Vol.3, No.3, 265-271 (2013) Open Journal of Ecology
http://dx.doi.org/10.4236/oje.2013.33030
Comparative study of the effect of Bacillus
thuringiensis on larval populations of Culex pipiens
L. (Diptera-Culicidae) of the City of Tlemcen (Algeria)
Nassima Tabti*, Karima Abdellaoui-Hassaïne
Laboratory of Valuation of the Actions of the Man for the Environmental Protection and the Application in Public Health, University
of Tlemcen, Tlemcen, Algeria; *Cor r e s p o n d i n g A u t h or :
Received 8 February 2013; revised 3 April 2013; accepted 30 May 2013
Copyright © 2013 Nassima Tabti, Karima Abdellaoui-Hassaïne. This is an open access article distributed under the Creative Com-
mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
ABSTRACT
In the cities of Algeria, Culex pipiens L. (Diptera:
Culicidae) is the mosquito which presents most
interest because of its wide geographical dis-
tribution and of its abundance which engender a
strong nuisance. Besides, its role of vector of
the virus West Nile arouses a particular interest
in the Mediterranean Basin. These insects are
generally controlled by conventional insecticid-
es for the greater part, chemicals which cause in
the long term side effects (effects on the not
aimed bodies and the resistance of the aimed
species). A research for the effect of the bioinsec-
ticide Bacillus thuringiensis (granulated com-
mercial shape in 200 IUT1/mg) was realized on
préimaginales populations of the artificial depo-
sits sites (taken directly of them natural the de-
posits sites) and cleansed populations (stem-
ming from a breeding) having never been han-
dled previously, taking into account local wea-
ther and physico-chemical conditions. Analyses
of variance, allowed to determine the combined
effect of the factor measure and of the factor
time which by increasing, increase the efficiency
of the product. The results of the rates of mor-
talities registered after treatment allowed to loo-
sen the DL50 and the DL90 for every embryonic
st age. From the results, we estimated the degree
of sensibility of the larva populations of Culex
pipiens which have proved heterogeneous in
partial tolerance in Bacillus thuringiensis for the
populations of the sensitive artificial and homo-
geneous the deposits sites for those stemming
from the breeding.
Keywords: Tlemcen; C ul ex pipiens; Bacillus
Thuringiensis israelensis; Nuisance; Bio Insecticide
1. INTRODUCTION
The situation of nuisance, caused by Culex pipiens L.
1758 and strongly felt in most cities in Algeria including
the city of Tlemcen [1], interested us to stud y its ecolog y,
for the sole purpose of establishing a more effective
fight.
The ecological plasticity of this species allows it to
grow in most deposits and to put up with the wider varia-
tions of ecological factors [2-6,11].
The creation of the deposits sites remains the result of
the man carelessness. The hardly controlled extension of
urban and industrial pollutant sector added to the sewage
and sanitation systems are generators of deposits of Cx.
pipiens [5].
The resistance of these insects to toxic chemical com-
pounds, non-biodegradable, prompts to continually review
the ways to fight [7].
Biological control is an alternative and an element of
the strategy defined element but difficult to imple-
ment.
Discovered during research aimed at developing new
biological agents for the fight against tropical disease
vectors (example: malaria and onchocerciasis...), Baci-
llus thuringiensis israelensis (Bti) showed a significant
larvicidal effect on many mosquito species. In 1985, 72
species of mosquitoes were susceptible to the action of
Bti. Thirteen years later, that number had risen to more
than 115 species [8].
In this context, this work aims to estimate and com-
pare the effect of this bio insecticide on Cx pipiens larval
populations from livesto ck and those taken directly from
the deposits, with toxicology tests performed according

N. Tabti, K. Abdellaoui-Hassaïne / Open Journal of Ecology 3 (2013) 265-271
266
to the method recommended by the WHO1 [9] in order to
demonstrate that the bacteriological and physicoche-
mical conditions may play a role in the effectiveness of
Bti.
Moreover, if the evaluation of the effect of insecticide
treatment on larval populations is usually done in the
field, in natural environments, this study analyzes the ef-
fects on populations from breeding after purification.
2. MATERIALS AND METHODS
Toxicological tests are conducted in two ways:
- Populations from livestock breeding: the larval hat-
chlings of hypogean deposits are grouped in lots of
20 larvae at stage 4 placed in cages. Laid eggs are
retrieved in 200 ml crystallizers. Adults (30 males
and 10 females) are evenly distributed in five diffe-
rent cages. The food, provided daily, of the larvae is
composed of a mixture of biscuit and dried yeast
(75/25). This is done in May at an ambient tempera-
ture of 25˚C and a photoperiod 14/10 hours [10].
