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conditions described previously (Céspedes
et al.
, 2000). Briefly, the artificial diet
(1 kg) used contained 800 ml of sterile
water, 10.0 g of agar, 50.0 g of soya meal,
96.0 g of corn meal, 40.0 g of yeast extract,
4.0 g of wheat germ, 2.0 g of sorbic acid,
2.0 g of choline chloride, 4.0 g of ascorbic
acid, 2.5 g of p-hydroxybenzoic acid
methyl ester, 7.0 ml of Wesson salt mix-
ture, 15.0 ml of Vanderzant vitamin mix-
ture for insects, 2.5 ml of formaldehyde,
0.1 unit of streptomycin, 5.0 g of aureo-
mycin and 20.0 g of milled ear of maize
grain, which was prepared by the proce-
dure described earlier (Céspedes
et al.
,
2000). Polystyrene multidishes (24-well)
were filled with the liquid diet and
allowed to solidify for 20 min at room
temperature under sterile conditions. The
3.4 ml wells measured 17 mm in depth
and 15 mm in diameter, with a 1.9 cm
2
culture area. All test extracts were dis-
solved in 95% ethanol and layered on top
of each well with the artificial diet at five
concentrations, using 1 ml 95% ethanol
as a control. For each concentration used
and for the controls, a single
S. frugiperda
neonate larva was placed on the diet
mixture in each well for 7 days. Thus
each treatment included 72 larvae in total
(i.e. three plates of 24 wells). After 7 days,
surviving larvae were measured and
weighed and then transferred to separate
vials containing fresh stock diet. Larval
weight gains and mortality were recorded
after 21 days of incubation because the
pupation average is 23 ± 1 day. Some life-
cycle data such as time to pupation, mor-
tality of larvae and adult emergence and
deformities were registered. All experi-
ments were carried out in a controlled
environmental chamber with an 18:6
light:dark (L:D) photoperiod, 19 and 25°C
night and day temperature, respectively,
and a relative humidity of 80 ± 5%. There
were three replicates for each treatment.
Controls contained the same numbers of
larvae, volume of diet and ethanol as
the test solutions (Céspedes
et al.
, 2000,
2005; Torres
et al.
, 2003; Céspedes and
Alarcon, 2011).
15.4.6 Not-choice test against
Drosophila melanogaster
Insecticidal activity against larvae of
Drosophila melanogaster
was assayed as fol-
lows (Miyazawa
et al.
, 2000): five concen-
trations (10.0, 20.0, 50.0 and 100.0 ppm of
sample) were used determining LD
50
values.
Test compounds were dissolved in 50 ml of
EtOH and mixed in 1 ml of artificial diet
(60 g of brewer's yeast, 80 g of glucose, 12 g
of agar, 8 ml of propionic acid and 1000 ml
of water). A control diet was treated with
50 ml of EtOH only.
The diet and 10 eggs were placed in a
Petri dish. Ten Petri dishes were prepared.
Then, they were placed at 25°C and relative
humidity >90% for 8 days. One day after
transplantation, the larvae hatched and were
fed with the different test extracts (water,
ethyl acetate, hexane and methanol) in the
different concentrations (10.0, 20.0, 50.0 and
100.0 ppm of sample) mixed with the artifi-
cial diet. At 25°C, larvae generally change to
pupae after 7 days. In each instance the
developmental stage was observed, and the
numbers of pupae were recorded and com-
pared with those of a control.
15.4.7 Bioassays with yellow meal
worm (
Tenebrio molitor
)
Larvae of
Tenebrio molitor
L. (Coleoptera:
Tenebrionidae) were fed with wheat bran in
plastic boxes at 24.0 ± 1°C, with a 16:8 L:D
photoperiod, these larvae maintained in a
chamber under these environmental condi-
tions were used in the test. Bioassays were
performed with last instar larvae of
T. molitor
based on live weight (103-160 mg). For each
compound, test solutions Me
2
CO/MeOH
(9.5:0.5 v/v) were topically applied to ventral
abdominal segments with a microsyringe 2 ml/
larva; equivalent to 0.2 mg/larva of the assayed
compounds for each one of concentrations
assayed. Controls were treated with the solvent
alone. For each individual compound there
were three replicates of 20 larvae each, and
the assay had three replicates. After treatment
the insects were placed in Petri dishes
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