Nationwide profiling of insecticide resistance in Aedes albopictus (Diptera: Culicidae) in Cameroon

The Asian mosquito, Aedes albopictus (Skuse), is an invasive mosquito which has become one of the most important vectors of dengue, Zika, and chikungunya viruses worldwide. This species was reported for the first time in Cameroon in early 2000s and became the dominant Aedes species in the urban areas in the southern part of Cameroon but remain poorly characterized. Here, we assessed the susceptibility profile of A. albopictus collected throughout Cameroon and investigated the potential resistance mechanisms involved. Immature stages of A. albopictus were collected between March and July 2017 in 15 locations across Cameroon and reared until G1/G2 generation. Larval, adult bioassays, and synergists [piperonyl butoxide (PBO) and diethyl maleate (DEM)] assays were carried out according to WHO recommendations. F1534C mutation was genotyped in field collected adults (Go) using allele specific PCR. All tested populations were susceptible to both larvicides, temephos and Bacillus thuringiensis israelensis (Bti), after larval bioassays. Adult bioassays revealed a high level of resistance of A. albopictus to 4% DDT with mortality rates ranging from 12.42% in Bafang to 75.04% in Kumba. The resistance was reported also in 0.05% deltamethrin, 0.25% permethrin, and 0.1% propoxur in some locations. A loss of susceptibility to 0.1% bendiocarb was found in one of three populations analysed. A full susceptibility to 1% fenitrothion were observed across the country. A full recovery or partial of susceptibility was observed in A. albopictus when pre-exposed to PBO or DEM and then to DDT and permethrin, respectively. The F1534C kdr mutation was not detected in A. albopictus. This study showed that the susceptibility profile of A. albopictus to insecticide vary according to the sampling location and insecticides used. These findings are useful to planning vector control program against arbovirus vectors in Cameroon and can be used as baseline data for further researches.


Introduction
The Asian tiger mosquito, Aedes albopictus (Skuse) 1894, originated from South East Asia has invaded all the five continents during the past four decades [1]. This mosquito species is one of the most important vectors of several arboviruses including dengue virus (DENV, Flaviviridae, Flavivirus) [2], chikungunya virus (CHIKV, Togaviridae, Alphavirus) and Zika virus (ZIKV, Flaviviridae, Flavivirus) [3,4] worldwide. During the last two decades, diseases caused by these viruses are increasingly reported in several regions of the world including Central Africa [3,[5][6][7][8][9][10][11][12][13] where the epidemics were formerly considered as scarce. Coincidentally, the emergence of arboviral diseases notably dengue and chikungunya has matched with the establishment of A. albopictus in the region. Indeed, A. albopictus was reported for the first time in Central Africa in Cameroon in early 2000s [14], and has rapidly colonized almost all countries of the subregion [15]. Nowadays, in Cameroon A. albopictus is the dominant Aedes species in most cities located under 6 N latitude [16]. This species was suspected as the main vector during dengue and chikungunya outbreaks in Cameroon in 2006 [6]. It was also recently demonstrated that A. albopictus populations collected in different ecological settings in Cameroon are able to transmit dengue 2 virus [17].
Control of A. albopictus relies on destruction of breeding sites and insecticide-based interventions. Indeed, the use of larvicides such as Bacillus thuringiensis var. israelensis (Bti) or temephos to treat water storage containers and space spraying of adulticides in emergency situations can help to reduce the density of Aedes mosquitoes [18,19]. Unfortunately, intensive and prolonged use of insecticides usually leads to the emergence of resistance in mosquito species under selection pressure, by decreasing the frequency of susceptible mosquitoes and reducing variability of field mosquitoes [20]. Thus, many vector control programmes are facing the challenge from the development of insecticide resistance in A. albopictus. Two main mechanisms are associated in insecticide resistance: target site due to the mutation that reduces or blocks the binding affinity between the insecticide and target site [21], and metabolic resistance [22]. Target site resistance is caused by the mutation in target genes such as the acetylcholinesterase (Ace-1), the GABA receptor, and the voltage-gated sodium channel (VGSC) causing knockdown resistance (kdr). Among target site resistance, kdr resistance is one of the main conferring resistance to both pyrethroids and dichlorodiphenyltrichloroethane (DDT). In A. albopictus, kdr mutation is less prevalent with only four VGSC mutations detected affecting two codons (1532 and 1534). Among these mutations only the F1534S variant has been shown to be moderately associated with resistance to DDT and pyrethroids [23,24]. Furthermore, metabolic resistance through upregulation of detoxification genes is a common resistance mechanism in A. albopictus. It is caused primarily by three main enzyme families, the monooxygenases (cytochrome P450s), glutathione S-transferases (GSTs) and carboxylesterases (COEs) [22,25]. CYP6P12 gene has been shown to be over-expressed in pyrethroid resistant A. albopictus in Asia [26]. In Cameroon, data on insecticide resistance in A. albopictus are very scarce apart preliminary studies which highlighted the high resistance of this species to DDT, and a loss of susceptibility to pyrethroids and carbamates [27,28]. As insecticide resistance is a dynamic process which can vary according geographical space and time, we undertook this study aiming to assess the susceptibility profile of A. albopictus nationwide and the potential resistance mechanisms involved.

