is defined as  a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species  ( irac 2007 ) . this study generated data that can serve as a reference point for future monitoring programs associated with h. zea , aiding in the detection of resistance alleles prior to field control failures . future monitoring programs will aid resistance management efforts , allowing the diamide insecticide class to continue to play an important role in crop protection strategies .
corn earworm , helicoverpa zea ( boddie ) , is a significant pest of agroecosystems in the midsouthern and southeastern regions of the united states . 
 these insects have developed resistance to , or inconsistent control has occurred with , most insecticide classes . with their unique mode of action 
 , insecticides in the diamide class have become a key component in management of agriculturally important lepidopteran pests . in this study , 
 field populations of h. zea were collected in the southern united states and compared to susceptible laboratory colonies to generate baseline concentration  mortality data . lc50 and lc90 values 
 were generated for flubendiamide and chlorantraniliprole using neonates . to achieve equivalent levels of mortality , a higher concentration of flubendiamide 
 was required compared to chlorantraniliprole . 
 flubendiamide lc50 values for h. zea ranged from 16.45 to 30.74  ng / ml , with a mean of 23.53  ng / ml . 
 chlorantraniliprole lc50 values for h. zea ranged from 2.94 to 4.22  ng / ml , with a mean of 3.66  ng / ml . 
 significant differences were observed for some field populations relative to the laboratory colony . for flubendiamide , five populations had greater lc50 values and two populations had lower lc50 values compared to the laboratory colony . for chlorantraniliprole , three populations had greater lc50 values and three populations had lower lc50 values compared to the laboratory colony . 
 the response of these populations most likely represents natural variability among populations and does not indicate a significant shift in susceptibility of this species .