Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia.

BACKGROUND: Indoor residual spraying and insecticide-treated nets are among the key malaria control intervention tools. However, their efficacy is declining due to the development and spread of insecticide resistant vectors. In Ethiopia, several studies reported resistance of An. arabiensis to multiple insecticide classes. However, such data is scarce in irrigated areas of the country where insecticides, pesticides and herbicides are intensively used. Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms were assessed in Arjo-Didessa sugarcane plantation area, southwestern Ethiopia.
METHODS: Adult An. gambiae s.l. reared from larval/pupal collections of Arjo-Didessa sugarcane irrigation area and its surrounding were tested for their susceptibility to selected insecticides. Randomly selected An. gambiae s.l. (dead and survived) samples were identified to species using species-specific polymerase chain reaction (PCR) and were further analyzed for the presence of knockdown resistance (kdr) alleles using allele-specific PCR.
RESULTS: Among the 214 An. gambiae s.l. samples analyzed by PCR, 89% (n = 190) were An. amharicus and 9% (n = 20) were An. arabiensis. Mortality rates of the An. gambiae s.l. exposed to deltamethrin and alphacypermethrin were 85% and 86.8%, respectively. On the other hand, mortalities against pirmiphos-methyl, bendiocarb, propoxur and clothianidin were 100%, 99%, 100% and 100%, respectively. Of those sub-samples (An. amharicus and An. arabiensis) examined for presence of kdr gene, none of them were found to carry the L1014F (West African) allelic mutation.
CONCLUSION: Anopheles amharicus and An. arabiensis from Arjo-Didessa sugarcane irrigation area were resistant to pyrethroids which might be synergized by extensive use of agricultural chemicals. Occurrence of pyrethroid resistant malaria vectors could challenge the ongoing malaria control and elimination program in the area unless resistance management strategies are implemented. Given the resistance of An. amharicus to pyrethroids, its behavior and vectorial capacity should be further investigated.

Background

Indoor residual insecticide spraying (IRS) and insecticide-treated nets (ITNs) are key strategies to prevent malaria transmission [1, 2]. However, these interventions are threatened due to the increasing occurrence of insecticide resistant malaria vectors [3-5]. Target site resistance, metabolic resistance, cuticular and behavioral resistance are among the resistance mechanisms in the vectors [4, 6, 7]. Knockdown resistance (kdr) is a target site resistance mechanism conferred by mutation(s) in the voltage-gated sodium channel (VGSC) gene. A single amino acid substitution of Leucine with Phenylalanine (L1014F/Kdr-West) and Leucine with Serine (L1014S/Kdr-East) at position 1014 of VGSC gene are the most common kdr mutations [8, 9].

The primary malaria vector in Ethiopia, An. arabiensis, has developed resistance to multiple insecticides such as DDT and pyrethroids, possibly due to their long term use for IRS and ITN [10-12]. Resistance in malaria vectors could be enhanced by the use of similar insecticides (chemicals) in agricultural practices [13, 14]. Small and large scale irrigation agricultural activities are increasing year after year to meet the food and economic demands of the population in Ethiopia [15, 16]. Arjo-Didessa sugarcane irrigation is one of the state owned macro-agroeconomic projects in the country [17, 18]. Insecticides, pesticides and herbicide are being extensively used for the control of malaria vectors, agricultural pests and weeds in the irrigation area and its surrounding.

Pyrethroid (deltamethrin and alpha-cypermethrin) impregnated LLINs and carbamate (propoxur and/or bendiocarb) based IRS are used to control adult malaria vectors while temephos (Abate formula) is applied for larval mosquito control in western Ethiopia including Arjo-Didessa irrigation area (Sources: Arjo-Didessa Sugar Factory malaria prevention and control department and Jimma-Arjo District Health Office). Chlorpyrifos (CPS) has been used as pesticide for the sugarcane plantation while Ametryn, Afratine and bound off (glyphosate) as herbicides against broad leaved and grass weeds in the irrigation and its surroundings (Personal communication: Local farmers and Mr. Getechew Etefa; Arjo-Didessa Sugar Factory, Agricultural Chemical’s Section Head). These chemicals may enhance development of insecticide resistance through creating selection pressure [4, 19–25]. Use of agriculture pesticide has been associated with An. gambiae s.l. resistance in West Africa [19, 21, 26], An. arabiensis resistance in Sudan [27] and An. gambiae s.l. resistance in Tanzania [6]. High metabolic resistance of An. arabiensis to pyrethroids [28] and increased frequencies of kdr mutation were attributed to massive use of DDT and pyrethroids in cotton growing farms [19-21]. However, similar mortalities of An. gambiae s.l populations were observed regardless of the pesticide use pattern in areas of varying agrochemical use in Côte D’Ivoire [29].

To avert the effect of multiple and widespread insecticide resistance, clothianidin: a novel neonicotinoid insecticide, has been recommended to supplement the current insecticide based interventions [30-32]. Thus, together with the existing insecticides, it is imperative to evaluate efficacy of clothianidin against An. gambiae s.l. [33] in field setups including in irrigation areas.

Although several studies reported widespread resistance in An. gambiae s.l. most importantly in An. arabiensis [10-12], data on insecticide resistance and associated mechanisms is scarce in irrigated areas of Ethiopia. Furthermore, despite multiple studies conducted on resistance of An. gambiae s.l. (primarily An. arabiensis), the susceptibility status of sibling species and minor/rare species (such as An. amharicus) are often overlooked [34]. Except some old information on its zoophilic behavior and little importance in malaria transmission [35, 36], the current role of An. amharicus in malaria transmission as well as its behavior and response to public health insecticides is unknown. To our knowledge, there was no study conducted to evaluate the susceptibility status of An. amharicus to available insecticides in Ethiopia and beyond. Therefore, this study was the first to investigate the susceptibility of this species in western Ethiopia where An. arabiensis and An. amharicus co-exist [18]. Most importantly, regular monitoring of insecticide resistance is critical for effective resistance management especially in areas where same/similar insecticide is used for both vector control and agricultural purposes [5].

