Mapping the distribution of Anopheles funestus across Benin highlights a sharp contrast of susceptibility to insecticides and infection rate to Plasmodium between southern and northern populations

Background. Malaria remains an important public health issue in Benin, with Anopheles gambiae s.l. and Anopheles funestus s.s being the predominant vectors. This study was designed to generate information on An. funestus distribution, molecular speciation, Plasmodium infection rate and insecticide susceptibility status across Benin. Methods. Mosquito samples were collected from December 2014 to January 2016 in 46 localities in Benin. These samples were mapped and An. funestus collected were speciated to the molecular level. Plasmodium infection rate was determined using a Taqman assay and susceptibility to insecticides was assessed using the WHO guidelines. The genotyping of the L119F- Gste2 mutation was also carried out. Results. An. funestus was found in 8 out of the 46 localities surveyed with a high presence in Tanongou (wet Sudanese ecological zone), Kpome, Doukonta and Pahou (sub-equatorial ecological zone). Molecular identifications revealed that only An. funestus s.s was present in southern Benin, whereas in Tanongou (northern Benin) An. funestus s.s. and An. leesoni were found in sympatry at proportions of 77.7% and 22.3% respectively. Plasmodium infection rate of An. funestus was higher in southern Benin at a range of 13 to 18% compared to 5.6% recorded in Tanongou. High DDT (8±0.5%) and permethrin (11±0.5%) resistance were observed in Doukonta, Kpome and Pahou, contrasting with relatively low resistance profiles: mortality-DDT=90±3.18% and mortality-permethrin=100% in Tanongou. Genotyping analysis revealed high frequency of the resistant 119F allele in the South (Kpome and Doukonta) compared to the North (Tanongou). Discussion and Conclusion. The high presence of An. funestus in the South compared to the North could be due to favorable environmental and climatic conditions found in both regions. A significant Plasmodium infection rate was recorded across the country. A high resistance profile was recorded in the southern Benin; this raises the need for further investigations on resistance selection factors.

significant infection rate was recorded across the country. A high Plasmodium resistance profile was recorded in the southern Benin; this raises the need for further investigations on resistance selection factors. Rousseau

Background
Malaria remains a major public health challenge in Benin, with the most vulnerable populations being children less than five years and pregnant women 1 . It accounts for around 37% of hospital consultations in the country 2 . Efforts to eradicate this disease in Africa have focused on treatment of diagnosed cases and preventive strategies, which are mainly based on vector control, such as the use of insecticide treated nets, indoor residual spraying of insecticides and larviciding 1 .
In the past decade, vector control interventions have massively contributed to the significant decrease observed in the burden of malaria across Africa, notably in Benin 3 . To sustain such progress,

Amendments from Version 1
We did not remove any of the already published information, rather few insertions were made in the manuscript based of the second reviewer's comments. Briefly, information inserted are as follows:

Methods
Comment 1: I recommend the authors to give the different sequences of the primers used (P. ovale, vivax, malariae and falciparum) for Plasmodium infection rate detection.

REVISED
national control programs need better knowledge on key malaria vectors nationwide, including their geographical distribution, susceptibility profile to insecticides and contribution to malaria transmission, as well as understanding the vectorial complexity of these species. Such information already exists for Anopheles gambiae across Benin 2,4,5 , but this is not the case for the other major vector An. funestus, for which only limited information is available, mainly from few coastal populations 6,7 . An. funestus Giles is one of the key malaria-transmitting mosquitoes in Africa. The vectorial capacity of this mosquito vector is close to and could exceed that of An. gambiae, the most documented malaria vector in some countries 8  This study aims to generate information on the distribution, Plasmodium infection rate and resistance status of An. funestus in the South-North transect of Benin to help control programs to have a better assessment of the contribution of this species nationwide and how best to control it.

