4. DISCUSSION

Previous studies undertaken in Burkina Faso established that the use of pesticides against crop pests, especially for cotton protection, contributes to the selection of insecticide resistance in malaria vector mosquitoes (Diabate et al. 2002, Gnankiné et al. 2013, Hien et al. 2017).
This original study aims to provide the impact of introduction of a new method of cotton cultivation, transgenic cotton, that is expected to reduce the amount of insecticides and the number of their treatments used against the pests of this crop (Gnankiné et al. 2018) compared to pre-existing cotton cultivation technologies, essentially conventional and to a lesser extent organic, on the insecticide susceptibility ofAnopheles mosquitoes. This study represents a great opportunity to further our knowledge on the effect of cotton growing systems on the insecticide resistance dynamics of the main malaria vector mosquitos, belonging to the An. gambiae complex, in connection with the eco-climatic regions of Burkina Faso.
Consecutive years of intensive use of chemicals in both agriculture and public health have led to the selection of resistant pests and vector populations as observed for An. gambiae in Burkina Faso (Gnankiné et al. 2013). Previous studies have also showed the presence of resistance in the tomato bollworm Helicoverpa armigera to Pyrethroids (Martin et al. 2000, 2005, Raj et al. 2002), the whiteflyBemisia tabaci to Pyrethroids, Organophosphates and neonicotinoids due to intensive use of PY and OP for crop protection (Gnankiné et al. 2013).
In regard to this situation, the cotton strategy has been modified in conventional cotton growing areas that consisted in reduction of PY and OP use and re-introduction of endosulfan insecticide (Martin et al. 2005). At the same time, nationwide surveys have shown phenotypic and genotypic resistance of An. gambiae s.l populations to PY and OP in agricultural settings including cotton growing areas in central and western Burkina Faso (Chouaïbou et al. 2016, Chabi et al. 2018).
In 2008, genetically modified cotton plants expressing both Cry1Ac and Cry2Ab toxins were implemented in Burkina Faso (Gnankiné et al. 2018). For transgenic cotton, pesticides belonging to the OP and PY families are not used in fields but pesticides from neonicotinoids class are used at the end of the cotton phenological stages (Gnankiné et al. 2018). As far as species of the An. gambiae complex concerned, An. gambiae s.s , An. coluzzii and An. arabiensis were identified and the cotton pest strategies do not affect the distribution of these species. Moreover, according to the previous studies investigated in the same climatic zones, the geographical distribution of these species did not change over the past 10 years (Diabate et al. 2004, Dabiré et al. 2012).
In this current study, the proportions of An. gambiae s.l. complex populations in Burkina Faso shows that An. gambiae s.spredominates in the west and southwest parts of the country and still lives in sympatry with An. coluzzii in proportions of 70% and 30% respectively. In central and east-central parts, An. coluzzii was prevalent and was found in sympatry with An. arabiensis, being most frequent in the northwest and east-central part of the country with frequencies between 16.7% and 51.5%.
Human activities leading to the creation of permanent streams and ponds have also favoured the emergence of An. coluzzii, a species typical of irrigated areas (Pages et al. 2007) as reported in the current study in the rice growing area of the Vallée du Kou. So far,An. arabiensis has changed in Soumousso (rural area) where its proportion has increased significantly in recent years (Namountougou et al. 2019) compared to the present study (from 2008 to 2014) during the same sampling period (e.g. rainy season). The expansion of An. arabiensis in this locality is attributed to global ecological changes (such as climate change) or local human activities (such as deforestation) that promote the colonization of this species.
Our phenotypic data exhibited resistance to DDT and permethrin (from 2008-2009) as well as deltamethrin (2008-2014) in all populations ofAn. gambiae s.l. sampled regardless of cotton pest control. The situation is quite different with Bendiocarb. Although resistance to this insecticide has been observed in populations collected in conventional growing areas, the level of susceptibility appears to have been partially restored from 2009 to 2014 at Dano (organic cotton area) and completely restored in Fada N’Gourma (transgenic cotton area) where the mortality rate recorded by bioassays has increased from 90 to 100 %.
