Journal of Environmental Protection, 2010, 1, 337-345
doi:10.4236/jep.2010.14040 Published Online December 2010 (http://www.SciRP.org/journal/jep)
337
Biological Activity of Bacillus thuringiensis
(Berliner) Strains on Larvae and Adults of
Ceratitis capitata (Wiedemann) (Diptera:
Tephritidae)
Houda Aboussaid1,2, Loubna El-Aouame1,2, Said El-Messoussi2, Khalid Oufdou1
1Laboratory of Biology and Biotechnology of Microorganisms, Cadi Ayyad University, Marrakech, Morocco; 2Laboratory of
Molecular Modeling and Ecophysiology, Faculty of Sciences-Semlalia, Cadi Ayyad University, Marrakech, Morocco.
Received August 9th, 2010; revised August 29th, 2010; accepted September 9th, 2010.
ABSTRACT
The objective of this study was to evaluate the efficiency of Moroccan Bt strains against neonate larvae, third instar
larvae and emerged adults of Ceratitis capitata. This Mediterranean fruit fly causes serious damages to Argan forest
and other agricultural plants. There is no successful control program of this pest fly in the endemic Argan forest in
Morocco. A single-dose test was performed on neonate larvae (25 µL/g) and adult (333.33 µL/g), when three doses of
Bt toxins (50 µL/g, 100 µL/g and 150 µL/g) were tested against third instar of C. capitata. Among the twenty-six Bt
strains examined, local Bt 13.4 and Bt A7 strains showed highest toxicity levels against larvae and adults, when com-
pared to the reference strain, Bacillus thuringiensis subsp. israelensis HD567 “code 4Q1”, and commercial product
“Skeetal”. One hundred percent mortality was observed against neonate larvae after 7 days of application by Bt 13.4
toxin. Third instar larvae were very susceptible to Bt A7 and Bt M-Ag 21.6 strains with 68% mortality (Lethal Concen-
tration: LC50 = 1.115) at a dose of 150 µL/g. The Bt A7 strain was also highly toxic to adults with 81.66% of mortality
after 7 days of application. This study demonstrated that some of our collection Bt strains can contribute to integrated
C. capitata management system with strong biological control components.
Keywords: Argan Forest, Bacillus thuringiensis, Biological Control, Ceratitis capitata, Diptera
1. Introduction
Control of the Mediterranean fruit fly or medfly Ceratitis
capitata (Wiedemann) (Diptera: Tephritidae); one of the
world’s most destructive insect, continues to rely on the
use of broad-spectrum chemical insecticides in bait
sprays [1]. While these chemical products are generally
effective, they are controversial because of concerns for
adverse effects on human health and non-target organ-
isms, environmental pollution and development of insec-
ticide resistance [2]. Therefore there is a need for alterna-
tive strategies such as bacterial entomopathogenic toxins:
Bacillus thuringiensis.
Commercial preparations of Bt have been shown to be
successful biological control products worldwide [3,4].
Bt insecticidal activity primarily lies in its parasporal
crystals. They are predominantly composed of one or
more types of proteins, known as δ-endotoxins: the Cry
proteins and the Cyt (cytolitic) proteins [5]. Nevertheless,
other factors may contribute to Bt toxicity, such as the
spore itself and extracellular soluble factors like β-exo-
toxins, VIP (vegetative insecticidal proteins) toxins,
phospholipases, proteases and chitinases [6].
Cry toxins are highly specific to their target insects,
are harmless to humans, vertebrates and plants, and are
easily biodegradable [7]. To date, several invertebrates
have been described to be susceptible to Bt strains,
mostly insects from Lepidoptera, Diptera and Coleoptera,
but also from other orders (Hymenoptera, Homoptera,
Orthoptera and Mallophaga) and also species from
nematodes, mites and protozoa. However, for many
crystal-bearing Bt strains, no toxic activity has been de-
tected yet [8]. Thus, in recent decades, there has been a
great interest in the screening of Bt collections to find
isolates useful for pest control [9-12]. Many dipteran
species have been found to be susceptible to Bt; these

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
338
include mosquitoes, blackflies, chironomids, tipulids,
muscids, sciarids, drosophilids [13] and tephritidis; such
as the olive fly Bactrocera oleae [14,15], the Mexican
fruit fly Anastrepha ludens [16,17] and the Mediterra-
nean fruit fly C. capitata [18-20].
