Investigation of DDT resistance mechanisms in Anopheles funestus populations from northern and southern Benin reveals a key role of the GSTe2 gene

Malaria remains the most severe infectious disease and a major public health challenge in sub-Saharan Africa [1]. The mortality and the loss of productivity due to the illness, has devastating effects on cognitive development in children surviving the disease, leaving many disabled for life [2]. Since the discovery of the connection between Anopheles vectors and malarial transmission in 1897, vector control strategies have been the most widely used malarial control measures [3]. These measures (based on insecticide use) are insecticide treated bed-nets (ITN) and indoor residual spraying (IRS), both of which have been shown to be effective for reducing malaria prevalence in Africa [4]. One of the insecticides of choice for IRS is DDT (dichloro-diphenyl-trichloro-ethane) because of its high insecticidal activity, low acute mammalian toxicity, wide spectrum use, low price, and long duration of activity.

The availability of dichlorodiphenyltrichloroethane (DDT) and other insecticides in the 1940s marked a new era for malarial control in the world. The effectiveness of DDT against indoor resting mosquitoes led to the adoption of the Global Eradication Programme of Malaria in 1955, coordinated and supported by the World Health Organization (WHO). Although the use of DDT raises concerns of potential harm to the environment and human health, mainly because of the persistent and bio-accumulative nature of DDT and its potential to magnify through the food chain, it continued to be used for pest control, for which exemptions were granted by the federal government and it is still available for public health use today [5].

Inevitably, the major malaria vectors, Anopheles gambiae and Anopheles funestus, have developed resistance to this insecticide. The basic mechanisms underlying insecticide resistance include insecticide target-site mutations, and increased metabolic detoxification of the insecticide through overproduction or elevated enzymatic activity [6]. Three enzyme families are primarily involved in insecticide detoxification: the carboxylesterases (COEs), glutathione-S-transferases (GSTs) and cytochrome P450s (P450s). DDT resistance in An. gambiae can be due either to a specific detoxification mechanism (glutathione-S-transferase) or to a nerve insensitivity resulting from a modification of the target site (sodium channel). The latter, governed by the kdr gene, reduces both the knockdown and lethal effects of DDT [7]. In West Africa, it induces a cross-resistance to pyrethroids, which also depends on kdr mutation [7, 8]. In contrary, no kdr mutation has been detected in An. funestus so far [9‐11]. Indeed, a single amino acid change in the binding pocket of the glutathione-S-transferase epsilon 2 (GSTe2) gene, coupled with increased transcription of this gene, confers a high level of DDT resistance and also cross-resistance to pyrethroids in An. funestus. Furthermore, analysis of GSTe2 polymorphism established that the L119F-Gste2 mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa [12]. Nevertheless, the strong contrast in the allele frequencies of the L119F-GSTe2 frequencies despite the similar resistance profile recorded in An. funestus populations from two localities in Ghana [13] suggest that possible barriers to gene flow could exist between populations of the same country. Such differences in the underlying resistance mechanisms should be taken into account when designing suitable insecticide resistance management strategies. In southern Benin (Kpome and Pahou), An. funestus was found to be highly resistant to DDT [14] [15] while the population from Tanongou was moderately resistant to DDT with 90% mortality [16]. Also, as GSTe2 gene has been associated with DDT resistance patterns across Africa, this study aimed to investigate the role of the GSTe2 gene in DDT resistance across Benin to fill the knowledge gap by checking if this resistance is driven by the same mechanism.

Insecticide resistance is a complex trait and factors involved vary depending on species, insecticide and population. This research was designed to assess the underlying molecular basis driving DDT resistance in the South-North transect of Benin to improve performances of malaria controls tools.

Physiological resistance to insecticides often involves either mutations in the insecticide target site (target-site resistance), or elevated activity of detoxifying enzymes that metabolise and/or sequester insecticides (metabolic resistance). In the absence of knockdown resistance mutations in the voltage-gate sodium channel in An. funestus [10, 11, 14], this study showed how DDT resistance in this mosquito species is a result of both target-site resistance and up-regulation of a DDT detoxifying enzyme. Overall, this study has revealed GSTe2 gene as a key gene implicated in DDT as a result of elevated expression rather than allelic variation through the GSTe2-L119F mutation. This is in line with previous studies [2‐7, 11, 12] showing that over transcription of the GSTe2 gene confers DDT resistance and cross-resistance to permethrin. It has also been shown that the overexpression of GSTe2 gene in DDT resistant strain of An. gambiae [8, 25] and the overproduction of this gene is very efficient at metabolizing DDT [26]. Also, the GSTe2 gene has been implicated in DDT resistance in Aedes aegypti species from Thailand [27] showing the important role of the up-regulation of this gene in DDT resistance. Such observations are in accordance with the resistance profile of Pahou and Kpome An. funestus populations, which are highly resistant to DDT compared to Tanongou closer to Burkina-Faso where moderate resistance was recorded. Beside the overexpression of the GSTe2 gene, it is acknowledged that the presence of L119F-GSTe2 mutation confers DDT resistance in An. funestus s.s. populations in West/Central and East Africa [12] and this is in line with the high allelic frequency of this mutation recorded in Kpome and Doukonta. High allelic frequency of the GSTe2-L119F mutation and up-regulation of the GSTe2 gene were observed in Kpome, Pahou and Doukonta while low regulation and low allelic frequency of the mutation were observed in Tanongou and Burkina-Faso in correlation with DDT profile observed. The consistent difference observed for this gene between the population of southern Benin (Kpome, Pahou and Doukonta) and that of Tanongou (North Benin) and Burkina-Faso suggest that possible barriers to gene flow exist between these populations. These barriers to gene flow could be due to geographic distance, because gene flow can be restricted by physical barriers separating the populations. Clarke [28] and Duke et al. [29]. also reported that habitat discontinuities may present barriers to gene flow. Furthermore, the genetic and behavioural divergence may be related to differences in the scale of vector control interventions between the regions or an effect of climate change could also explain this phenomenon. Such anti-vector interventions have been found to impact population size of vector populations [30]. Insecticide resistance in vector populations has been widespread with large scale exposure resulting in altered abundance, behavioural shifts and general ecology of major vector populations (e.g. An. funestus, An. gambiae) [31]. However, this observation needs to be confirmed in future studies.

Analysis of the full-length GSTe2 gene shows a possible association between the GSTe2 polymorphism and observed DDT resistance in the 6 localities. The 119F resistant allele is fixed in highly DDT-resistant Benin mosquitoes especially in Pahou, Kpome and Doukonta and in Nigeria (Akaka Remo), but very low in moderate resistant mosquitoes in Tanongou and Burkina-Faso showing the key role of this mutation in DDT resistance as reported by [12]. This study revealed that southern Benin and Nigeria populations of An. funestus are more genetically differentiated as they form a unique cluster compared to North populations. This pattern of genetic diversity of the GSTe2 gene observed in this study support the contrast in resistance patterns between populations of An. funestus. In addition, a significant shift in the over-expression profile of this gene was detected across a South/North transect of Benin in line with the DDT resistance profile observed, showing that the L119F-GSTe2 mutation coupled with up-regulation of this gene confer a high level DDT resistance in An. funestus [12]. The consistent differences between the An. funestus population across Benin is likely to impact the design and implementation of resistance management strategies in Benin.

Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. This study shows that molecular basis of DDT resistance in southern Benin An. funestus is associated with L119F-GSTe2 mutation and over-expression of this gene. The variations observed between southern and northern populations of An. funestus could suggest the presence of barriers to gene flow that are affecting the spread of resistance and associated genes.