Ethical statement
No ethical permit was required for this study. However, there was a focus group discussion with the community and household heads where verbal consent was obtained for mosquito collections in the community after the study aims and objectives were explained. During this research study, we did not perform insecticide spraying, night collections, or human bait for mosquito collection. All mosquitoes were sampled during daytime using electrical aspirators activated with batteries.
Study sites and mosquito collection Study site description. Benin lies between the Equator and the Tropic of Cancer at latitudes ranging from 6°30′ N to 12°30′ N and longitude from 1° E to 3°40′ E. This country shares boundaries with Togo in the West, Burkina Faso and Niger in the North, and Nigeria in the East. Four main climatic zones are found in the country. The North Sudanese climatic region, which is characterized by one long dry season and a short rainy season, with low relative humidity and rainfall that is the lowest in the country (800 to 1000 mm per year). Large water bodies are found in this region and temperatures are the highest, and could reach 45°C during dry seasons. The second region is the wet Sudanese climatic zone (Atacorian). This climatic region is dominated by hills of up to 800 m of altitude and several small water bodies, which makes the region colder. Annual rainfall ranges from 1200 to 1300 mm per year, the vegetation is partially of wet savanna type, the temperature in this part of the country is the lowest. The third region is the sub-Sudanese climatic region that covers the center of the country and part of the South. This climatic region has one long rainy season and one short dry season. Rainfall is between 900 and 1200 mm, the region is less hilly and the vegetation is of wet savanna type. The fourth region is the southern sub-equatorial climatic region that spans the southern part of the country and extends up to coastal areas of Benin. This region is made up of two rainy seasons and two dry seasons. The relative humidity is high, temperatures are relatively low and the vegetation is a mosaic of coastal, wetlands, forest, and wet savanna type. Several water bodies join together in this part of the country before being channeled into the sea (Figure 1).

Mosquito sampling.
From December 2014 to January 2016, indoor collections of adult female mosquito were made between 06 to 10am in several localities along South-North transect of Benin using four electric aspirators. Mosquito collections were carried out in different localities and the GPS was used to determine the latitude and longitude for each sampled locality. Maps of surveyed sites and the distribution of An. funestus in Benin were developed using recorded latitudes and longitudes. For each surveyed locality, a minimum of 30 rooms were randomly selected for mosquito aspirations. These rooms were selected in a way to cover the various ecologies found in each locality. At least three days were spent in each surveyed site but for localities where An. funestus were found, the number of days was extended to five days to obtain a good number of mosquitoes to be used for various analyses. Aspirated mosquitoes were identified morphologically 9 , counted and the total number for each species was recorded. All blood-fed and gravid An. funestus (F 0 ) collected inside houses were taken to the IITA insectary in Cotonou (Benin), where they were kept in small cups until fully gravid. The forced egg laying technique described by Morgan et al. 26 was then used to induce female An. funestus to lay eggs. Egg batches and emerging larvae from the same female mosquito were reared together and later pooled with larvae from other females, if these females were found belonging to the same molecular species. The insectary condition was at a temperature and relative humidity of 25-27°C and 80±5% respectively. Larvae were fed daily with Tetramin ™ baby fish food and the water of each larvae bowl was changed every two days to reduce the mortality. The F 1 adults generated were randomly mixed in cages for subsequent experiments.

Seasonal estimation of mosquito densities per room
Mosquito densities per room (m/r) were estimated during four annual climatic seasons: rainy season, transition from rainy to dry season, dry season and transition from dry to rainy season. This estimation was based on the total number of An. funestus s.l. collected during each season divided by the number of rooms surveyed for mosquito collections in that season. Seasonal variations of An. funestus densities were determined per room in two localities in Benin: the locality of Tanongou in the North (wet Sudanese/ Atacorian climatic region) and the locality of Kpome in the South (subequatorial climatic region). Kpome and Tanongou were selected to represent the southern and northern regions respectively, due to the high density of An. funestus recorded in these localities.