According to our knowledge, there is no available data
on the effect of Moroccan Bt strains isolated especially
from the endemic Argan forest which is strongly infested
by C. capitata.
In the present study, we evaluate the biological ento-
mopathogenic activity of Bt strains isolated from Marra-
kech region and Argan region in Morocco. We test the
insecticidal effects of Bt strains against neonate larvae,
third instar larvae and emerged adults of C. capitata.
2. Materials and Methods
2.1. Insect Rearing
The insects used in the bioassays came from the labora-
tory colony of C. capitata, maintained at the Faculty of
Sciences Semlalia, Cadi Ayyad University, Marrakech
(Morocco).
The laboratory conditions used for rearing and bioas-
says were 25 ± 1℃, 60 ± 10% relative humidity (RH)
and 12 h of light and 12 h of darkness. Eggs laid through
a net placed on one side of an insect-rearing cage were
collected in a plastic container that was filled with dis-
tilled water. Larvae from 0.5 ml of eggs were raised on an
artificial diet containing 250 g wheat bran, 70 g sucrose,
36.3 g brewer’s yeast, 2.8 g (methyl 4-hydroxybenzoate;
Sigma), 2.8 g Nipasol (propyl 4-hydroxybenzoate; Sigma)
nipagin, 2.5 g Benzoic acid and 600 mL distilled water.
Third-instar larvae were transferred into a pupation
chamber with sand and were maintained there for at least
1 week before they were collected using a sieve. Adult
flies were fed an artificial diet comprised of 20% (wt/wt)
yeast autolysate and 80% (wt/wt) sucrose. Water was
provided to the flies via a damp yellow sponge.
2.2. B. thuringiensis Strains
Samples were collected from different areas of Mar-
rakesh region and endemic argan forests (southwest of
Morocco) (Table 1). Sampling was performed from het-
erogeneous sources: soils underground surface (to avoid
spore inactivation caused by UV radiation), Citrus Phyll-
plane, Farm animal excrement, wastewater and sludge of
wastewater treatment system.
Bt isolation was carried out as described previously by
Bel et al. [21]. Colonies with characteristic Bt morphol-
ogy and presence of parasporal inclusions in the sporan-
gium were selected and plated again for single colony
isolation. After a second microscopic observation to ver-
Table 1. Local Bt strains isolated from different habitats in
Morocco.
Localisation Habitat No. of
samples
No. of Bt
isolates
Agadir Argan soils 8 13
Essaouira Argan soils 2 4
Olive-cultivated soil 1 3
Bean-cultivated soil 1 1
Farm animal
excrement 1 1
Citrus phylloplane 1 1
Wastewater 1 1
Marrakech
Sludge of wastewater
treatment system 2 2
Total 17 26
ify presence of protein inclusions, strains were cultured
in CCY (Casein Casein Yeast) liquid medium [22], for 2
days until sporulation. Vegetative cells were eliminated
by heating the sample at 70℃ for 20 min. Aliquots were
taken and stored in 25% glycerol at −20℃.
The standard strains, other than those isolated in this
study were Bacillus thuringiensis subsp. israelensis HD
567 (code 4Q1, Bacillus Genetic Stock Center, B.G.S.C.,
Ohio State University, Columbus, OH, U.S.A.) and
Skeetal, a commercial product based on B. thuringiensis
subsp. israelensis (Valent Biosciences Corporation, Lib-
ertyville, U.S.A.).
2.3. Culture Conditions and Production of
Spore – Crystal Biomass
A loopful of cells from a single colony of each strain
grown in CCY agar medium were inoculated in 4.5 ml of
liquid CCY medium (pre-culture) and grown for 48 h at
28℃ and agitated at 200 rpm. An aliquot was taken to
verify spore and crystal formation (over 90% sporulation
is optimum), and the pre-culture was incubated for 20
min at 70℃ to eliminate vegetative cells (synchroniza-
tion).
