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Review of Temephos Discriminating Concentration for Monitoring the Susceptibility of Anopheles labranchiae (Falleroni, 1926), Malaria Vector in Morocco

Review of Temephos Discriminating Concentration for Monitoring the Susceptibility of Anopheles... SAGE-Hindawi Access to Research Malaria Research and Treatment Volume 2010, Article ID 126085, 5 pages doi:10.4061/2010/126085 Research Article Review of Temephos Discriminating Concentration for Monitoring the Susceptibility of Anopheles labranchiae (Falleroni, 1926), Malaria Vector in Morocco 1 1 1 2 2 3 C. Faraj, E. Adlaoui, M. Elkohli, T. Herrak, B. Ameur, and F. Chandre Laboratoire d’Entomologie M´edicale, Institut National d’Hygi`ene, 27 Avenue Ibn Batouta, Agdal, Rabat 10090, Morocco Service de lutte Antivectorielle, Direction de l’Epid´emiologie et de Lutte contre les Maladies, Agdal, Rabat 10080, Morocco Institut de Recherche pour le D´eveloppement (IRD), UR016, Caract´erisation et Controle ˆ des Populations de Vecteurs, Montpellier Cedex 5, France Correspondence should be addressed to C. Faraj, chafikaf@gmail.com Received 30 September 2010; Accepted 3 December 2010 Academic Editor: Abdoulaye Diabate ´ Copyright © 2010 C. Faraj et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In Morocco, the resistance monitoring of Anopheles labranchiae larvae to temephos is done using discriminating concentration of 0.125 mg, which is half of the WHO recommended dose for Anopheles. However, this dosage seemed to be too high to allow an early detection of the resistance and its revision was found necessary. The present study was carried out during May-June 2008 and 2009 in nine provinces from the north-west of the country. The aim was to determine the lethal concentrations LC100 of temephos for the most susceptible populations and to define the discriminating dosage as the double of this value. The bioassays were conducted according to WHO standard operating protocol to establish the dose-mortality relationship and deduct the LC50 and LC95. The results of this study indicated that the LC100 obtained on the most susceptible populations was close to 0.05 mg/L. Therefore, the temephos discriminating dosage for susceptibility monitoring of An. labranchiae larvae in Moroccowas settobe 0.1 mg/L. 1. Introduction component of the NMCP. This activity has started with the launch of the programme following WHO protocol, using In Morocco, last autochthonous malaria case was registered discriminating dosage [3]. The first susceptibility tests of in 2004. Since then a vector control program was established Anopheles labranchiae to temephos were carried out before to prevent a possible return of malaria transmission. It its introduction for larval control using concentrations is mainly based on the larval control of the main vector: supplied by WHO (0.005, 0.025, 0.125, 0.625 mg/L). Results of these tests, carried out on natural populations, showed Anopheles labranchiae (Falleroni 1926) [1]. This control includes integrated management using environmental meth- that the lowest dosage involving regularly 100% mortality ods and larvivorous fish (Gambusia holbrooki) as biological was 0.125 mg/L. This concentration was considered as a specific discriminating dose for Moroccan An. labranchiae control. Insecticides are used as the last option but they take an important place in the National Malaria Control and was used in routine resistance monitoring. Although Programme (NMCP). this dose represents half of the diagnostic concentration Larval treatments started in the 1950’s using DDT [2]. In recommended by WHO (0.25 mg/L) [4]for Anopheles, this 1978, an organophosphate insecticide, the temephos (Abate is higher than the operational dosage of temephos that is still 500 EC) was introduced, and since then, it has been the only effective for larval control. Indeed, operational treatments insecticide used for An. labranchiae larval control. are made using a dosage of 50 g/ha, corresponding to a The monitoring of An. labranchiae susceptibility to concentration of 0.05to 0.1 mg/L for breeding sites, respec- tively, of about 10 and 5 cm of depth. The diagnostic dose of insecticides used in larval and adult control is an essential 2 Malaria Research and Treatment 0.125 mg/L seems to be too high to allow an early detection sets with mortalities different from 0 and 100% or with of the resistance, and so its revision was found necessary. mortality in control higher than 20% were not considered The present