Comparative study of the effect of <i>Bacillus thuringiensis</i> on larval populations of <i>Culex pipiens</i> L. (Diptera-Culicidae) of the City of Tlemcen (Algeria)

doi:10.4236/oje.2013.33030

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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 : [email protected]

Received 8 February 2013; revised 3 April 2013; accepted 30 May 2013

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.

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267

Figure 1. Test Results of Bti larvae L4, L3, L2 and L1 of purified populations.

OPEN A CCESS

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(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.

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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

N. Tabti, K. Abdellaoui-Hassaïne / Open Journal of Ecology 3 (2013) 265-271

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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