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Field Evaluation Against Aedes aegypti Larvae of Aluminum-

Carboxymethylcellulose-Encapsulated Spore-Toxin Complex
Formulation of Bacillus thuringien....

Article  in  Journal of Economic Entomology · June 2010

DOI: 10.1603/EC09372 · Source: PubMed

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 BIOLOGICAL AND MICROBIAL CONTROL

Field Evaluation Against Aedes aegypti Larvae of Aluminum-

Carboxymethylcellulose-Encapsulated Spore-Toxin Complex
Formulation of Bacillus thuringiensis Serovar israelensis
O. AGUILAR-MEZA,1 M. RAMÍREZ-SUERO,1 J. S. BERNAL,2 AND M. RAMÍREZ-LEPE1,2,3

J. Econ. Entomol. 103(3): 570Ð576 (2010); DOI: 10.1603/EC09372

ABSTRACT The insecticidal activity after Þeld exposure of an aluminum-carboxymethylcellulose
microencapsulated formulation of Bacillus thuringiensis israelensis (Bti) spore-toxin complex, with
malachite green as photoprotective agent, was evaluated using third-instar Aedes aegypti (L.) larvae
in laboratory bioassays in Veracruz, México. Four insecticide treatments and an untreated control
were compared at low and high doses over 96 d of Þeld exposure under full sun or full shade conditions:
1) microencapsulated Bti spore-toxin complex, 2) nonmicroencapsulated Bti spore-toxin complex, 3)
a commercial Bti formulation, 4) a commercial formulation of temephos, and 5) an untreated control.
The low and high doses corresponded to the LC50 and LC90 concentrations for the Bti insecticides
and to 0.5 and 1.0 mg/liter for temephos; the corresponding values for the microencapsulated Bti and
commercial Bti, estimated in this study, were 0.061 and 0.14 mg/ml and 0.13 and 0.30 mg/ml,
respectively. Overall, the study demonstrated that microencapsulation with aluminum-carboxymeth-
ylcellulose improved the activity against Ae. aegypti larvae of B. t. israelensis spore-toxin complex over
that of a nonmicroencapsulated spore-toxin complex and that the improvement was particularly
important under full sun and high dose. Moreover, insecticidal activity of the microencapsulated B.
thuringiensis israelensis spore-toxin complex was superior to that of a commercial B. thuringiensis
israelensis formulation and comparable to that of the chemical insecticide temephos. Finally, it was
suggested that the microencapsulated B. thuringiensis israelensis formulation should be evaluated for
Þeld use in Veracruz because its activity against Ae. aegypti larvae remained high through 31 d and
this would allow halving of the current insecticide application frequency.

KEY WORDS microbial control, Bti, aluminum-carboxymethylcellulose, microencapsulation,