study was carried out to determine a more [12]. accurate diagnostic dose, for the larvae of An. labranchiae Results analysis was made using log-probit analysis soft- from Morocco, if possible lower than the operational dose ware (WinDL version 2.0) developed by CIRAD-CA/MABIS to early detect a significant reduction of insecticide suscepti- [13]. It allows calculating LC50, LC95, and their confidence bility among field populations. This study will also serve as intervals. reference data which could be applied in countries around Diagnostic concentration was calculated as the double of Mediterranean sea, particularly Algeria, Tunisia, Italy, and the observed LC100 of the most susceptible populations. France where An. labranchiae is present and could constitute a risk for malaria transmission [5–8]. 3. Results 2. Material and Methods A total of more than 7,000 larvae were tested for 18 different tests conducted in 12 villages. Among these bioassays, only 2.1. Study Areas. An. labranchiae is an eurygamic species. 12 were valid. Among the six tests considered as invalid, It is almost impossible to rear in insectarium and hence mortality in control was superior to 20% in 2 tests, and there difficult to have a susceptible reference strain to estimate its were 4 tests involving less than four sets with mortalities baseline susceptibility. So, we attempted to search for a wild different from 0 and 100%. population as susceptible as possible, in regions where the Results of bioassays are presented in Table 2. selection pressure by insecticide treatment was low. Pressure The LC95 varies from 0.036 to 0.105 mg/L among of organophosphate (OP) used in public health is low as the different populations, representing a ratio less than temephos is the first and the only OP used in Anopheles 3 folds. The most susceptible populations were collected, larvae control, and DDT was the only insecticide used in respectively, in Benslimane and Meknes. The least susceptible adult control. However, pressure of insecticides used in populations were collected in Khemisset and Larache. agriculture could be important because An. labranchiae is The lowest concentration involving 100% of mortality on abundant in the agricultural zones particularly in the north the natural populations is of 0.0625 mg/L. It was obtained western of the country [9] where the main culture practices on Ben Slimane and Boucharen populations. As the highest are wheat, corn, sugar cane, and rice cultivation. To mitigate tested concentration producing a mortality less than 100% this way, larvae were collected in sites as far as possible from is 0.025 mg/L, the real LC100 should be situated between cultivation areas. Hence, the study was carried out in nine 0.025 mg/L and 0.0625 mg/L. provinces (Figure 1). Concentration of 0.125 mg/L did not involve 100% mortality in the population of Sbih (Sidi Kacem) suggesting the emergence of a beginning of resistance. 2.2. Mosquitoes. Because Morocco is in a phase of prevention of malaria reintroduction, the vector An. labranchiae is submitted to regular controls. Consequently, its density is 4. Discussion low and it was not easy to find many positive breeding sites in sufficient density to realize bioassays. We were then forced to The standardized bioassays on larvae or adults using a realize tests in sites where density was allowable, generally far discriminating dosage of insecticides are largely employed for from villages. Larvae were collected, using standard dipping the resistance monitoring of mosquito populations targeted method, in different biotopes (swamps, rivers, rice fields) by vector control programmes [14]. This method has the during May-June 2008 and 2009 (Table 1). Specimens were advantage of being simply used, fast, and inexpensive, as identified morphologically [10]. well as of giving reproducible results and requiring only a small number of specimens compared to the conventional 2.3. Bioassays. Bioassays were carried out following WHO bioassays. standard procedures to establish the dose-mortality relation- The discriminating dosage corresponds to the theoretical ship and to calculate lethal concentrations LC50 and LC95 LC100 of susceptible individuals obtained by extrapolating (concentrations involving, resp., the death of 50% and 95% the regression curve (probit mortality/log dosage) to a of the tested population) [11]. Tests were carried out on the mortality rate of 99.9% [15]. In practice andtokeepasafety third and fourth instar larvae. Ranges of 5 to 6 concentrations