Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, Anopheles funestus

Background: Metabolic resistance is a serious challenge to current insecticide-based interventions. The extent to which it affects natural populations of mosquitoes including their reproduction ability remains uncharacterised. Here, we investigated the potential impact of the glutathione S-transferase L119F-GSTe2 resistance on the mating competitiveness of male Anopheles funestus, in Cameroon. Methods: Swarms and indoor resting collections took place in March, 2018 in Tibati, Cameroon. WHO tube and cone assays were performed on F 1 mosquitoes from indoor collected females to assess the susceptibility profile of malaria vectors. Mosquitoes mated and unmated males collected in the swarms were genotyped for the L119F metabolic marker to assess its association with mating male competitiveness. Results: Susceptibility and synergist assays, showed that this population was multiple resistant to pyrethroids, DDT and carbamates, likely driven by metabolic resistance mechanisms. Cone assays revealed a reduced efficacy of standard pyrethroid-nets (Olyset and PermaNet 2.0) with low mortality (<25%) whereas synergist PBO-Nets (Olyset Plus and PermaNet 3.0) retained greater efficacy with higher mortality (>80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in this An. funestus population at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21 An. funestus couples were isolated from those swarms. A comparative genotyping of the L119F-GSTe2 mutation between mated and unmated males revealed that heterozygote males 119L/F-RS were less able to mate than homozygote susceptible (OR=7.2, P<0.0001). Surprisingly, heterozygote mosquitoes were also less able to mate than homozygote resistant (OR=4.2, P=0.010) suggesting the presence of a heterozygote disadvantage effect. Overall, mosquitoes bearing the L119-S susceptible allele were significantly more able to mate than those with 119F-R resistant allele (OR=2.1, P=0.03). Conclusion: This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.


Amendments from Version 1
The main change in this version of the manuscript is only on the result of Plasmodium infection in An. gambiae s.l. Compared to the first version where we presented the infection rate in both An. gambiae and An. coluzii combined, here the result is presented for each species. In addition we have corrected some English mistakes as suggested by the reviewer.

Background
Despite significant reduction of malaria burden in the past decade, this disease remains a major public health concern in Africa. Recent reports of increase cases of malaria 1 is a further indication that more is needed to control this disease. The scale up of vector control measures, in particular long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS), has been the main driver of this reduction of malaria burden with about 78% of all gains achieved since 2000 attributed to these methods 2 . However, resistance is spreading in malaria vectors in Africa including Anopheles funestus for the four classes of insecticides used in public health, compromising the effectiveness of these interventions 3 . Providing adequate information about the mechanisms of resistance and more importantly its impact on key traits of mosquito biology, ecology and behaviour such as their mating ability in the presence of resistance can help in planning and implementing suitable insecticide resistance management strategies.
Insecticide resistance management strategies including rotation of insecticide rely on the assumption that insecticide resistance alleles are very often detrimental in the absence of insecticide selection pressure 4,5 . The adaptive allele in this case might be associated with modification of physiological processes or resource availability 6 which can lead to decreased performance and fitness disadvantage of resistant mosquitoes 7-9 and therefore, a reversal to susceptibility is expected in the absence of selection pressure from the specific insecticide. However, little is currently known on such fitness costs in field populations of malaria vectors notably for metabolic resistance mechanism because of a lack of DNA-based markers. A previous study using a laboratory strains of An. gambiae, demonstrated fewer copulations in dieldrin resistant males when compared with their susceptible counterparts 6 . Berticat et al. demonstrated also the disadvantage in competitive mating ability of Culex pipiens males with the target-site resistance Ace1R genotype, when compared with susceptible males, pointing to its potential impact on the spread and persistence of resistant alleles. In contrast, for malathion resistance in the beetle Tribolium castaneum it was noticed that resistance enhanced male reproductive success. If this last case is observed in malaria vectors, it will be a great concern for control program as it will prevent the implementation of resistance management strategies based on the rotation of insecticides. Currently, there is little information on the impact of metabolic insecticide resistance on the mating ability of natural populations of major malaria vectors in Africa. So far the only study on this topic reported a lack of impact of metabolic resistance on male competitiveness of An. gambiae field population in Burkina Faso 10 . The study assessed only the global transcription profiling of mated and unmated mosquitoes since a lack of DNA marker for metabolic resistance prevented a direct genotyping correlation with mating status. However, recent progress made in elucidating the molecular basis of metabolic resistance had identified a single amino acid change (L119F) in the glutathione S-transferase epsilon 2 (GSTe2) conferring pyrethroid/DDT resistance in An. funestus 11 . This new marker now provide the opportunity to directly investigate the impact of metabolic resistance on mating male competitiveness. However, assessing the impact of resistance on the mating of malaria vectors through swarm's collection in natural populations of mosquitoes requires a good knowledge of the mating places and also the mating behaviour of these vectors.
Concerning the mating behaviour of insects, it was reported that most of them mate in swarms, whereby dispersed populations aggregate at specific times and places 12,13 . In mosquitoes including malaria vectors, swarming occur very often around visual markers such as vegetation and brick piles on the ground 14-16 . This knowledge on mating places and behaviour can also help to reduce mosquito densities or interrupt the mating thus helping to reduce pathogen transmission in vector populations 17 . This technique has been effective against some Anopheles mosquitoes in Burkina Faso but on a limited scale 18 . Little information is currently available for other vector species like An. funestus. In An. gambiae s.s. mating is limited to a very short period at dusk. In this species, males always swarming before and disbanding after copulation 19 . Females approach a swarm, promptly acquire a male and leave in copula 19,20 . Mating behavior, which is one of the most important aspects of reproduction 21 , remains widely under-investigated in many malaria vectors. While many studies were conducted on An. gambiae swarms in Western Africa 10,20,22,23 , observations have rarely been reported in Eastern, Southern and Central Africa. Prior to this current report, there has been little information available on the swarms in Cameroon. Improved understanding of mosquito mating systems, and more importantly how insecticide resistance mechanisms affects the mating success in field population of malaria vectors such as An. funestus, could possibly give new tools for vector control implementation.
In this study, after characterizing the mating swarms of An. funestus, we investigated the resistance profiling and molecular basis of insecticide resistance in a natural populations of An. funestus in Cameroon. Furthermore, we investigated the potential impact of metabolic resistance on mating male competitiveness by assessing the association between the L119F-GSTe2 metabolic resistance marker and the mating success of An. funestus mosquitoes in field conditions.

