Research Article, J Genet Disor Genet Rep Vol: 6 Issue: 3
Chlorpyrifos Resistance Characteristics of Culex pipiens (Diptera: Culicidae) from Northern Tunisia
*Corresponding Author : Tabbabi A
Laboratory of Genetics, Faculty of Medicine of Monastir, Monastir University, Monastir-5019, Tunisia
Tel: +216-97 085 424
E-mail: [email protected]
Received: May 22, 2017 Accepted: June 13, 2017 Published: June 20, 2017
Citation: Daaboub J, Tabbabi A, Lamari A, Feriani M, Boubaker C, et al. (2017) Chlorpyrifos Resistance Characteristics of Culex pipiens (Diptera: Culicidae) from Northern Tunisia. J Genet Disor Genet Rep 6:3. doi: 10.4172/2327-5790.1000161
We collected four field populations of Culex pipiens larvae from Northern Tunisia to study their chlorpyrifos resistance characteristics. Assays were performed using ethanol solutions of chlorpyrifos and data were analyzed using a log-probit program. All samples were resistant to chlorpyrifos (RR>1, p<0.05) and a large variation in the tolerance to this insecticide was observed. The sample 3 (Northeast) was the most resistant with a level higher than 10,000-fold (RR50=43,174). The level of resistance was lower, not exceeding 5-fold in samples 1 (North West). CYP450, EST and/or GST enzymes accounted for only a small part of the observed resistances. Synergists tests confirmed biochemical study where four esterases were detected in studied samples with low and average frequencies (0.03%-0.68%). Moreover, the mortality due to propoxur was significantly correlated with the LC50 of chlorpyrifos (P<0.01) indicated an insensitive AChE 1.
Keywords: Culex pipiens; Chlorpyrifos; Resistance characteristics; Esterases; Acetylcholinesterase 1; Northern Tunisia
In addition to their nuisance in urban areas, Culex pipiens is the suspected vector in the transmission of West Nile Virus in Tunisia. In order to be able to use insecticides in vector control, the target species must be effectively sensitive to these products under field conditions. Laboratory tests have been commonly recorded resistance to insecticides in many vector populations throughout the world. These resistances can be due to the detoxification of the product by enzymes or to a mutation at the targeted site: the sodium channel for DDT and pyrethroids (kdr) or acetylcholinesterase (AChE 1) for organophosphates (OP) and carbamates [1-9]. Because of their lack of accumulation in the organism, OPs including chlorpyrifos have been used on a large scale since 1935 as insecticides in place of organochlorines. The previous studies realized on Culex pipiens populations of some Tunisian areas showed that these mosquitoes have developed high chlorpyrifos resistance levels [10,11] hence the aim of this study was to evaluate chlorpyrifos resistance status of Culex pipiens from Northern Tunisia.
Materials and Methods
Four field populations of Culex pipiens larvae were collected from Northern Tunisia (Figure 1). We stored some adults mosquitoes for biochemical study. We used S-Lab as a susceptible strain without any resistance genes , and two resistant strains (SA2, SA5) selected for A2-B2 and A5-B5 esterases, respectively .
Assays were performed as described by Raymond et al. , using ethanol solutions of chlorpyrifos (99.5% [AI]), brought from laboratory Dr Ehrenstorfer, Germany, and propoxur ( 99.9% [AI], Bayer AG, Leverkusen , Germany). Chlorpyrifos bioassays included 5-9 concentrations providing between 0 and 100% mortality and 3-5 replicates per concentration on sets of 20 early 4th instars in a total volume of 100ml of water containing 1 ml of ethanol solution of the tested insecticide. The effect on chlorpyrifos resistance of 2 synergists, the DEF (98% [AI], Chem Service, England), and the PBO ( 94% [AI], Laboratory Dr Ehrenstorfer, Germany) , was studied by exposing larvae to a standard sublethal doses of 0.08 mg/liter for DEF , and 2.5 mg/liter for Pb, 4h before the addition of the insecticide. The DEF is known to inhibit esterases (EST) and/or glutathione-S-transferases (GST) while PBO inhibits cytochrome P450-dependent monooxygenases (CYP450). Propoxur bioassays included one dose (1mg/liter) and five replicates. This concentration kills all susceptible mosquitoes.