- Populations sampled from deposits: the larvae from
the four stages are taken directly from three hypogean
deposits (crawl spaces) which are located in different
neighborhoods of Tlemcen urban group. Water intake
is putrid, nauseating and heavily loaded with organic
matter from the leakage of defective pipes (rate of
conductivity).
The bio insecticide used is the standard commercial
formula Vectobac at 200 IUT/mg, granules. The tests were
repeated three times on the same larval stage and were
performed on the same number of larvae (50 larvae of the
same stage), placed in the same volume of water in 1000
ml test tubes, at an ambient temperature between 20 and
25˚C.
The water used is distilled and seven do ses decreasing
from 35 to 2 mg/L were tested on each stage of larvae
from breeding and from 100 to 10 mg/L for those taken
from the deposits. For each test a control group (50 larvae
of the same stage) was placed.
Dead larvae are removed at regular time intervals (30
minutes) until the death of all larvae (100% mortality).
The results are analyzed by a statistical treatment, de-
veloped by the software Minitab12. Mortalities are ex-
pressed as m ean a n d st an dard deviatio n, calculated on t he
percentage mortalities of the three tests and corrected [1 1],
this allows to eliminate th e natural mortality an d to know
the actual larvicide toxicity. Variance analyses with con-
trolled factor were used to demonstrate the effect of time,
the effect of dose and the effect of larval stage. The re-
gression lines are based on Swaroop & Uemera (1966)
method to determine the LD50 and LD90 of the different
larval stages, with a probability of 95%.
3. RESULTS
3.1. Determination of the Efficiency of Bti
For all doses tested, the bio insecticide treatment caus-
es a significant lengthening of the duration effect (p <
0.001). It goes from 330 minutes in high doses to 510
minutes for the dose of 50 mg/L.
The Bti time effect is shorter on populations from
breeding than on populations taken directly from depo-
sits (Figur es 1 and 2).
The analysis of variance shows that under the effect of
the same doses, the mortality rates recorded 360 minutes
after treatment of larvae are high (Figures 1 and 2). In
the four larval stages (p < 0.005), there wa s a highly sig-
nificant difference between the average mortality for all
instars.
Taking into account the statistical analysis, it is clear
that the Bti has effects on larval mortality, especially for
higher doses, where the larvae are all dead.
The first stage treated larvae, appear to be more sen-
sitive to Bti, due to mortality rates recorded for the dif-
ferent concentrations. For larval stages L2, L3 and L4,
doses act the same way on the mortality of larvae as well
as purified populations of artificial deposits.
The effectiveness of the two factors “dose” and “larval
stage” is demonstrated through an analysis of variance
with two controlled factors, encompassing the four larval
stages and the different doses tested. This analysis shows
the impact of each of the two factors; the larval stage and
the different doses used, significantly, affect larval mor-
tality of Cx. pipiens: probability p < 0.005 (Table 1).
3.2. Determination of Lethal Dose (Table 2)
The coefficients of the carried out eight regression
lines show that there is a significant relation ship between
the dead larvae and the Bti different dosages.
The probit analysis allowed us to retain 50% lethal
dose (LD50) of Bti which is of the order of 15.87 mg/L
for the first larval stage of the breeding population col-
lected from polluted deposits, its upper and lower limits
are respectively upper 14.74 and 17.09 m g/L. for t his same
level of rearing larvae from the LD50 is 2.53 mg/L, its
upper and lower limits are 2.26 to 2.83 mg/L.
For larvae of the second stage, the LD50 of larvicide is
20.36 mg/L. Confidence intervals of LD50 are from 12.05
to 34.41 mg/L (larvae from polluted deposits). For larvae
from breeding, the LD50 is 3.11 mg/L and confidence
intervals are from 2.8 to 3.45 mg/L.
The lethal dose for 50% mortality for larvae stage 3 is
19.44 mg/L. The lower limit is 10.56 mg/L and the upper
limit is 35.76 mg/L, this for individuals collected directly
from shelters. LD50 populations from livestock is 5.3
1international toxicity units.
2lethal dose for 50% mortality.
3World Health Organization.

N. Tabti, K. Abdellaoui-Hassaïne / Open Journal of Ecology 3 (2013) 265-271 267
Figure 1. Test Results of Bti larvae L4, L3, L2 and L1 of purified populations.