Study area
Initially, the surveys covered two villages, Tibati (6°28' N, 12°37' E) and Mibellon (6°46'N, 11° 70'E) (Adamawa Region, Cameroon) but we eventually focused on just one village (Tibati) according to the density of the swarms. The main malaria vectors in Tibati are An. funestus during the dry season and An. gambiae s.l during the rainy season, whereas in Mibellon An. funestus is the predominant species 24 . The dominance of An. funestus in these areas is due to the presence of multiple lakes known as suitable breeding sites for this species. LLINs is the main vector control approach in Cameroon. The villages included in this study benefited from universal LLIN distribution in 2011 and 2016. Because of high selection pressure of insecticide contained in the LLINs, the main malaria vectors have developed resistance to pyrethroids used in the nets 25 . The communities rely mainly on substance farming, agriculture but also fishing.
Detection and collection of An. funestus swarms Swarm collections were undertaken on 12 evenings in February and March 2018. The search for potential swarms in each village started in the first evening at sunset around 5.30 pm, and then each swarm located were characterized and/or collected throughout the study. Swarms were searched in various places (around the potential breeding sites, closer to habitations, the farms, on the street) with the presence of potential markers assessed. For all swarms identified, different characteristics such as i) heights ii) starting time of swarming, iii) time at night when the swarms became invisible and iv) the behaviour of mosquitoes in the swarms were recorded. Swarms were then sampled using sweep nets as described previously 10,20,23 . All couples of An. funestus (mated) were extracted from the swarms and each couple manually transferred into a clean cartoon cup. Samples of the remaining males that did not mate in the same swarms were collected. All mosquitoes sampled were separated into unmated males, mated males and mated females and stored in RNA-later solution for further analysis.
Indoor female collections and F 1 rearing For the purpose of assessing the susceptibility profile to various public health insecticides and WHO recommended bed nets, F 1 females were generated from indoor-resting blood-fed (F 0 ) females collected using electric aspirators. Collected mosquitoes were morphologically identified using the key of Gillies and De Meillon (1968). After sampling, female mosquitoes were transferred to the insectary of the Centre of Research in Infectious diseases (CRID) in Yaoundé, Cameroon. Female mosquitoes collected were kept in standard insectary conditions of 25 ± 2°C, 80 ± 10% relative humidity and fed with 10% sugar solution for at least four days and then left to oviposit using the forcedegg laying method 26 . F 1 larvae were reared to adults using the protocol previously described 26 .

Species identification
Genomic DNA was extracted from 40 F 0 An. gambiae s.l. and 102 F 0 An. funestus s.l female mosquitoes (head and thorax) using the Livak protocol 27 which includes grinding of mosquito in a Livak buffer, followed by a 65°C incubation for 30 min and then centrifugation. Further steps involved an incubation on ice (30min) followed by centrifugation steps, precipitation with alcohol (100% and 70%) 27 . Mosquito species was identified using the Koekomoer cocktail Polymerase Chain Reaction (PCR) assay for An. funestus group and the SINE PCR assay for An. gambiae s.l. 28,29 .

