Organophosphate and carbamate susceptibility profiling of Anopheles gambiae sl. across different ecosystems in southern Benin

BackgroundTo overcome the spread of high pyrethroid resistance in the main malaria vectors and malaria disease persistence, it is crucial to look for effective and better resistance management strategies. Understanding the phenotypic profile ofAnopheles gambiae sl. against alternatives insecticides like organophosphates and carbamates is crucial.MethodsAnopheles larvae and pupae were collected from the breeding sites in rice fields, pineapple crop areas, and peri-urban areas. WHO susceptibility tests were conducted on unfed female mosquitoes aged 3–5 days old. Mosquitoes were exposed to malathion 5%, pirimiphos-methyl 0.25%, and bendiocarb 0.1% using the standard WHO protocol. Polymerase chain reaction (PCR) techniques were used to detect species,kdr andAce-1 mutations.ResultsAnopheles gambiae sl. from Sèdjè-Dénou rice field population was resistant to bendiocarb (0.1%) with a mortality rate of 72.2% whereasAnopheles gambiae sl. populations from Zinvié-Dokomey (rice field), Zè-Tozounmè (pineapple field), and Adjagbo (peri-urban area) were suspected to be resistant with mortality rates of 90%, 93.5%, 95.4% respectively. However, all of them were susceptible to organophosphates (malathion and pirimiphos-methyl) with a mortality rate of 100%. PCR assay revealed that 100% of the mosquitoes tested wereAnopheles coluzzii. The frequencies ofAce-1R mutation in allAnopheles coluzzii populations tested were low (3–27%).ConclusionsOrganophosphates (malathion and pirimiphos-methyl) have maintained their efficacy againstAnopheles coluzzii populations from Sèdjè-Dénou (rice field), Zè Tozounmè (pineapple field), Zinvié Dokomey (rice field), or Adjagbo (peri-urban area). The good efficacy of these organophosphates againstAnopheles coluzzii populations from the southern part of Benin are observed in the current study. The use of pirimiphos-methyl for IRS in this part of the country would be a successful alternative for malaria control in this area.

BackgroundThe detection of insecticide resistance in natural populations of Anopheles vectors is absolutely necessary for malaria control. In the African region, the WHO insecticide susceptibility test is the most common method for assessing resistance status. In order to search for a simple, rapid and more reliable technique in the assessment of insecticide resistance in malaria vectors, we compared the WHO tests with the CDC bottle bioassay in the Ouemé province of southern Benin where insecticide resistance has been widely reported.MethodsLarvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Ouemé. WHO and CDC susceptibility tests were conducted simultaneously on unfed female mosquitoes aged 2–5 days old. WHO bioassays were performed with impregnated papers of deltamethrin (0.05%) and bendiocarb (0.1%), whereas CDC bioassays were performed with stock solutions of deltamethrin (12.5 μg per bottle) and bendiocarb (12.5 μg per bottle). PCR techniques were used to detect species, Kdr and Ace-1 mutations. CDC biochemical assays using synergists were also conducted to assess the metabolic resistance.ResultsA slight decrease in mortality rates was observed with 97.95% and 98.33% obtained from CDC and WHO bioassays respectively in populations of mosquitoes from Adjara and Dangbo. PCR revealed that all specimens tested were Anopheles gambiae s.s. The Kdr mutation was found at high frequency in all populations and both the Kdr mutation and mono-oxygenase enzymes were implicated as mechanisms of pyrethroid resistance in An. gambiae from Misserete.ConclusionThis study emphasizes that both WHO and CDC bioassays give similar results with regards to the susceptibility of mosquitoes to insecticides in southern Benin. There were complementarities between both methods, however, some specificity was noted for each of the two methods used. Both Kdr and metabolic mechanisms were implicated in the resistance.