México

Bacillus thuringiensis subsp. israelensis has been stud-
ied in great detail over the past 25 yr because of its
insecticidal properties. The insecticidal activity of this
bacterium is derived from crystalline inclusions pro-
duced during sporulation, which are toxic to dipteran
larvae, such as mosquitoes and black ßies (de Barjac
1978). The inclusions, or ␦-endotoxins, are ingested by
susceptible larvae, solubilized in the high pH larval
midgut, and activated by proteases. The gut cells swell
and lyse, and the insects stop feeding and eventually
die (Soberón et al. 2007). Bacillus thuringiensis is-
raelensis is currently used in mosquito control pro-
grams worldwide. Some advantages of using ␦-endo-
toxin-based insecticides include that they are safe to
mosquito natural enemies and vertebrates, biodegrad-
able, receptive to genetic engineering and large-scale
production, highly effective, and not likely to rapidly
1 Instituto Tecnológico de Veracruz UNIDA, Laboratorio de Ge-
nética, Av. Miguel A. de Quevedo #2779, Colonia Formando Hogar,
Veracruz, Ver. México C.P. 91897.
2 Biological Control Laboratory, Department of Entomology, Texas
A&M University. College Station, TX 77843-2475.
3 Corresponding author, e-mail: lepe@itver.edu.mx.
0022-0493/10/0570Ð0576$04.00/0 䉷 2010 Entomological Society of America
produce resistant insect populations (Satinder et al.
2006). However, exposure to environmental variables
such as solar radiation, dust and sand particles, organic
material, elevated temperatures, tannins, and water
depth, among others, decrease the efÞcacy and insec-
ticide activity of B. thuringiensis israelensis formula-
tions (Dunkle and Shasha 1989, Yu-Tien et al. 1993,
Elcin et al. 1995, Vilarinhos and Monnerat 2004). Con-
sequently, a variety of formulations have been devel-
oped to overcome the effect of such variables, includ-
ing granules (Batra et al. 2000), pellets (Mulla et al.
2004), briquettes (Kase and Branton 1986), water-
dispersible granular formulations (Su and Mulla 1999),
ßowable concentrates (Lee et al. 1996), wettable pow-
ders (Thiéry et al. 1999), and tablet formulations
(Melo-Santos et al. 2001; Armengol et al. 2006), among
others.
A prior study showed that a formulation of B. thu-
ringiensis israelensis consisting of spore-toxin complex
microencapsulated with aluminum-carboxymethyl-
cellulose (CMC) and photoprotective agents was sta-
ble and protected the ␦-endotoxin against UV light
under laboratory conditions (Ramṍrez-Lepe et al.
June 2010 AGUILAR-MEZA ET AL.: FIELD EVALUATION OF Bti AGAINST Ae. aegypti
2003). In the current study, the residual insecticidal
activity of low- and high-dose suspensions of that
formulation was assessed in laboratory bioassays in-
volving third-instar larvae of Aedes aegypti (L.) after
Þeld exposure to full sun and full shade conditions in
Veracruz, México. For comparison, a commercial for-
mulation of B. t. israelensis (Vectobac AS), a nonen-
capsulated formulation of B. t. israelensis spore toxin-
complex, and the chemical insecticide temephos
(Abate) were evaluated alongside the microencapsu-
lated B. thuringiensis israelensis formulation.
Materials and Methods
Insecticides, Insolation, and Doses. The insecticidal
treatments were as follows: aqueous suspensions of 1)
a microencapsulated B. t. israelensis spore-toxin com-
plex (Ramṍrez-Lepe et al. 2003) (hereafter MeBti), 2)
a nonmicroencapsulated B. thuringiensis israelensis
spore-toxin complex (Ramṍrez-Lepe et al. 2003)
(hereafter Bti), 3) a commercial B. t. israelensis for-
mulation (Vectobac AS; Adventis, México City, Méx-
ico) (herafter CommBti, 4) an aqueous solution of
temephos (Abate; Agrevo, México City, México), and
5) an untreated control (water). All insecticidal treat-
ments were evaluated under two insolation levels,
namely, full sun and full shade, and at two nominal
doses, namely, low and high. The combination of in-
secticide, sunlight, and dose variables yielded 20 treat-
ments. Full sun corresponded to exposure to direct
sunlight, and full shade to no exposure to direct sun-
light (under a roofed, wall-less structure, 6 by 5 by 3 m,
length by width by height) during the entire evalua-
tion period, 96 d (see below). Low and high doses
corresponded to LC50 and LC90 values for the B. t.
israelensis treatments and to 0.5 and 1.0 mg/liter for