OPEN A CCESS

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268
(P20: test 1; P21: test 2; P22: test 3) We began again th e same test 3 ti mes for P22: test 3 every larvae stage L4, L3, L2 and L1.
Figure 2. Test Results of Bti on larvae L4, L3, L2 and L1of artificial deposits.

N. Tabti, K. Abdellaoui-Hassaïne / Open Journal of Ecology 3 (2013) 265-271 269
Table 1. Ef fects of Bti on each instar f or seven h ours (aver age of thr ee trials followed by standard deviation). (a) Immature populatio ns
from breeding; (b) Immature populations taken directly from the field.
(a)
Stade/Dose (mg/L) 1 2 3 4 5 6 7
L1* 60.0 ± 5.0 61.6 ± 5.7 75.0 ± 5.0 86.6 ± 5.7 86.6 ± 5.7 95.0 ± 5.0 100
L2* 58.3 ± 5.7 80 ± 8.6 93.3 ± 5.0 80.0 ± 8.6 95 ± 5.0 98.3 ± 2.9 100
L3* 50.0 ± 5.0 73.3 ± 7.6 80.0 ± 8.6 81.6 ± 2.8 86.6 ± 2.9 95.1 ± 5.0 98.3 ± 2.8
L4* 13.3 ± 2.8 31.6 ± 5.7 41.6 ± 2.9 50.0 ± 5.0 61.6 ± 2.8 73.3 ± 7.6 83.3 ± 2.9
Tot al * 45.4 ± 3.9 61.6 ± 6.3 72.4 ± 5.3 74.5 ± 4.9 82.4 ± 4.7 90.4 ± 4.5 95.4 ± 0.9
(b)
Stade/Dose (mg/L) 1 2 3 4 5 6 7
L1* 10.6 ± 2.3 28.0 ± 7.2 86.6 ± 5.0 86.6 ± 14.4 75.3 ± 7.5 95.3 ± 4.1 100
L2* 25.3 ± 6.1 65.3 ± 15.5 66.0 ± 2.0 87.3 ± 7.0 88 ± 9.1 93.3 ± 3.0 100
L3* 24.6 ± 11.5 64.0 ± 8.0 63.3 ± 11.3 86.6 ± 11.3 86.6 ± 3.0 86.6 ± 1.1 96.6 ± 4.1
L4* 10.6 ± 1.1 20.6 ± 12.0 32.0 ± 5.2 76.0 ± 5.2 84.0 ± 7.2 84.6 ± 11.0 96.6 ± 4.1
Tot al* 17.7 ± 4.7 44.4 ± 10.1 61.9 ± 5.3 84.1 ± 8.9 83.4 ± 6.1 89.9 ± 4.2 98.3 ± 1.5
(a) *For each in star, the asteri sk indi cates a h ighl y signif icant d ifference (P < 0.001) by ANOVA. (b) *For each inst ar, the asterisk in dicates a hig hly si gnifican t
difference (P < 0.001) by ANOVA.
Table 2. 50% Lethal doses expressed in mg/L (ITU between
brackets) and the fudicial limits.
Larval stage L1 L2 L3 L4
Purified populations 2.53 (506) 3.11 (622) 5.3 (1060) 9.65 (1930)
Populations sampled
from deposits 15.87 (3174) 20.36 (4072) 19.44 (3888) 40.82 (8164)
Proportion 1/6 1/6 1/4 1/4
Larval
stage L1 L2 L3 L4
Limits Lower Higher Lower Higher Lower Higher LowerHigher
Purified
populations 1.45 12.93 2.01 16.71 5.64 28.94 16.6345.62
Populations
sampled
from
deposits
14.74 17.09 12.05 34.41 10.56 35.76 26.562.87
mg/L, its upper and lower limits are 4.18 to 5.83 mg/L.
The results of the fourth larval stage are: for larvae col-
lected lodges, the LD50 was 40.82 mg/L, its limits are
26.50 to 62.87 mg/L. for larvae from breed ing, the LD50
is 9.65 mg/L and confidence intervals are 8.93 to 10.42
mg/L.
Lethal doses LD50 and L90 are relatively lower for
breeding populations. It should be noted that only 1/5th
of doses for the same mortality between larvae reared in
clean water and those taken from the deposits. This dif-
ference is significant for all doses and all larval stages.
