Putative pleiotropic effects of the knockdown resistance (L1014F) allele on the life-history traits of Anopheles gambiae

Background Existing mechanisms of insecticide resistance are known to help the survival of mosquitoes following contact with chemical compounds, even though they could negatively affect the life-history traits of resistant malaria vectors. In West Africa, the knockdown resistance mechanism kdrR (L1014F) is the most common. However, little knowledge is available on its effects on mosquito life-history traits. The fitness effects associated with this knockdown resistance allele in Anopheles gambiae sensu stricto (s.s.) were investigated in an insecticide-free laboratory environment. Methods The life-history traits of Kisumu (susceptible) and KisKdr (kdr resistant) strains of An. gambiae s.s. were compared. Larval survivorship and pupation rate were assessed as well as fecundity and fertility of adult females. Female mosquitoes of both strains were directly blood fed through artificial membrane assays and then the blood-feeding success, blood volume and adult survivorship post-blood meal were assessed. Results The An. gambiae mosquitoes carrying the kdrR allele (KisKdr) laid a reduced number of eggs. The mean number of larvae in the susceptible strain Kisumu was three-fold overall higher than that seen in the KisKdr strain with a significant difference in hatching rates (81.89% in Kisumu vs 72.89% in KisKdr). The KisKdr larvae had a significant higher survivorship than that of Kisumu. The blood-feeding success was significantly higher in the resistant mosquitoes (84%) compared to the susceptible ones (34.75%). However, the mean blood volume was 1.36 µL/mg, 1.45 µL/mg and 1.68 µL/mg in Kisumu, homozygote and heterozygote KisKdr mosquitoes, respectively. After blood-feeding, the heterozygote KisKdr mosquitoes displayed highest survivorship when compared to that of Kisumu. Conclusions The presence of the knockdown resistance allele appears to impact the life-history traits, such as fecundity, fertility, larval survivorship, and blood-feeding behaviour in An. gambiae. These data could help to guide the implementation of more reliable strategies for the control of malaria vectors.

Since an effective malaria vaccine is yet to become available, vector control remains the main strategy for the prevention of malaria transmission [6]. Indeed, longlasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) remain the backbone of malaria vector control and have been shown to contribute to malaria control through the reduction of human-vector contact [7]. Unfortunately, insecticide resistance to pyrethroids (permethrin, deltamethrin) and other classes of insecticides has been reported in An. gambiae, the main malaria vector in several African countries [8][9][10][11][12][13][14]. The major insecticide resistance mechanisms in An. gambiae consist of target sites insensitivity (ace-1 R and kdr R ) and increased metabolic activity of detoxifying enzymes [15][16][17][18][19][20]. In An. gambiae s.s., mutations related to pyrethroids and dichlorodiphenyltrichloroethane (DDT) resistance are located mainly at codon 1014 within the transmembrane segment 6 of domain II in the Voltagegated sodium channel (Vgsc) gene. These mutations lead to a change of leucine to either phenylalanine (L1014F) or serine (L1014S) [21,22]. Further, additional mutation at position 1575 of the linker between domains III-IV in the Vgsc resulting in asparagine-to-tyrosine substitution (N1575Y) has been found occurring solely on a L1014Fbearing haplotype [23]. Recent studies carried out in Benin [24], Ivory Coast [25] and Burkina Faso [26] have shown that the L1014F allele frequency is almost fixed in wild An. gambiae mosquitoes. However, little is known about the fitness cost induced by this homozygous resistance allele in the malaria vector An. gambiae.
Although resistance alleles confer the potential of surviving particular insecticide exposures to mosquitoes, it is often assumed that they may also influence various fitness-related traits of mosquitoes (e.g., trophic behaviour, fecundity, fertility, parasite transmission, longevity, and larval survivorship) in the presence or absence of insecticide selection pressure [27]. Therefore, better understanding the effects of resistance alleles on the most important life-history traits of mosquitoes appears crucial to improve malaria vector control interventions.
Several studies have shown that insecticide resistance mechanisms can confer detrimental effects on reproductive fitness, host-seeking, feeding and mating behaviours in Anopheles mosquitoes [28][29][30] as well as in some Aedes [31][32][33] and Culex mosquitoes [34][35][36]. Decreased longevity and increased larval survivorship have also been observed in insecticide-resistant strains of Aedes aegypti, Culex pipiens and An. gambiae [31,[37][38][39][40]. A study carried out by Platt et al. [30] revealed that kdr R heterozygous males An. coluzzii were more likely to successfully mate than homozygote-resistant ones, illustrating a deleterious effect of homozygote-resistant kdr R allele on An. coluzzii paternity success. Also, they were more competitive compared to homozygous-susceptible mosquitoes indicating a heterozygous fitness advantage [30]. Furthermore, it was demonstrated that pupae of An. gambiae homozygous for ace-1 R (G119S) allele were more likely to die during the pupation stage than those of the susceptible strain [40]. All these studies highlight the variability of mosquito life-history traits according to species and the effects of specific insecticide resistance mechanisms on these traits.
Herein, the relative effects of kdr R (L1014F) allele on reproductive success, larval survivorship, blood-feeding behaviour, and adult survivorship post-blood meal in An. gambiae s.s. were evaluated.