The first studies on the resistance of An. gambiae s.l. to pyrethroids in West Africa and more particularly in Burkina Faso began in the late 1990s when Chandre et al. (1999) reported cross-resistance to permethrin and DDT in An. gambiae s.l. populations (but these populations were then susceptible to deltamethrin) in south-western Burkina Faso. Three years later, this resistance was subsequently confirmed by the works undertaken by Diabaté et al. (2002) in several parts of the country. Other studies covering different regions of the country have shown that the kdr L1014F mutation is the main mechanism involved in cross-resistance to DDT and pyrethroids inAn. gambiae s.l. populations from Burkina Faso. This resistance mechanism was detected firstly in An. gambiae s.s and thenAn. coluzzii inherited it by genetic introgression from An. gambiae s.s (Diabate et al. 2004). Recent phenotypic and genotypic analysis have shown that resistance to both DDT and pyrethroids is now widespread among the three species of the An. gambiae complex occured in Burkina Faso (Dabiré et al. 2014, Hien et al. 2017). These malaria vectors, although present in sympatric in most of areas, have divergent ecological preferences and behaviours (Diabaté et al. 2008, Coulibaly et al. 2016). Thus, An. Arabiensis,, due to its exophagic and exophilic biting behaviours, would spare to the selective insecticide pressure from the main vector control tools (essentially LLINs) as exerted on An. gambiae s.s and An. coluzzii . Beyond the use of insecticides in public health, particularly through LLINs, the use of insecticides in cotton growing areas also favoured the emergence of this cross-resistance to DDT/pyrethroids in An. gambiae s.l. populations (Diabate et al. 2002). This causality may be explained by intense and permanent selection pressure throughout the rainy season, that is exerted on the entire mosquito population at the larval stage in breeding sites and not only on the proportion of biting females. In fact, an increase of insecticide resistance in mosquito vectors was observed during insecticide treatments periods in cotton growing areas (Dabiré et al. 2012). However, by comparing the frequencies of kdr L1014F by cotton growing area (transgenic, organic and conventional), no significant differences were found during the four years follow-up, except in the transgenic cotton growing area in 2008 and 2013. Indeed, a slight reduction in the frequency of the kdr L1014F mutation in this area was detected (from 0.6 to 0.45 in An. gambiae s.l. between 2009 and 2013) coupled with the bednet campaign. This reduction should improve the susceptibility ofAn. gambiae s.l. to both DDT and pyrethroids as the kdrL1014F mutation is known to be the main mechanism of crossed resistance to DDT/pyrethroids. Unfortunately, this reduction was observed during a very short period of time since an increase of the frequency of thekdr L1014F mutation was immediately observed the following year (2014). The high levels of pyrethroids resistance in malaria vectors maintained in transgenic and organic cotton growing areas, both characterized by low insecticide use, are believed to be due to the fact that the kdr alleles are widespread in all areas of southern and central Burkina Faso and almost fixed. If the kdr mutation is fixed, its frequency will not vary even though the selection pressure of pyrethroid treatments in cotton fields decreases due to its low genetic cost; except in the case of a large influx of migrants who can bring new genetic variants, which seems unlikely given its expansion throughout the West African region.
Moreover, the transgenic and especially organic cotton fields investigated are close to conventional cotton growing areas, or even vegetable areas where agricultural insecticide applications are also practiced. Insecticides residues from conventional or vegetable areas can retain significant selection pressure on neighboring An. gambiae sl populations. Furthermore, it is not excluded that some cotton farmers by fear of seeing developing some secondary pests not targeted by transgenic Bt , treat in secret their fields by OP and PY chemicals except those recommended by the technical route.
In addition to molecular resistance mechanism (target site modification), other mechanism of insecticide resistance like metabolic resistance may be involved in Anopheles s.l. populations wherekdr gene was almost fixed as shown recently by Namountougou et al. (2019). These authors found that most of populations of An. gambiae s.s from cotton growing areas (Conventional cotton) of western part of Burkina Faso exhibited high level of GSTs and NSEs enzymes with a level of enzymes might vary in connection with cotton growing areas.
This resistance status is worrying as cotton farmers are heavy consumers of insecticides that have been and continue to be used at large scale in the country that may have an negative impact on the vector control strategies based on insecticides applications (LLINs or IRS). This situation of insecticide resistance requires regular annual monitoring for a better vector control management. This could already lead to predict a possible temporal rotation of the insecticidal classes, especially in IRS, but also the need to find new insecticides or innovative methods to prevent and control vector-borne diseases as recommended by WHO (WHO 2012, 2019).