margin, this dosage corresponds to twice the LC99.9% or of temephos and control were prepared to determine the preferentially twice the lowest dosage giving 100% of mortal- LC50 and LC95 for each population. For each dilution, 3 to 4 ity of a fully susceptible population, since the LC99.9% has a replicates were done, with 15 to 25 larvae each. Larvae were low statistical value when the slope of probit line is low or the placed in 99 mL of water; 1 mL of adequate concentration of population is heterogeneous. temephos was then added. After 24 hours of exposition at The temephos discriminating dosage, established by ambient temperature (21-22 C) without feeding, alive and WHO, for the genus Anopheles is 0.25 mg/L. Afterward, it dead larvae were counted. When it was possible, 2 tests were was stated locally for a limited number of species [16]. conducted on the same population in the same experimental For An. Hyrcanus, this dose was set at 0.025 mg/L and it conditions in 2 different days. Tests involving less than four was 0.625 mg/L for An. sacharov that belongs to the same Malaria Research and Treatment 3 Mediterranean sea Larache 0 100 200 400 (Km) Sidi Kacem Rabat Skhirat Fes Temara Sale Meknes Khemisset Ben Slimane 0 20 40 80 120 160 (Km) Figure 1: Map of Morocco showing provinces where An. labranchiae populations were sampled. Table 1: Details of larval breeding sites which were sampled. Geographical coordinates Population Province Breeding site Type of breeding site Longitude Latitude ◦  ◦ Sidi Allal Msader Khemisset 06 02W33 44NRiver Permanent ◦  ◦ Beggara Larache 06 06W35 10 N Swamps Permanent ◦  ◦ Boucharen Larache 06 04W35 07NRicefield Temporary ◦  ◦ Ain Aghbal Meknes 05 27W33 40NRiver Permanent ◦  ◦ Ben Slimane Ben Slimane 07 04W33 38NDaya Temporary ◦  ◦ Skhirat Skhirat-Temara 07 05W33 50 N Swamps Permanent ◦  ◦ Ain Elouali Fes 004 54W34 07NRiver Permanent ◦  ◦ Rabat Rabat 06 46W33 58 N Swamp Temporary ◦  ◦ Sbih Sidi Kacem 05 26W34 24 N Swamps Permanent ◦  ◦ Sehoul Sale 06 35W33 51NSwamps Temporary complex as An. labranchiae. In Morocco, before the first Bioassays carried out by resistance monitoring sentinel use of temephos, 0.025 mg/L did not involve systematically sites of Khemisset, Meknes, and Tetouan between 2004 100% mortality on populations having never been in contact and 2008 showed that several populations used to have a with this product, and so they were expected to be fully LC100 sometimes equal to 0.025 mg/L or slightly higher. susceptible. The discriminating dosage would have been Accordingly, the LC100 of the most susceptible populations around or higher than 0.05 mg/L. As a compromise the in Morocco was found to be 0.05 mg/L. This dose allows concentration of 0.125 mg/L was chosen and was probably for determining the diagnostic dosage of temephos for superior to the real discriminating dosage. An. labranchiae to be 0.1 mg/L, which is finally very close Atlantic ocean 4 Malaria Research and Treatment Table 2: Susceptibility of An. labranchiae larvae to temephos in mg/L. Province Population LC50 (CI) LC95 (CI) LC100 0.0198 (0.0052–0.0947) 0.1058 (0.0407–0.4210) >0.05 Khemisset Sidi Allal Msader 0.0196 (0.0016–0.0295) 0.0676 (0.0413–0.3013) >0.05 0.0098 (0.0074–0.0121) 0.0853 (0.0571–0.1632) >0.05 Sale Shoul 0.0094 (0.0078–0.0112) 0.0693 (0.0500–0.1090) >0.05 Boucharen 0.0079 (0.0061–0.0101) 0.0586 (0.0391–0.1088) 0.0625 Larache Beggara 0.0186 (0.0127–0.0262) 0.1002 (0.0622–0.2301) 0.1250 0.0084 (0.0072–0.0098) 0.0362 (0.0288–0.0491) 0.0625 Ben Slimane Ben Slimane 0.0208 (0.0186–0.0233) 0.0537 (0.0447–0.0692) 0.1250 Skhirat-Temara Skhirat 0.0067 (0.0055–0.0080) 0.0485 (0.0366–0.0703) 0.1250 Rabat Rabat 0.0188 (0.0073–0.0343) 0.0688 (0.0368–0.9785) 0.1250 Sidi Kacem Sbih 0.0301 (0.0223–0.0399) 0.0967 (0.0663–0.1961) 0.2500 Meknes Ain Aghbal 0.0178 (0.0160–0.0197) 0.0371 (0.0314–0.0480) 0.1250 LC50/LC95: lethal concentrations for 50% and 95% of larvae with confidence intervals (CI) at 5% level. LC100: observed concentration involving 100% of mortality. to the diagnostic concentration previously used by the References national control programme and which could be higher than [1] C. Faraj, E. Adlaoui, S. Ouahabi, M. Rhajaoui, D. Fontenille, operational dose. and M. Lyagoubi, “Entomological investigations in the region Results of this study show that, with the exception of Sidi of the last malaria focus in Morocco,” Acta Tropica, vol. 109, Kacem population where a low resistance was suspected, all no. 1, pp. 70–73, 2009. tested populations are