PCR species identification
For each locality, female mosquito specimens that were morphologically identified as belonging to An. funestus group 9 were subjected to DNA extractions using Qiagen DNeasy Kit followed by PCR for species identification, as described by Koekemoer et al. 31 .

Insecticide susceptibility tests
Protocols and standard insecticide treated papers supplied by WHO 33 were used to test for insecticide susceptibility of An. funestus from selected localities in the northern and southern where there was a consistent number of ovipositing females. These selected localities were Tanongou, northern Benin in the wet Sudanese climatic region (Atacorian region), and Doukonta, southern Benin in the sub-equatorial climatic region. We assessed the susceptibility pattern of An. funestus s.s. from both localities to two insecticides of public health interest: pyrethroids type I permethrin (0.75%) used for insecticide treated nets (ITNs), and organochlorines DDT (4%) used in insecticide residual spraying (IRS). Exposed mosquitoes were fed with 10% sugar solution after 1hr of insecticide exposure after which mortalities were recorded 24hrs post exposure to insecticide treated papers 33 . The wild population of An. funestus was exposed to non-treated insecticide papers as a control 33 due to lack of susceptible strains of An. funestus, (An. funestus FANG). Prior to the experiment, the effectiveness of insecticide treated papers purchased from the Vector Biology Department, Liverpool School of Tropical Medicine (LSTM), UK was confirmed by exposing the susceptible strain An. gambiae kisumu to insecticide impregnated papers. Tests were conducted at a temperature and relative humidity of 25-27°C and 80±5% respectively. WHO criteria were used to determine resistance status with mortality between 98-100% indicating susceptibility, 90-97% potential resistance, and less than 90% resistance 33 .
Distribution of L119F-GSTe2 resistance allele using TaqMan assay To assess the role of L119F mutation in DDT resistance, wild female An. funestus s.s. collected from each selected location were genotyped using the Taqman assay, as previously demonstrated 30 .
The reaction was performed in a 10µl final volume containing 1×SensiMix (Bioline, London, UK), 800 nM of each primer and 200 nM of each probe using an Agilent MX3005P machine. The following cycling conditions were used: 10 min at 95°C, 40 cycles of 15s at 92°C and 1 min at 60°C. Two probes labelled with fluorochromes FAM and HEX were used. The FAM was used to detect the mutant allele, while the HEX detected the wild type allele.

Data analysis
MedCalc easy-to-use online statistical software 34 using the Fisher's exact test was used to test for significant difference of Plasmodium infection rate and L119F-GSTe2 genotyping data in the South compared to the North of Benin.

Distribution of Anopheles funestus species in a South-North transect of Benin
Out of the 46 surveyed localities (Figure 1 and Supplementary  found in Benin. In the wet Sudanese climatic region, and more specifically in Tanongou An. funestus s.s. from Tanongou were exposed to permethrin and DDT in 5 replicates (average pool of 20 mosquitoes). Results revealed low mortalities to DDT (8±0.5%) and permethrin (11±0.5%) for An. funestus s.s. from Doukonta, whereas the Tanongou population had higher mortality rates to DDT (90±3.18%) and permethrin (100%). This shows that there is a higher resistance in Doukonta compared to Tanongou (Figure 3). Similarly, high resistance levels have been previously documented in southern localities of Pahou and Kpome 6,7 .

Screening of L119F-GSTe2 mutation in a wild population of Anopheles funestus from Benin
Genotyping of the L119F-Gste2 mutation in wild An. funestus population from each of the selected locations revealed the presence of the resistant 119F allele at a high frequency: 96% in Kpome 7 , 83.2% in Doukonta (southern Benin), while in Tanongou (North Benin), 35% mutant allelic frequency was recorded.
No susceptible allele (SS) was observed either in Kpome or Doukonta mosquitoes, showing that the 119F gene is close to fixation in the An. funestus populations of these two locations in the southern Benin. A significant difference (P≤0.0001) was observed between the 119F allelic frequency recorded in Kpome and Doukonta, where a high resistance to DDT was observed compared to Tanongou (Figure 4).