Mosquito strains and rearing
Two laboratory reference strains of An. gambiae s.s. were used. The insecticide-susceptible reference strain Kisumu, sampled from Kenya the early 1950s and was maintained at insectary [41]. The KisKdr strain, which is homozygous [kdr RR ] for the L1014F allele and resistant to both pyrethroids and organochlorines, was obtained by introgression of the kdr R (L1014F) allele into the Kisumu genome [42]. This strain has the same genetic background as Kisumu [kdr SS ] and was free of metabolic resistance.
In order to investigate the role of kdr R (L1014F) allele in An. gambiae s.s. blood-feeding behaviour, heterozygote [kdr RS ]-resistant mosquitoes were obtained by crossing Kisumu females [kdr SS ] with KisKdr males [kdr RR ] and Kisumu males with KisKdr females encoded F1-1 (♀Kisumu X ♂KisKdr) and F1-2 (♂Kisumu X ♀KisKdr), respectively. For routine rearing in the insectary at the Regional Institute of Public Health/ University of Abomey-Calavi (Benin), these strains were reared under soft conditions (insecticide-free laboratory environment) in a climate-controlled room at a temperature fixed at 27 °C (± 0.2), a relative humidity of 70% (± 8) and 12:12 light and dark period. Larvae were reared in plastic trays (about 30 × 20 cm) and fed with TetraMin Baby fish food. Pupae were collected and placed in small plastic cups inside a fresh cage for adult emergence. Adult mosquitoes were kept in 30 × 30x30 cm insect cages (produced locally) and continuously supplied. Mosquitoes were fed ad libitum on 10% honey solution (made with deionized water) until they were ready to be used for further assays. Female individuals were blood-fed on laboratory rabbits (used for the purpose of blood-feeding mosquitoes) twice a week. Gravid females were allowed to oviposit in plastic petri dishes containing a water-soaked cotton covered with filter paper. The eggs were collected and put in plastic trays containing dechlorinated water (1 L per tray) for hatching.

Female reproductive success assessment
Three days after emergence from the larval-rearing conditions described, 180 An. gambiae females of both KisKdr (n = 90) and Kisumu (n = 90) strains were bloodfed on a laboratory rabbit. The gravid mosquitoes of each strain were individually transferred into plastic cups containing wet Whatman filter paper for oviposition. They were allowed to feed on 10% honey solution until egg laying. The number of females that laid eggs was recorded and the eggs were counted under a stereomicroscope (Leica Microsystems EZ4HD). Egg batches (from individual females) were transferred in separate plastic trays (about 10 cm diameter) filled with dechlorinated water and the number of hatched larvae was recorded. The experiments were performed two times.

Larval survival assessment
The larvae from each mosquito strain reared in insecticide-free laboratory conditions as described, were used for the survival assays. To assess larval mortality associated with kdr R (L1014F) allele in each mosquito strain, assays were performed as described by Yahouédo et al. [43]. In total, 480 first instar larvae (L1) of each mosquito strain were used. For each replicate, 32 larvae were pipetted into a 50 mL graduated plastic beaker (9 cm diameter). The beaker was filled with dechlorinated water to the 32 mL mark and larvae were then poured into a new petri dish. The petri dishes remained covered with the lids and their positions were changed every day to compensate for any localized differences that may exist on the rack. Petri dishes were used in order to reduce variation in larval growth rate. Every day, the larvae of each petri dish were fed with 640 µg of TetraMin Baby fish food. Water was changed every two days to reduce the effect of pollution. The petri dishes containing larvae were inspected once daily and the dead pupae or larvae were recorded and removed. Daily mortality of larvae was monitored until the last one reached pupal stage. The experiments were performed three times.