Thus, susceptibility status of An. arabiensis and An. amharicus (sibling species of the gambiae complex in Ethiopia) to commonly used insecticides and clothianidin (a new candidate insecticide), and presence of West African knockdown resistance gene (kdr-west) were investigated at Arjo-Didessa sugarcane irrigation area and its surrounding villages.

Methods and materials

Study area

The study was conducted at Arjo-Didessa sugarcane irrigation area and its vicinity, Oromia Region, Ethiopia (Fig 1), from September to November 2019. The study setting and socio-demographic characteristics of the inhabitants has been described elsewhere in previous studies [17, 18, 37]. Currently, the sugarcane irrigation area covers about 5,000 hectare (ha) of land with huge future expansion plans [18, 37]. The area is malarious [17, 37] where diverse Anopheles species including An. arabiensis, An. coustani, An. pharoensis and An. amharicus co-occur [18].

Fig 1

Major Anopheles larvae collection sites, Arjo-Didessa sugarcane irrigation area and its surrounding, southwestern Ethiopia, September to November, 2019.

[This map was made using ESRI ArcGIS Pro2.8 with publicly available datasets from NASA, OpenStreetMap, and field surveys].

The irrigation area has been classified into 11 agricultural commands (clusters by the International Center of Excellence for Malaria Research (ICEMR) project in which this study was part). For this study, eight clusters were randomly selected for larval/pupal collection on the basis of anopheline larvae availability, larval density and habitat distribution. These were Command-2, command-3, Command-4, Command-5, Command-6 and Abote Didessa (from the irrigation scheme), and from Kerka and Didessa clusters (clusters outside the irrigation area) (Fig 1). The minimum distance between villages from the irrigation area was about 3 kilo meter.

Anopheline larvae collection, rearing and identification

Anopheline larvae and pupae were collected from the breeding habitats of eight clusters and reared to adults in Arjo-Didessa ICEMR Insectary. The main habitat types in the irrigated clusters were manmade ponds, tyre tracks, sugarcane farm ditches, hippopotamus trenches, hoof prints of hippopotamus and seepages from irrigation canals and gate valves (Fig 2). Whereas, the major habitats outside the irrigated clusters include animal hoof prints, stagnant water, swamps/marshes, river edges and stream seepages.

Fig 2

Major Anopheles breeding habitat types in Arjo-Didessa sugarcane irrigation area, southwestern Ethiopia, September to November 2019.

Anopheline larvae and pupae were collected using dipper (350 ml, Bio Quip Products, Inc. California, USA), transported to ICEMR Insectary and reared to adults in enamel trays (27×16×6.5 cm). Larvae were fed with finely ground fish food (Tetramin baby) while pupae were transferred to cages and allowed to emerge to adults. The emerged adults were provided 10% sucrose solution. The mosquitoes were kept under standard conditions (25 ± 2°C temperature, 70% ± 10% relative humidity) [38]. Adult female Anopheles mosquitoes were identified to species morphologically [39] and An. gambiae s.l. were further identified to sibling species using species-specific polymerase chain reaction (PCR) assay [18, 40].

Insecticide susceptibility tests

Knock-down and mortality of An. gambiae (s.l.) females resulting from tarsal contact with insecticide impregnated papers with discriminating doses were assessed using WHO susceptibility test kits [5]. Deltamethrin (0.05%), alphacypermethrin (0.05%), bendiocarb (0.1%), propoxur (0.1%), and pirimiphos-methyl (0.25%) impregnated papers (obtained from Vector Control Research Unit, School of Biological Sciences, Malaysia) and clothianidin (2%) impregnated papers (Sumitomo chemical, Japan; Lot: CL190805) were tested for their efficacy against adult An. gambiae s.l. females (later identified to sibling species). In the selection of the test insecticides, current and previous insecticide usage profile of the national malaria control program was considered.

Four replicates of 24–25 non-blood fed, 3–5 days old females mosquitoes were exposed to insecticide impregnated papers for 1hour. A minimum of 99 and a maximum of 100 mosquitoes were exposed and fifty were used as a control for each insecticide tested. The number of knocked down mosquitoes were recorded at 10, 15, 20, 30, 40, 50 and finally after 60 minutes (1hour) exposure time [5]. In parallel, two replicates of mosquitoes (25x2) were exposed to control papers impregnated with silicone oil as control for pyrethroids and olive oil for organophosphate/carbamate insecticides. For clothianidin, water impregnated untreated papers obtained from similar company (Sumitomo chemical, Japan, Lot: UC190815) were used as negative controls.

After one-hour exposure time, mosquitoes were transferred back into holding tubes, provided with 10% sugar solution and the proportion of surviving and dead mosquitoes were recorded 24 hours post exposure. However, for clothianidin tests, 10% sugar solution was changed every 12 hours and mortality was recorded daily until 100% mortality was obtained. All the tests were performed at 25°C ±2°C and 70% ± 10% relative humidity. The quality of each insecticide impregnated paper was checked on a known susceptible laboratory colony of An. arabiensis obtained from Sekoru insectary, Tropical and Infectious Diseases Research Centre, Jimma University (JU TIDRC), Ethiopia.

From each test, randomly selected samples of dead and surviving mosquitoes were preserved individually in Eppendorf tubes over silica-gel and kept in a freezer (-21°C) for subsequent molecular species identification and kdr allele detection [5].

Identification of An. gambiae s.l. sibling species

About 36% (n = 214: 185 dead and 29 survived) of the 598 adult An. gambiae s.l samples tested for susceptibility to insecticides were identified to sibling species using polymerase chain reaction (PCR) assay following the methods of Scott et al. [40]. In brief: genomic DNA was extracted from legs and wings of individual mosquito using DNA extraction kit (Qiagen, Sigma Aldrich, USA). The extracted DNA product was amplified by PCR using universal primer (UN: 5’-GTGTGCCCCTTCCTCGATGT-3’) and species specific primers for An. gambiae s.s (GA: 5’-CTGGTTTGGTCGGCACGTTT-3’), An. arabiensis (AR: 5’-AAGTGTCCTTCTCCATCCTA-3’) and An. amharicus (QD: formerly An. quadriannulatus B; 5’-CAGACCAAGATGGTTAGTAT-3’). Then the amplicon was loaded on 2% agarose gel stained with ethidium bromide and run for gel electrophoresis. Anopheles arabiensis from Sekoru Insectary colony and previously confirmed An. amharicus [18] were used as positive controls.