Infection of malaria vectors by Plasmodium parasites
Plasmodium infection rate was estimated by Taqman (401400, Santa Clara, CA, USA) assay using the head and thorax of F 0 field-collected mosquitoes as previously described 30 . 102 females An. funestus sensu stricto (s.s.) and 40 An. gambiae s.l were used for the detection of Plasmodium falciparum (falcip+) and/or Plasmodium ovale, Plasmodium vivax, and Plasmodium malariae (OVM+) sporozoites.

Synergist assays
To assess the contribution of cytochrome P450 and GST enzymes in the resistance profile, synergist assays were performed with PBO (Piperonyl Butoxide) and DEM (Diethyl Maleate) with An. funestus s.s. Four replicates of 20-25 adult mosquitoes (2-5 day old) were immediately exposed to permethrin (0.75%), deltamethrin (0.05%), or DDT (4%) for 60 minutes after pre-exposed to PBO or DEM impregnated papers (4 %) for 1 hour. In addition, control assays using only the synergist impregnated papers for 60 minutes were also performed and mortality recorded 24 hours after. The mortality rate obtained were compared with those without synergist's exposure using a chi square test.
Assessment of bed net efficacy using cone assays In order to assess the impact of resistance on insecticide-based interventions against the malaria vectors of this location, we checked the efficacy of common bed nets recommended by WHO against the Tibati's An. funestus population. 3 minute cone bioassays were carried out following the WHO guidelines 31 . Five batches of ten F 1 females (2-5 days old) were placed in plastic cones attached to 5 commercial nets: PermaNet ® 2.0 (deltamethrin 1.8 g/kg) (Vestergaard, Lausanne, Switzerland), PermaNet ® 3.0 (side of the net; deltamethrin 2.8g/kg) (Vestergaard, Lausanne, Switzerland), PermaNet ® 3.0 (top of the net; deltamethrin 4.0 g/kg plus 25g/kg piperonyl butoxide (PBO)) (Vestergaard, Lausanne, Switzerland), Olyset ® (2 % permethrin) (Sumitomo Chemical UK PLC, London, UK) and Olyset ® plus (2 % permethrin plus 1 % PBO) (Sumitomo Chemical UK PLC, London, UK). Genotyping of resistance marker and assessment of the impact on the mating male competitiveness of An. funestus field population L119F-GSTe2 metabolic and A296S-RDL target-site resistance markers, involved in DDT/permethrin and dieldrin resistance in An. funestus respectively were genotyped in order to assess the effect of these resistance mechanisms on the mating ability of An. funestus field population as there is no evidence of kdr in An. funestus 32 . The L119F-GSTe2 was genotyped using an allele-specific (AS)-PCR and the A296S-RDL by TaqMan assay (Santa Clara, CA, USA). A296S-RDL TaqMan reaction was performed as previously described 33 PCR reactions (10 μl) contained 1 μl of genomic DNA, 5μl of SensiMix DNA kit (catalog: SM2-717104), 0.125μl of the A296S-RDL probe and 3.875 μl of sigma water. Samples were run on a Mx3000P™ (Stratagene) using the temperature cycling conditions of: 10 minutes at 95°C followed by 40 cycles of 95°C for 10 seconds and 60°C for 45 seconds. We designed a new allele specific PCR to genotype the L119F-GSTe2 mutation 9 . Two pairs of primers were used for the AS-PCR (two outer and two inner primers, Table 1). Specific primers were designed manually to match the mutation and an additional mismatched nucleotide was added in the 3 rd nucleotide from the 3′ end of each inner primer to enhance the specificity. PCR was carried out using 10 mM of each primer and 1ul of genomic DNA as template in 15 μl reactions containing 10X Kapa Taq  Furthermore, in an effort to characterize the broad dynamic of resistance to insecticides in this location, we also genotyped the L1014F target-site knockdown resistance (Kdr w) associated with DDT/pyrethroid resistance in An. gambiae using a Taqman (Santa Clara, CA, USA) method as previously described 34 . PCR reactions (10 μl) contained 1 μl of genomic DNA, 5μl of SensiMix DNA kit (catalog: SM2-717104), 0.125μl of the L1014F-kdrw probe and 3.875 μl of sigma water. Samples were run on a Mx3000P™ Multiplex quantitative PCR system (Stratagene) using the temperature cycling conditions of: 10 minutes at 95°C followed by 40 cycles of 95°C for 10 seconds and 60°C for 45 seconds.