still susceptible to temephos. Although [2] WHO, “Malaria in Morocco: Relentless efforts towards the temephos is used in vector control in Morocco within goal of elimination,” WHO-EM/MAL/345/E, 2007. the framework of a resistance management programme [3] C. Faraj, E. Adlaoui, C. Brengues, D. Fontenille, and M. which privileges the environmental control and the use of Lyagoubi, “Resistanc ´ e d’Anopheles labranchiae au DDT au larvivorous fishes as biological control, it is essential that the Maroc: Identification des mecanismes ´ et choix d’un insecti- NMCP have replacement insecticides before the emergence cide de remplacement,” Eastern Mediterranean Health Journal, of Anopheles labranchiae resistance to temephos and the vol. 14, no. 4, pp. 776–783, 2008. failure of the present vector control program. Attention [4] OMS, R´esistance aux pesticides des vecteurs et r´eservoirs de maladies. Dixi`eme rapport du comit´eOMS d’expertsdela must be steered towards the use of microbial control biologie des vecteurs et de lute antivectorielle, vol. 737 of S´erie agents and insect growth inhibitors taking into account cost de rapports Techniques, Organisation Mondiale de la Sante, ´ effectiveness, feasibility, and respect of the nontarget fauna Geneva, Switzerland, 1986. and the environment. [5] R. Romi, “Anopheles labranchiae, an important malaria vector The adoption of this discriminating dosage will allow in Italy, and other potential malaria vectors in Southern simplifying the procedure for monitoring the susceptibility Europe,” European Mosquito Bulletin, vol. 4, pp. 8–10, 1999. of An. labranchiae to temephos in the different provinces of [6] C. Toty, H. Barre, ´ G. Le Goff et al., “Malaria risk in Corsica, Morocco. former hot spot of malaria in France,” Malaria Journal, vol. 9, no. 1, article 231, 2010. [7] M. K. Chahed, A. Bouratbine, G. Krida, and A. Ben Hamida, Conflict of Interests “ec ´ eptivite ´ de la Tunisie au paludisme apres ` son eradication: The authors declare that they have no conflict of interests. Analyse de la situation pour une adequation ´ de la surveil- lance,” Bulletin de la Soci´et´edePathologieExotique, vol. 94, no. 3, pp. 271–276, 2001. Acknowledgments [8] D. Hammadi, S. C. Boubidi, S. E. Chaib et al., “Le paludisme au Sahara alger ´ ien,” Bulletin de la Societe de Pathologie The authors wish to thank Dr. Abraham Mnzava from Exotique, vol. 102, no. 3, pp. 185–192, 2009. WHO/EMRO for his support. They are thankful to the [9] C. Faraj, S. Ouahabi, E. Adlaoui, and R. Elaouad, “Current staff of Medical Entomology Laboratory and Vector Control status of the knowledge on Moroccan anophelines (Diptera: Service: Souad Ouahabi, Elhousseine Lakraa, Mohammed Culicidae): Systematic, geographical distribution and vectorial ElRhazi, Hassan Nachi, Mohammed Bouhrara for their competence,” Epidemiology and Public Health,vol. 58, no.5, assistance in field work and rearing mosquitoes. They thank pp. 349–357, 2010. Omar Lahlou (Khemisset), Amal Aboulkacem (Meknes), [10] J. Brunhes, A. Rhaim, B. Geoffroy, G. Angel, and J. P. Abderrahim (Larache), Abdelhakim El Ouali Lalami (Fes), Hervy, “Les moustiques de l’Afrique medit ´ erraneenne. ´ Logi- (Benslimlane), and Abdelhaq Grine (Sidi Kacem) for assis- ciel d’identification et d’enseignement,” Montpellier, France, tance in the field surveys. This investigation received financial IRD & IPT, CD-Rom collection didactique, Editions IRD; assistance from WHO. Malaria Research and Treatment 5 [11] WHO, “Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides,” Report of the WHO Expert Committee on Resistance of Vectors and Reser- voirs of Diseases to Pesticides, WHO, Geneva, Switzerland, [12] W. S. Abbott, “A method of computing the effectiveness of an insecticide,” Journal of Economic Entomology, vol. 18, pp. 265– 267, 1925. [13] M. Giner, J. M. Vassal, C. Vassal, F. Chiroleu, and Z. Kouaik, Logiciel, CIRAD. URBI/MABIS, Montpellier, France. [14] F. Chandre, R´esistance d’Anopheles gambiae Giles et de Culex pipiens quinquefasciatus Say aux insecticides en Afrique de l’Ouest et implications op´erationnelles, Ph.D. thesis, Universite ´ Paris XII Val-de-Marne, Paris, France, 1998. [15] WHO, “Guidelines for laboratory and field testing of mosquito larvicides,” WHO/CDS/WHOPES/GCDPP/2005, WHO, Geneva, Switzerland, 2005. [16] WHO, “Vector resistance to pesticides,” 15th report of the WHO Expert Committee on Vector Biology and Control, Technical Report Series 818, WHO, Geneva, Switzerland, 1992. 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Review of Temephos Discriminating Concentration for Monitoring the Susceptibility of Anopheles labranchiae (Falleroni, 1926), Malaria Vector in Morocco