Assessment of blood-feeding behaviour
Membrane feeding assays (MFAs) previously described by Kristan et al. [44] were performed to blood-feed the mosquitoes. The 3-5-days old females of Kisumu (n = 495), KisKdr (n = 200) and those from the crossings, namely F1-1 (n = 95) and F1-2 (n = 105), were used in three different experiments. Mosquitoes were glucose-starved (with access to water-soaked cotton) for 24 h and the batches of 25 individuals were separately exposed for 30 min to membrane feeders containing the blood sample pre-heated following procedures described in [45]. The fully blood-fed mosquitoes were scored 24 h later and were kept for survivorship assessment post-blood feeding.
A portion of the blood-fed mosquitoes was used to assess the blood meal size using a spectrophotometer (MULTISCAN GO, Thermo Scientific) as previously described [46]. Each experiment using at least 30 individuals per strain, was performed three times.

Mosquito longevity post-blood meal
After the blood-feeding assays, successfully blood-fed females from Kisumu (n = 172), KisKdr (n = 168), F1-1 (n = 71) and F1-2 (n = 90) were transferred into brandnew disposable paper cups (an average 10 females per cup) and were allowed to feed on 10% honey solution. The mortality was recorded daily until the death of the last mosquito.

Data analysis
Data were recorded in appropriate designed forms, entered into Microsoft Excel for data cleaning and exported to R statistical software version 3.4.4 [47] and GraphPad Prism 8.0.2 software (San Diego, CA, USA) for analysis. The normality of data distribution was checked using Shapiro Wilk test [48].
Fecundity of each mosquito strain was assessed as the total number of eggs over the total number of females that contributed to oviposition. A correlation between kdr R genotype and fecundity was calculated using negative binomial model (NBM) defined as follow: log (Ov) = Genotype + ε where Ov is the number of eggs/ female; Genotype is the two-level factor corresponding to the different genotypes tested; ε is the error parameter which follows a negative binomial distribution. For each mosquito strain, fertility was evaluated as percentage of hatched larvae by dividing the total number of first instar larvae over the total number of eggs. A correlation between kdr R genotype and fertility was calculated using NBM, defined as follow: log (Ha) = Genotype + ε where Ha is the percentage of larvae/egg batch. Descriptive statistics were used to calculate pupation percentage (number of pupae/number of first instar larvae), blood-fed mosquito percentage (number of blood-fed mosquitoes/number of exposed mosquitoes). The Chi-square independence test was performed to compare proportions using the R statistical software [47]. The Mann-Whitney procedure was used to compare the means between mosquito strains. For the larval and blood-fed females survivorships, differences in the computed survival curves of Kisumu and KisKdr strains were analysed using Kaplan-Meier pair-wise comparisons [49]. The Log-rank test was performed to evaluate the difference in survival time between the mosquito strains [50]. Differences in larval survival time and in adult survival time post-blood meal between the two genotypes were tested using Cox proportional hazards regression model (Cox model) with a binomial error distribution. The models were calculated as follows: Survival = Genotype + ε, where Survival is a proportion of dead larvae or adults; Genotype is the two-level factor corresponding to the different genotypes tested; ε is the error parameter which follows a binomial distribution. The pupae were censored in the larval survivorship analysis. The significance of differences in blood-feeding rates between the genotypes was assessed with the following generalized linear models (GLM): Fed = Genotype + ε, where Fed is the blood-fed status; Genotype is a three-level factor corresponding to the different genotypes tested ([kdr SS ], [kdr RS ] and [kdr RR ]); ε is the error parameter which follows a binomial distribution. All these analyses were set at significance threshold of p < 0.05.

Reproductive success
The mean number of eggs laid per mosquito female (fecundity) and the average larval hatching rate (fertility) were significantly different between the two strains

Larval survivorship
The median survival times of Kisumu and KisKdr larvae were, respectively, 10 days and 11 days (Fig. 3A).
However, the survival time of Kisumu larvae was significantly shorter than that of KisKdr larvae (Log-rank test: χ 2 = 110, Δdf = 1, p = 2.10 -16 ). Furthermore, more than 50% of KisKdr larvae were still alive and have reached the pupal stage at the end of the larval following-up period (Fig. 3A).  (Fig. 3B).