Statistical analysis
Association between the GSTe2 mutation and mating success was assessed by calculating the odds ratio of mating between the homozygous resistant, heterozygous and susceptible for each gene in mated males compared to unmated group with statistical significance based on the Fisher's exact probability test. All analyses were conducted using GraphPad Prism version 7.00. Collection of the An. funestus swarms 15 swarms with considerably large size (more than 100 mosquitoes/swarm) were observed in Tibati, while very few swarms (with small size, less than 50 mosquitoes/swarm) were observed in Mibellon. Most mating swarms were located close to human habitations compared to other places and swarming started with two to three mosquitoes congregating after sunset around 6.05pm, and flying above a swarm place. The number of mosquitoes increased in the swarms over the next 5 to 10 minutes and slowly decreased in size then disappeared after 30 minutes when the sky became dark. Flying mosquitoes were observed by viewing them against the sky after sunset. Males of An. funestus swarmed at the height of 2.5m from the ground. Concerning the mating behaviour, when a female coupled with a male, they immediately left the swarms, flying at 1.5m from the ground. It is at that moment that the couples were extracted from the swarms using the sweep nets. There was no clear physical marker for An. funestus swarm's position in Tibati but the commonest place for swarming were just an empty space close to habitations and most of the swarm locations remained the same for several days. Throughout this survey, we observed and collected a total of 21 copulation events in Tibati. Furthermore, we collected more than 1000 male mosquitoes from those remaining in the swarms after a mating period (that most likely did not mate). The low number of collected couples suggests a low number of females in these swarms but could also indicate that mating was also taking place in other swarms not detected in this study.
Analysis of An. gambiae s.l. mosquitoes revealed that this population was generally more resistant than An. funestus with lower mortality rates observed for DDT (23.6% ± 2.6 mortality), permethrin (1.75% ± 1.75) and deltamethrin (10.0% ± 5.8%) ( Figure 1C).   Figure 3A). Moderate frequency of the 119F resistant allele in all samples, was recorded in mated (23.8%) compared to unmated males (33.5%) ( Table 2). Direct comparison of the frequency of each genotype between mated and unmated males revealed no significant differences between all groups of mosquitoes (P≥ 0.16). However, an assessment of the association of each genotype with mating success using odds ratio (OR) revealed that the heterozygote genotype (L119F-RS) showed a significantly lower chance of mating than both homozygous resistant (OR = 4.2 IC: 1.49-11.9; P< 0.01) and homozygous susceptible mosquitoes (OR = 7.2 IC: 3.1 -16.8; P < 0.0001) ( Table 3; Figure 3A). In contrast, no significant difference was observed between Homozygote resistant and homozygote susceptible mosquitoes (OR=1.77; IC 0.77-3.7; P=0.22). The impact of the resistant allele 119F on the mating competitiveness was also supported by the significantly greater likelihood of not mating when possessing this resistant allele than the susceptible L119 (OR=2.1; CI 1.1-4.0; P=0.03) ( Table 4).
Genotyping of A296S-RDL target-site resistance in An. funestus s.s Genotyping of A296S-RDL mutation associated with dieldrin resistance in mated and unmated males revealed that the 296S    Figure 3B). These results were confirmed by the full susceptibility observed with dieldrin in the bioassay test. For this reason, no further comparison was performed for this mutation about its impact on mating success.

Discussion
Elucidating the malaria vector ecology and behaviour is crucial for the implementation of alternative control measures in order to achieve the aim of malaria elimination. Mating is one component of mosquito behaviour that remains poorly characterized.
After characterizing an An. funestus population in Cameroon including insecticide resistance profiling and swarm patterns, we took advantage of the recent detection of the glutathione S-transferase L119F-GSTe2 marker in An. funestus 11 to investigate the potential influence of metabolic resistance on mating competitiveness of male An. funestus mosquitoes. As reported in other studies 12,50 , mosquito swarms in Tibati occurred perpetually in the same locations at approximately the same time each day. This phenomenon needs to be assessed in other parts of Africa, which may allow the swarm to be targeted as an alternative control measure for malaria prevention. It is also unknown if An. funestus mate in fewer large swarms or in multiple small swarms. The number of mosquitoes in swarms as reported by Charlwood et al. 46 were also relatively low, and on average less than 50 adults/swarm in Mibellon. In contrast, as reported by Harper 48 , about 300-500 mosquitoes were present in each swarm in Tibati during the collecting period.
Association between GSTe2-mediated metabolic resistance and mating success of An. funestus This study revealed a negative impact of L119F-GSTe2 DDT/ pyrethroid resistance on the mating competitiveness of males An. funestus as possessing the 119F resistant allele reduced the likelihood of mating. This is the first report of such negative impact of metabolic resistance on the mating success of field malaria vectors. The reduced fitness of L119F resistant mosquitoes observed in this study may suggests that the L119F mutation in the GSTe2 gene potentially affects some physiological traits in resistant mosquitoes including mobility, perception of stimuli or even the olfactory system as the target site resistance 4,5 . However, heterozygote mosquitoes were more affected by this negative impact than homozygote resistant individuals suggesting a heterozygote disadvantage effect. In contrast, the study conducted in Vallée du Kou in Burkina Faso on the male Table 4. Assessment of the association of different genotypes at L119F-GSTe2 mutation with mating success; *, significant difference.