Ethics statement
This study was approved by the Cameroonian national ethics committee for human health research N˚2017/05/911/CE/CNERSH/SP including using of rabbits. Oral consent to inspect the potential breeding sites was obtained in the field from household or business occupants.
Mosquito sampling. Aedes albopictus mosquitoes were sampled as larvae or pupae between March and July 2017 in 15 locations across Cameroon (Fig 1) Immature stages (field generation, G0) were collected from different potential breeding sites: domestic (e.g. jars, tanks), peri-domestic (e.g. used tires, discarded tanks), and natural (e.g. tree holes). In each location, larvae or pupae from 20 positive larval breeding places were collected, stored in plastic boxes and transferred to insectary, pooled according to the location and reared to adult stage for identification using taxonomic keys [29,30]. Mosquitoes identified as A. albopictus were reared until generation G1 for adult bioassays and G2 for larval bioassays. Mosquito populations were maintained at insectary conditions (27˚C ± 2˚C; relative humidity 80% ±10%), and females were fed on rabbits to complete their gonotrophic cycle. The A. albopictus susceptible strain from the Malaysia Vector Control Research Unit (VCRU) lab strain coming from Malaysia was used as reference strain.
Larval bioassays. Larval bioassays were performed according to WHO guidelines [31] using G2 generation larvae. The susceptibility of larvae was evaluated against technical grade temephos (Sigma Aldrich-Pestanal1, Seelze, Germany) and a formulated Bacillus thuringiensis israelensis (Bti) product (Vectobac1 12AS, Illinois, USA). First, stock solutions and serial dilutions were prepared in 95% ethanol for temephos and distilled water for Bti and stored at 4˚C. Six doses of concentrations ranging from 0.08 to 0.2 mg/l for Bti, and from 0.001 to 0.006 mg/l for temephos have been used. 80 to 100 larvae per concentration (with three to four replicates, depending on the sample and the number of larvae available) were tested. Late-third or early-fourth instars larvae of A. albopictus were placed in plastic cups with 99 ml of distilled mineral water, and 1 ml insecticide solution at the required concentration was added.
Control groups were run systematically with larvae exposed to 1 ml of ethanol for temephos or 1 ml of distilled water for Bti. No food was provided to larvae during the bioassays, which were run at 27 ± 2˚C and 80 ±10% relative humidity. Mortality was determined after 24 hrs of exposure to the insecticide. Mortality rates were corrected with Abbott's formula [32], when the mortality of controls was > 5%. All data were analysed with Win DL v. 2.0 software [33]. Lethal concentrations (LC50 and LC95) were calculated with their 95% confidence intervals (CIs). Resistance ratios (RR50 and RR95) were calculated by comparing the LC50 and LC95 for each species with those of susceptible strain, as RR50(95) = LC50(95) of studied population/LC50(95) susceptible strain and RR95 = LC95(95) of studied population / LC95(95) reference strain. The resistance levels were ranked into three categories: susceptibility (RR 50 < 5), medium or moderate resistance (5 � RR 50 � 10), and high resistance (RR 50 > 10) [31].
Synergist assays. In order to investigate the potential role of oxidases and glutathioneStransferases (GSTs) in the metabolic resistance mechanism, synergist assay was performed, when number of mosquitoes permitted, using 4% piperonyl butoxide (PBO) and/or 8% diethyl maleate (DEM) respectively. 3-5-days-old adults were pre-exposed for one hour to PBO-or DEM-impregnated papers and after that immediately exposed to the selected insecticide. Mortality was scored 24 hrs later and compared to the results obtained with each insecticide without synergist according to the WHO standards [34]. The comparison of mortality rate after pre-exposure of mosquitoes to synergist and without pre-exposure to synergist was done using Chi-square test. The difference was statistically different when P-value was inferior to 0.05.
Investigating of F1534mutation using allele specific PCR. Genomic DNA of 30 specimens of A. albopictus per populations was extracted using Livak protocol [35]. This DNA was used to genotype the F1534 mutation using allele specific (AS) PCR as described previously

Larval bioassays to A. albopictus
Due to the limited number of larvae available, larval bioassays were performed for six populations from Bertoua, Douala, Bafoussam, Tibati, Bafang, and Edéa with temephos, and four populations from Bertoua, Bafoussam, Tibati, and Edéa with Bti (Tables 1 and 2). Analysis revealed that for both larvicides and populations tested, RR 50 and RR 95 were less than 5 suggesting that A. albopictus from these locations are susceptible to Bti and temephos.