Esterases of high activity were characterized on homogenates of adult thorax and abdomen by studying esterase activity in the presence of α-and-β-naphtyl acetate after protein separation by starch-gel electrophoresis (TME 7,4 buffer system) as described by Pasteur et al.  and were identified by comparing their electrophoretic mobility to that of known over-produced esterases.
Larval mortality was recorded after 24-h exposures, and data were analyzed using a log-probit program of Raymond et al.  based on Finney . This program tests the linearity of a dose-mortality response, computes the different lethal doses (LCs) and their confidence interval (CI) at the chosen probability (here P=95%). In this study, the ratios computed were: the resistance ratio (RR) comparing each sample to the reference strain (S-Lab), and the synergism ratio (SR) comparing mortality data observed in presence of the insecticide alone to mortality data observed in presence of the insecticide plus the synergist in each sample. RR and SR are considered significant (P<0.05) when their 95% CI does not include the value 1. To test whether a synergist was more efficient in the field population than in the S-Lab, relative synergism ratios (RSR) were determined. The RSR is equal to the RR for insecticide alone divided by the RR for insecticide plus synergist. A RSR>1 indicates that the synergist has a stronger effect in the field population than in the S-Lab, that is, that the detoxifying mechanism synergized is enhanced in the field population; a RSR<1 shows that the two samples compared are not different as far as the mechanism inhibited by the synergist is concerned .
The linearity of the dose/mortality response is accepted (p>0.05) for reference strain (S-Lab) and rejected for other studied populations. All samples were resistant to chlorpyrifos (RR>1, p<0.05) and a large variation in the tolerance to this insecticide was observed (Table 1). The sample 3 was the most resistant (RR50=43,174). The level of resistance was lower, not exceeding 5-fold in samples # 1.
|Population||Chlorpyrifos||Chlorpyrifos +DEF||Chlorpyrifos +PBO|
|LC50 in µg/l||Slope||RR50||LC50 in µg/l||Slope||RR50||SR50||RSR||LC50 in µg/l||Slope||RR50||SR50||RSR|
|(a)||± SE||(a)||(a)||± SE||(a)||(a)||(a)||± SE||(a)||(a)|
|Susceptible lab strain||0.56||9.0||-||0.17||2.85||-||1.4||-||0.45||1.16||-||0.53||-|
|(0.53-0.58)||± 1.04||(0.14-0.20)||± 0.26||(1.08-1.8)||(0.17-1.3)||± 0.43||(0.35-0.79)|
|(0.43-3.3)||± 0.17||(1.3-3.3)||(0.15-0.48)||± 0.09||(1.3-2.3)||(2.7-6.1)|
|(72-257)||± 0.13||(176-344)||(2.9-28)||± 0,14||(35.8-83.2)||(10.0-22.1)||(11-140)||± 0.30||(49.5-167)||(2.0-5.4)|
|(14100-79700)||± 0.29||(26885-69330)||-||± 0.51||(101266-784669)||(0.16-1.50)||(4070-5460)||± 0.21||(7170-15523)||(3.2-8.0)|
|(20-84)||± 0.15||(53.7-104)||(108-437)||± 0.13||(915-1600)||(0.14-0.28)||(4.1-86)||± 0.24||(21.6-83.0)||(1.3-3.6)|
RR50: resistance ratio at LC50 (RR50=LC50 of the population considered/LC50 of Slab); SR50: synergism ratio (LC50 observed in absence of synergist/LC50 observed in presence of synergist); RR and SR considered significant (P<0.05) if their 95%CI did not include the value 1;
RSR: relative synergism ratio (RR for insecticide alone / RR for insecticide plus synergist).
Table 1: Chlorpyrifos resistance characteristics of Tunisian Culex pipiens in presence and absence of synergists DEF and PBO.