L119F-GSTe2
Odds ratio P-value showed some level of resistance to carbamates: bendiocarb and propoxur similar to reports in Northern Cameroon 43 . The common used Olyset and Permanet 2.0 LLINs presented a very low bioefficacy against An. funestus in cone assays. The low efficacy of this two nets, treated with permethrin and deltamethrin only, is wide-spread in An. funestus populations across the continent 33,37,44 . This loss of efficacy of these pyrethroid-only nets correlates well with the very high permethrin and deltamethrin resistance observed for this species. However, the greater efficacy with PBO-based nets (OlysetPlus and PermaNet 3.0) possibly provides an alternative solution to control this species for which resistance is mainly metabolic with an important role played by cytochrome P450 as shown by the synergist PBO assay. However, the spread or increased frequency of GSTe2-mediated resistance could limit the efficacy of such PBOnets in the future. This is supported by the only partial recovery of susceptibility observed with (Olyset Plus), coupled with the increased mortality with the DEM synergist assay. The impact of GST-mediated resistance on efficacy of PBO-based nets will need to be assessed particularly in situations where such mechanism become predominant, as reported in Benin 11,45 .
of An. coluzzii mosquitoes reported a heterozygote advantage for the target site resistance mechanisms. It was observed that kdr heterozygote males were more likely to mate than homozygote resistant counterparts and heterozygote RDL R /RDL S were also more likely to mate than homozygote-resistant males. It may be that heterozygote individuals are not affected in the same way by target site mutation and metabolic resistance driven by GSTe2 enzymes. To confirm the lower mating ability of heterozygote mosquitoes compared to other genotypes as observed in this study, more work is needed in other locations to confirm such heterozygote disadvantage effect as the low sample size of L119F It has previously been reported that metabolic resistance mechanisms, such as the overproduction of carboxylesterases as observed in resistant C. pipiens, could confer a significant fitness cost on mosquitoes life-traits. It was noticed in this species that resistant individuals displayed a reduced locomotive performance compared to the susceptible ones. It was suggested that such reduced performance was caused by a resource depletion linked to the overproduction of carboxylesterases 54 . Prior to this study the only report of the impact of metabolic resistance on mating ability of malaria vector was conducted in 2015 in An. gambiae. Mating competitiveness in this species was not found to be significantly influenced by metabolic resistance mechanisms. However, that study 10 did not use a molecular marker for metabolic resistance, but a genome-wide microarray-based transcription analysis. The reduced performance of resistant mosquitoes in mating could contribute to slow the speed of increase in the frequency of resistant alleles in the wild, and will also prevent or delay the fixation of the resistance genes in the population. It is thus necessary that such studies are extended for other metabolic resistance mechanisms and in other locations with larger sample sizes in order to help implement successful management strategies.

Conclusion
This study revealed a high and multiple resistance to insecticides, coupled with low efficacy of LLINs without PBO in An. Funestus, highlighting the threat that insecticide resistance poses on the efficacy of existing vector control tools. Interestingly, this study revealed that An. funestus swarms can be detected and characterized in the field providing the opportunity for mating swarms of this species to be targeted to implement alternative vector control strategies. Furthermore, this study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competitiveness in resistant mosquitoes. As a negative fitness costs could influence the evolution of insecticide resistance in field populations of mosquitoes like the speed of increase or reversal to susceptibility in vector populations it is crucial that such impacts are understood and taken into consideration when designing and implementing future insecticide resistance management strategies.