The main culture (40 ml) was inoculated with 1/1,000
volumes of synchronized pre-culture and grown as men-
tioned above. Optimal crystal formation was checked by
phase-contrast microscopy (DM2500, Leica Microsys-
tems, Germany). The total number of cells in a culture
was determined by plating 10-fold serial dilutions on
CCY plates. Then the whole culture was centrifuged at
9,000 ×g for 10 min.
The pellet was washed once with ice-cold 1 mol/l
NaCl, 10 mmol/l EDTA solutions. Finally, the pellet was
suspended in 1 ml of 10 mmol/l KCl. Optical Density

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
339
(OD) was measured at 600 nm and the suspensions were
stored at −20℃ until bioassay. All steps after centrifuga-
tion were done on ice to limit proteolysis.
2.4. Bioassays
In order to determine and optimize the toxicity of 26 Bt
strains, three kinds of bioassays were carried out:
2.4.1. Activity against First Instar Larvae
A single-dose test was performed; about 50 µL of the
culture of each strain corresponding to 1.9·108 UFC/mL
added onto 2 g of the artificial diet surface contained in
24-well polystyrene plates. One-first instar larvae of C.
capitata was added per well, and the mortality was re-
corded after 7 days of incubation at 25℃ in dark condi-
tions. Two replicates of 24 larvae were conducted for
each Bt strain.
B. thuringiensis subsp. israelensis HD567 strain (Bti)
(B.G.S.C. code 4Q1) and Skeetal, a commercial product
based on Bti and reported to be active against dipterans,
were used as standard controls. Sterile distilled water was
used as a negative control.
2.4.2. Activity against Third Instar Larvae
Five third instar larvae were transferred to plastic recipi-
ents (30 mm diam × 70 mm depth) containing 10 g of
diet (see insect rearing) mixed with the toxin, except for
the control group, which had just received water with the
diet. Three doses of Bt spore-crystals (50 µL/g, 100 µL/g
and 150 µL/g) were used in five repetitions for each
strain to confirm the results. The recipients were covered
with nylon mesh, and fixed around the edges with a rub-
berband.
After 48 h of exposure, the number of dead larvae was
counted and the pupae were separated from diet and then
transferred to Petri dish (9 cm), until adult emergence.
Bioassay tests were carried out to determine LC50 values
of the Bt strains against C. capitata larvae.
2.4.3. Activity against C. capitata Adults
20 adults newly emerged (1-2 days old) were kept in the
cage (25 cm by 25 cm) containing artificial diet (see in-
sect rearing) mixed with 333.33 µL/g of spores and crys-
tals mixture. The sweet substances (hydrolyzed protein
and sugar) was used in the bioassay to attract the adults
recently emerged to feed Bt spore-crystal. Water was
provided to the flies via a damp yellow sponge. For each
toxin, a negative control was prepared with distilled wa-
ter and fly feeding. Three replicates per assay were car-
ried out. Fly mortality was recorded daily for 7 days.
2.5. Statistical Analysis
The mortality assay was calculated according to the Ab-
bot’s formula:
Mortality corrected % = (1 − n in T after treatment/n in
Co after treatment) * 100
Where: n = Insect population, T = treated, Co = con-
trol.
The LC50 regression equation and the 95% confidence
limit were calculated by using Probit analysis. Percent-
ages of mortality obtained from bioassay tests were ana-
lyzed using one-way analysis of variance (ANOVA, p <
0.05). Tukey’s test (p < 0.05) was used to analyze f sig-
nificant differences among the Bt strains tested against C.
capitata.
3. Results
3.1. Activity against First Instar Larvae
Results of the larvicidal effectiveness of 26 Bt strains are
shown in Table 2.
The spore-crystal mixture of selected strains showed
significantly larvicidal activity (df: 27, F: 10.42, p <
0.005 at 95%). The mortality rate was ranging from 8.33
to 100%, obtained after 7 days of treatment with single
dose 25 µL/g. The toxin produced by Bt 13.4 strain
showed the highest effect, with 100% of corrected mor-
tality. The effect of Bt 2.1 strain was the lowest, with
8.33% as percentage of larval mortality. The reference
strain 4Q1 (45.83%) and Skeetal (60.83%) caused mor-
tality to the same degree with some of our strains or low
like Bt M-Ag 2.7 (47.92%).