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Copyright © 2010 C. Faraj et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SAGE-Hindawi Access to Research Malaria Research and Treatment Volume 2010, Article ID 126085, 5 pages doi:10.4061/2010/126085 Research Article Review of Temephos Discriminating Concentration for Monitoring the Susceptibility of Anopheles labranchiae (Falleroni, 1926), Malaria Vector in Morocco 1 1 1 2 2 3 C. Faraj, E. Adlaoui, M. Elkohli, T. Herrak, B. Ameur, and F. Chandre Laboratoire d’Entomologie M´edicale, Institut National d’Hygi`ene, 27 Avenue Ibn Batouta, Agdal, Rabat 10090, Morocco Service de lutte Antivectorielle, Direction de l’Epid´emiologie et de Lutte contre les Maladies, Agdal, Rabat 10080, Morocco Institut de Recherche pour le D´eveloppement (IRD), UR016, Caract´erisation et Controle ˆ des Populations de Vecteurs, Montpellier Cedex 5, France Correspondence should be addressed to C. Faraj, chafikaf@gmail.com Received 30 September 2010; Accepted 3 December 2010 Academic Editor: Abdoulaye Diabate ´ Copyright © 2010 C. Faraj et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In Morocco, the resistance monitoring of Anopheles labranchiae larvae to temephos is done using discriminating concentration of 0.125 mg, which is half of the WHO recommended dose for Anopheles. However, this dosage seemed to be too high to allow an early detection of the resistance and its revision was found necessary. The present study was carried out during May-June 2008 and 2009 in nine provinces from the north-west of the country. The aim was to determine the lethal concentrations LC100 of temephos for the most susceptible populations and to define the discriminating dosage as the double of this value. The bioassays were conducted according to WHO standard operating protocol to establish the dose-mortality relationship and deduct the LC50 and LC95. The results of this study indicated that the LC100 obtained on the most susceptible populations was close to 0.05 mg/L. Therefore, the temephos discriminating dosage for susceptibility monitoring of An. labranchiae larvae in Moroccowas settobe 0.1 mg/L. 1. Introduction component of the NMCP. This activity has started with the launch of the programme following WHO protocol, using In Morocco, last autochthonous malaria case was registered discriminating dosage [3]. The first susceptibility tests of in 2004. Since then a vector control program was established Anopheles labranchiae to temephos were carried out before to prevent a possible return of malaria transmission. It its introduction for larval control using concentrations is mainly based on the larval control of the main vector: supplied by WHO (0.005, 0.025, 0.125, 0.625 mg/L). Results of these tests, carried out on natural populations, showed Anopheles labranchiae (Falleroni 1926) [1]. This control includes integrated management using environmental meth- that the lowest dosage involving regularly 100% mortality ods and larvivorous fish (Gambusia holbrooki) as biological was 0.125 mg/L. This concentration was considered as a specific discriminating dose for Moroccan An. labranchiae control. Insecticides are used as the last option but they take an important place in the National Malaria Control and was used in routine resistance monitoring. Although Programme (NMCP). this dose represents half of the diagnostic concentration Larval treatments started in the 1950’s using DDT [2]. In recommended by WHO (0.25 mg/L) [4]for Anopheles, this 1978, an organophosphate insecticide, the temephos (Abate is higher than the operational dosage of temephos that is still 500 EC) was introduced, and since then, it has been the only effective for larval control. Indeed, operational treatments insecticide used for An. labranchiae larval control. are made using a dosage of 50 g/ha, corresponding to a The monitoring of An. labranchiae susceptibility to concentration of 0.05to 0.1 mg/L for breeding sites, respec- tively, of about 10 and 5 cm of depth. The diagnostic dose of insecticides used in larval and adult control is an essential 2 Malaria Research and Treatment 0.125 mg/L seems to be too high to allow an early detection sets with mortalities different from 0 and 100% or with of the resistance, and so its revision was found necessary. mortality in control higher than 20% were not considered The present study