Data availability
Underlying data Underlying data is available from Open Science Framework This is an interesting manuscript that provides some comprehensive and potentially useful bionomic data for and, to a lesser extent, and in Cameroon. My comments Anopheles funestus An. gambiae An. coluzzii on its contents are as follows: The title does not adequately reflect the full scope of the content of this manuscript. Mating competitiveness is only one component of the data presented here.
In the introduction (page 3) it is stated that little is known on the fitness costs of metabolic resistance mechanisms in anophelines owing to a lack of DNA based markers. I suggest that such markers, although of course useful, are not essential for such evaluations as fitness costs can also be assessed using biological characteristics such as life table analyses including fecundity and fertility. These likely give better indications of fitness cost. The authors should also think about what parameter they have actually measured here: was it mating competitiveness or propensity to mate? The former primarily speaks to physiological fitness while the latter is a behavioural issue. These terms may be interchangeable but there are subtle differences. Basically, less able to mate = reduced mating competitiveness; less inclined to mate = reduced propensity. These terms may be interchangeable but there are subtle differences. Basically, less able to mate = reduced mating competitiveness; less inclined to mate = reduced propensity.
For the insecticide susceptibility assays, it should be noted that the standard method for assessing resistance intensity is by using increasing concentrations of the insecticide(s) in question. I assume the authors chose to use extended exposure times instead because of a shortage of test samples (F1s) but the problem with this method is that there is no clearly elucidated method for assessing the operational implications of increased intensity where detected using data generated in this way.
The data on mating competitiveness are somewhat ambivalent because the genotype frequencies for the L119F-GSTe2 resistant homozygotes (RR) were equivalent between mated and unmated males. It is therefore difficult to see how this mutation negatively impacts mating competitiveness/propensity. As the only real difference was the frequency of heterozygotes in each group, an alternative explanation may point to an associative negative heterotic effect on propensity to mate in males that is not caused by the L119F-GSTe2 mutation itself, but rather by linkage disequilibrium between this locus and other deleterious alleles i.e. a negative pleiotropic effect.
Is 'Koekemoer cocktail Polymerase Chain Reaction assay' an official name for this assay?

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.

If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
Are the conclusions drawn adequately supported by the results? Partly No competing interests were disclosed. We really appreciate this comment from the reviewer. We just want to highlight that a high frequency of 1014F kdr resistance allele found was for mosquitoes since for instance, An. gambiae there is no evidence of in . For this reason, we could not assess the interaction of Kdr An. funestus the presence of and in relation to the mating competition in . However, as kdr GSTe2 An funestus the and are both present in this species we wanted to assess the cumulative impact of RDL GSTe2 these genes on mating competition but this was not possible because of the very low frequency of the A296S-RDL mutation in this population. The title of this paper is somewhat of a misnomer. It is probably described thus to attract a wider audience than a more somewhat mundane title describing the resistance status of the Anopheles funestus from a village in Cameroon. The paper describes in considerable detail the resistance status of the mosquito more than it does the effect of this on mating success. Thus, the results concerning mating success concern just 21 males caught in copula whilst the overall resistance status covers a number of tests from standard WHO tests to tests with the synergist PBO and bioassays on different net types.

No competing interests
The authors discuss the fact that results on mating competitiveness of resistant mosquitoes from a small number of studies give confusing results. Indeed they have participated in studies where five times the number of mating mosquitoes have been investigated. Some indicate that there may actually be an advantage whilst others give different results. It obviously is a field that can be explored further. But in the present case I feel that the authors are confusing statistical significance with biological significance. They do point out that further studies would be useful but given their title,which others may take for a fact, this is a problem.
Given that the swarms the authors observed occurred close to houses it would seem that the males Given that the swarms the authors observed occurred close to houses it would seem that the males rested inside houses (as they do elsewhere). It is a shame that they did not examine the proportions of the resistance genotype among these insects. Indeed, it might have been possible to collect resting insects and to examine their terminalia to determine the degree of rotation so that the effect of age on survival and resistance status among the males could have been investigated.
The authors write 'Concerning the mating behaviour, when a female chose a male, they immediately left the swarms, flying at 1.5m from the ground.' But it is by no means certain that female mosquitoes 'choose' their mates (which implies that sexual selection is taking place). With respect to their earlier paper it is perhaps worth pointing out that (as far as I know) there is no evidence of olfaction playing a part in the mating behaviour of Anopheles mosquitoes.
Given that the swarms seen were large (more than 100 individual males in a swarm) it begs the question as to how many mating pairs were seen and how many were successfully collected. If after 12 nights of observation of such large swarms only 21 pairs were formed it begs the question 'Is this all the mating that was taking place?' Either there were other sites where a lot more mating was taking place or something else is going on. I do not know.
The place used by the mosquitoes to swarm is similar to that described from Mozambique. There the mosquitoes actively avoid markers if they are introduced under the swarm. Again the characteristics of the location remain undefined -why they swarm where they swarm is still an enigma but as the authors point the insects remain consistent and swarm in the same place night after night.
Given the time and effort that resistance tests require their extensive data on resistance should allow them to suggest what is the optimum or minimal method that could be used to determine the resistance status in other populations. This is perhaps a better focus for their paper since much of the information that they provide is irrelevant to the title of the paper. If the paper continues with the same theme then much of the data that they present could, in fact be provided as supplemental files.
Given the advantage conferred by resistance alleles over susceptible ones and given the very high level of resistance it is strange that these genes are not fixed in the population. It also means that if in the absence of insecticide pressure resistant insects are at a disadvantage compared to the wild type (perhaps because they have to divert resources from eggs to cuticle) the presence of susceptible insects implies that once insecticide pressure is removed the population will revert back. How this might affect transmission is moot.
The English in the paper could be improved. For example, in my opinion, the very first sentence reads better thus: 'Despite an increase in the last two years1 significant progress has been made in recent decades in malaria vector control. This has contributed to a significant reduction in the burden of disease caused by this parasite'. Additionally, if the title if it is retained, can be rearranged thus 'Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, '