According to sensitivity tests [12], préimaginales po-
pulations of Cx. pipiens polluted lodges are heteroge-
neous populations, a party may be highly sensitive and
one tolerant. Larvae from breeding proved consistent and
sensitive to Bti (Table 3).
4. DISCUSSION
Time, dose and larval stages effects are functional in
the same way for the two populations of studied Culex
pipiens.
Factors such as environmental parameters (larval den-
sity, water temperature...) can significantly affect the ef-
fectiveness of Bti.
Mosquito species show different levels of suscepti-
bility to Bti crystals. In general, Culex larvae are the
most sensitive, and the larvae o f Aedes and Ochlerotatus
are equal or slightly less sensitive and Anopheles larvae
are more resistant when exposed to the same amount of
Bti crystals. This difference in susceptibility within the
same genus (e.g. species belonging to the genera Culex,
Aedes, Anopheles or Ochlerotatus) is caused by beha-
vioral [13] and physio logical changes of the various sp e-
cies, but it is clearly linked to the behavior of crystals in
the environment [14-16].
Although a difference in the type and a number of “re-
ceptors” may exist between the various mosquito spe-
cies [17], the same number of Bti crystals induce a low-
er mortality rate in cold water than in hot water [17,18].
This toxicity decrease is due to a reduction of metabolic
activity (reduction of ingestion and enzymatic activity)
observed when insect is exposed to temperatures appro-
aching the minimum temperature at which it is normally
found in the environment. It should be noted that at low
temperatures, some formulations show a low rate of mix-
ing and dispersion, which reduces the availability of Bti
crystals.
Generally, in most of studied species, the youngest
larvae are more susceptible than the older [19]. Ageing,
the larvae become significantly less sensitive to the

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270
Table 3. Results of sensitivity tests to Bti on the four Cx. pipiens instars. (a) Immature populations sampled in the belowground
deposits; (b) Immature people from breeding.
(a)
Stage LD50 LD90
K = corrected
LD50/LD50 basi s P = corrected LD90/corrected LD50 Interpretation
Stage 1 15.87 48.35 0.86 3.04 Heterogeneous partial tolerance
Stage 2 20.36 55.84 1.11 2.74 Heterogeneous partial tolerance
Stage 3 19.44 92 1.06 4.73 Heterogeneous partial tolerance
Stage 4 40.82 133 2.23 3.25 Heterogeneous partial toler ance
(b)
Stage LD50 LD90
K = corrected
LD50/LD50 basisP = corrected LD90/corrected LD50 Interpretation
Stage 1 2.53 6.16 0.14 2.43 Sensitive homogeneous
Stage 2 3.11 7.65 0.17 2.45 Sensitive homogeneous
Stage 3 5.30 13.15 0.29 2.48 Sensitive homogeneous
Stage 4 9.65 21.25 0.52 2.20 Sensitive homogeneous
same number of Bti crystals. In general, stage 2 larvae
are 1.5 to 5 times more sensitive than stage 4 larvae [15 ,
18]. Stage 4 larvae feed very little, as they start pupation
[19].
Generally, the more the settlement contains organic
matter and colloidal matters in suspension, the more the
number of Bti crystals must be high for the same mor-
tality rate [20,21]. The adsorption of the crystals on the
particles, followed by a slow precipitation, reduces the
availability of Bti crystals. In addition, larvae exposed to
high concentrations of “nutritious” particles can prove
reduced ingestion rates, suggesting that they have reach-
ed the level of satiety [15]; so the larvae will ingest less
crystals causing a decrease in mortality. The presence of
organic pollution also reduces the toxic activity [17].
According to the sensitivity test, the larvae in con-
taminated deposits proved to be heterogeneous with par-
tial tolerance. The product does not take effect on a regu-
lar basis on all individuals within a population and even
within a single generation. Some died in the first two
hours and some others, however, others took a much long-
er time up to 16 hours. For the more advanced stages, L3
and L4, we noted that approximately 3% of the indivi-
duals tested are highly tolerant. Thus, there is a time
effect that can be explained by bacterial spores that pro-
liferate gradually in epithelial cells.
Larvae from livestock are homogeneous and susce-
ptible, these individuals reared in clean water would have
a less effective immune system than the larvae grown in
contaminat ed wa ter r ic h in microorganisms.
Preimaginal populations, living in wastewater highly
loaded in bacteria in particular, require lethal doses at 50
and 90%, significantly higher than those from livestock.
These larvae thus have a certain tolerance towards Bti.
The water pollution would be respon sible for the decrea-
se of the effect of Bti crystals [1 7,22-24].
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