was carried out to determine a more [12]. accurate diagnostic dose, for the larvae of An. labranchiae Results analysis was made using log-probit analysis soft- from Morocco, if possible lower than the operational dose ware (WinDL version 2.0) developed by CIRAD-CA/MABIS to early detect a significant reduction of insecticide suscepti- [13]. It allows calculating LC50, LC95, and their confidence bility among field populations. This study will also serve as intervals. reference data which could be applied in countries around Diagnostic concentration was calculated as the double of Mediterranean sea, particularly Algeria, Tunisia, Italy, and the observed LC100 of the most susceptible populations. France where An. labranchiae is present and could constitute a risk for malaria transmission [5–8]. 3. Results 2. Material and Methods A total of more than 7,000 larvae were tested for 18 different tests conducted in 12 villages. Among these bioassays, only 2.1. Study Areas. An. labranchiae is an eurygamic species. 12 were valid. Among the six tests considered as invalid, It is almost impossible to rear in insectarium and hence mortality in control was superior to 20% in 2 tests, and there difficult to have a susceptible reference strain to estimate its were 4 tests involving less than four sets with mortalities baseline susceptibility. So, we attempted to search for a wild different from 0 and 100%. population as susceptible as possible, in regions where the Results of bioassays are presented in Table 2. selection pressure by insecticide treatment was low. Pressure The LC95 varies from 0.036 to 0.105 mg/L among of organophosphate (OP) used in public health is low as the different populations, representing a ratio less than temephos is the first and the only OP used in Anopheles 3 folds. The most susceptible populations were collected, larvae control, and DDT was the only insecticide used in respectively, in Benslimane and Meknes. The least susceptible adult control. However, pressure of insecticides used in populations were collected in Khemisset and Larache. agriculture could be important because An. labranchiae is The lowest concentration involving 100% of mortality on abundant in the agricultural zones particularly in the north the natural populations is of 0.0625 mg/L. It was obtained western of the country [9] where the main culture practices on Ben Slimane and Boucharen populations. As the highest are wheat, corn, sugar cane, and rice cultivation. To mitigate tested concentration producing a mortality less than 100% this way, larvae were collected in sites as far as possible from is 0.025 mg/L, the real LC100 should be situated between cultivation areas. Hence, the study was carried out in nine 0.025 mg/L and 0.0625 mg/L. provinces (Figure 1). Concentration of 0.125 mg/L did not involve 100% mortality in the population of Sbih (Sidi Kacem) suggesting the emergence of a beginning of resistance. 2.2. Mosquitoes. Because Morocco is in a phase of prevention of malaria reintroduction, the vector An. labranchiae is submitted to regular controls. Consequently, its density is 4. Discussion low and it was not easy to find many positive breeding sites in sufficient density to realize bioassays. We were then forced to The standardized bioassays on larvae or adults using a realize tests in sites where density was allowable, generally far discriminating dosage of insecticides are largely employed for from villages. Larvae were collected, using standard dipping the resistance monitoring of mosquito populations targeted method, in different biotopes (swamps, rivers, rice fields) by vector control programmes [14]. This method has the during May-June 2008 and 2009 (Table 1). Specimens were advantage of being simply used, fast, and inexpensive, as identified morphologically [10]. well as of giving reproducible results and requiring only a small number of specimens compared to the conventional 2.3. Bioassays. Bioassays were carried out following WHO bioassays. standard procedures to establish the dose-mortality relation- The discriminating dosage corresponds to the theoretical ship and to calculate lethal concentrations LC50 and LC95 LC100 of susceptible individuals obtained by extrapolating (concentrations involving, resp., the death of 50% and 95% the regression curve (probit mortality/log dosage) to a of the tested population) [11]. Tests were carried out on the mortality rate of 99.9% [15]. In practice andtokeepasafety third and fourth instar larvae. Ranges of 5 to 6 concentrations