Susceptibility of the 17 insect colonies of S. frugiperda mentioned above was determined using two methods: leaf tissue and diet-incorporated bioassays.
Leaf tissue bioassays used leaf tissue that was excised from fully-expanded leaves of plants in the V5-V8 phenological stage. In the bioassays, three pieces of Bt or non-Bt leaf tissue that were approximately 3 cm long were placed in each well of a 32-well bioassay tray (C-D International, Pitman, NJ) and then, four newly-hatched larvae (<24 h) of an insect colony were released on the surface of the leaf tissue in each well and confined using vented covers (Bio-CV-4, C-D International, Pitman, NJ) (Figure 5.2A). Fresh leaf tissue was added to each well on days 3 and 5. Four replicates were performed for each combination of maize hybrid and insect colony, each replicate consisting of 32 larvae (8 wells), so that the total number of larvae tested per colony and maize hybrid was n=4×32=128.
In the case of diet-incorporated bioassays, for each colony three Cry1F concentrations (3.16, 10.00 and 31.60 μg/g) and a control solution (no Bt protein) were prepared by diluting the appropriate amount of Cry1F protein in distilled water, and then each solution was mixed with the appropriate amount of the same meridic diet used for insect rearing (Ward’s Stonefly Heliothis diet, Rochester, NY). The use of these concentrations in the bioassays was based on the results of the past studies and the average Cry1F concentration expressed in the leaves of Herculex® I maize plants (event TC1507) in the V9 growth stage, which was estimated to be 12.1 ng/mg of dry weight (EPA, 2005). In the bioassays, approximately 1 g of treated or control diet was placed in each cell of a 128-cell bioassay tray (C-D International, Pitman, NJ), and then a neonate larva (<24 h)
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was placed on the diet surface in each cell and confined using a cover that allowed air circulation (Bio-CV-16, C-D International, Pitman, NJ) (Fig. 5.2B). Four replicates of 25 larvae per replicate were run for each combination of insect colony and Cry1F concentration, so that a total of 100 larvae were tested per Cry1F concentration and colony (n= 4×25=100).
Figure 5.2. Susceptibility bioassays. Leaf tissue (A) and diet-incorporated (B) bioassays.
For both assay methods, bioassay trays were placed in growth chambers and maintained at 28 ± 0.5 °C, 50% rh and a 16:8 h (L:D) photoperiod for a week. The number of surviving larvae in each replicate was checked 7 days after larval release. A larva was considered dead if it did not move when prodded with a fine hair brush. Surviving larvae were weighed and the number of larvae that had a body weight of <6 mg/larva was also recorded for each replicate.
5.2.6. Statistical analysis
For each replicate, mortality was measured as ‘practical mortality’, which was calculated based on the total number of dead larvae plus the number of larvae weighing less than 6 mg (Huang et al., 2014). Practical mortality (thereafter called mortality in this chapter) was corrected according to the method described in Abbott (1925), and then transformed using the arcsine√x to normalize the data. A one-way analysis of variance (ANOVA) was performed using the General Linear Model procedure with insect colony as the main factor. Treatment means based on
A B
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insect colonies were separated using Tukey’s HSD tests at α=0.05 level (SAS Institute, 2010).
Sex linkage of Cry1F resistance was evaluated separately for PR and FL by comparing differences in the susceptibility of the two F1 colonies obtained from the reciprocal crosses between SS and each resistant colony. If significant differences in the mortalities between the two F1 reciprocal colonies were observed, resistance was considered to be sex-linked. Otherwise, if the mortalities were similar, resistance was considered to be autosomal.
Effective dominance level (DML), which evaluates the relative mortality level for an indicated insecticide concentration, was measured for each resistant colony according to the method described in Bourguet et al. (2000b), so that:
DML = (MLRS – MLSS) / (MLRR – MLSS)
where MLRS is the mortality level at a given insecticide concentration for the heterozygotes; MLSS is the mortality level at a given insecticide concentration for the susceptible homozygotes; and MLRR is the mortality level at a given insecticide concentration for the resistant homozygotes. DML was calculated for the leaf tissue and diet-incorporated bioassays at each of the three Cry1F concentrations tested. Values of DML can range between 0 (completely recessive) and 1 (completely dominant).
Larval mortality data of F2 and backcrossed colonies on the Cry1F-leaf tissue and Cry1F-treated diet were analyzed with chi-square tests to determine if the Cry1F resistance in PR and FL fitted the Mendelian single gene model (Tabashnik, 1991). For the diet-incorporated bioassays, the test for the monogenic model was performed for Cry1F concentrations at 10.00 and 31.60 μg/g only, because the concentration of 3.16 μg/g was not a good dose to discriminate heterozygotes from homozygotes. Given that leaf tissue bioassays were not conducted for the backcrossed colony related to FL, the corresponding chi-square test for fitting the monogenic model was not conducted for this colony.
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Complementation tests for allelism were performed to find out whether the two resistant strains share a resistance locus (Tabashnik et al., 1997). This hypothesis was evaluated by comparing the mortalities of the two F1 reciprocal colonies generated from the intercolony crosses of PR and FL (F1PR♀FL♂ and F1PR♂FL♀) with that of the parental colonies. A similar performance in the susceptibility assays between F1 colonies and their parents (i.e. PR and FL) would indicate a similar genetic basis between PR and FL, since resistance alleles located at different loci would restore susceptibility to the toxin in F1 offspring.
5.3. Results
5.3.1. Overall larval mortalities of the insect colonies on Cry1F leaf