Anopheles gambiae, which is the main malaria vector in Benin has developed high level of resistance to pyrethroid insecticides. This raises serious concerns to the future use of long- lasting insecticidal nets (LLIN) and indoor residual spraying (IRS). It is therefore important to seek better and effective resistance management strategies which will use organophosphates or carbamates as alternatives against pyrethroid resistant malaria vectors in the field. Larvae and pupae of A. gambiae s.l. mosquitoes were collected from the breeding sites in Dassa-Zoume and Zogbodomey districts. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with fenitrothion 1%, pirimiphos-methyl 0.25%, and bendiocarb 0.1%. Polymerase chain reaction (PCR) techniques were used to detect species and Ace-1 mutations. A. gambiae Dassa-Zoume populations were susceptible to bendiocarb 0.1% with mortality rate of 99%. A. gambiae Zogbodomey populations were susceptible to pirimiphos-methyl 0.25% and fenitrothion 1% with mortality rates of 98.96 and 99%, respectively. PCR assay revealed that 100% of mosquitoes tested were A. gambiae s.s. The frequencies of Ace-1R mutation in A. gambiae Dassa-Zoume and Zogbodomey were 0%. Carbamates (bendiocarb) and organophosphates (fenitrothion and pirimiphos-methyl) have maintained their efficiency against A. gambiae Dassa-Zoume and Zogbodomey populations. Carbamates (bendiocarb) and organophosphates (fenitrothion and pirimiphos-methyl) have proven to be powerful alternatives against pyrethroid resistant malaria vectors such as A. gambiae Dassa-Zoume and Zogbodomey populations. The use of any of these three compounds in the centre Benin would be successful in malaria vector control.   Key   words:   Anopheles gambiae,   Ace-1,   resistance,   fenitrothion,   pirimiphos-methyl, bendiocarb, Benin.

The current study was aimed to investigate on dynamics of propoxur resistance in Anopheles gambiae s.l. populations from N’ dali district in northern Benin (West Africa) and also to investigate on dynamics of malathion resistance in Anopheles gambiae s. l. populations from Toffo district in southern Benin. Larvae and pupae of Anopheles gambiae s. l . mosquitoes were collected from the breeding sites in Borgou and Atlantic departments in 2015 and 2019. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with propoxur 0.1% and with malathion 5%. PCR techniques were used to detect species and Ace-1 mutations in 2015. Anopheles gambiae s. l. populations from N’dali were resistant to propoxur in 2015 and were still remained resistant to this product in 2019. Regarding Anopheles gambiae s. l. populations from Toffo, they were susceptible to malathion in 2015 whereas the malathion resistance status of these mosquitoes requires further investigation in 2019. PCR revealed that all specimens tested were Anopheles gambiae s. s. The presence of Ace-1R at very low frequency (0.01) was observed in Anopheles gambiae s. l. populations from both districts. This study shows that propoxur resistance detected in An. gambiae s. l. populations from N’ dali needs to be monitored for insecticide resistance in this area.

BackgroundPyrethroids are the main insecticides used in vector control for malaria. However, their extensive use in the impregnation of long-lasting insecticidal nets (LLINs) and indoor residual spraying has led to the development of resistance, threatening its success as a tool for malaria control. Baseline data prior to large scale distribution of LLINs are important for the implementation of efficient strategies. However, no data on the susceptibility of malaria vectors is available in the Moyen-Ogooué Province in Gabon. The aim of this study was to assess the susceptibility to pyrethroids and organochlorides of malaria vectors from a semi-urban and rural areas of the province and to determine the frequency of insecticide resistance genes.MethodsLarvae were collected from breeding sites in Lambaréné and Zilé and reared to adults. Three to five-day old female Anopheles gambiae sensu lato mosquitoes were used in cone tube assays following the WHO susceptibility tests protocol for adult mosquitoes. A subsample was molecularly identified using the SINE200 protocol and the frequency of Vgsc-1014 F and − 1014 S mutations were determined.ResultsAnopheles gambiae sensu stricto (s.s.) was the sole species present in both Lambaréné and Zilé. Mosquito populations from the two areas were resistant to pyrethroids and organochlorides. Resistance was more pronounced for permethrin and DDT with mortality lower than 7% for both insecticides in the two study areas. Mosquitoes were statistically more resistant (P < 0.0001) to deltamethrin in Lambaréné (51%) compared to Zilé (76%). All the mosquitoes tested were heterozygous or homozygous for the knockdown resistance (Kdr) mutations Vgsc-L1014F and Vgsc-L1014S with a higher proportion of Vgsc-L1014F homozygous in Lambaréné (76.7%) compared to Zilé (57.1%).ConclusionThis study provides evidence of widespread resistance to pyrethroids in An. gambiae s.s., the main malaria vector in the Moyen-Ogooué Province. Further investigation of the mechanisms underlining the resistance of An. gambiae s.s. to pyrethroids is needed to implement appropriate insecticide resistance management strategies.