margin, this dosage corresponds to twice the LC99.9% or of temephos and control were prepared to determine the preferentially twice the lowest dosage giving 100% of mortal- LC50 and LC95 for each population. For each dilution, 3 to 4 ity of a fully susceptible population, since the LC99.9% has a replicates were done, with 15 to 25 larvae each. Larvae were low statistical value when the slope of probit line is low or the placed in 99 mL of water; 1 mL of adequate concentration of population is heterogeneous. temephos was then added. After 24 hours of exposition at The temephos discriminating dosage, established by ambient temperature (21-22 C) without feeding, alive and WHO, for the genus Anopheles is 0.25 mg/L. Afterward, it dead larvae were counted. When it was possible, 2 tests were was stated locally for a limited number of species [16]. conducted on the same population in the same experimental For An. Hyrcanus, this dose was set at 0.025 mg/L and it conditions in 2 different days. Tests involving less than four was 0.625 mg/L for An. sacharov that belongs to the same Malaria Research and Treatment 3 Mediterranean sea Larache 0 100 200 400 (Km) Sidi Kacem Rabat Skhirat Fes Temara Sale Meknes Khemisset Ben Slimane 0 20 40 80 120 160 (Km) Figure 1: Map of Morocco showing provinces where An. labranchiae populations were sampled. Table 1: Details of larval breeding sites which were sampled. Geographical coordinates Population Province Breeding site Type of breeding site Longitude Latitude ◦  ◦ Sidi Allal Msader Khemisset 06 02W33 44NRiver Permanent ◦  ◦ Beggara Larache 06 06W35 10 N Swamps Permanent ◦  ◦ Boucharen Larache 06 04W35 07NRicefield Temporary ◦  ◦ Ain Aghbal Meknes 05 27W33 40NRiver Permanent ◦  ◦ Ben Slimane Ben Slimane 07 04W33 38NDaya Temporary ◦  ◦ Skhirat Skhirat-Temara 07 05W33 50 N Swamps Permanent ◦  ◦ Ain Elouali Fes 004 54W34 07NRiver Permanent ◦  ◦ Rabat Rabat 06 46W33 58 N Swamp Temporary ◦  ◦ Sbih Sidi Kacem 05 26W34 24 N Swamps Permanent ◦  ◦ Sehoul Sale 06 35W33 51NSwamps Temporary complex as An. labranchiae. In Morocco, before the first Bioassays carried out by resistance monitoring sentinel use of temephos, 0.025 mg/L did not involve systematically sites of Khemisset, Meknes, and Tetouan between 2004 100% mortality on populations having never been in contact and 2008 showed that several populations used to have a with this product, and so they were expected to be fully LC100 sometimes equal to 0.025 mg/L or slightly higher. susceptible. The discriminating dosage would have been Accordingly, the LC100 of the most susceptible populations around or higher than 0.05 mg/L. As a compromise the in Morocco was found to be 0.05 mg/L. This dose allows concentration of 0.125 mg/L was chosen and was probably for determining the diagnostic dosage of temephos for superior to the real discriminating dosage. An. labranchiae to be 0.1 mg/L, which is finally very close Atlantic ocean 4 Malaria Research and Treatment Table 2: Susceptibility of An. labranchiae larvae to temephos in mg/L. Province Population LC50 (CI) LC95 (CI) LC100 0.0198 (0.0052–0.0947) 0.1058 (0.0407–0.4210) >0.05 Khemisset Sidi Allal Msader 0.0196 (0.0016–0.0295) 0.0676 (0.0413–0.3013) >0.05 0.0098 (0.0074–0.0121) 0.0853 (0.0571–0.1632) >0.05 Sale Shoul 0.0094 (0.0078–0.0112) 0.0693 (0.0500–0.1090) >0.05 Boucharen 0.0079 (0.0061–0.0101) 0.0586 (0.0391–0.1088) 0.0625 Larache Beggara 0.0186 (0.0127–0.0262) 0.1002 (0.0622–0.2301) 0.1250 0.0084 (0.0072–0.0098) 0.0362 (0.0288–0.0491) 0.0625 Ben Slimane Ben Slimane 0.0208 (0.0186–0.0233) 0.0537 (0.0447–0.0692) 0.1250 Skhirat-Temara Skhirat 0.0067 (0.0055–0.0080) 0.0485 (0.0366–0.0703) 0.1250 Rabat Rabat 0.0188 (0.0073–0.0343) 0.0688 (0.0368–0.9785) 0.1250 Sidi Kacem Sbih 0.0301 (0.0223–0.0399) 0.0967 (0.0663–0.1961) 0.2500 Meknes Ain Aghbal 0.0178 (0.0160–0.0197) 0.0371 (0.0314–0.0480) 0.1250 LC50/LC95: lethal concentrations for 50% and 95% of larvae with confidence intervals (CI) at 5% level. LC100: observed concentration involving 100% of mortality. to the diagnostic concentration previously used by the References national control programme and which could be higher than [1] C. Faraj, E. Adlaoui, S. Ouahabi, M. Rhajaoui, D. Fontenille, operational dose. and M. Lyagoubi, “Entomological investigations in the region Results of this study show that, with the exception of Sidi of the last malaria focus in Morocco,” Acta Tropica, vol. 109, Kacem population where a low resistance was suspected, all no. 1, pp. 70–73, 2009. tested populations are still