3.2. Activity against Third Instar Larvae
Screening for toxicity against C. capitata adults was
performed using spore-crystal mixture. In Table 3, the
results of bioassays on the biological activity of Bt
strains tested against C. capitata third instar larvae are
presented.
The susceptibility of third instar of C. capitata, evalu-
ated as percentage of larvae that did not survive to
adulthood, varied significantly between 0-8%, 8-48%
20-68% at dosages of 50 µL/g, 100 µL/g and 150 µL/g
respectively (Table 3). At 150 µL/g, Bt A7 and Bt M-Ag
21.6 strains proved most effective activity (68%)
whereas Bt A10 strain showed the lowest corrected mor-
tality (20%).
The LC50s determined after 48 h of application were
ranging between 1.115 µL/g for Bt A7 and 3.199 µL/g
for Bt A14. Significant differences were noted with
tested doses (df: 2, F: 49.97, p < 0.005 at 95%).
3.3. Activity against C. capitata Adults
Table 4 shows biological activity of the spore-crystal
mixture of Bt strains in newly emerged adults of C.

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
340
Table 2. Entomopathogenic activity of the spore-crystal mixture of Bt strains against newly emerged larvae of C. capitata.
95% Confidence Limits
Bt strains Mortalitya (%) ± SE
Lower Upper
Skeetal 60.83 ± 0.04824bcd 0.778 0.6725
4Q1 45.83 ± 0.05924abd 0.3316 0.6228
Bt A7 95.83 ± 0.02915a 0.8997 0.8997
Bt M-Ag 4.1 85.42 ± 0.05148abc 0.7506 0.9577
Bt 32.3 87.50 ± 0.04824abc 0.778 0.972
Bt A14 66.67 ± 0.06876hi 0.528 0.805
Bt A10 77.08 ± 0.06131i 0.6475 0.8942
Bt M-Ag 2.2 85.42 ± 0.03531abc 0.8665 1.0085
Bt M-Ag 26.4 77.08 ± 0.06131bcd 0.6475 0.8942
Bt M-Ag 21.6 77.08 ± 0.06131bc 0.6475 0.8942
Bt M-Ag 2.4 87.50 ± 0.04824abc 0.778 0.972
Bt A9 75.00 ± 0.06316cde 0.6229 0.8771
Bt A11 66.67 ± 0.06876efg 0.5283 0.805
Bt M-Ag 2.1 8.33 ± 0.04031fgh 0.0022 0.1644
Bt M-Ag 2.7 47.92 ± 0.07287ghi 0.3326 0.6258
Bt 7.16 62.50 ± 0.07062efg 0.4829 0.7671
Bt 11.3 66.67 ± 0.0719cde 0.4387 0.728
Bt 32.7 75.00 ± 0.06316cde 0.6229 0.8771
Bt 10.3 95.83 ± 0.02915abc 0.8997 1.017
Bt 13.4 100.00 ± 0.0000abc 1.000 1.000
Bt 16.1 79.17 ± 0.05924bcd 0.6725 0.9108
Bt B1 81.25 ± 0.05693bcd 0.698 0.927
Bt B6 83.33 ± 0.04824fgh 0.778 0.972
Bt B9 83.33 ± 0.05436fgh 0.724 0.9427
Bt M-Ag 1.7 66.67 ± 0.06876abc 0.5283 0.805
Bt M1 79.17 ± 0.04824abc 0.778 0.972
Bt M5 68.57 ± 0.06761hi 0.5515 0.8235
Bt M12 87.50 ± 0.04824cdi 0.778 0.972
a: a,b Mean values from the same row with different letters in superscript are significantly different according to Tukey test (p < 0.05). Means followed by the
same letter in columns are not different from each other by the Tukey’s test at 5% significance.

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
341
Table 3. Entomopathogenic activity of the spore-crystal mixture of Bt strains against last instar larvae of C. capitata.