BackgroundIn 2011, Benin’s National Malaria Control Programme (NMCP) organized a nationwide mass distribution campaign of LLINs throughout the country. Following this intervention, it was important to assess whether the level of susceptibility of malaria vectors to insecticides had remained the same as compared to the pre-intervention period. The current study investigated this.MethodsLarval collections were conducted in Ifangni, Sakété, Pobè and Kétou districts located in Plateau department, Southeastern Benin before (2009) and after (2012–2013) LLIN distribution. Anopheles gambiae sensu lato (s.l.) larvae from the 4 study districts were reared to adulthood and WHO susceptibility tests were conducted. The insecticides tested were deltamethrin (0.05%), permethrin (0.75%), bendiocarb (0.1%) and DDT (4%). Molecular species identification as well as, the characterization of the kdr L1014F mutation were also performed in the An. gambiae s.l. complex using PCR method.ResultsOverall, a significant decrease in mortality rates of An. gambiae s.l. to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) was observed post-LLIN distribution, respectively: (100% vs 80.9%, p < 0.0001), (77.5% vs 70%, p = 0.01) and, (47.8% vs 4.4%, p < 0.0001). By contrast, susceptibility of vectors to bendiocarb (0.1%) remained the same (100% mortality in the WHO susceptibility tube tests) pre- and post-intervention. An increase in the kdr L1014F frequency was observed post-LLIN distribution [F(kdr) = 0.91)] compared to the pre-intervention period [F(kdr) = 0.56], p < 0.0001. Anopheles coluzzii and An. gambiae were the two molecular species identified in the study area.ConclusionThe decrease susceptibility to pyrethroids and DDT as well as, the increase in the frequency of the kdr L1014F mutation after the intervention stressed at the time, the need for the development and implementation of effective insecticide resistance management strategies. At present, an update of the vectors resistance status in the area is also necessary for decision-making.

BackgroundMalaria vector control in Tanzania is based on use of long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS), which both rely on the use of chemical insecticides. The effectiveness of these control tools is endangered by the development of insecticide resistance in the major malaria vectors. This study was carried out to monitor the susceptibility status of major malaria vectors to insecticides used for IRS and LLINs in mainland Tanzania.MethodsMosquito larvae were collected in 20 sites of Tanzania mainland in 2015. Phenotypic resistance was determined using standard WHO susceptibility tests. Molecular assay were used to determine distribution of Anopheles gambiae sub-species. A microplate assay approach was used for identifying enzyme levels on single mosquitoes from each sites compared with a susceptible reference strain, An. gambiae sensu stricto (s.s.) Kisumu strain.ResultsAnopheles arabiensis was the dominant malaria specie in the country, accounting for 52% of the sibling species identified, while An. gambiae s.s. represented 48%. In Arumeru site, the dominant species was An. arabiensis, which was resistant to both pyrethroids (permethrin and deltamethrin), and pirimiphos-methyl, and had significant elevated levels of GSTs, non-specific esterases, and oxidase enzymes. An. arabiensis was also a dominant species in Kilombero and Kondoa sites, both were resistant to permethrin and deltamethrin with significant activity levels of oxidase enzymes. Resistance to bendiocarb was recorded in Ngara site where specie composition is evenly distributed between An. gambiae s.s. and An.arabiensis. Also bendiocarb resistance was recorded in Mbozi site, where An. gambiae s.s. is the dominant species.ConclusionsOverall, this study confirmed resistance to all four insecticide classes in An. gambiae sensu lato in selected locations in Tanzania. Results are discussed in relation to resistance mechanisms and the optimization of resistance management strategies.

