2.2.1.1. Sesamia nonagrioides
In November 2014, six populations of S. nonagrioides were collected in commercial non-Bt maize fields adjacent to Bt maize fields in two different zones of the Ebro Valley, which were separated geographically by the river Cinca, a tributary of the river Ebro, and by a strip over 10 km wide with no maize fields.
Samplings were carried out in three locations per zone: Zone 1 included populations from the municipalities of Vencillón (Huesca), Alfés and Almacelles (Lleida),
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whereas Zone 2 included a population collected in the municipality of Bujaraloz (Zaragoza) and two populations from Candasnos (Huesca) (Fig. 2.1; Table 2.1).
Figure 2.1 Geographical origin of S. nonagrioides and M. unipuncta populations.
Populations of S. nonagrioides were collected in six sampling locations ( ), whereas M. unipuncta populations came from two sampling locations ( ).
Table 2.1 Sampling locations of the S. nonagrioides populations. All populations were collected in November 2014.
Zone Population Province GPS coordinates Initial number of larvae
1
Vencillón Huesca N 41º40’55.8” E 0º18’53.3” 139 Alfès Lleida N 41º29’53.4” E 0º37’20.3” 126 Almacelles Lleida N 41º44’27.4” E 0º29’35.4” 158
2
Bujaraloz Zaragoza N 41º29’00.9” W 0º03’50.4” 78 Candasnos I Huesca N 41º32’31.5” E 0º02’43.4” 149 Candasnos II Huesca N 41º29’20.0” E 0º05’37.6” 143
Each field population included between 78 and 158 larvae that were obtained by slicing up maize stalks with signs of borer damage. Only one larva was collected per plant to avoid collection of siblings, which could bias the results. All larvae were in the last developmental instars when they were collected, and most of them had entered diapause. Individuals from each population were placed in ventilated
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boxes in groups of 40-60 larvae and provided with pieces of maize stalk for transportation to the laboratory. Fresh plant material was added daily to guarantee larvae were appropriately fed during the transportation period.
Upon arrival in the laboratory, all larvae were dipped in a 1% bleach solution to eliminate external pathogens and then they were allowed to dry before they were transferred to ventilated plastic boxes (21 x 16 x 4 cm) provided with a meridic diet prepared as described in González-Núñez et al. (2000). A thin layer of vermiculite was added to the bottom of each box to facilitate pupation. All boxes were maintained in growth chambers (SANYO MLR-352 PE, Tokyo, Japan) at 16 1 ºC and a 12:12 (L:D) photoperiod. These environmental conditions were aimed to maintain diapause. Every 3-4 days, fresh diet was added to all boxes. When an increase in the pupation rate was observed in a given population, the environmental conditions were shifted to 25 1ºC and a 24:0 (L:D) photoperiod to disrupt diapause. Pupae were removed and transferred to ventilated boxes (Ø 11.5 cm x 4.5 cm high), which were kept in growth chambers at a temperature of 25 1 ºC and a 16:8 (L:D) photoperiod. As adults emerged, they were placed in cages for mating and oviposition, which took place at the same environmental conditions.
To study interpopulation variation in susceptibility to Cry1Ab protein, groups of approximately six pairs of adults of the same population were placed in oviposition cages, consisting of a pot with 8-10 maize seedlings confined by a ventilated methacrylate cylinder closed on top by a mesh cloth adjusted tightly by rubber bands. Between five and ten oviposition cages of this type were set up per population (Fig. 2.2A).
Intrapopulation variation in susceptibility to Cry1Ab protein was studied in three out of the six field populations: Candasnos I, Alfés and Almacelles, the three populations in which the highest number of adults emerged. For this purpose, in each of these populations between 10 and 13 additional oviposition cages containing just three maize seedlings were set up for mating of single pairs of adults.
Seven days later, all adults were removed from both kinds of oviposition cages and the maize stalks were inspected for egg masses. Egg clusters were carefully
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removed and placed on top of moistened filter paper in ventilated plastic boxes (Ø 9 cm x 3 cm high), that were kept in growth chambers at 25 1ºC and a 16:8 (L:D) photoperiod until the eggs hatched.
Susceptibility bioassays were performed on neonate larvae (<24 h) of the first generation (F1) of all six field populations, on offspring of both multiple-pairs and single-pair mating. These assays had to be repeated on second generation neonates (F2) of some single adult pairs due to the poor adjustment of the results obtained on F1 larvae to a regression line.
2.2.1.2. Mythimna unipuncta
A population collected in Galicia (northwest Spain) was studied as representative from an area where Bt maize had never been sown, whereas one originally from Lleida (northeast Spain) was evaluated as representative from a high Bt maize adoption area (Fig. 2.1).
The Galicia population was started from a lot of 66 L6 larvae and pupae kindly provided by Dr. A. Butrón and Dr. R.A. Malvar, of the Misión Biológica de Galicia (MBG, CSIC) (Pontevedra, Spain). This population was collected in September and October 2015 in an experimental non-Bt maize field located in an area where Bt maize had never been sown commercially, in the MBG (northwestern Spain).
On the other hand, the Lleida population was established from a batch of 92 L6 larvae kindly provided by Dr. M. Eizaguirre (Universitat de Lleida, Lleida, Spain) in October 2015. All individuals were first generation (F1) offspring of adults captured in light traps in the Universitat de Lleida - Institut de Recerca i Tecnologia Agroalimentàries (Lleida, Spain), in September 2015.
All the larvae received from Galicia and Lleida were transferred to 6-well plates (BD Falcon, Erembodegen, Belgium) and reared individually to avoid cannibalism.
Larvae were fed with fresh pieces of non-Bt maize leaves until pupation, whereupon pupae were transferred to ventilated boxes (Ø 11.5 cm x 4.5 cm high) for adult emergence. Mating and oviposition took place in ventilated plastic boxes (28 cm x 22 cm x 15 cm) at room conditions. Between 5 and 10 couples of adults were placed
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in each box and provided with a solution of watery honey as nourishment and test tubes covered by thin strips of Parafilm M® (Bemis NA, Wisconsin, USA) as a suitable stand for oviposition (Fig. 2.2B). Every 2-3 days, new test tubes were added to the oviposition box and those with egg masses were transferred to plastic boxes (Ø 11.5 cm x 4.5 cm high) with moistened filter paper for egg hatching. Rearing of M. unipuncta took place in growth chambers at 23 ± 1 ºC and a 16:8 (L:D) photoperiod.
Susceptibility assays were performed on neonate larvae (<24 h) on the first generation after the arrival of the populations to the laboratory, which corresponded to F1 for Galicia and F2 for Lleida, provided that field collected individuals constituted the parental generation (F0). To study whether susceptibility to Cry1Ab protein of a population from a high Bt maize adoption area decreases after the selection pressure has been removed, larvae of the population collected in Lleida that were not used in the assays performed on F2 neonates were reared in the same fashion for three generations and the assay was repeated on neonates of the F5. For comparison purposes, the same procedure was followed for the population collected in Galicia, in which the susceptibility bioassay was repeated in the F4.
Figure 2.2 Multiple-pair mating oviposition cages in S. nonagrioides (A) and oviposition cages in M. unipuncta (B).
42 2.2.2. Source of Cry1Ab protein
The Cry1Ab protein used in the assays was synthesized in Dr. Juan Ferré’s laboratory (Universitat de Valencia, Spain) and had a purity of 78.88%. It was obtained from an Escherichia coli culture transformed with a plasmid containing the gene Cry1Ab provided by Dr. Ruud de Maagd (Wageningen University, Netherlands).