Detection of knock down resistance gene mutation

Detection of knock down resistance gene, kdr-west (L1014F), mutation was carried out on 141 (n = 115 dead and n = 26 surviving) randomly selected An. amharicus and An. arabiensis (PCR identified) samples as described by Martinez-Torres et al. [41].

Briefly, genomic DNA extracted from individual mosquito (susceptible and resistant samples) was genotyped using allele specific primers [41]. Four allele specific primers namely Agd1 (5’-ATAGATTCCCCGACCATG-3’), Agd2 (5’-AGACAAGGATGATGAACC-3’), Agd3 (5’-AATTTGCATTACTTACGACA-3’) and Agd4 (5’-CTGTAGTGATAGGAAATTTA-3’) were used for the PCR amplification of kdr-west gene. The PCR reaction conditions were 94°C/5min x 1 cycle, (94°C/1min, 48°C/2min, 72°C/2min) x 40 cycles, 72°C/10min x 1 cycle, 4°C hold cycling condition. The amplicon was run on a 2% agarose gel and stained with ethidium bromide. Resulting fragments (bands) were interpreted as: 293pb internal control, 195bp resistant and 137bp susceptible/wild type mosquitoes [38, 41]. Susceptible An. arabiensis strains from Sekoru insectary colony of Jimma University, Tropical and Infectious Diseases Research Center, Ethiopia, was used as control.

Data analysis

Data entry and analysis were made using Microsoft Excel (Version 2016, Microsoft Corp, USA) and IBM SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) statistical software packages. The status of susceptibility/resistance to insecticides after 24 hours post exposure was determined using percentage mortality. Mosquitoes’ phenotypic resistance status was interpreted according to WHO criteria (i.e. mortality rate ≥98% as susceptible; mortality rate between 90–97%, suspected/potential resistance; and mortality <90%, resistant) [38]. The KT50 and KT90 (time to knockdown 50% and 90% mosquitoes) values were calculated for each insecticide using log-probit analysis using SPSS v20.0 for windows statistical software.

Ethical clearance

Ethical clearance was obtained from the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Ethiopia (Ref. No. ALIPB/IRB/012/2017/18). Permission was also obtained from East Wollega and Buno Bedele Zonal Health Offices, and Arjo-Didessa Sugar factory, Oromia Regional State, Ethiopia. Oral consent was taken from the interviewees.

Results

Mosquito species composition

Among a total of 598 An. gambiae s.l. mosquitoes tested for their susceptibility to different insecticides, 569 (95.15%) of them died and 29 (4.85%) survived. Of 214 randomly selected An. gambiae s.l. (n = 185; 35.5% of dead and n = 29; 100% of survivors) samples analyzed using species-specific PCR, about 89% (n = 190) were An. amharicus and the remaining 9% (n = 20) were An. arabiensis (Fig 3). In the PCR analysis, about 98% of the samples were successfully amplified.

Fig 3

Results of PCR gel electrophoresis: Lane 1; 315kb An. arabiensis, Lanes 2–5 & 7–12 were An. Amharicus (153kb); Lanes 6 and 13 were 100kb DNA ladders.

Anopheles amharicus was predominant species for every type of insecticide tested followed by An. arabiensis (Table 1). Among the An. gambiae s.l. sub-samples analyzed with PCR, the proportions of An. amharicus tested against propoxur were 100% (n = 26/26), pirmiphos-methyl 90% (n = 28/31) and clothianidin 91.5% (n = 43/47). The molecular distribution of An. amharicus and An. arabiensis tested against different insecticide classes is shown in Table 1.

Table 1

Composition and insecticide susceptibility status of An. arabiensis and An. amharicus to insecticides at Arjo-Didessa irrigation scheme and its surrounding, southwestern Ethiopia, September-November, 2019.

Insecticide (%)	# Tested (PCR)	An. arabiensis		An. amharicus		UA (%)
		Resistant (%)	Susceptible (%)	Resistant (%)	Susceptible (%)
Deltamethrin (0.05)	48	5 (10.4)	0 (0.0)	10 (20.8)	33 (68.8)	0 (0.0)
Alphacypermethrin (0.05)	40	0 (0.0)	0 (0.0)	10 (25.0)	29 (72.5)	1 (2.5)
Bendiocarb (0.1)	22	1 (4.5)	8 (36.4)	0 (0.0)	12 (54.5)	1 (4.5)
Propoxur (0.1)	26	0 (0.0)	0 (0.0)	0 (0.0)	25 (96.2)	1 (3.8)
Pirmiphos-methyl (0.25)	31	0 (0.0)	3 (9.7)	0 (0.0)	28 (90.3)	0 (0.0)
Clothianidin (2)	47	0 (0.0)	3 (6.4)	0 (0.0)	43 (91.5)	1 (2.1)
Total	214	6 (2.8)	14 (6.5)	20 (9.3)	170 (79.4)	4 (1.9)

UA: Unamplified; three resistant & one susceptible samples

Insecticide susceptibility status of Anopheles gambiae s.l.

Mortality rates of An. gambiae s.l. exposed to deltamethrin and alphacypermethrin impregnated papers were 85% and 86.8%, respectively (Table 2). On the other hand, pirmiphos-methyl induced 100% mortality, bendiocarb 99% mortality and propoxur 100% mortality. In all the control populations tested together, mortality rates were < 5%, and therefore, Abbott’s formula to correct mortality rate was not necessary during data analysis. Anopheles arabiensis controls from Sekoru insectary colony, JU TIDRC, were susceptible to each test insecticide used.