temephos (Mulla et al. 2004). The LC50 and LC90
values for the MeBti and CommBti treatments were
unknown, so they were measured in this study (see
below). For the Bti treatment, they were 0.05310 ⫾
0.0026 and 0.1074 ⫾ 0.0083 mg/liter aluminum-CMC,
respectively (Ramṍrez-Lepe et al. 2003). The strain of
B. thuringiensis israelensis used for the MeBti and Bti
treatments was obtained from the Pasteur Institute
(Paris, France) (Ramṍrez-Lepe et al. 2003).
LC50 and LC90 of Microencapsulated and Commer-
cial B. t. israelensis Formulations. Bioassays were car-
ried out following the procedure described by
McLaughlin et al. (1984) to determine the LC50 and
LC90 values of MeBti and CommBti. The bioassays
were conducted in a room maintained at 24 Ð28⬚C
using third-instar larvae of Ae. aegypti, which were
obtained from colonies maintained at the insectary of
the Ministry of Health of Veracruz state, México, in
Veracruz. To prepare suspensions of B. t. israelensis, 50
mg of product were placed in 5 ml of deionized water
and agitated in vortex yielding a 1% (wt:vol) suspen-
sion, which was then diluted serially 10-fold in a total
volume of 5 ml, giving suspensions of 0.1, 0.01, and
0.001 mg/liter. A preliminary test was conducted with
three replicates of each of four concentrations be-
tween 0.5 and 0.01 mg/liter to determine the activity
571
interval. Subsequently, Þve concentrations were pre-
pared for each assay. In each assay, 20 mosquito larvae
were placed in a container holding 100 ml of one
concentration; one container without MeBti or Com-
mBti served as a control. Mortality was determined
24 h after exposing mosquito larvae to a MeBti or
CommBti concentration. Mortality data were sub-
jected to probit analysis (Finney 1971) by using
SPSS12 (SPSS Inc. 2005) to calculate LC50 and LC90
values for MeBti and CommBti.
Field Exposure and Laboratory Bioassays.
Insecticides were exposed in the Þeld at low or high
dose to full sun or full shade conditions in 5-liter
containers as suspensions or solutions in 3 liters of
water. Each of the 20 treatments was replicated three
times (N ⫽ 60 5-liter containers). Samples of 100 ml
were taken from each container at 1, 2, 3, 10, 17, 24, 31,
46, 72, and 96 d and used for laboratory bioassays on
third-instar Ae. aegypti larvae. The bioassays consisted
of placing 20 Ae. aegypti third-instar larvae inside a
glass beaker holding a 100-ml sample from the Þeld
and counting the number of dead larvae after 24 h. The
100-ml samples were returned to the corresponding
container after each bioassay, after removing all Ae.
aegypti larvae. In addition, approximately twice per
week distilled water was added to each container to
compensate for evaporation and adjust its volume to
3 liters.
The number of dead larvae per container was con-
verted to a proportion, which was arcsine 公x-trans-
formed for analyses. Analyses consisted of repeated
measures (exposure intervals) analysis of variance
(ANOVA) of 20 treatments (because the design was
unbalanced, i.e., controls did not have “high” nor “low”
doses), followed by orthogonal contrasts to compare
group means of interest (see Table 1). Subsequently,
because the effects of dosage (low dose, high dose)
and insolation level (full shade, full sun) were signiÞcant
(see Results), repeated measures (exposure intervals)
ANOVA was applied to insecticidal treatments within all
insolation level and dose combinations, namely, full
shade⫹low dose, full shade⫹high dose, full sun⫹low
dose, and full sun⫹high dose. In every insolation level-
dose combination insecticidal treatment ⫻ sample day
interactions were signiÞcant, so one-way ANOVA was
applied to speciÞc exposure intervals of interest, namely,
days 1, 10, 31, and 46, followed by TukeyÕs tests to sep-
arate treatment means as appropriate. All analyses were
conducted using Statistix 9.0 software (Analytical Soft-
ware 2008) and are described in Zar (1996).
Results
The LC50 and LC90 values corresponding to MeBti
and CommBti for third-instar Ae. aegypti larvae are
presented in Table 1. These values were used as the
low- and high-dose levels, respectively, in the Þeld
exposure experiment and bioassays.
The treatments in the Þeld exposure experiment
had a highly signiÞcant effect on Ae. aegypti larval
572 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 103, no. 3