Discussion
This research was designed to map the distribution of An. funestus in Benin and compare the insecticide resistance profile of this malaria vector in the North-South transect, as well as their infection rates with Plasmodium species, for improved knowledge on this malaria vector and enhanced performances of current malaria control tools.
Distribution of An. funestus and its implication in malaria transmission in the various geo-climatic settings of Benin An. funestus was mainly found in the southern and the northwestern localities of Benin in this study. In these two geo-climatic regions, there seem to be a high tendency of this species to colonize the western areas of the country (north and southwestern). The relatively high presence of this vector in the western part of Benin could be explained by the humidity, relatively low temperatures associated with the hilly landscape, and the presence of rivers and streams covered with vegetation 35 . This shows that this species prefers more permanent water bodies with vegetation usually found along rivers, streams and lakes 36 , whereas An. gambiae tends to oviposit in temporary breeding sites, such as puddles and animal foot prints 37 . Very little or no population of An. funestus was found in the dry Sudanese climatic region of northeastern Benin. The low presence of this mosquito species in this dry hot region (low rain falls and temperature reaching 45°C during dry seasons) is either due to the period of sampling or the low presence of permanent fresh water bodies covered with vegetation coupled with dryness of the region 38 .    Comparative insecticides susceptibility tests of An. funestus s.s. from southern (Doukonta) and northern (Tanongou) localities of Benin Comparative analysis of insecticide resistance profiles in An. funestus populations from Doukonta (southern Benin) and Tanongou (northern Benin) reveals that An. funestus s.s. from Doukonta are relatively more resistant to DDT and permethrin (mortality rates of 8±0.5 and 11±0.5%, respectively) than those from Tanongou, where only a moderate resistance was observed to DDT (mortality rate of 90±3.18%) and a full susceptibility to permethrin (100%). High resistance to DDT and permethrin had previously been reported in populations of An. funestus from two other localities of southern Benin, Pahou and Kpome 6,7 . In addition to the use of agricultural insecticides in both the northern and southern surveyed sites, the high insecticide resistance observed in the South could be associated with environmental factors, such as urbanization, which increases the level of xenobiotics (pollution) in Anopheles breeding sites and could favor the selection of cross resistance to permethrin and DDT in southern Benin compared to northwestern Benin with less urbanization and pollution 45 . Recorded resistance profiles could also be associated with a relatively high flow of genes among An. funestus populations in southern Benin compared to the North, particularly if there are some barriers to gene flow, which needs to be investigated further. Other factors of resistance selection, such as the relatively high use of ITNs/IRS (use of public health insecticides) in the southern Benin compared to the North, might have also contributed to observed high resistance profile of mosquitoes 6,46-50 . Similar observations have been documented on An. gambiae s.l. in the North and South of Benin where increased pyrethroid resistance is also prevalent in An. gambiae s.l. species in South Benin 51-53 than in the North, mirroring the pattern that was observed here for An. funestus. Resistance to DDT and permethrin is also widely distributed in An. gambiae in Benin 4,54 .
Distribution of L119F-GSTe2 mutation in An. funestus populations in Benin The high frequency of the 119F-GSTe2 resistant allele in Kpome and Doukonta where high phenotypic resistance to DDT was also observed; both results suggest that this mutation plays an important role in DDT resistance in West Africa, as previously documented 30 . Indeed, consistent frequencies of this resistance allele were also recorded in other DDT resistant populations in Central and West Africa notably in Cameroon (52%), Ghana (44%) and Burkina Faso (25%) in accordance with the previously reported prevalence of DDT resistance in these countries 27-29 . The resistant 119F allele was detected in An. funestus populations from Tanongou, but with a relatively low frequency (35%), reflecting the moderate level of DDT resistance recorded. This result is in line with the detection of low frequencies of this resistant allele in the eastern African An. funestus of Uganda (20.4%) and Kenya (7.8%), which is associated with a moderate level of DDT phenotypic resistance observed in this region 19,26 . However, this observation is different in southern Africa where this mutation is completely absent despite recent reports of DDT resistance 25 , suggesting that DDT resistance in southern Africa is driven by a different mechanism to that observed in West and Central Africa. These heterogeneities in L119F frequencies suggest that there are different mechanisms responsible for the DDT resistance in An. funestus populations across Africa.