Table 2

Susceptibility status of Anopheles gambiae s.l. to insecticides in Arjo-Didessa sugarcane irrigation area, southwestern Ethiopia, September-November, 2019.

Insecticide (DC)	Insecticide class	Number exposed (n)	Number dead (n)	Mortality (%)	Susceptibility	Interpretation
Deltamethrin (0.05%)	Pyrethroid	100	85	85.0	Resistant	Confirmed
Alphacypermethrin (0.05%)	Pyrethroid	99	86	86.8	Resistant	Confirmed
Pirmiphos-methyl (0.25%)	Organophosphate	100	100	100.0	Susceptible	Confirmed
Bendiocarb (0.1%)	Carbamate	100	99	99.0	Susceptible	Confirmed
Propoxur (0.1%)	Carbamate	99	99	100.0	Susceptible	Confirmed
Clothianidin (2%)	Neonicotinoid	100	100	100.0†	Susceptible	Confirmed

DC: Discriminatory Concentration,

†100% mortality was recorded after 48 hours post exposure

Knockdown time50 and knockdown time90 values

The KT50 and KT90 values for deltamethrin were 20.6 and 80.6 minutes, respectively and the corresponding values for alphacypermethrin were 14.4 and 44.7 minutes, respectively (Table 3). The number of mosquitoes knocked down after 60-minute exposure times were eighty-four for deltamethrin and ninety-six for alphacypermethrin.

Table 3

Knockdown effects of deltamethrin and alphacypermethrin against An. arabiensis and An. amharicus mosquito species, Arjo-Didessa sugarcane irrigation scheme and its surrounding, southwestern Ethiopia, 2019.

Insecticide (%)	Wild/field mosquitoes (An. arabiensis & An. amharicus)		Insectary colony (An. arabiensis)		KT Ratio (Wild vs Colony)
	KT50* (95% CI)	KT90* (95% CI)	KT50* (95% CI)	KT90* (95% CI)	KT50	KT90
Deltamethrin (0.05)	20.6 (18.2–23.0)	80.6 (66.1–106.0)	16.34 (3.03–29.8)	36.51 (17.75–89.62)	1.26	2.21
Alphacypermethrin (0.05)	14.4 (12.5–16.1)	44.7 (39.1–53.1)	14.91 (1.14–29.89)	35.17 (13.18–97.51)	0.97	1.27

KT: Knockdown time; CI: Confidence interval;

*Time is in minute

Susceptibility status of An. gambiae s.l. to clothianidin

Clothianidin induced 48% (n = 48/100) knockdown after 60minutes exposure time and 94% (n = 94/100) mortality effects 24 hour post exposure against the wild An. gambiae s.l. The field collected An. gambiae s.l. (later identified as An. amharicus and An. arabiensis) exposed to clothianidin reached 100% mortality within 48 hours (2 days) post exposure while An. arabiensis from Sekoru insectary colony took 96 hours (4 days) to reach 100% mortality (Fig 4). From 47 An. gambiae s.l. sub-samples analyzed with species specific PCR assay, 91.5% (n = 43) were identified as An. amharicus while only 6.4% (n = 3) were An. arabiensis.

Fig 4

Mortality rates of the An. gambiae s.l. (field vs laboratory spp.) exposed to clothianidin, Arjo-Didessa sugarcane irrigation scheme and its surrounding, Ethiopia, September-November, 2019.

Detection of kdr (L1014F) gene mutation in An. arabiensis and An. amharicus

A total of 141 PCR confirmed (124 An. amharicus and 27 An. arabiensis) samples from dead (n = 115) and survived (n = 26) mosquitoes were examined for the occurrence of L1014F (West African kdr) allelic mutation. Among these, 97.2% (n = 137) were successfully amplified while only 2.8% (n = 4) were unamplified. Of those samples analyzed for presence of kdr gene, none of them were positive for kdr (L1014F) allele mutation.

Discussion

The present study revealed the susceptibility status of An. amharicus and An. arabiensis to existing and new insecticide classes of public health use at national and continental level. Of the An. gambiae s.l. sub-population collected and tested from the irrigation fields and its surrounding, the main species identified was An. amharicus (~89%) while few (9%) were identified as An. arabiensis. Previous study from the same area also revealed the co-existence of these two sibling species in the sugarcane irrigation settings although An. arabiensis was the predominating species [18]. This study is the first to detect pyrethroid resistance and characterize resistance mechanisms in An. amharicus and An. arabiensis particularly in areas of agro-chemical use for sugarcane irrigation activities in Ethiopia.

The dominant An. amharicus and An. arabiensis in the Arjo-Didessa sugarcane irrigation area and its surrounding villages were resistant to deltamethrin and alphacypermethrin. This could challenge the ongoing malaria vector control and elimination program as these chemicals are among the intensively used insecticides for public health and agricultural purposes in the area. This is in agreement with studies from northwestern [10], central, southwestern [10, 11, 42] and other parts of Ethiopia [12, 43] which documented resistance of An. arabiensis against deltamethrin. Although the mortality rates against deltamethrin (85%) and alphacypermethrin (86.8%) were comparable with a study in Gorgora, Ethiopia [10], it was relatively higher than studies in other parts of the country (were 9–75% mortalities) [10, 11, 43]. This could be attributed to the higher abundance of An. amharicus in our study population. Knock down time (KT50 and KT90) were elevated (e.g. for deltamethrin, KT90 ratio = 2.2 compared to Lab strains) which indirectly indicate the reduced efficacy of their rapid knockdown effect which was in agreement with a study in Gorgora [10] but contrasted with a study around Gilgel-Gibe hydroelectric dam, Ethiopia [11] which could be attributed to the mixed population of the present study.