Resistance in malaria vectors is likely to be caused by the massive use of insecticides in agriculture. Anopheles gambiae s.l. collected from breeding grounds in two cabbage growing areas within Accra were assessed for levels of resistance to 0.75% permethrin, 0.05% deltamethrin, 5% malathion and 4% DDT using standard WHO susceptibility test kits. Pyrethroid and organophosphate residue levels in soil and run-off water from these cabbage farms were determined and possible association between resistance and residue levels were established. Compared to the susceptible ‘Kisumu’ strain, both Korle-Bu and Airport populations were highly resistant to DDT and gave resistance levels which were over nine-fold for permethrin and over 2.5-fold for deltamethrin. Both wild and susceptible populations showed full susceptibility to malathion. The S and M forms of A. gambiae s.s. were found to occur in sympatry in the two study sites with a higher frequency of S form in the Airport area. Toxicity testing of extracts of soil and run-off water from these cabbage farms, using brine shrimp lethality tests, showed high level of toxicity, indicative of the presence of residues of insecticides. Differential fractionation of these extracts using solid phase extractor (SPE) suggests that the bulk of residues in these extracts may be pyrethroids and organophosphates. No correlation was observed between either residue levels or residual bioactivity in soil and run-off water, and resistance levels in A. gambiae s.l. populations, collected from breeding grounds within the farms under investigation. It is proposed that resistance in A. gambiae larvae in these breeding sites contaminated with agricultural insecticides may have occurred over time due to continuous exposure to sub-lethal doses.

BackgroundVector control is an effective way of reducing malaria transmission. The main vector control methods include the use of insecticide-treated bed nets and indoor residual spraying (IRS). Both interventions rely on the continuing susceptibility of Anopheles to a limited number of insecticides. However, insecticide resistance, in particular pyrethroid-DDT cross-resistance, is a challenge facing malaria vector control in Africa because pyrethroids represent the only class of insecticides approved for treating bed nets and DDT is commonly used for IRS. Here baseline data are presented on the insecticide susceptibility levels of malaria vectors prior to The Gambian indoor residual spraying intervention programme.MethodsAnopheles larvae were collected from six malaria surveillance sites (Brikama, Essau, Farafenni, Mansakonko, Kuntaur and Basse) established by the National Malaria Control Programme and the UK Medical Research Council Laboratories in The Gambia. The mosquitoes were reared to adulthood and identified using morphological keys and a species-specific polymerase chain reaction assay. Two- to three-day old adult female mosquitoes were tested for susceptibility to permethrin, deltamethrin and DDT using standard WHO protocols, insecticide susceptibility test kits and treated papers.ResultsAll Anopheles mosquitoes tested belonged to the Anopheles gambiae complex. Anopheles arabiensis was predominant (54.1%), followed by An. gambiae s.s. (26.1%) and Anopheles melas (19.8%). Anopheles gambiae s.s. and An. arabiensis were found at all six sites. Anopheles melas was recorded only at Brikama. Mosquitoes from two of the six sites (Brikama and Basse) were fully susceptible to all three insecticides tested. However, DDT resistance was found in An. gambiae from Essau where the 24 hours post-exposure mortality was <80% but 88% for permethrin and 92% for deltamethrin.ConclusionThis current survey of insecticide resistance in Anopheles provides baseline information for monitoring resistance in The Gambia and highlights the need for routine resistance surveillance as an integral part of the proposed nation wide IRS intervention using DDT.