Table 1. Toxicity of MeBti and a commercial CommBti to
third-instar Ae. aegypti in 24-h laboratory bioassays
LC50 ⫾ SD, LC90 ⫾ SD,
Formulation
mg/liter mg/liter
MeBti 0.0612 ⫾ 0.0059 0.1448 ⫾ 0.0380
CommBti 0.1265 ⫾ 0.0051 0.3001 ⫾ 0.0456
LC50 and LC90 values were used, respectively, as “low” and “high”
doses for subsequent bioassays.
mortality in the bioassays (P ⬍ 0.001) (Table 2a).
Orthogonal contrasts showed signiÞcant effects (P ⬍
0.0001 in all cases) and greater Ae. aegypti mortality in
1) the MeBti treatment relative to the Bti and Com-
mBti treatments combined; 2) full shade relative to full
sun; 3) high dose relative to low dose; 4) MeBti in
full sun relative to Bti in full sun; 5) high-dose MeBti
in full sun relative to high-dose Bti in full sun; 6)
MeBti in full sun relative to MeBti in full shade;
and7) temephos relative to MeBti, Bti, and Com-
mBti combined (Table 2b).
Because the effects of dosage (low dose, high dose)
and insolation level (full shade, full sun) were signif-
icant (both P ⬍ 0.0001) (Table 2b), subsequent anal-
yses focused on insecticide treatment effects under
the four combinations of those variables: low
dose⫹full shade, low dose⫹full sun, high dose⫹full
shade, and high dose⫹full sun. Both treatment and
days ⫻ treatment effects were highly signiÞcant under
all four combinations (P ⬍ 0.001 in all cases) of dosage
and insolation levels (Table 3, aÐ d). Thus, compari-
sons among treatments were made at four exposure
intervals: 1, 10, 31, and 46 d.
Treatment effects were signiÞcant at all exposure
intervals under low dose⫹full shade conditions (P ⬍
0.0001; Table 3a). The control treatment yielded 0%
Ae. aegypti mortality at every exposure interval (Fig.
1a). At 1 d, temephos produced signiÞcantly higher
mortality larvae (100%) than CommBti, Bti, and
MeBti, which produced similar degrees of mortality
(⬇61%) (Fig. 1a). At 10 d, temephos produced sig-
niÞcantly higher mortality (72%) than Bti (58%) and
CommBti, and MeBti, which produced similar degrees
of mortality (⬇49%). At 31 d, temephos and Bti pro-
Slope ⫾ SD duced signiÞcantly higher mortalities of Ae. aegypti
larvae (⬇66%) compared with MeBti (47%) and Com-
3.63 ⫾ 0.67
3.54 ⫾ 0.64 mBti (30%). At 46 d, temephos, Bti and MeBti pro-
duced signiÞcantly higher mortalities (⬇36%) than
CommBti (12%).
Treatment effects were signiÞcant at all exposure
intervals under low dose⫹full sun conditions (P ⬍
0.0001) (Table 3b). The control treatment yielded the
lowest Ae. aegypti mortality at every exposure interval,
and varied between 0 and 5% (Fig. 1b). At 1 d, teme-
phos produced signiÞcantly higher mortality (97%)
than CommBti, Bti, and MeBti, which produced sim-
ilar degrees of mortality (⬇61%) (Fig. 1b). At 10 d,
MeBti produced signiÞcantly higher mortality (59%)
than CommBti and temephos, which produced similar
degrees of mortalities (⬇46%), and Bti (28%). At 31 d,
temephos produced signiÞcantly higher mortality
(62%) than MeBti (40%), commercial Bti (18%), and
Bti (7%). At 46 d, temephos produced signiÞcantly
higher mortality (37%) than MeBti (15%), and Bti and
CommBti, which produced similar mortalities (⬇4%).
Treatment effects were signiÞcant at all exposure
intervals under high dose⫹full shade conditions (P ⬍
0.0001) (Table 3c). The control yielded 0% mortality
of Ae. aegypti larvae at every exposure interval (Fig.
2a). At 1 d, temephos, CommBti, Bti, and MeBti pro-
duced similar degrees of mortality (⬇61%) (Fig. 2a).
At 10 d, temephos, MeBti, and CommBti produced
similar degrees of mortality (⬇95%) and signiÞcantly
higher than Bti (65%). At 31d, temephos, CommBti,
Bti, and MeBti produced similar degrees of mortality
(⬇85%). At 46 d, temephos and MeBti produced sim-
ilar levels of mortality (⬇81%), which was signiÞ-
cantly higher than mortalities produced by Bti and
CommBti (⬇16%).
Treatment effects were signiÞcant at all exposure
intervals under high dose⫹full sun conditions (P ⬍
0.0001) (Table 3d). The control treatment yielded the