susceptible to temephos. Although [2] WHO, “Malaria in Morocco: Relentless efforts towards the temephos is used in vector control in Morocco within goal of elimination,” WHO-EM/MAL/345/E, 2007. the framework of a resistance management programme [3] C. Faraj, E. Adlaoui, C. Brengues, D. Fontenille, and M. which privileges the environmental control and the use of Lyagoubi, “Resistanc ´ e d’Anopheles labranchiae au DDT au larvivorous fishes as biological control, it is essential that the Maroc: Identification des mecanismes ´ et choix d’un insecti- NMCP have replacement insecticides before the emergence cide de remplacement,” Eastern Mediterranean Health Journal, of Anopheles labranchiae resistance to temephos and the vol. 14, no. 4, pp. 776–783, 2008. failure of the present vector control program. Attention [4] OMS, R´esistance aux pesticides des vecteurs et r´eservoirs de maladies. Dixi`eme rapport du comit´eOMS d’expertsdela must be steered towards the use of microbial control biologie des vecteurs et de lute antivectorielle, vol. 737 of S´erie agents and insect growth inhibitors taking into account cost de rapports Techniques, Organisation Mondiale de la Sante, ´ effectiveness, feasibility, and respect of the nontarget fauna Geneva, Switzerland, 1986. and the environment. [5] R. Romi, “Anopheles labranchiae, an important malaria vector The adoption of this discriminating dosage will allow in Italy, and other potential malaria vectors in Southern simplifying the procedure for monitoring the susceptibility Europe,” European Mosquito Bulletin, vol. 4, pp. 8–10, 1999. of An. labranchiae to temephos in the different provinces of [6] C. Toty, H. Barre, ´ G. Le Goff et al., “Malaria risk in Corsica, Morocco. former hot spot of malaria in France,” Malaria Journal, vol. 9, no. 1, article 231, 2010. [7] M. K. Chahed, A. Bouratbine, G. Krida, and A. Ben Hamida, Conflict of Interests “ec ´ eptivite ´ de la Tunisie au paludisme apres ` son eradication: The authors declare that they have no conflict of interests. Analyse de la situation pour une adequation ´ de la surveil- lance,” Bulletin de la Soci´et´edePathologieExotique, vol. 94, no. 3, pp. 271–276, 2001. Acknowledgments [8] D. Hammadi, S. C. Boubidi, S. E. Chaib et al., “Le paludisme au Sahara alger ´ ien,” Bulletin de la Societe de Pathologie The authors wish to thank Dr. Abraham Mnzava from Exotique, vol. 102, no. 3, pp. 185–192, 2009. WHO/EMRO for his support. They are thankful to the [9] C. Faraj, S. Ouahabi, E. Adlaoui, and R. Elaouad, “Current staff of Medical Entomology Laboratory and Vector Control status of the knowledge on Moroccan anophelines (Diptera: Service: Souad Ouahabi, Elhousseine Lakraa, Mohammed Culicidae): Systematic, geographical distribution and vectorial ElRhazi, Hassan Nachi, Mohammed Bouhrara for their competence,” Epidemiology and Public Health,vol. 58, no.5, assistance in field work and rearing mosquitoes. They thank pp. 349–357, 2010. Omar Lahlou (Khemisset), Amal Aboulkacem (Meknes), [10] J. Brunhes, A. Rhaim, B. Geoffroy, G. Angel, and J. P. Abderrahim (Larache), Abdelhakim El Ouali Lalami (Fes), Hervy, “Les moustiques de l’Afrique medit ´ erraneenne. ´ Logi- (Benslimlane), and Abdelhaq Grine (Sidi Kacem) for assis- ciel d’identification et d’enseignement,” Montpellier, France, tance in the field surveys. This investigation received financial IRD & IPT, CD-Rom collection didactique, Editions IRD; assistance from WHO. Malaria Research and Treatment 5 [11] WHO, “Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides,” Report of the WHO Expert Committee on Resistance of Vectors and Reser- voirs of Diseases to Pesticides, WHO, Geneva, Switzerland, [12] W. S. Abbott, “A method of computing the effectiveness of an insecticide,” Journal of Economic Entomology, vol. 18, pp. 265– 267, 1925. [13] M. Giner, J. M. Vassal, C. Vassal, F. Chiroleu, and Z. Kouaik, Logiciel, CIRAD. URBI/MABIS, Montpellier, France. [14] F. Chandre, R´esistance d’Anopheles gambiae Giles et de Culex pipiens quinquefasciatus Say aux insecticides en Afrique de l’Ouest et implications op´erationnelles, Ph.D. thesis, Universite ´ Paris XII Val-de-Marne, Paris, France, 1998. [15] WHO, “Guidelines for laboratory and field testing of mosquito larvicides,” WHO/CDS/WHOPES/GCDPP/2005, WHO, Geneva, Switzerland, 2005. [16] WHO, “Vector resistance to pesticides,” 15th report of the WHO Expert Committee on Vector Biology and Control, Technical Report Series 818, WHO, Geneva, Switzerland, 1992. 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