Widespread of insecticide resistance amongst the species of the Anopheles gambiae complex continues to threaten vector control in Senegal. In this study, we investigated the presence and evolution of the Ace-1 and Gste2 resistance genes in natural populations of Anopheles gambiae s.l., the main malaria vector in Senegal. Using historical samples collected from ten sentinel health districts, this study focused on three different years (2013, 2017, and 2018) marking the periods of shift between the main public health insecticides families (pyrethroids, carbamates, organophosphates) used in IRS to track back the evolutionary history of the resistance mutations on the Ace-1 and Gste2 loci. The results revealed the presence of four members of the Anopheles gambiae complex, with the predominance of An. arabiensis followed by An. gambiae, An. coluzzii, and An. gambiae–coluzzii hybrids. The Ace-1 mutation was only detected in An. gambiae and An. gambiae–coluzzii hybrids at low frequencies varying between 0.006 and 0.02, while the Gste2 mutation was found in all the species with a frequency ranging between 0.02 and 0.25. The Ace-1 and Gste2 genes were highly diversified with twenty-two and thirty-one different haplotypes, respectively. The neutrality tests on each gene indicated a negative Tajima’s D, suggesting the abundance of rare alleles. The presence and spread of the Ace-1 and Gste2 resistance mutations represent a serious threat to of the effectiveness and the sustainability of IRS-based interventions using carbamates or organophosphates to manage the widespread pyrethroids resistance in Senegal. These data are of the highest importance to support the NMCP for evidence-based vector control interventions selection and targeting.

BackgroundThe emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported.MethodsThe protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace-1 mutations were determined by PCR.ResultsStrong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (≤ 5%).ConclusionThis study showed a widespread resistance to permethrin in An. gambiae populations from southern Benin, with a significant increase of kdr frequency compared to what was observed previously in Benin. The low frequency of Ace-1 recorded in all populations is encouraging for the use of bendiocarb as an alternative insecticide to pyrethroids for IRS in Benin.

Background Insecticide resistance among the vector population is the main threat to existing control tools available. The current vector control management options rely on applications of recommended public health insecticides, mainly pyrethroids through long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). Regular monitoring of insecticide resistance does not provide information on important factors that affect parasite transmission. Such factors include vector longevity, vector competence, feeding success, and fecundity. This study investigated the impacts of insecticide resistance on longevity, feeding behaviour, and egg batch size of Anopheles gambiae s.l. Method The larval sampling was conducted in rice fields using a standard dipper (350 ml) and reared to adults in field insectary. A WHO susceptibility test was conducted using standard treated permethrin (0.75%) and deltamethrin (0.05%) papers. The susceptible Kisumu strain was used for reference. Feeding succession and egg batch size were monitored for all survivors and control. Results The results revealed that mortality rates declined by 52.5 and 59.5% for permethrin and deltamethrin, respectively. The mortality rate for the Kisumu susceptible strain was 100%. The survival rates of wild An. gambiae s.l. was between 24 and 27 days. However, the Kisumu susceptible strain blood meal feeding was significantly higher than resistant colony (t = 2.789, df = 21, P=0.011). Additionally, the susceptible An. gambiae s.s. laid more eggs than the resistant An.gambiae s.l. colony (Χ2 = 1366, df = 1, P ≤ 0.05). Conclusion It can, therefore, be concluded that the wild An. gambiae s.l. had increased longevity, blood feeding, and small egg batch size compared to Kisumu susceptible colonies.