Table 2. Repeated measures ANOVA of mortality (percentage) of Ae. aegypti larvae caused by five insecticidal treatments at two dose
levels (low dose, high dose) and two insolation levels (full sun, full shade), which were exposed outdoors for 96 da

df F P
a. Factor
Treatment 83 ⬍0.001
Days 10 35.59 ⬍0.001
Error (interaction) 190 11.35 ⬍0.001
Error 440
Total 659
b. Contrastb Means ⫾ SE SchefféÕs F P
MeBti vs. Bti⫹CommBti 57.9 ⫾ 0.2 vs. 34.5 ⫾ 0.1 10.54 ⬍0.0001
Full shade vs. full sun 50.7 ⫾ 0.1 vs. 45.3 ⫾ 0.1 11.77 ⬍0.0001
High dose vs. low dose 58.8 ⫾ 0.6 vs. 36.9 ⫾ 0.1 19.21 ⬍0.0001
MeBti full sun vs. Bti full sun 58.5 ⫾ 0.5 vs. 27.6 ⫾ 0.4 11.99 ⬍0.0001
MeBti full sun, high dose vs. Bti full sun, high dose 87.2 ⫾ 0.6 vs. 30.9 ⫾ 0.6 7.41 ⬍0.0001
MeBti full sun vs. MeBti full shade 58.5 ⫾ 0.5 vs. 57.3 ⫾ 0.3 46.02 ⬍0.0001
Temephos vs. MeBti⫹Bti⫹CommBti 65.3 ⫾ 0.2 vs. 42.1 ⫾ 0.1 46.08 ⬍0.0001

a
Insecticides were MeBti, Bti, CommBti, temephos (a chemical insecticide), and a control (untreated water). Low doses correspond to LC50
values for the B. t. israelensis and to 0.5 mg/liter for temephos, and high dose corresponds to LC90 values and 1.0 mg/liter, respectively.
b
Post repeated measures ANOVA orthogonal contrasts between groups of treatments are as described in Factor (a) information.
June 2010 AGUILAR-MEZA ET AL.: FIELD EVALUATION OF Bti AGAINST Ae. aegypti 573