Long term use of public health insecticides and agricultural chemicals (pesticides, herbicides and fertilizers) might have contributed for the resistance of An. arabiensis and An. amharicus against pyrethroids in the study area. Studies from cotton growing areas in Burkina Faso [44] and Northern Benin [21], West Africa and southern Côte d’Ivoire [45] suggested that agricultural use of pesticides selects pyrethroid resistance within An. gambiae s.l. populations. In Tanzanian agricultural settings where An. arabiensis was the predominant species, a significant correlation was found between adult mosquitoes resistance to deltamethrin and pesticide use for agricultural activity [46]. Furthermore, high resistance of An. gambiae s.l. populations to pyrethroids was observed from Okyereko rice irrigation site of Ghana [14] similar to our finding. These mosquitoes exposed to sub-lethal doses of herbicides, pesticides, fertilizers or pollutants at their larval stage become more tolerant to insecticides (e.g. pyrethroides) possibly due to over expression of their detoxifying enzymes and selection of resistance genes [22, 44, 47, 48]. The interaction between xenobiotics (e.g. herbicides such as glyphosate and atrazine) present in mosquito breeding sites and the expression of mosquito genes encoding detoxification enzymes could exert the selection pressure [22, 49, 50]. Supporting our finding, a study by Oliver and Brooke, [51] demonstrated that larval exposure to glyphosate (herbicide) induced insecticide resistance in the major malaria vector, An. arabiensis. There was about 4.7 fold increase in deltamethrin tolerance among adult An. arabiensis with fertilizer exposure at their larval stage which can also be translated to an increase in pyrethroid resistance intensity due to fertilizer use [52].

In spite of the observation of phenotypic resistance to deltamethrin and alphacypermethrin, there was no kdr gene mutation (kdr-w/L1014F allele) detected among An. arabiensis and An. amharicus. However, this cannot rule out a potential involvement of other resistance mechanisms (target site, metabolic or cuticular) in the study area. For example, N1575Y mutation has been recently emerged within domains III-IV of VGSC of pyrethroid resistant An. gambiae population [53, 54] which might be considered as possible target site resistance mechanism in the area. Involvement of enzymatic mechanisms had also been reported in western Kenya agro-ecosystems where there were pyrethroid resistant An. gambiae s.l. (An. arabiensis) but without kdr allele detection [55]. Elevation of monooxygenases and esterases enzymatic activities were observed in those resistant An. gambiae mosquitoes exposed to permethrin and deltamethrin [55]. A more recent study further strengthens that An. arabiensis with increased phenotypic resistance to pyrethroids was found with lowest kdr-w allelic frequency [56]. The increased number of An. amharicus in our test population; a species with previously unknown kdr frequency and with less insecticide exposure due to its exophilic and zoophagic (prefer animal shelters) behavior [57, 58], might contribute for the absence of kdr mutation in our study.

Anopheles arabiensis and An. amharicus were susceptible to propoxur, bendiocarb and pirmiphos-methyl insecticides. This is supported by a previous nationwide study that documented susceptibility of An. arabiensis to these insecticides [12] and a study in southwestern Ethiopia, susceptible to propoxur [11]. Unlike our study, bendiocarb resistance was detected in malaria vectors from Ethiopia [12] as well as rice irrigation areas of southern Côte d’Ivoire [45]. Similarly, the mosquito population were fully susceptible to the new novel insecticide, clothianidin (2%), with 100% mortality. However, 100% mortality was achieved within 48 hours (2 days) post exposure for the field strains while 96 hours (4 days) to reach 100% mortality for the laboratory strains. A similar trend was reported by a study in Ethiopia where field population of An. arabiensis was more susceptible to clothianidin reaching 100% mortality by day two compared to the laboratory strain reaching 100% mortality by day three [32]. Such increased susceptibility in the field strains may result from fitness cost due to presence of resistance and cross-resistance traits found in the wild populations [32, 59]. Similar studies in Ethiopia [32] and Africa [60] reported clothianidin susceptibility of malaria vectors. From its efficacy, clothianidin is being highly recommend as viable candidate to replace the current insecticides used in IRS for the control of insecticide resistant malaria vectors [30, 61, 62]. Therefore, clothianidin can be utilized as alternative/supplement for malaria vector control and elimination operations by National Malaria Elimination Program in Ethiopia.

The findings from this study strongly suggest for implementation of inter-sectoral integrated insecticide resistance management strategies involving public health, agricultural and environmental sectors, by incorporating novel chemicals such as clothianidin. This could help to reduce insecticide resistance in malaria vectors at such irrigation settings.

Limitations of the study

Although target site (kdr-w) resistance mechanism was investigated, other mechanisms such as metabolic, cuticular and behavioral resistances were not determined in this study. This calls for the need for further investigations in these areas. In addition, insecticide resistance status of mosquitoes from non-irrigated (control) villages was not determined due to the critical shortage of positive larval habitats for the number of bioassay tests during the study period.

Conclusion and recommendations

In the Arjo-Didessa sugarcane irrigation area and its surrounding villages, An. amharicus (for the first time) and An. arabiensis were observed to be resistant to pyrethroid insecticides. This brings additional challenge on current malaria vector control programs in the irrigation areas. Integrated resistance management strategies are critically needed to improve malaria vector control. Susceptibility of the study population against carbamates and organophosphate insecticides could help to exploit them as alternative chemicals for insecticide resistance management. Given the resistance of An. amharicus to pyrethroids, its behavior, blood feeding pattern and vectorial capacity should be further investigated. Although kdr-w gene mutation was not detected in our study, other resistance mechanisms including kdr-e should not be ruled out.

Uncropped (raw) image of gel electrophoresis result (blot).

(PDF)

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20 Oct 2021

PONE-D-21-30434Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, EthiopiaPLOS ONE

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Reviewer #2: Yes

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Reviewer #1: Review manuscript PONE-D-21-30434

Indoor residual spraying and insecticide-treated nets helped to significantly reduce the malaria burden. However, their efficacy is declining due to the development and spread of insecticide resistant vectors which is causing by massive use of insecticides in vector control and selection from agricultural pesticides. It is there important to monitoez the extent of resistance in malaria vector in order to implement suitable resistance management strategies

In this study, The evaluated the Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms in ArjoDidessa sugarcane plantation area, southwestern Ethiopia.