The addition of DEF decreased significantly the tolerance to chlorpyrifos in S-Lab (SR50=1.41, p<0.05) (Table 1). It also decreased significantly the resistance (SR50>1, p<0.05) of 2 among 4 field samples, and the SR was significantly higher than that recorded in S-Lab in the 2 samples (1 and 2). So, the increased detoxification by EST and/or GST was responsible, at least in part, for chlorpyrifos resistance in these samples. This mechanism accounts for only a part of the observed resistances since the RR50 remained significant (p<0.05) in the presence of the DEF. The role of the EST (and/or GST) in the chlorpyrifos resistance was relatively important in sample 2 (RR50=54, p<0.05, RSR=4.8) while it was minor in the other sample. The addition of DEF to Chlorpyrifos bioassays did not decrease significantly the resistance (RSR<1) in samples 3 which manifested the highest chlorpyrifos resistance levels in absence of DEF.
The addition of PBO to chlorpyrifos bioassays significantly increased the tolerance of S-Lab (SR=0.53, p<0.05) and decreased the resistance of 3 among 4 field samples (#2, 3, and 4) (Table 1). The recorded SR in these samples was significantly higher than that observed in S-Lab. However, oxidative metabolism accounted for only a small part of the observed resistances because chlorpyrifos resistance ratios remained significant in the presence of the PBO (e.g., chlorpyrifos RR50>10,000-fold in samples # 3).
Mortality caused by propoxur ranged from 0% in samples 3 which showed the highest resistance levels to studied insecticide to 87% in sample 1. The mortality due to propoxur was significantly correlated with the LC50 of chlorpyrifos (P<0.01) indicated an insensitive AChE 1.
Four esterases were detected in studied samples. The A2-B2 esterases were revealed in 3 samples with a frequency ranged from 0.06 in sample 2 to 0.33 in sample 3. The A4-B4 (and / or A5-B5) esterases were present in all samples with a frequency ranged from 0.03 in sample 1 to 0.68 in sample 3. The B12 and C1 esterases were observed in 2 and 3 samples, respectively.
The resistance levels were very high in sample # 3 (RR50>10,000). This could be explained by the massive mosquitoes control using chemical insecticides in Tunisia (Table 2). A strong resistance of Tunisian Culex pipiens population to OP chlorpyrifos (410 000-folds) was recorded by Pasteur et al. . The rate of resistance to this insecticide ranged from 800-folds to 4-folds in the world [19-26]. The same situation was recorded in Northern Tunisia despite the small area of study. Liu et al.  showed the ability of Culex quinquefasciatus to develop resistance to many group of insecticides including chlorpyrifos (OP) and permethrin (pyrethroid). Likewise, Ben Cheikh et al.  investigated the resistance of Culex pipiens collected between 1990 and 1996 to different OPs insecticides. Resistance to chlorpyrifos was highly variable and reached the highest level (>10,000-folds) recorded worldwide. In contrast, resistance to temephos (OP) was very low not exceeding 10-folds in the samples that showed the highest resistance to chlorpyrifos.
|Code||Locality||Breeding site||Date of collection||Mosquito control (used insecticides)||Agricultural pest control|
|1||Krib||River||0ct. 2005||Occasional (P)||Yes|
|2||Belli||River||Aug. 2003||Rare (C,D)||Yes|
|3||Tazarka||River||May 2005||Very frequent (C, T, Pm, F, P, D)||Yes|
|4||Sidi khalifa||Water pond||July 2004||None||None|
Table 2: Geographic origin of Tunisian Culex pipiens populations, breeding site characteristics, and insecticide control.
CYP450, EST and/or GST enzymes accounted for only a small part of the observed resistances. Synergists tests confirmed biochemical study where four esterases were detected in studied samples with low and average frequencies (0.03%-0.68%). Moreover, the mortality due to propoxur was significantly correlated with the LC50 of chlorpyrifos (P<0.01) indicated an insensitive AChE 1. These results are consistent with those found in the world: resistances to OPs insecticides can be due to the detoxification of the product by enzymes or /and to a mutation at the targeted site, AChE 1[11,23,28-43].
Other groups of mosquitoes should be used to test their resistance to different chemical insecticides so that the control of vectors will successfully help in the reduction of incidence of vector-borne diseases.
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