Bt Research 2015, Vol.6, No.5, 1-10
8
larvae, at four days of age, were fed with
Bt
plants
(maize or rice). Exposure of
S. frugiperda
larvae to the
genetically modified plants (rice or maize) was performed
in acrylic plates (35 mm diameter) whose bottom was
covered with soaked filter paper discs and plant leaves.
The same procedure was adopted in treatments T1 and
T3, but with the transgenic plants being replaced with
the non-transformed cultivar IRGA 424 or conventional
Dekalb® corn.
The
S. frugiperda
larvae from all treatments remained
exposed to the transgenic or non-transgenic plants,
and the moisture in the filter paper was regularly
monitored. Replacement of the leaves was performed
daily, and the number of dead and living larvae and
the timing of parasitoid emergence were recorded. The
acrylic plates (35 mm diameter) containing the larvae
(
S. frugiperda
) were maintained in a B.O.D. chamber,
set at a temperature of 25°C, a 12 hour photoperiod
and an RH of 65%.
3.4 Mortality bioassay of
Spodoptera frugiperda
parasitized by
Campoletis flavicincta
and exposed
to bacterial suspensions of the monogenic strains
Bt thuringiensis
4412 (Cry1B) and
Bt kurstaki
HD1
(Cry1Ab)
These assays were performed according to the method
described above in section 3.5 (Figure 1), except that
the plants were replaced with suspensions of the
monogenic strains
Bt kurstaki
HD1 (Cry1Ab) and
Bt
thuringiensis
4412 (Cry1B), which were provided by
the Institute Pasteur (Paris). For the assays, the strains
were grown in Glucosed Usual Medium (Barjac, 1976)
at 28°C and 180 rpm for 48 hours.
Then, the suspensions were centrifuged at 5,000 rpm
for 15 min, after which the supernatant was discarded,
and the pellet was diluted in sterile distilled water. The
concentration was determined and standardized to 8.6
x 10
6
cells/mL for
Bt thuringiensis
4412 and to 1 x 10
9
cells/mL for
Bt kurstaki
HD1 (Polanczyk et al., 2000)
using a Neubauer chamber at optical microscopy. A
100µL aliquot of the suspension was applied to the
surface of the artificial diet (Poitout et al., 1974),
previously conditioned in mini-acrylic plates (35 mm
diameter).
After the evaporation of the excess of moisture, a total
of 30 larvae of
S. frugiperda
from 2nd instar were
individually introduced per treatment. In the control,
sterile distilled water was applied at a volume equivalent
to that in the treatment groups. Three repetitions were
performed, totalizing 360 larvae assessed in this
bioassay. The experiment was conducted in a B.O.D.,
set at a temperature of 25°C, a ± 65% RH, and 12
hours of photoperiod.
3.5 Biology of
Campoletis flavicincta
descendants
emerging from
Spodoptera frugiperda
that were
exposed or not exposed to the monogenic strain
Bt
subesp
.
thuringiensis
strain 4412 (Cry1B)
In the evaluation of the descendants of
C. flavicincta,
parasitized larvae of
S. frugiperda
were exposed to
Bt
thuringiensis
4412 as described above. As a control,
parasitized larvae that were not exposed to Cry
proteins were used. For each treatment, 100 larvae
were assayed to ensure the emergence of the required
number of parasitoids. From these insects, the parasitoids
obtained from larvae that were either exposed to the
Cry
proteins or not exposed were divided into two
pairs (two days old) for each treatment.
Each pairs was maintained for 24 h in a glass jar (11
cm high and 7 cm diameter). After this period, 50
larvae from the 2nd instar were exposed to each pair
of parasitoids for 24 h. The larvae were fed with the
artificial diet, and the parasitoids were fed with 10%
glucose. This procedure was repeated three times, and
the larvae were then isolated according to the experimental
procedure described above. Each track was monitored
daily, evaluating: (a) the date of formation of the
parasitoid pupa, (b) the date of emergence, (c) sex,
and (d) adult parasitoid longevity.
Figure 1 Schematic representation of the methodology
followed for the mortality bioassays with
Spodoptera
frugiperda
parasitized by
Campoletis flavicincta
and
treated with
Bt
plants (rice or maize) or
Bt
bacterial
suspensions (
Bt thuringiensis
4412 or
Bt kurstaki
HD1).
3.6 Statistical analysis
Using the statistical program Systat, the log
10
-transformed
mortality data from each treatment were subjected to
one-way analysis of variance (ANOVA), followed by
the Tukey test at a 5% probability. To assess the
frequency of parasitoid emergence from infected and
uninfected larvae and the average life cycle of these
parasitoids, the data were subjected to the
2
-square
and t-test at a 5% significance level.