. They found that mosquitoes collected were susceptible to pyrethroids (deltamethrin and alphacypermethrin) but suceptible to all other classes tested. They did not detected the presence of Kdr W

The results obtained can guide in option for resistance management for example, the Susceptibility of the study population against carbamates and organophosphate insecticides could help to exploit them as alternative chemicals for insecticide resistance management. However, I have a major concerning the some of the aspect of this work

- Why monitoring the distribution of Kdrw while Ethiopia is in the eastern part where we expected to have more kdrE (the L1014S mutation). This need to be investigated

- Why testing two pyrethroids type II. It will be more informative to tested at least one type I pyrethroid such as permethrin

- The synergistic testing with PBO is missing which can inform about potential resistance mechanisms involved in the observed resistance

- The authors need to evaluate the intensity of resistance using the 5x and 10x concentrations of insecticides as recommended by WHO

- Having the results of the bioefficacy of bed nets could have provide more information about the impact of the observed resistance on the efficacy of control tools

Reviewer #2: See comments in the attached file. In general the manuscripts addresses an important is vector control issue withe reference to An amuharicus. The authors should indicate the difference in transmission capacity between An. amuharicus and An. arabiensis.

**********

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Reviewer #1: No

Reviewer #2: Yes: Dr. Andrew K. Githeko PhD

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Submitted filename: Review Remarks Githeko Ak.docx

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5 Nov 2021

Author’s Responses to Reviewers and Editor Comments

Title: Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia. PONE-D-21-30434

Authors list:

Assalif Demissew (assalid@yahoo.com),

Abebe Animut (animut2004@yahoo.com),

Solomon Kibret (s.kibret@gmail.com),

Arega Tsegaye (2003arega@gmail.com),

Dawit Hawaria (hawaria.dawit@gmail.com)

Teshome Degefa (teshedege@gmail.com),

Hallelujah Getachew (hgetachew4@gmail.com),

Ming‑Chieh Lee (mingchil@uci.edu),

Guiyun Yan (guiyuny@hs.uci.edu),

Delenasaw Yewhalaw (delenasawye@yahoo.com)

Author’s response to reviewers:

Editor comments on journal requirements: when submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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Author’s Response: Dear editor in chief, we are very grateful for your academic edits and fast response. We prepared the manuscript according to the PLOS ONE style using the guideline/s given. Please look at the clean version of our manuscript for the format and editing requirments.

Editor’s comments:

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

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Author’s Response: Thank you. We submitted the figures separately on “Figure Submission” section and the original blot/gel image as supporting information; labeled as S1_raw_image. [Supporting information section: Page 29: Line#: 590-591].

We also indicated in our cover letter that the blot/gel image data is in the supporting information.

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Author’s Response: Dear Editor, thank you for the comment. We understand and fully aware that copyright is a serious issue if one uses a product without permission. However, the figure in our manuscript is free of copyright issues. It (Figure 1) was done by one of the co-authors, Dr. Ming-Chieh Lee (mingchil@uci.edu), using ESRI ArcGIS Pro 2.8 software with publicly available datasets from NASA (SRTM DEM), OpenStreetMap (major road and river), and field surveys (settlement and habitat types). We took the geo-points (GPS) by ourselves and we prepared the map from those coordinates. Therefore, we confirm that figure 1 does not contain any proprietary data. The copyright of figure 1 does not belong to any other 3rd party.

Also, look at under caption of figure 1, we put the statement: “This map was made using ESRI ArcGIS Pro 2.8 with publicly available datasets from NASA, OpenStreetMap, and field surveys”. [Page 6, Line# 123-125]

Reviewer Comments to the Authors (Responses highlighted with blue and red colors)

NB: The Pages and Line numbers indicated in Author’s response are within Track Change MS.

Reviewer #1 general remarks: Review manuscript PONE-D-21-30434

Indoor residual spraying and insecticide-treated nets helped to significantly reduce the malaria burden. However, their efficacy is declining due to the development and spread of insecticide resistant vectors which is causing by massive use of insecticides in vector control and selection from agricultural pesticides. It is there important to monitoez the extent of resistance in malaria vector in order to implement suitable resistance management strategies.

In this study, the evaluated the Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms in Arjo Didessa sugarcane plantation area, southwestern Ethiopia. They found that mosquitoes collected were susceptible to pyrethroids (deltamethrin and alphacypermethrin) but suceptible to all other classes tested. They did not detect the presence of Kdr W.

Reviewer #1 comment:

The results obtained can guide in option for resistance management for example, the Susceptibility of the study population against carbamates and organophosphate insecticides could help to exploit them as alternative chemicals for insecticide resistance management.

However, I have a major concerning the some of the aspect of this work

1. Why monitoring the distribution of Kdrw while Ethiopia is in the eastern part where we expected to have more kdrE (the L1014S mutation). This need to be investigated

Author’s Response: we thank the reviewer for the comments, suggestions and concerns. It’s true that Kdr-E (L1014S) is distributed in East African while Kdr-W (L1014F) is for West African mosquito populations and the authors are well aware of this situation.

In this regard, Ethiopian’s case can be considered as an exception because unlike other East African An. gambiae s.l mosquito population, KDR-E is not yet detected in the country whereas KDR-W is widely distributed among the target site resistance mechanisms. Therefore, we looked for Kdr-W on the basis of previous studies [References below] in which no study could detect KDr-E in Ethiopia (i.e. KDR-E is not yet detected in the country).

Some Evidences on this: A study by Yewhalaw et al., (2010); one of the senior author in this research, confirmed the first evidence of West African KDr (L1014F) mutation in Ethiopia/in East Africa. Similarly, another study from central, northern and south west Ethiopia by Balkew and his colleagues (2010), detected only KDr-W among the mosquitoes tested. A more recent nationwide resistance study by Messenger et al., (2017) between 2012-2016 also failed to detect Kdr-E in Ethiopia. A study by Alemayehu et al., (2017) on mapping insecticide resistance and characterization of resistance mechanisms in Anopheles arabiensis in Ethiopia could not detect KDR-E in the country.