Mortality (%) ± SE
Strains
50 µL/g 100 µL/g 150 µL/g
LC50 95% Confidence Limits
Skeetal 0 ± 0.0000 36 ± 1.30384 52 ± 1.14018 1.328 1.143 ± 1.650
4Q1 0 ± 0.0000 28 ± 0.89443 40 ± 0.0000 1.351 1.144 ± 1.833
Bt A7 8 ± 54772 48 ± 54772 68 ± 54772 1.115 0.938 ± 1.370
Bt M-Ag 4.1 1 ± 0.0000 40 ± 0.0000 60 ± 0.0000 1.250 1.067 ± 1.554
Bt 32.3 4 ± 0.44721 36 ± 0.44721 60 ± 0.0000 1.268 1.028 ± 1.833
Bt A14 4 ± 0.44721 12 ± 0.54772 24 ± 0.83666 3.199 1.738 ± % 100000002.000E + 12*
Bt A10 0 ± 0.0000 8 ± 0.54772 20 ± 0.70711 2.394 1.656 ± 4815083.000
Bt M-Ag 2.2 4 ± 0.44721 44 ± 0.44721 60 ± 0.0000 1.212 1.007 ± 1.592
Bt M-Ag 26.4 8 ± 0.54772 28 ± 0.54772 40 ± 0.0000 1.852 1.298 ± 11.893
Bt M-Ag 21.6 0 ± 0.0000 44 ± 0.44721 68 ± 0.54772 1.164 1.001 ± 1.381
Bt M-Ag 2.4 8 ± 0.54772 36 ± 0.44721 60 ± 0.70711 1.268 1.028 ± 1.833
Bt A9 0 ± 0.0000 28 ± 0.54772 40 ± 0.70711 1.604 1.297 ± 2.886
Bt A11 0 ± 0.0000 24 ± 0.44721 32 ± 0.54772 1.847 1.413 ± 5.420
Bt M-Ag 2.1 0 ± 0.0000 28 ± 0.54772 32 ± 0.54772 1.164 1.001 ± 1.381
Bt M-Ag 2.7 0 ± 0.0000 16 ± 0.44721 32 ± 0.54772 1.872 1.458 ± 6.085
Bt 7.16 0 ± 0.0000 20 ± 0.70711 36 ± 0.44721 1.743 1.390 ± 3.959
Bt 11.3 0 ± 0.0000 16 ± 0.44721 52 ± 0.54772 1.464 1.273 ± 2.005
Bt 32.7 0 ± 0.0000 16 ± 0.44721 53 ± 0.54772 1.464 1.273 ± 2.005
Bt 10.3 4 ± 0.44721 24 ± 0.83666 60 ± 0.70711 1.360 1.137 ± 1.877
Bt 13.4 0 ± 0.0000 32 ± 0.54772 64 ± 0.44721 1.264 1.094 ± 1.532
Bt 16.1 4 ± 0.44721 16 ± 0.44721 52 ± 0.54772 1.548 1.260 ± 2.603
Bt B1 4 ± 0.44721 20 ± 0.70711 52 ± 0.54772 1.515 1.231 ± 2.490
Bt B6 0 ± 0.0000 8 ± 0.54772 44 ± 0.44721 1.574 1.376 ± 2.400
Bt B9 0 ± 0.0000 12 ± 0.54772 40 ± 0.70711 1.654 1.391 ± 3.203
Bt M-Ag 1.7 0 ± 0.0000 36 ± 0.83666 56 ± 0.44721 1.313 1.117 ± 1.695
Bt M1 0 ± 0.0000 28 ± 0.54772 56 ± 0.44721 1.362 1.169 ± 1.768
Bt M5 0 ± 0.0000 8 ± 0.54772 28 ± 0.54772 1.947 1.529 ± 18.077
Bt M12 4 ± 0.44721 12 ± 0.54772 52 ± 0.54772 1.580 1.288 ± 2.723
a: a,b Mean values from the same row with different letters in superscript are significantly different according to Tukey test (p < 0.05). Means followed by the same letter in columns
are not different from each other by the Tukey’s test at 5% significance. *: Upper limits greater than or equal to 1.E20 are really infinite.

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
342
Table 4. Entomopathogenic activity of the spore-crystal mixture of Bt strains against emerged adults of C. capitata.