Anopheles funestus
It is also a bit of a shame that the sporozoite data for the s.l. were not given by Anopheles gambiae species. With such a high rate both members of the complex would be expected to be infected. If only one species was then this would not only boost the rate -to very high levels -but would also indicate that there might be differences in vectorial capacity between the species that merit further investigation. It would seem reasonable to assume that they were dealing with ageing populations in decline after the rainy season which was responsible for the high sporozoite rates. The title of this paper is somewhat of a misnomer. It is probably described thus to Comment 1: attract a wider audience than a more somewhat mundane title describing the resistance status of the from a village in Cameroon. The paper describes in considerable detail the Anopheles funestus resistance status of the mosquito more than it does the effect of this on mating success. Thus, the results concerning mating success concern just 21 males caught in copula whilst the overall resistance status covers a number of tests from standard WHO tests to tests with the synergist PBO and bioassays on different net types.
We sincerely appreciate this comment from the reviewer. The main aim of this study Response: was to evaluate the impact of insecticide resistance on mating competition using the L119F-GSTe2 resistance marker hence the title: ''Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males , Anopheles funestus African malaria vector''. Because prior to this study the resistance profile of malaria vectors in the locality was unknown, it was important to start by elucidating the resistance profile and the potential mechanisms driving the resistance to insecticide in the study site. So the title was not chosen to attract a wider audience as the reviewer is noticing but refers to the main question we wanted to address when designing this study. Also by using the word "investigation" in the title, we are acknowledging that more work is needed to fully establish this impact on mating but this provides initial useful observations on this topic.
The authors discuss the fact that results on mating competitiveness of resistant Comment 2: mosquitoes from a small number of studies give confusing results. Indeed they have participated in studies where five times the number of mating mosquitoes have been investigated. Some indicate that there may actually be an advantage whilst others give different results. It obviously is a field that can be explored further. But in the present case I feel that the authors are confusing statistical significance with biological significance. They do point out that further studies would be useful but given their title, which others may take for a fact, this is a problem.
We thank again the reviewer. We agree that there is a risk of confusing statistical Response: significance with biological significance. But we believe that we have avoided this situation by presenting each result as we saw it. We noticed when using the Odd-ratio comparison that mosquitoes with the 119F resistant allele displayed significantly lower mating competitiveness compared to those with the L119 susceptible allele and we provided the statistical significance associated with the observation. Then we discussed the potential meaning of the result but taking a cautionary approach by stating that although it could mean that there is a biological meaning that only further studies will confirm this. This is the general message of this manuscript and we agree with the reviewer that such confusion should be avoided that is why we mentioned in discussion that further studies are needed in other locations and other resistant markers to widely appreciate the impact of resistance on mating competitiveness.
Given that the swarms the authors observed occurred close to houses it would seem Comment 3: that the males rested inside houses (as they do elsewhere). It is a shame that they did not examine the proportions of the resistance genotype among these insects. Indeed, it might have been possible to collect resting insects and to examine their terminalia to determine the degree of rotation so that the effect of age on survival and resistance status among the males could have been investigated.
We thank the reviewer for noting this. Our main aim as mentioned above was mainly to Response: see if insecticide resistance has any impact on mating competition through swarm collection. That is why we did not collect resting males to examine their terminalia and determine the degree of rotation as pointed by the editor but this is a good idea that will be taken into consideration in future studies.
The authors write 'Concerning the mating behaviour, when a female chose a male, Comment 4: they immediately left the swarms, flying at 1.5m from the ground.' But it is by no means certain that female mosquitoes 'choose' their mates (which implies that sexual selection is taking place). With respect to their earlier paper it is perhaps worth pointing out that (as far as I know) there is no evidence of olfaction playing a part in the mating behaviour of Anopheles mosquitoes.
We agree with the reviewer that there is very little information on the impact of olfaction Response: on the mating behavior of Anopheles mosquitoes but in this study we noticed that when a female coupled with a male, they immediately left the swarms, flying at 1.5m from the ground for copulation although the mechanisms involved remain unknown. To avoid confusion we have replaced "chose" by "coupled with".
Given that the swarms seen were large (more than 100 individual males in a swarm) Comment 5: it begs the question as to how many mating pairs were seen and how many were successfully collected. If after 12 nights of observation of such large swarms only 21 pairs were formed it begs the question 'Is this all the mating that was taking place?' Either there were other sites where a lot more mating was taking place or something else is going on. I do not know.
It is true that given the size of the swarms 21 couples collected were very low but this Response: can be explained by the fact that females are not necessarily participating to the swarming; that is why the number of unmated males was very high. But the hypothesis that this was not all the mating taking place cannot be rejected since when the night started it was not easy to observe the couples. We have now added this sentence to highlight it: "The low number of collected couples suggests a low number of females in these swarms but could also indicate that mating was also taking place in other swarms not detected in this study." The place used by the mosquitoes to swarm is similar to that described from Comment 6: Mozambique. There the mosquitoes actively avoid markers if they are introduced under the swarm. Again the characteristics of the location remain undefined -why they swarm where they swarm is still an enigma but as the authors point the insects remain consistent and swarm in the same place night after night.
We appreciate this comment from the reviewer. Compared to complex Response: An. gambiae which swarm around markers such as brick piles, rice fields, banana trees, burnt ground, garbage heaps and ant hills swarms we observed in this study appeared to avoid ground An. funestus markers. So it will be important in the future to assess the ecological and physiological parameters enhancing mating.

