Bt Research 2015, Vol.6, No.5, 1-10
7
issues to be analyzed in this work include whether
parasitoids of insect pests can be used to increase the
efficiency of pest control in the areas of refuges (no
Bt
plants) . Also, the results of this study suggested that
proteins Cry1B may have a direct effect on
C.
flavicincta
. The occurrence of direct effects of Cry
proteins on a hymenopteran parasitoid, such as
C.
flavicincta
, merits further research because of the
importance of these parasitoids as natural enemies in
agroecosystems.
3 Materials and Methods
3.1. Insects
3.1.2
Spodoptera frugiperda
S. frugiperda
larvae were obtained from rearing
conditions established in a controlled room at the
UNISINOS Laboratory of Microbiology and Toxicology,
with a temperature of 25ºC, a relative humidity (RH)
of ± 65%, and photoperiod of 12 hours. Adults
obtained from material collected in rice fields were
placed in cages with a substrate for oviposition and
glucose-based feeding. The eggs collected daily, were
transferred to Petri dishes lined with moistened filter
paper. After hatching, the larvae were individually
placed in PVC cups containing an artificial diet
(Poitout et al., 1974) developed specifically for the
rearing of noctuids, until the pupae had formed, when
they were separated by sex and placed in glass jars.
The adults were maintained in cages under controlled
conditions as described above.
3.1.3 Rearing of
Campoletis flavicincta
in the
laboratory
The laboratory culture of
C. flavicincta
parasitoids was
initiated from larvae parasitized by
S. frugiperda
collected in the field. The larvae were individually
placed in 50 mL polypropylene vials containing an
artificial diet (Poitout et al., 1974), until the emergence
of adults or parasitoid.
The emerged parasitoids were multiplied through the
exposure of 20 larvae to a pair of parasitoids in glass
jars (11 cm high and 7 cm diameter) for approximately
24 hours. The larvae were fed with the artificial diet,
and the parasitoids were fed with a 10% glucose
solution. The larvae were subsequently individually
placed in 50 mL plastic pots containing the artificial
diet and evaluated until the formation of pupae or the
emergence of the parasitoids. Both larvae and parasitoids
were kept in B.O.D. chambers, adjusted to a temperature
of 25°C, a 12 h photoperiod, and ±65% RH.
3.2
Bt
plants
The genetically modified rice plants (
Bt
rice) used in
this study belong to the IRGA 424 cultivar, which was
transformed using
Agrobacterium tumefaciens
carrying
the
cry1B
gene of
B. thuringiensis
with a constitutive
ubi
(
ubiquitin
) promoter, as described by Pinto et al
(2013). The vegetative growth of
Bt
rice was maintained
at the Laboratory of Microbiology and Toxicology in
Biochemical Oxygen Demand (B.O.D) chambers under a
temperature of 25°C, a RH of 65% and a photoperiod
of 12 hours.
The
Bt
maize hybrids used in this study, which are also
resistant to
S. frugiperda
, belong to the commercial
cultivar DKB-350 (YieldGard® Corn Borer-YGCB),
which was transformed with the
cry1Ab
gene of
B.
thuringiensis
(recommended for planting in Brazil)
and the near isogenic negative checks (recommended
for planting in midwestern Brazil). The
Bt
maize was
grown under the same environmental conditions as the
rice in PVC pots 15 cm diameter containing 3seeds
per pot). In these experiments, seeding was performed
weekly, and the plants were used between 2 and 6
weeks of vegetative growth. In these treatments, no
fertilizers or chemical treatments were used.
3.3 Mortality bioassays of
Spodoptera frugiperda
parasitized by
Campoletis flavicincta
and fed with
genetically modified plants (
Bt
maize or rice)
In these experiments, adapted from (Dequech et al.,
2005),
S. frugiperda
larvae were exposed to the following
treatments for three or ten days: (a results obtained by
Ramirez-Romero et al. (2007) suggested that Cry1Ab
is not acutely lethal, and mortality depends on level
(dosage) and length of exposure) (T1) without parasitism
or
Bt
plants (control); (T2) exposed to
Bt
plants (
Bt
rice
or maize); (T3) only exposed to parasitism by
C.
flavicincta
; and (T4) exposed to both
Bt
plants (
Bt
rice
or maize) and parasitism (Figure 1). For each treatment,
30 larvae from the 2nd instar were used, and three
repetitions performed, for a total of 360 assessed larvae.
The period of exposure was 3-10 days to compare the
mortality rates of the larvae. To obtain the insects to
be used for treatments T3 and T4, 2nd instar larvae of
S. frugiperda
were exposed to parasitoids in acrylic
cages for 24 hours. After this period, both T4 and T2