95% Confidence Limits
Strains Mortality (%) ± SE
Lower Upper
Skeetal 48.33 ± 0.2801bc 0.7967 1.9652
4Q1 33.33 ± 0.25332bc 0.424 1.4808
Bt A7 81.66 ± 0.52834a 1.4217 3.6259
Bt M-Ag 4.1 80 ± 0.43799a 1.2292 3.0565
Bt 32.3 70 ± 0.371c 1.3213 2.8691
Bt A14 68 ± 0.55838c 1.2162 3.5457
Bt A10 60 ± 0.347ca 1.1333 2.581
Bt M-Ag 2.2 80 ± 0.53282a 1.3647 3.5876
Bt M-Ag 26.4 70 ± 0.4555c 1.4784 3.3787
Bt M-Ag 21.6 80 ± 0.4555a 1.4784 3.3787
Bt M-Ag 2.4 80 ± 0.59074a 0.9106 3.3751
Bt A9 60 ± 0.4555ca 1.4784 3.3787
Bt A11 56.66 ± 0.347d 1.1333 2.581
Bt M-Ag 2.1 68.66 ± 0.32819ca 1.1249 2.4941
Bt M-Ag 2.7 78.66 ± 0.371a 1.3213 2.8691
Bt 7.16 75 ± 0.56625a 1.1522 3.5145
Bt 11.3 70 ± 0.45848c 1.3293 3.2421
Bt 32.7 75 ± 0.59074a 0.9106 3.3751
Bt 10.3 81.66 ± 0.30971a 1.6397 2.9318
Bt 13.4 80 ± 0.53282a 1.3647 3.5876
Bt 16.1 80 ± 0.4555a 1.4784 3.3787
Bt B1 80 ± 0.4555a 1.4784 3.3787
Bt B6 75 ± 0.40908a 1.4324 3.139
Bt B9 80 ± 0.26342a 1.8791 2.978
Bt M-Ag 1.7 81.66 ± 0.4555a 1.4784 3.3787
Bt M1 81.66 ± 0.53282a 1.3647 3.5876
Bt M5 60 ± 0.347ca 1.1333 2.581
Bt M12 80 ± 0.33503a 1.7297 3.1274
a: a,b Mean values from the same row with different letters in superscript are significantly different according to Tukey test (p < 0.05). Means followed by the
same letter in columns are not different from each other by the Tukey’s test at 5% significance.

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
343
capitata.
The difference of fly’s mortality was significantly
higher between those exposed to the spore-crystal mix-
tures and the ones exposed to the water control.
The majority of Bt strains killed more than 50% of C.
capitata adults. The mortality values varied significantly
between strains. They produced significant mortalities
from 56.66 to 81.66% (df: 27, F: 0.68, p < 0.005 at 95%);
when using both spore-crystal suspensions of Bt toxin
after 7 days exposure time. The more toxic Bt strains
caused adult mortality starting from the 2nd day or the 3rd
day of ingestion.
The commercial product based on Bti (Skeetal) and
the reference Bti strain (code 4Q1) were also tested
against C. capitata adults. They were in general less effi-
cient, showing corrected mortality about 48.33% and
33.33% respectively (Table 4).
4. Discussion
During sporulation, the Gram-positive bacterium, Bt
forms crystalline protein inclusions, which possess insec-
ticidal activity. These parasporal inclusions differentiate
this species from other related species such as Bacillus
cereus and Bacillus mycoides [23]. The crystals are gen-
erally smaller than the spores and can represent ±30% of
the dry weight of the cell [24].
A total of 26 Bt strains were tested on both larvae and
adults; and it was found that 100% of strains were toxic
against adults of C. capitata, 92% on neonate larvae on
adults, and 60% on third instar larvae. Neonate larvae
and adults were more susceptible than third instar larvae
of C. capitata; in fact, several strains produced high
mortality only after 3 days of exposure. The toxicity of
spore-crystal mixtures against third larvae was seldom
high but some strains yielded 68% mortality after 48
hours of application at the doses used in these experi-
ments.
The biological activity of Bt strains has been studied
by many authors, who determined the insecticide poten-
tial of obtained toxins using both spore and crystal. Our
results are similar to that reported by Gingrich [18]. This
author have tested 94 strains of Bt and found that 15 of
them killed at least 80% of C. capitata adults that fed
pellets for 9 days.