However, we share the reviewers’ concern because, although it is not yet detected in Ethiopia, still we cannot rule out KDr-E in the study area and we recommend this for further investigation and included in the recommendation section. [Page 21, Line#: 383];”….other resistance mechanisms including kdr-e should not be ruled out”

References (Studies at regional and national level):

• Balkew et al. (2010). Insecticide resistance in Anopheles arabiensis (Diptera: Culicidae) from villages in central, northern and south west Ethiopia and detection of kdr mutation. Parasites Vectors 3, 40. https://doi.org/10.1186/1756-3305-3-40

• Yewhalaw et al. (2010). First Evidence of High Knockdown Resistance Frequency in Anopheles arabiensis (Diptera: Culicidae) from Ethiopia. Am J Trop Med Hyg. 83(1)

• Messenger et al. (2017) Insecticide resistance in Anopheles arabiensis from Ethiopia (2012–2016): a nationwide study for insecticide resistance monitoring. Malar J 16: 1-14)

• Alemayehu et al. (2017). Mapping insecticide resistance and characterization of resistance mechanisms in Anopheles arabiensis (Diptera: Culicidae) in Ethiopia. Parasite Vectors 10.

Reviewer #1 comment:

2.- Why testing two pyrethroids type II. It will be more informative to tested at least one type I pyrethroid such as permethrin

Author’s Response: Thank You. In the methods and materials section, we stated our insecticide selection criteria as “In the selection of the test insecticides, current and previous insecticide usage profile of national malaria control program was considered” [Page 7, Line# 157-159]. Therefore, our test insecticide selection criteria was their current and previous use (local and national operational application/utilization) in the area for either IRS or LLINs or both. PermaNet (deltamethrin) and MagNet (alphacypermethrin) are incorporated in LLINs operations as principal chemicals and pirimiphos-methyl, propoxur and bendiocarb are among the insecticides being used for IRS in Ethiopia. Due to this reason, we hypothesize that there could be a selection pressure due to utilization of the above mentioned insecticides. We also believe that there are several studies conducted on Type-I pyrethroids in the country and decisions can be made based on those studies together with our research outcome.

Reviewer #1 comment:

3. The synergistic testing with PBO is missing which can inform about potential resistance mechanisms involved in the observed resistance

Author’s Response: we share the reviewer’s comment because PBO could grossly suggest the possible biochemical/enzymatic resistance mechanism in the field. We didn’t conduct PBO assay at the field because of critical shortage of mosquitoes in the study setting. We took almost three months to complete this study (September to November, 2019) due to this problem. In the area, although there were positive habitats for Anopheline mosquitoes (An. caustani complex, An. funestus group and An. phoroensis), there was shortage of An. gambiae s.l mosquito larva and pupa to conduct both PBO and intensity assays. We put this on our limitation of the study as “other mechanisms such as metabolic, cuticular and behavioral resistances were not determined in this study” on [Page 20, Line#: 369-370] indicating the need of biochemical/enzymatic investigation by other researchers.

Reviewer #1 comment:

4.-The authors need to evaluate the intensity of resistance using the 5x and 10x concentrations of insecticides as recommended by WHO

Author’s Response: We thank the reviewer for the comment. We didn’t conduct the intensity assay due to mainly logistic problem (lack of 5x and 10x concentration kits) in addition to mosquito shortage. However, from our results, the population was susceptible for majority (four) of the insecticides tested and were resistant only for deltamethrin and alphacypermethrin. For these insecticides (the pyrethroids), we believe that the mosquito mortality at the diagnostic concentration (85% and 86.8% mortality) was relatively higher compared to multiple studies in the country. This might indicate that the population had less resistance intensity. We include this in the discussion section of the original manuscript [Page 17-18, Line#:303-310].

Reviewer #1 comment:

5.-Having the results of the bioefficacy of bed nets could have provide more information about the impact of the observed resistance on the efficacy of control tools.

Author Response: As indicated by the reviewer, information on bio-efficacy of insecticide treated bed nets could have strengthened the result. However, this was not in our objectives.

Reviewer #2: Dr. Andrew K. Githeko (PhD)

See comments in the attached file. In general, the manuscripts address an important is vector control issue with reference to An. amharicus. The authors should indicate the difference in transmission capacity between An. amharicus and An. arabiensis.

General remarks:

The manuscript addresses an important vector control operational issues. The data generated will be useful to the Ethiopian National Malaria program.

Author’s response: We thank the reviewer for the careful review and positive remarks.

Reviewer #2 comment:

1. It would be useful to provide some information on the relative importance of An. amharicus as a vector relative to An. arbiensis.

Author’s response: We thank the reviewer for this valuable suggestion. We provide information on the vectorial importance and behaviour of An. Amharicus in the background: “Except some old information on its zoophilic behavior and little importance in malaria transmission, the current role of An. amharicus in malaria transmission as well as its behavior and response to public health insecticides is unknown” [Page 4-5, Line#: 100-103].

Because of the lack of current information, we recommend further investigation on the vectorial capacity and behaviour of An. amharicus as: “Given the resistance of An. amharicus to pyrethroids, its behavior, blood feeding pattern and vectorial capacity should be further investigated”. [Page 21, line:380-382]

Reviewer #2 comment:

2. Indicate which of the two species is more anthropophilic and thus more likely to come into contact with insecticides indoors.

Author’s Response: As we explained in the previous response (we also indicated this in the discussion), there is old information indicating An. amahricus to be zoophilic and exophagic [Page, 19, Line#:344-345]. “An. amharicus…. less insecticide exposure due to its exophilic and zoophagic (prefer animal shelters) behaviour”. However, current evidence on its feeding and resting behaviour is required. Therefore, we put a recommendation for further study as explained in the previous response [Page 21, line:380-382].

Reviewer #2

Specific remarks

Anopheles amharicus was predominant species for every type of insecticide tested followed by

An. arabiensis (Table 1). The proportions of An. amharicus tested for propoxur was 100% (n=26/26), for pirmiphos-methyl 90% (n=28/31) and for clothianidin 91.5% (n=43/47). The distribution of An. amharicus and An. arabiensis tested again.

