First report of L1014F-kdr mutation in Culex pipiens complex from Morocco

Mosquitoes of the Culex pipiens complex are potential vectors of Rift Valley fever virus (RVFV) and West Nile virus (WNV). RVFV is a Phlebovirus of the family Bunyaviridae, considered as an emerging zoonotic vector-borne disease representing a threat to animal and human health, and livestock production mainly in sub-Saharan Africa [1]. It causes abortions and high mortalities in newborn animals [2, 3], and in humans, it gives different symptoms varying from a flu-like syndrome to hemorrhagic manifestations with a case fatality rates as high as 50% [4]. Besides, WNV is an arbovirus of the family Flaviviridae and the genus Flavivirus. It has an extensive distribution throughout Africa, the Middle East, southern Europe, western Russia, south-western Asia and Australia.

Widely spread in North Africa, Culex pipiens complex is a competent vector of several pathogens affecting human and/or animals such as WNV [5] and RVFV [6]. In Morocco, Culex pipiens mosquitoes have been strongly suspected as being the vectors of WNV during epizootics in 1996 with 42 dead horses [7–9] and in 2003 [10]. In the Maghreb region, WNV was repeatedly responsible for several outbreaks: Algeria (1994), Tunisia (1997, 2003, 2010–2012), and Morocco (1996, 2003 and 2010) [10–12].

Culex pipiens includes two forms, pipiens and molestus, which are morphologically identical but genetically different. They are also distinguishable by their physiological and behavior differences. Pipiens form is anautogenous (needs a blood meal for eggs development), ornithophilic (feeds on birds), heterodynamic (enters into diapause in winter), and eurygamous (prefers mating in large and open spaces), whereas molestus form is autogenous (lays the first egg batches without feeding on blood), mammophylic (feeds on mammals), homodynamic (is active throughout the year), and stenogamous (mates in closed areas) [13]. In the absence of effective vaccines, the control of mosquito populations remains the unique measure to limit pathogen transmission. Thus, the use of insecticides plays a major role in the prevention and control of vector-borne diseases. However, the frequent use of insecticides (mainly pyrethroids and organophosphates) has contributed to select several resistance mechanisms in targeted mosquito populations. There are two mechanisms of resistance: (i) increased production of detoxifying enzymes such as cytochrome P450 oxidases or glutathione-S-transferases; and (ii) modification of insecticides targets as the synaptic acetylcholinesterase (AchE1) encoded by ace-1 gene, the ?-aminobutyric acid (GABA) receptor gene encoded by Rdl and the voltage-dependant sodium channel encoded by kdr [14, 15]. Pyrethroids (PYR) target Sodium channels; this neurotoxin insecticide binds to the Na?+?channel and then prolongs depolarization [16–18]. The magnitude of the PYR effect depends on the type of insecticide molecule: the type I insecticide (e.g. permethrin) does not present a cyano group compared to the type II insecticide (e.g. lambda-cyalothrin and delmathrin). The type II insecticides induce a more acute effect as they produce longer depolarization [17]. Phenotypically, Na?+?channels inactivation results in a rapid knockdown (kd) of mosquitoes leading in some cases to death. Resistance to kd is caused by a mutation L1014F, the substitution of a leucine at position 1,014 by a phenylalanine conferring the kdr phenotype [19], leading to a lower sensitivity of receptors to these insecticides and modifying the potential action of the channel [18, 20].

In Morocco, the mechanisms responsible for insecticide resistance in Cx. pipiens remain unknown; the only data available describes the level of larval resistance to the OP insecticide temephos [21]. Knowing that insecticide resistance remains a global issue for the control of mosquito-borne diseases, this study aims to investigate the L1014F kdr mutation frequencies in different forms of Culex pipiens complex collected in three regions in Morocco: Tangier, Casablanca and Marrakech.