An. funestus
Given the time and effort that resistance tests require their extensive data on Comment 7: resistance should allow them to suggest what is the optimum or minimal method that could be used to determine the resistance status in other populations. This is perhaps a better focus for their paper since much of the information that they provide is irrelevant to the title of the paper. If the paper continues with the same theme then much of the data that they present could, in fact be paper continues with the same theme then much of the data that they present could, in fact be provided as supplemental files.
Thank you for your comments. We cannot suggest what is the optimum or minimal Response: method that could be used to determine the resistance status in other populations since there are WHO's recommendations on how to assess the resistance profile in a given population and we were just following those instructions. We suggest keeping the whole data as it is as we explained above the insecticide work was necessary in order to explore the impact of the GSTe2 resistance allele on mating competition. Given the advantage conferred by resistance alleles over susceptible ones and given Comment 8: the very high level of resistance it is strange that these genes are not fixed in the population. It also means that if in the absence of insecticide pressure resistant insects are at a disadvantage compared to the wild type (perhaps because they have to divert resources from eggs to cuticle) the presence of susceptible insects implies that once insecticide pressure is removed the population will revert back. How this might affect transmission is moot.
Thanks to the reviewer for this remark. Some resistance genes are fixed in mosquito's Response: populations like in Southern Africa, the L119F-GSTe2 in Benin for and the Cy6p9a/b An. funestus L1014F-Kdr in many African populations. However fixation of resistance alleles An. gambiae depends on other factors and the mode of selection of these alleles. That is why to it is crucial to study the impact of resistance on mosquito's life such mating competitiveness before implementing any resistance management strategy based on rotation because when the resistance becomes fixed in the population there is little chance to revert to susceptibility.
The English in the paper could be improved. For example, in my opinion, the very Comment 9: first sentence reads better thus: 'Despite an increase in the last two years1 significant progress has been made in recent decades in malaria vector control. This has contributed to a significant reduction in the burden of disease caused by this parasite'. Additionally, if the title if it is retained, can be rearranged thus 'Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, '

Anopheles funestus
We have double-checked the whole manuscript for English grammar and mistakes.

Response:
It is also a bit of a shame that the sporozoite data for the s.l.

Comment 10:
Anopheles gambiae were not given by species. With such a high rate both members of the complex would be expected to be infected. If only one species was then this would not only boost the rate -to very high levelsbut would also indicate that there might be differences in vectorial capacity between the species that merit further investigation. It would seem reasonable to assume that they were dealing with ageing populations in decline after the rainy season which was responsible for the high sporozoite rates.

An. gambiae
An. coluzzii However, among the 5 mosquitoes infected with Plasmodium sporozoites2 were [2/7 An. coluzzii infected (28.5%)] and 3 were [3/33 infected 9.1%)]. However, the low sample size of An. gambiae means that this rate is not comparable. Nevertheless we have now presented the An. coluzzii infection rate for each species in the manuscript.