Discussion
In the dominant malaria vector An. gambiae, pyrethroid resistance is spreading over time and space on the African continent, supported by several point mutations in the Voltage-gated sodium channel gene [21,23,51]. It was demonstrated that alleles conferring resistance in mosquito populations allow the mosquito to survive longer in an area of insecticide pressure but may alter some vector life-history traits [30,31,52] in an insecticide-free environment. Understanding and documenting the effects of kdr allele on life-history traits of An. gambiae, is a key for developing evidencebased resistance management strategies, including suppression of the insecticide selection pressure that allows the susceptible alleles to become more predominant [53]. This study has investigated the pleiotropic effects associated with the presence of the West African knockdown resistance allele (L1014F) on the reproductive success, larval and adult survivorships and blood-feeding success in laboratory An. gambiae s.s. by comparing the susceptible and resistant strains (homozygous kdr genotype) which share the same genetic background but differ in the presence or absence of the kdr R (L1014F) allele. Reduced egg production and egg hatchability have been reported in other insecticide-resistant mosquito species, including Ae. aegypti [31,54,55]. However, in An. funestus, the egg production rates between pyrethroid-resistant and susceptible strains did not vary significantly [56]. The current study reported significantly lower fecundity and fertility in the homozygous KisKdr individuals compared to susceptible Kisumu strain mosquitoes. These results suggest that the kdr R allele negatively affected the ability for egg production and hatchability in resistant homozygote [kdr RR ] An. gambiae. Consequently, reduced larval production would reduce adult density and lead to a decreased level of malaria parasite transmission in the resistant An. gambiae mosquitoes.
This study revealed that the kdr R (L1014F) allele confers a high larval-to-pupal survivorship and pupation rate in KisKdr mosquitoes compared to the susceptible strain. This suggests that both life-history traits are positively affected by the presence of the kdr allele. Relatively long larval development time and reduced survival time have previously been observed in insecticide-resistant An.
gambiae [57]. It was recently demonstrated that insecticides in the larval environment (containing a lower dose of pyrethroid insecticide with a variation of food availability) could significantly influence the immune response of adults An. gambiae [58]. Indeed, the exposure of pyrethroid-resistant larvae (having escaped potential predators) to sub-lethal doses of insecticide residues during their aquatic developmental stage, especially in agricultural areas, could further affect the adult life-history traits. Such a phenomenon could drive the emergence of new outcomes related to the infection with specific mosquito-borne pathogens and the persistence of insecticide-resistant An. gambiae, which is still an essential impediment to the malaria vector control measures.
The results from this work indicate a significant association between harbouring of kdr R allele and the high blood-feeding success in An. gambiae s.s.. This result suggests that the L1014F kdr allele may increase the ability of An. gambiae to blood-feed. By contrast, an absence of association was observed for the blood meal volume. Previous work on insecticide resistance markers has shown an association between the CYP6P9a gene (a marker of cytochrome P450, which mediates metabolic resistance again pyrethroid insecticides) and the feeding success and blood meal size in An. funestus [59]. These findings highlight the need for further studies to improve knowledge of the influence of multiple insecticide resistance markers harbouring on the propensity of malaria vectors to blood feed. However, heterozygous KisKdr F1-1

Fig. 5
Parents and first generation female longevity after blood-feeding. Dotted lines are 95% confidence intervals (CIs) around the respective survival curve. Arrows indicate the median survival time and F1-2 mosquitoes ingested higher blood volume compared to Kisumu specimens.
Gametocyte-infected mosquitoes must survive long enough to become infectious and transmit sporozoites to a new host [60]. One of the key factors modulating malaria transmission is the vector longevity after bloodfeeding. This study demonstrates that the presence of kdr R allele seems to increase the longevity of heterozygote KisKdr mosquitoes while no survival advantage was observed in homozygous individuals compared to the susceptible strain Kisumu. This benefit in heterozygote [kdr RS ] over homozygote [kdr RR ] makes the kdr an over-dominant gene for this specific trait. The heterozygote mosquitoes survived until 24 days post-blood meal. Thus, these specimens have sufficient lifespan to allow an extrinsic incubation period of Plasmodium parasites if they ingest gametocyte-infected blood. However, further investigations are needed to evaluate the cost of Plasmodium infection to heterozygote-resistant KisKdr mosquito survivorship.

Conclusion
In order to generate valuable predictions of malaria transmission, the impact of resistance mechanisms on the vector life-history traits needs to be taken into consideration. The data presented here indicate that kdr R allele induces a cost on fecundity and fertility in adult An. gambiae. Remarkably, this allele positively affects the larval survivorship, pupation rate, blood-feeding success in homozygote-resistant mosquitoes, and increases the post-blood feeding survivorship, especially in heterozygote individuals. It would be interesting to characterize the fitness effects of kdr R allele in natural populations of An. gambiae and identify the potential synergist genes.