Hassani and Gaouar Benyelles [19] have tested the ef-
fect of the preparation of Bti on wild third instar larvae
and adults of C. capitata isolated from Citrus fruit or-
chards in Algeria, and observed toxicity in high doses
(100 mg/g) with a reduction in average emergence
(84.62%), concluding that the stage L3 and adults of the
pest are very susceptible at this dose of Bti product.
Molina et al. [25] have tested sporulated cultures of
115 bacterial strains of Bacillus pumilus (4.65 × 108 to
1.45 × 107 CFU/mL) against adults and neonate larvae of
C. capitata. None of these strains were caused significant
mortality of C. capitata adults compared with the nega-
tive controls. The mortality rates with the 115 bacterial
strains at the end of the experiment ranged from 0% to
40%, while the average mortality rates with the negative
controls ranged from 5% to 30%. Similar results were
obtained in bioassays with larvae, the maximal corrected
mortality rates with the 115 bacterial isolates ranged
from 0% to 36%, while the average mortality rates with
the negative controls ranged from 1% to 12% after 15
days, at the end of the experiment. After this toxicity
screening, they obtained a novel Bacillus pumilus strain,
which is highly toxic to C. capitata larvae. The mortality
rate for C. capitata larvae ranged from 68 to 94% de-
pending on the conditions under which the culture was
kept before the bioassay.
Karamanlidou et al. [14] and Yamvrias and Anagnou
[26] have reported a mortality > 80% when they used
various Bt strains against old larvae of the dipteran olive
fruit fly, B. oleae (Gmelin). Robacker et al. [16] evalu-
ated the action of 55 Bt strains on larvae of Mexican fruit
fly, Anastrepha ludens (Loew). Only 7 strains were
found toxic on adults; the centrifuge pellets or precipi-
tates of Bt strains have killed > 50% of larvae. The mor-
tality rates varied between 4 to 62% after application of
the pellets of these strains against adult flies. Only 5 of
them killed 65-80% of adults in 10 days compared with
2.7% mortality in controls. The other killed 40% of
adults in the same experiment.
Alberola et al. [15] studied the Bt activity against sec-
ond instar of B. oleae larvae and new emerged adults.
These authors have reported that both spore-crystal mix-
tures (109/mL) caused 70% mortality for larvae in 72
hours and 80% of mortality for adult flies in 6 to 10 days
of application.
The obtained results during our study showed that
some Bt strains were most toxic against adults than on
larvae and vice versa. For example, the insecticidal activ-
ity of Bt M-Ag 2.1 against larvae of C. capitata is 8.33,
whereas the activity of this strain towards C. capitata
adults is 68.66 (Tables 2 and 4). The genes which coding
for insecticidal activity against the two stages of the
medfly, could be different [15,16]. Alberola et al. [15]
have also reported that there are some different chemical
conditions in the gut of the two stages of the insect that
can control the activation or activity of the protoxin.
On the other hand, some Bt strains expressed high ac-
tivities against the both stages of C. capitata such as Bt

Biological activity of Bacillus thuringiensis (Berliner) strains on larvae and adults of Ceratitis capitata (Wiedemann)
(Diptera: Tephritidae)
344
A7, Bt 13.4, Bt M-Ag 4.1, Bt M-Ag 2.2, Bt M-Ag 21.6
(Tables 2 and 4).
5. Conclusions
Sustainable agriculture will rely increasingly to biologi-
cal control of pests such as C. capitata that is considered
as a quarantine pest over the world and particularly in the
Argan area. No control program has been undertaken
until now against C. capitata in the Argan forest. The use
of biopesticides is environmentally friendly and reduces
the contact of human to chemical pesticides. The present
study provides evidence for the insecticidal activity of
Moroccan Bt strains against C. capitata. Some Bt strains
showed a great activity against neonate larvae, third in-
star larvae and especially towards adults of C. capitata.
A number of our collection Bt strains showed high insec-
ticidal activity against C. capitata in comparison to that
noted for the Bti HD567 strain and the commercial
product Skeetal previously used in the control of harmful
fruit flies. Some of our Bt strains can be used in the bio-
logical control system to fight against C. capitata and
may contribute to reduce the use of chemical insecticides
harmful to the consumers and the environment.
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