High malaria prevalence remains a major problem in Ruangwa District, despite the high coverage rates of malaria control interventions. The objective of this study was to assess the level of insecticide resistance in malaria vectors in southern, Tanzania. The study was conducted in three villages of Likangara, Nandagara and Chienjere. Resistance level to insecticides in An. gambiae s.l was evaluated using a standard WHO Susceptibility Test Kit. These mosquitoes were reared from larvae collected in varoius breeding sites in a major rice, vegetable and leguminous plant cultivation area in which pesticides use is intensive. Each test was run in four replicates of 25 adult non-blood fed female Anopheles gambiae s.l mosquitoes per tube. In total, 100 specimens were exposed for standard concentration of 1% fenitrothion, 0.1% bendiocarb, 0.75% permethrin and 0.05% deltamethrin using WHO susceptibility test kit. The number of knocked down mosquitoes were recorded at 10,15,20,30,50 and 60 min and mortality rates were determined after 24 hours and results were classified according to WHO guidelines, as susceptible (97-100%), possible resistance (90%-98%) and resistance (&lt;90%). The results indicate suggestive levels of mosquitoes resistance to Bendiocarb at (75%), possible resitance levels of Deltamethrin at (95%), Permethrin at (97%) and Fenitrothion at (96%) (p &lt; 0.05). This implies that there is reduced effectiveness of insecticides used in vector control interventions in the district. Superior insecticides should made available and introduced in order to promote the sustainable management of malaria vectors and elimination malaria transmission in the district.

To measure pyrethroid susceptibility in populations of malaria vectors and nuisance-biting mosquitoes in Tanzania and to test the biological efficacy of current insecticide formulations used for net treatment. Anopheles gambiae Giles s.l., An. funestus Giles s.l. and Culex quinquefasciatus Say were collected during three national surveys and two insecticide-treated net (ITN) studies in Tanzania. Knockdown effect and mortality were measured in standard WHO susceptibility tests and ball-frame bio-efficacy tests. Test results from 1999 to 2004 were compared to determine trends in resistance development. Anopheles gambiae s.l. and An. funestus s.l. were highly susceptible to permethrin (range 87-100%) and deltamethrin (consistently 100%) in WHO tests in 1999 and 2004, while Culex quinquefasciatus susceptibility to these pyrethroids was much lower (range 7-100% and 0-84% respectively). Efficacy of pyrethroid-treated nets was similarly high against An. gambiae s.l. and An. funestus s.l. (range 82-100%) while efficacy against Cx. quinquefasciatus was considerably lower (range 2-100%). There was no indication of development of resistance in populations of An. gambiae s.l. or An. funestus s.l. where ITNs have been extensively used; however, susceptibility of nuisance-biting Cx. quinquefasciatus mosquitoes declined in some areas between 1999 and 2004. The sustained pyrethroid susceptibility of malaria vectors in Tanzania is encouraging for successful malaria control with ITNs. Continued monitoring is essential to ensure early resistance detection, particularly in areas with heavy agricultural or public health use of insecticides where resistance is likely to develop. Widespread low susceptibility of nuisance-biting Culex mosquitoes to ITNs raises concern for user acceptance of nets.

This study was conducted during the rainy season of 2017 in the agricultural areas at Mouila, with the aim to assess the determination of susceptibility of Anopheles gambiae complex to insecticides and the detection of Ace 1R mutation. Mosquito larvae were collected by using the dipping method and nursed to adult stage. The susceptibility of adult Anopheles gambiae s.l. populations to organophosphates and carbamates was assessed using the standard WHO susceptibility test protocol. The tested mosquitoes species were identified by PCR. These tested mosquitoes were used to search for the Ace 1R mutation. The insecticides used were pirimiphos-methyl 0.25% and 1.25%, fenitrothion 1%, malathion 5%, propoxur 0.1%, bendiocarb 0.1%, 0.5% and 1%. In the prospected areas, An. gambiae s.l. included An. gambiae s.s. and An. coluzzii sympatric in their larval habitats. However, An. gambiae s.s. was predominant in all studied areas (99.1%) comparatively to An. coluzzii Moreover, the susceptibility tests of Anopheles to organophosphates and carbamates revealed mortality rates of 100% regardless of the origin of the mosquitoes. PCR diagnosis of Ace 1R mutation showed that none tested mosquito (An. gambiae s.s. and An. coluzzii) from prospected areas harbored the Ace 1R gene in all prospected areas. The lack of Ace 1R mutation in An. gambiae s.l. reveals the current effectiveness of organophosphates and carbamates for the control of these mosquitoes. Bboth these classes of insecticides could be used for the anopheline populations control in the agricultural spaces at Mouila.