Table 3. Repeated measures ANOVA of mortality (percentage) of insecticidal activity of B. thuringiensis-based bio-
of Ae. aegypti larvae caused by five insecticidal treatments that were logical insecticides (Morris 1983, Pozsgay et al. 1987,
exposed outdoors for 96 d at (a) low dose under full shade, (b) low
dose and full sun, (c) high dose and full shade, and (d) high dose and Chen et al. 2004). Thus, UV light has been an impor-
full suna tant variable limiting the effective Þeld-use of those
insecticides, and numerous attempts have been made
Dose/insolation Factor df F P to develop protectants against UV radiation for B.
(a) Low dose/full thuringiensis-based biological insecticides (Dunkle
shade and Shasha 1989, Prasad et al. 2003, El-Sharkawy et al.
Total Days 10 2009). Our previous work in the laboratory showed
Treatments 4 26.84 ⬍0.0001
Days ⫻ treatments 40 14.01 ⬍0.0001
that microencapsulation with aluminum-CMC and
Day 1 Treatments 4 144.02 ⬍0.0001 sunscreens protected B. t. israelenis spore-toxin com-
Day 10 156.19 ⬍0.0001 plex from UV radiation (Ramṍrez-Lepe et al. 2003).
Day 31 121.20 ⬍0.0001 The current study showed that microencapsulation
Day 46 21.98 ⬍0.0001
(b) Low dose/full
with aluminum-CMC protected a B. thuringiensis is-
sun raelenis spore-toxin complex from UV radiation over
Total Days 10 an extended exposure period in the Þeld. For example,
Treatments 4 21.92 ⬍0.0001 under full sun MeBti (i.e., microencapsulated Bti)
Days ⫻ treatments 40 11.37 ⬍0.0001
Day 1 Treatments 4 96.00 ⬍0.0001
produced more than two-fold greater mortality of Ae.
Day 10 227.48 ⬍0.0001 aegypti larvae than Bti (i.e., nonmicroencapsulated
Day 31 66.28 ⬍0.0001 Bti) over the entire Þeld exposure period of 96d, par-
Day 46 6.02 ⬍0.0001 ticularly at high doses (Table 1). Moreover, after 31d
(c) High dose/full
shade
of Þeld exposure under full sun MeBti at high dose
Total Days 10 produced 90% mortality, whereas Bti and CommBti at
Treatments 4 29.68 ⬍0.0001 high doses produced ⬇40% mortality (Fig. 2b). The
Days ⫻ treatments 40 6.43 ⬍0.0001 high level of mortality, 90%, produced by MeBti after
Day 1 Treatments 4 50.14 ⬍0.0001
Day 10 37.73 ⬍0.0001
31 d of Þeld exposure is signiÞcant because in the study
Day 31 42.30 ⬍0.0001 area, Veracruz, México, Ae. aegypti-infested water
Day 46 10.75 ⬍0.0001 bodies are every 2 wk, and usually with a chemical
(d) High dose/full insecticide, such as temephos (Sesver 2005).
sun
Total Days 10
The insecticidal activity of MeBti was maintained
Treatments 4 17.38 ⬍0.0001 over extended exposure periods but was affected par-
Days ⫻ treatments 40 16.12 ⬍0.0001 ticularly by dosage levels and less so by insolation
Day 1 Treatments 4 95.52 ⬍0.0001 level. For example, at high dose Ae. aegypti mortality
Day 10 127.71 ⬍0.0001
Day 31 48.08 ⬍0.0001
levels caused by MeBti fell below 90% after 31 d in-
Day 46 41.67 ⬍0.0001 dependently of insolation level (Fig. 2a and b),
whereas at low dose they never reached 60% inde-
a
In aÐ d, the interaction term was signiÞcant (P ⬍ 0.0001), so a pendently of insolation level (Fig. 1a and b). Impor-
one-way ANOVA was applied to speciÞc exposure periods, namely, 1, tantly, at high dose and under full sun the mortality
10, 31, and 46 d (post hoc comparisons for among treatments at these
periods are shown in Figs. 1 and 2). The treatments were a microen- produced by MeBti was greater than that produced by
capsulated B. t. israelensis spore-toxin complex, a nonmicroencapsu- the commercially available B. thuringiensis israelensis
lated B. t. israelensis spore-toxin complex, a commercial B. t. israelensis formulation CommBti by 17 d of Þeld exposure and
spore-toxin formulation, temephos (a chemical insecticide), and a was similar to that produced by the chemical insec-
control (untreated water). Low doses corresponded to LC50 values
for the B. t. israelensis and to 0.5 mg/liter for temephos, and high dose ticide temephos through 31 d (Fig. 2b). CommBti at
corresponded to LC90 values and 1.0 mg/liter, respectively. high dose and under full shade produced mortality
levels comparable to those of MeBti only through 24 d
lowest mortality at every exposure interval, and varied (Fig. 2a). Thus, the Ae. aegypti mortality levels pro-
between 0 and 5% (Fig. 2b). At 1 d, temephos, CommBti, duced by MeBti are competitive with those by teme-
Bti, and MeBti produced similar degrees of mortality phos under the local treatment frequency of ⬇30 d
(⬇92%) (Fig. 2b). At 10 d, temephos, MeBti, and Com- and are superior to those produced by the locally
mBti produced similar degrees of mortality (⬇95%) and available commercial formulation of B. t. israelensis.
signiÞcantly higher than produced by Bti (47%). At 31 d, Previous studies showed variable activity levels of B.
temephos and MeBti produced similar degrees of mor- t. israelensis formulations against Ae. aegypti larvae in
tality (⬇91%), which were signiÞcantly greater than pro- the Þeld. For example, Maldonado et al. (2007)
duced by Bti and CommBti (⬇41%). At 46 d, temephos showed Ae. aegypti control (⬎80%) for 21 d, whereas
produced signiÞcantly higher mortality (90%) than Armengol et al. (2006) showed control for 12 wk and
MeBti (47%), and greater than Bti and CommBti, which Araújo et al. (2007) and Melo-Santos et al. (2009) for
produced similar degrees of mortalities (⬇1%). 6 mo. The evident differences in activity levels may be
due to the particular formulations, doses, level of in-
solation, among other environmental variables (Lacey
Discussion
2007). Aside from dosage and insolation levels, other
Numerous studies over the last ⬇25 yr have shown environmental variables may have contributed to the
that UV light is an important causative factor in the loss differences in Ae. aeypti activity levels between our
574 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 103, no. 3

Fig. 1. Mortality (% ⫾ SE) of Ae. aegypti third-instar larvae in laboratory bioassays after exposure (intervals in days) of
insecticides at low dose in the Þeld under (a) full shade or (b) full sun conditions. Treatments indicated in legend boxes are
MeBti, microencapsulated B. t. israelensis spore-toxin complex; Bti, nonmicroencapsulated B. t. israelensis spore-toxin complex;
CommBti, commercial B. t. israelensis spore-toxin formulation; temephos (a chemical insecticide); and control, untreated
water. Different lowercase letter indices among treatments within legend box or among treatment means within plot indicate
signiÞcant differences (P ⬍ 0.05). Low dose corresponds to LC50 values for each of the B. t. israelensis-based treatments (see
text) and to 0.5 mg/liter for temephos.