Adult bioassays
Bioassays performed with susceptible strain revealed that this A. albopictus strain was fully susceptible to almost all insecticides tested excepted DDT for which 80.7% mortality rate were found.
Analysis revealed that 14 populations of A. albopictus analysed across Cameroon exhibited a high level of resistance to organochlorine DDT with mortality rates ranging from 12.42% in Bafang to 90.03% in Melong (Figs 2 and 3). Resistance was also observed against pyrethroids notably to permethrin (type I) for which nine populations out of 13 were found resistant with  (Figs 2 and 3). In three populations tested with bendiocarb (carbamate), two from Foumban and Kumba were full susceptible and a loss of susceptibility was reported in Bertoua samples. All tested populations across Cameroon exhibited a full susceptibility toward the organophosphate fenitrothion (Fig 4).
Using different doses of permethrin (0.25, 0.5 and 0.75%), results showed that the mortality rate increased with dose of permethrin used (Fig 5). Populations of Bafang did not have a full susceptibility even with 2X concentrated permethrin as 100% mortality observed only with 3X. However, population from Edéa exhibit 100% mortality at 2X permethrin.

F1534 kdr genotyping
After genotyping of 420 specimens of A. albopictus from 14 locations across Cameroon no resistant individual was detected. We observed 100% mosquitoes homozygote for the susceptible F1534 allele.   45,46]. A significant level of resistance to pyrethroid type I permethrin was reported in nine A. albopictus populations while for the pyrethroid type II deltamethrin, the resistance was suspected only in five populations. The striking difference of the resistance pattern in both pyrethroids tested could be due to the fact that diagnostic dose used for deltamethrin 0.05% is high than 0.03% recommended for Aedes [34] as suggested previously [44]. Indeed, the assessing of intensity of resistance to permethrin using 0.75% permethrin which is the dose recommended for Anopheles as 0.05% deltamethrin, populations tested exhibited the full susceptibility. However, the loss of the susceptibility has been previously reported to deltamethrin in Cameroon and Central African Republic [27,28,38]. Although data on permethrin remain scarce in Central Africa preliminary studies in Yaoundé Cameroon highlighted the resistance of this compound [28] using the Anopheles diagnostic dose 0.75%. Previous studies in other parts of the world using the diagnostic dose recommended for Anopheles had shown that the level of resistance of A. albopictus to permethrin was higher than those of deltamethrin [41,47]. In contrast, the more recent studies in China highlighted that A. albopictus is more resistant to deltamethrin than permethrin [43,48]. As in this study, the loss of susceptibility to propoxur and bendiocarb was reported in certain locations across the country [28]. Similar results were observed in Central Africa Republic [38] and outside Africa in A. albopictus [41,49,50].
The reduced susceptibility to both pyrethroids tested in A. albopictus may poses a serious threat for vector control programs, because pyrethroids are mainly recommended for the control of adult Aedes mosquitoes [51,52]. In addition, it was demonstrated that A. albopictus was the dominant Aedes species in urban area in southern Cameroon [16] and could be considered as main arbovirus vectors in these areas. The source of selection driving the observed resistance to DDT, permethrin, deltamethrin, propoxur and bendiocarb in some A. albopictus populations remains unclear notably as the use of insecticides against A. albopictus is limited in the region [27,28,44]. As suggested previously [28,38], domestic used of insecticides through the indoor spraying and impregnating bed nets, and agriculture use could be the main source of resistance selection in Aedes vectors in Central Africa. Indeed, the use of pesticides in agriculture for the protection of market gardening could also promoted the emergence of resistance in mosquitoes by contamination of breeding sites and resting places of mosquitoes. As A. albopictus was firstly reported in Cameroon in early 2000s [14], we cannot exclude the possibility that the invading populations possessed the resistance background, as previously suggested [27,38,44].
A partial or full recovery of susceptibility to permethrin and DDT after pre-exposure to synergist PBO or DEM suggests that the cytochrome P450 monooxygenases and glutathione-Stransferases are playing an important role in the resistance in these populations role in the observed resistance which is consistent with previous data from the sub-region [28,38,44] and in other regions such as in Malaysia where a major P450, CYP6P12 was associated with pyrethroid resistance [26]. None of the specimens of A. albopictus genotyped possesses the 1534C allele suggesting this mutation is not currently involved in pyrethroid resistance in populations of this species across Cameroon. Nevertheless, this mutation has been detected in A. albopictus from several countries outside Africa like Brazil, India, Greece, Singapore, and China [53]. It will be interesting to investigate the role of other kdr mutations such as V1016I/G which was described in A. albopictus in Europe and Asia [54] and investigate the genes involved in metabolic resistance in A. albopictus.

Conclusions
Our result showed that the resistance profile varied significantly depending on the insecticides and populations tested. The full susceptibility reported to organophosphates in both larval stages (temephos) and adult stages (fenitrothion) as well as to Bti suggests that theses insecticides are suitable for control A. albopictus across the country. These findings could help to design and implement the best strategies of insecticide-based interventions in Cameroon against arbovirus vector A. albopictus.