Reviewer #2 comment:

1. Is the above sentence referring to proportion mortality? Please clarify.

Author’s Response: We thank the reviewer for the comment. We give clarification as: “Among the An. gambiae s.l. sub-samples analysed with PCR, the proportions of An. amharicus tested against propoxur were 100% (n=26/26), pirmiphos-methyl 90% (n=28/31) and clothianidin 91.5% (n=43/47)” [Page 11, Line #: 234-236].

Clarification on Table 1: it shows the proportions of An. amharicus and An. arabiensis among sub samples of An. gambiae s.l. analysed with PCR (the proportion of PCR confirmed An. amharicus and An. arabiensis species).

Reviewer #2 comment:

KT50 and KT90 values

2. Spell out KT in full in the subheading.

Author’s Response: Thank you. We correct accordingly. [Page 14, Line #: 255]

Reviewer #2 comment:

Previous study from the same area also revealed the co-existence of these two sibling species in the sugarcane irrigation settings although An. arabiensis was the predominating species [18].

3. Could the species abundance in this and in the previous studies have been affected by seasonality? An gambiae and An. arabiensis abundance in sympatric populations, is influenced by seasonality elsewhere in Africa.

Author’s Response: Yes, from our previous study, species abundance was highly affected by seasonality in the area. About 86% of the total Anopheles mosquitoes were collected during the wet seasons and the remaining 14% (n = 295) collected during the dry seasons and the difference was statistically significant (χ2 = 70.423, df = 4, P < 0.001). In the wet seasons, indoor and outdoor density of An. gambiae s.l. was the highest in the irrigated clusters [18].

Depending on this result, we made our study period between September and November to get a good density of mosquitoes. However, we didn’t determine mosquito abundance in this study because our objective was to determine the insecticide susceptibility status of An. gambiae s.l. within the study period.

Reviewer #2 comment:

Long term use of public health insecticides and agricultural chemicals (pesticides, herbicides and

fertilizers) might have contributed for the resistance of An. arabiensis and An. amharicus against

pyrethroids in the study area.

4. Is there a history of the use of DDT for vector control in the study area ?. This could explain the cross resistance to pyrethroides.

Author’s Response: DDT had been used for more than four decades at the national level, and also in particular in Arjo-Didessa (the study area) as a principal part of IRS chemical. However, nationally, DDT spraying was discontinued in 2009 (nearly 12 years before) and was replaced by deltamethrin. Therefore, the selection pressure and cross resistance (with pyrethroids) might be minimum although this needs further investigation.

5. Please provide any evidence that herbicides and fertilisers are linked to insecticide resistance and in particular to pyrethroides. The authors need to discuss the mechanisms involved. The studies in west Africa (Côte d’ Ivoire ) and Tanzania did not provide the mechanism involved.

Author’s Response: We thank the reviewer for the suggestion. We include the following evidences in the discussion section: “These mosquitoes exposed to sub-lethal doses of herbicides, pesticides, fertilizers or pollutants at their larval stage become more tolerant to insecticides (e.g. pyrethroides) possibly due to over expression of their detoxifying enzymes and selection of resistance genes [Hien et al. (2017; Akogbeto et al., 2006; Nkya et al., 2013; David et al., 2010]. The interaction between xenobiotics (e.g. herbicides such as glyphosate and atrazine) present in mosquito breeding sites and the expression of mosquito genes encoding detoxification enzymes could exert the selection pressure [Poupardin et al., 2008; Riaz et al., 2009; Nkya et al., 2013]. Supporting our finding, a study by Oliver and Brooke, [Oliver and Brooke, 2018] demonstrated that larval exposure to glyphosate (herbicide) induced insecticide resistance in the major malaria vector, An. arabiensis. There was about 4.7fold increase in deltamethrin tolerance among adult An. arabiensis with fertilizer exposure at their larval stage which can also be translated to an increase in pyrethroid resistance intensity due to fertilizer use [Jeanrenaud et al., 2019]”. [Page 18, Line #: 319-329].

6. Indicate the class of insecticides for clothianidin after its first mention: neonicotinoid

Author’s Response: Thank you. We corrected accordingly after first mention in the background as: “To avert the effect of multiple and widespread insecticide resistance, clothianidin: a novel neonicotinoid insecticide, has been recommended to supplement the current insecticide based interventions” [Page 4; Line#: 91-92].

Similar studies in Ethiopia [32] and Africa [52] reported clothianidin susceptibility of malaria vectors. From its efficacy, clothianidin is being highly recommend as viable candidate to replace IRS for...

7. It is nor IRS that is replaced, rather it is the insecticide such as DDT, deltamethrin, etc that are being replaced.

Author’s Response: we thank the reviewer for the correction. We correct as: “From its efficacy, clothianidin is being highly recommend as viable candidate to replace the current insecticides used in IRS for the control of insecticide resistant malaria vectors” [Page 20; Line #: 360-361]

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Dec 2021

Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia

PONE-D-21-30434R1

Dear Dr. Demissew,

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: No further comments. The authors have responded to all the remarks adequately. It is understood that the study is based on issues arising from the ongoing vector control around the sugar irrigation area of Ethiopia rather than a fundamental study of insecticide resistance. The authors have explained that there is little data on the vdectorial capacity of An. amharicus and they have recommended further studies in this subject. The authors have acknowledged the previous use of DDT for IRS in the area, and this could patiala explain the origin of resistance to pyrethroids.

The authors have provided supporting information to their proposition that the use of herbicides and ferterlizers can select for resistance in vectors at the larval stage. Advanced water chemistry studies are require to answer this yet to be understood issue.

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Reviewer #1: No

Reviewer #2: Yes: Dr. Andrew K. Githeko. PhD

6 Jan 2022

PONE-D-21-30434R1

Evidence of pyrethroid resistance in Anopheles amharicus and Anopheles arabiensis from Arjo-Didessa irrigation scheme, Ethiopia

Dear Dr. Demissew:

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