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Bt Research 2013, Vol.4 No.2 8-13
ISSN 1925-1939
http://bt.sophiapublisher.com
11
containing 25 2
nd
instar larvae of
A. aegypti
or
C.
quinquefasciatus
.
B.
thuringiensis
subspecies
israelensis
(
Bti
) T14001, provided by the Institute
Pasteur, Paris, France, was used as a standard and
positive control. The test with larvae of
A. aegypti
was
evaluated after 24 hours, and
C. quinquefasciatus
after
48 hours, when the numbers of deaths were
determined. The strains that caused 50% mortality or
more were considered pathogenic (Monnerat et al.,
2005), and submitted to selective and dose bioassays
with larvae of
Simulium
spp. Mosquito toxicity was
used as predictive of blackly toxicity to restrict the
number of strains to be taken forward to the more
labour-intensive
Simulium
assay.
2.3
Simulium
spp. larval bioassay
Insects: The insects were collected daily from a river
at an experimental farm belonging to EMBRAPA,
located in Brasília (Distrito Federal). The identification
of the
Simulium
species collected was based on
morphological characteristics of larvae and pupae
according to the taxonomic key of Coscarón (1987).
Selective bioassay: For the selective bioassay, we used
two Amazon strains that killed more than 50% of the
larvae of
C. quinquefasciatus
and
A. aegypti
and 5
strains from the Collection of EMBRAPA Genetic
Resources and Biotechnology from different serotypes:
B. thuringiensis israelensis
(S1785),
B. thuringiensis
aizawai
(S1576), 2
B. thuringiensis kurstaki
(S1905
and S1450) and
B. thuringiensis tenebrionis
(S1122).
As standards we used
B. thuringiensis jegathesan
T28
A001 and
B. thuringiensis israelensis
T14001
(provided by Institute Pasteur, Paris, France) and
B.
thuringiensis medellin
163-0131 (provided by CIB,
Medellin, Colombia). Bacteria were cultivated in
EMBRAPA medium (Monnerat et al., 2007) for 72
hours with shaking at 200 rpm and 28
℃
.
The tests were performed by adding 1 mL of final
broth to a 500 mL beaker containing 100 mL of water
from the river and 25 larvae of the second instar of
Simulium
spp. The beakers were then placed in a
shaking incubator at 130 rpm at 28
℃
and the
experiments were performed in triplicate. As a
negative control, we used only water without the
addition of the bacteria. Data for larval mortality were
evaluated 24 hours after the beginning of the test.
Dosage bioassay: Strains that killed 50% or more of
total larvae in the bioassays were subjected to
selective dose bioassays against larvae of
Simulium
spp. to determine the lethal concentration required to
kill 50% of the population tested (LC
50
). To perform
this assay, the strains were grown in EMBRAPA
medium as before, then spores were harvested by
centrifugation at 12 000 g for 30 min. Then they were
frozen overnight, and lyophilized in a Labconco
model Lyphlock lyophilizer for 18 h. Final doses
ranged from 2.5 mg/mL to 500 mg/mL. The tests were
performed in triplicate and a control was left without
the addition of the bacteria. Counting the number of
dead larvae was performed 24 hours after the start of
the test. Mortality data were analyzed using Probit
Analysis (Finney, 1971) to determine the LC
50
(lethal
concentration required to kill 50% of larvae tested).
2.4 Characterization of toxic strains
Serological characterization: The Amazon strains were
serotyped according to the protocol described by de
Barjac and Frachon (1991). Strains S1785, previously
serotyped as
Bt
israelensis
, and the
Bti
T14001, were
used as positive controls.
Characterization of proteins by SDS-PAGE: The
biochemical characterization of the strains was
performed by protein electrophoresis in a denaturing
polyacrylamide gel (SDS-PAGE 10%). Protein
extraction was performed according to the protocol
described by Lecadet et al. (1991). Samples were
denatured at 100
℃
for 5 minutes and loaded on a
denaturing 10% polyacrylamide SDS-PAGE gel. The
gel was stained in a solution of Coomassie Blue (40%
methanol, 10% acetic acid and 25% blue Coomassie
250R) for 16 hours and placed in destain solution
(40% methanol and 10% acetic acid) to complete
visualization of protein bands.
Molecular characterization: The selected strains were
characterized for the presence of genes encoding Cry
proteins by PCR (polymerase chain reaction) using
primers specific for detection of
cyt1
,
cyt2
,
cry4A
,
cry4B
,
cry10
and
cry11
genes (Bravo et al., 1998;
Ibarra et al., 2003). DNA extraction was carried out as