Conclusion
This study has generated key relevant information on the bionomics of An. funestus in Benin, including its seasonal distribution in a South-North transect, its Plasmodium infection rate and its resistance profiles to permethrin and DDT in the southern and northern ecological zones. The contrasting profiles observed between southern and northern populations of An. funestus were evident in the present study in terms of density, contribution to malaria transmission and resistance to insecticides. The factors behind these differences need further investigation. Overall, the high density of An. funestus in the south and northwestern Benin coupled with the consistent high Plasmodium infection level of this Anopheles species and its high resistance to insecticides in the South strengthens the need for more research on this species for improved performances of malaria control programs in Benin.

Data availability
Raw data are available at the Open

Data analysis
In this section, it should be mentioned the used statistical tests

Screening of L119F-GSTe2….section Tables and figures
In all , for clarity please add the collection dates in the title Tables and figures Figure 3: The data represent mortality mate? means? medians? with 95 % confidence intervals? Please make clear. Figure 4: What does f (S) and f(R) represent? Please specify in the title.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. For this analysis as described in the methodology, primers and probes were used: Forward (F), PlasF (5'-GCTTAGTTACGATTAATAGGAGTAGCTTG-3') and reverse (R), PlasR (5'-GAAAATCTAAGAATTTCACCTCTGACA-3') primers. Specific probes for species plasmodium detection: 5'-TCTGAATACGAATGTC-3' labelled with FAM for detection and P. falciparum 5'-CTGAATACAAATGCC-3' labelled with HEX for or and detection P. ovale P. vivax P. malariae were also used.

condition of test (relative humidity and temperature) and insectary conditions.
Insectary and Bioassay room were at a temperature of 25-27°C and relative humidity of 80±5%.

Comments 3 & 4:
In this section, it should be mentioned the used statistical tests.

3.
Plasmodium Fisher's exact test was used to determine the significant levels (P-values) of the Plasmodium infection rates and L119F-GSTe2 allelic frequency of in the South compared Anopheles funestus to the North of Benin.
to the North of Benin.

Comment 5:
In all , for clarity please add the collection dates in the title Tables and figures This comment has been addressed in the latest version of the article. See titles of figures and tables.
: The data represent mortality rate? means? medians? with 95 % confidence Figure 3 intervals? Please make clear.
The data on Figure 3 represents the mortality rates of mosquitoes subjected to insecticide susceptibility tests and error bars represent the standard deviation of the mean.
: What does f(S) and f(R) represent? Please specify in the title. Figure 4 : Allelic frequency of the L119F-GSTe2 mutation in wild populations Figure 4 Anopheles funestus (F0) from Kpome (South Benin), Doukonta (South Benin) and Tanongou (North Benin). f(S) represents frequency of susceptible allele and f(R) represents frequency of resistant allele in the species population.
We declare no competing interest Competing Interests: 19  The research question is clearly defined and the study design appropriate.
The method section provides sufficient details to allow the repeatability of the work. In overall, the paper is scientifically good and provide relevant information on bionomy in Benin. However, Anopheles funestus there is one minor change required; In the Methods section, susceptibility test paragraph, line 14, the author states that : 'The wild population of . was exposed to nontreated insecticide papers as An funestus a control '. The author should due to lack of susceptible strains of , ( FANG) An. funestus An. funestus remove the underlined sentence as from WHO guideline, it is not required to use the susceptible strain as control.

Comment/question
Why has the author limited only to the determination of seasonal variation and infection rate?
The author could have estimated passive aggression by dividing the number of mosquitoes collected per