study and previous studies. For example, during the
exposure period the containers in our experiment ac-
cumulated leaves, organic matter, insects, sand, and
other debris, which settled at the containerÕs bottom;
e.g., ⬇1Ð2% of sand, by volume, accumulated in con-
tainers in full shade, and 4 Ð 6% in containers in full sun
(data not shown).
Environmental variables seemingly affected the in-
secticidal activity against third-instar Ae. aegypti larvae
of temephos in our study. The strong insecticidal ac-
tivity of temephos at high dose under both full sun and
full shade conditions (⬎90% mortality through 46 d)
(Fig. 2a) is lower than reported in previous studies
(Mulla et al. 2004; Thavara et al. 2004; Palomino et al.
2006). However, the insecticidal activity of teme-
phos in our study did not seem to be affected by
insolation, as suggested by the mortality patterns
caused by temephos at high dose under full sun and
full shade (Fig. 2).
On the basis of the Ae. aegypti mortality levels over
time caused by both BtiME and temephos at high
doses, it seems that these insecticides are suitable for
use in management programs targeting this species in
the study area. Management of Ae. aegypti in the study
area is based upon treatment of breeding areas at 2-wk
intervals with an insecticide, such as temephos (Ses-
ver 2005). Thus, because at high dose the Ae. aegypti
mortality caused by both MeBti and temephos are
similarly high (⬎90%) through 31 d (Fig. 2a and b), it
is likely that adequate management of Ae. aegypti
larval populations could be achieved by applying ei-
ther insecticide at 4-wk intervals, although this re-
mains to be investigated. Over the course of 1 yr, a
4-wk application frequency would lead to 13 applica-
tions, compared with 26 applications under a 2-wk
application frequency. Moreover, because MeBti and
temephos produced similar levels of Ae. aegypti mor-
tality, the choice of one insecticide versus the other for
use in a management program against this insect
should consider their environmental (e.g., nontarget
impacts) and pest management (e.g., selection of in-
secticide resistant Ae. aegypti populations) impacts,
and costs, among other variables.
Overall, the results of our study showed that mi-
croencapsulation with aluminum-carboxymethylcel-
lulose improved the activity against Ae. aegypti larvae
of a B. thuringiensis israelenis spore-toxin complex over
that of a nonmicroencapsulated spore-toxin complex,
and that the improvement was particularly important
under full sun and at high dose. Moreover, insecticidal
activity of the microencapsulated B. t. israelensis
spore-toxin complex was superior to that of a com-
mercial B. thuringiensis israelensis formulation, and
comparable to that of the chemical insecticide teme-
phos. Finally, our results indicate that the microen-
capsulated B. thuringiensis israelensis spore-toxin for-
June 2010 AGUILAR-MEZA ET AL.: FIELD EVALUATION OF Bti AGAINST Ae. aegypti 575

Fig. 2. Mortality (% ⫾ SE) of Ae. aegypti third-instar larvae in laboratory bioassays after exposure (intervals in days) of
insecticides at high dose in the Þeld under (a) full shade or (b) full sun conditions. Treatments indicated in legend boxes
are MeBti, microencapsulated B. t. israelensis spore-toxin complex; Bti, nonmicroencapsulated B. t. israelensis spore-toxin
complex; CommBti, commercial B. t. israelensis spore-toxin formulation; temephos (a chemical insecticide); and, control,
untreated water. Different lowercase letter indices among treatments within legend box or among treatment means within
plot indicate signiÞcant differences (P ⬍ 0.05). High dose corresponds to LC50 values for each of the B. t. israelensis-based
treatments (see text) and to 1.0 mg/liter for temephos.

mulation evaluated in our study should be evaluated
for Þeld use in Veracruz, México, because the mor-
tality it caused in Ae. aegypti larvae remained high
through 31 d, and this would allow halving of the
current insecticide application frequency in that area.
Acknowledgments
We thank Leopoldo Hidalgo Sosa and Celestino Erubiel
Prior (Ministry of Health of Veracruz State) for technical
assistance. O.A.-M. and M.R.-S. were supported by fellowship
from Programa de Mejoramiento para el Profesorado and
Direccion General de Educacion Superior Tecnologica
(DGEST), respectively. This research was supported by
DGEST and Consejo Nacional de Ciencia y Tecnologia
(México).
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Received 31 October 2009; accepted 20 January 2010.