Field Plots

In May 2005 transgenic cotton varieties containing the Bollgard®, Bollgard II® and WideStrike® traits were planted in research plots in the Mississippi Delta near Stoneville, MS (Table 1). Plots consisted of 2 rows (1.0 m centers) × 10.67 m. All plots were arranged in a randomized complete block design with each variety replicated 4 times (once in each block). Only insecticides not active on Lepidoptera were applied to all plots throughout the season as dictated by local management practices. In Mar 2007 transgenic cotton varieties containing the Bollgard, Bollgard II® and WideStrike® traits were planted in strip plots in the lower Rio Grande Valley of Texas near Weslaco, TX (Table 1).

Insects

All Lepidoptera utilized in these studies were obtained from laboratory colonies maintained at the USDA, ARS located in Stoneville, MS or Weslaco, TX.

Bioassays with Larvae

In 2005 bioassays were conducted with only fall armyworm larvae. A single neonate was placed into individual 9-cm diameter petri dishes (8) that each contained a moistened filter paper and a single lower leaf obtained from all plots for a total of 32 larvae per variety. Cotton plants were at peak bloom. The plates were covered with corresponding lids. After 5 d, surviving larvae were carefully transferred with a camel-hair brush into new petri dishes containing fresh filter paper and a new leaf. This procedure continued until pupation. At 7 and 10 d, larvae were prodded with a camel-hair brush and considered alive if coordinated movement was observed. Beginning at 15 d, plates were checked daily for the presence of pupae. Percent survival was analyzed by REML-ANOVA, and means were separated according to Fisher’s Protected LSD (Littell et al. 1996; PROC MIXED, SAS Institute 2001).

In 2007 leaves obtained from various sections of the plant containing various transgenic traits were assayed for bioactivity against beet and fall armyworms. Individual leaves were placed into a 50 × 9-mm Tight-Fit Lid sealing Petri dish (BD Falcon® #351006, VWR International). Beet armyworms (5) were placed in a dish containing a terminal (upper canopy) leaf or a mid-canopy leaf (10 dishes per variety) for a total of 50 larvae per variety. Fall armyworm bioassays were conducted identically, except only mid-canopy leaves were used. Leaves were also collected from various times during the growing season. At 5 d after exposure to cotton tissue, larvae were prodded with a camel-hair brush and considered alive if coordinated movement was observed. Percent mortality by trait was analyzed by REML-ANOVA, and means were separated according to Fisher’s Protected LSD (Littell et al. 1996; PROC MIXED, SAS Institute 2001).

Bioassays with Egg Masses

Inoculations of beet and fall armyworm egg masses to leaves located in various sections of the plant were conducted in 2007. In the laboratory, egg masses were deposited on nylon cloth placed on the top of adult rearing cages (3.79-L cardboard containers). For each inoculation, an egg mass of equal size (ca. 100-300 eggs per 2.54 cm² cloth sample) was pinned to the underside of a leaf for all traits and covered with a cage that consisted of a condiment cup (118 mL) (Solo Co., Highland Park, IL) coupled with a hard plastic lid (Fig. 1). Five d after inoculations, the infested leaf and corresponding cages were harvested and transported to the laboratory. Leaf damage (0-5) was estimated with a categorical rating scale where 0% indicated no leaf damage, while 80-100% of leaf consumption was given a value of 5. Damage ratings were analyzed by non-parametric statistics (SAS Institute 2001).

Bioassays with Larvae

Both Bollgard II® and WideStrike® had significantly higher efficacy against fall armyworms than Bollgard® (Fig. 2 and Fig. 3); however, in both 2005 and 2007, WideStrike® had typically higher efficacy compared to Bollgard II®. It is interesting to note that larval development to pupation was observed for fall armyworms when fed Bollgard® or Bollgard II®, but not WideStrike®. In WideStrike® isolines containing only Cry1F or Cry1Ac, Adamczyk & Gore (2004) showed that the Cry1F protein provided greater efficacy compared to Cry1Ac against this pest. In addition, previous studies have shown little efficacy of Cry1Ac against fall armyworms (Adamczyk et al. 1998). We believe that the greater efficacy of WideStrike® compared to Bollgard II® against fall armyworms is primarily due to the action of the Cry1F protein. Although we did not evaluate isolines containing only Cry1F or Cry2Ab, our data suggests greater efficacy of Cry1F versus Cry2Ab against this pest.

The expression of the Cry1F protein affects beet armyworm survival both temporally and spatially. When using mid-canopy leaves sampled throughout the season, no significant differences (P > 0.05) were observed between the survival of beet armyworms fed Bollgard II® and WideStrike® (Fig. 4). However late in the season when beet armyworms were fed terminal leaves located in the upper part of the plant (i.e., upper-canopy leaves), survival of larvae on WideStrike® was very high (>%60) (Fig. 5). Siebert et al. (2009) reported that expression of Cry1F protein is greatest in mature leaves as compared to other structures such as terminal leaves, squares, flowers, and bolls, and that protein levels in mature leaves increases with age. This expression profile for Cry1F is very different than what was reported for Cry2Ab in Bollgard II®, where expression in all tissue appears to remain relatively constant over time Adamczyk et al. (2001). In addition, mortality at >109 DAP of beet armyworms on WideStrike® terminal leaves was similar to what was observed with Bollgard® which also contains a Cry1Ac-like transgene that provides little efficacy against both beet and fall armyworms (Stewart et al. 2001; Adamczyk et al. 1998; Adamczyk & Gore 2004). We believe that low levels of Cry1F protein reported for new foliage (e.g., young terminal leaves) as compared to mature, fully expanded leaves may partially explain the low mortality observed against beet armyworms. Furthermore, our field studies with leaves collected from different portions of the plant supports these temporal and spatial conclusions. More damage was observed from beet armyworms feeding on WideStrike® leaves compared to Bollgard II® leaves in the upper canopy compared to the middle canopy (Fig. 6). Even with the presumed greater efficacy of Cry1F compared to Cry2Ab against the fall armyworm, this same trend was observed (Fig. 7). Regardless of transgenic trait, it is interesting to note that more damage was observed for both armyworm species caged on leaves located in the upper canopy. This may be partially explained by greater amounts of secondary plant compounds, as well as a thicker leaf cuticle, for leaves located lower in the plant canopy. In some situations, we believe that beet armyworms may need supplemental foliar insecticides for controlling outbreak populations that may be feeding on young tissues especially late in the season where Cry1F levels do not have the time to build to provide adequate control.

Various transgenes that are used to produce cotton with the WideStrike® trait may partially explain the observed protein expression profile of Cry1F. In particular, the cry1F trait in WideStrike® is regulated by the (4ocs) DeltaMas 2’ Promoter. Previous studies have shown that the dual promoter of Agrobacterium tumefaciens mannopine synthase (mas) genes is regulated by plant growth hormones, and that the activity of the mas dual promoters increases basipetally in developing plants; it has also been reported that the apical meristem contains a factor that inhibits stimulation of mas promoter activity (Langridge et al. 1989). However, it is important to note that WideStrike® traits contain enhancers that make the promoter more constitutive, whereby losing sensitivity to hormones (US Patent: 5,955,646). Thus, further research is needed to determine the various plant mechanisms that confer protein expression for the WideStrike® traits.