Insecticide resistance in Aedes aegypti from Tapachula, Mexico: Spatial variation and response to historical insecticide use.

BACKGROUND: Insecticide use continues as the main strategy to control Aedes aegypti, the vector of dengue, Zika, chikungunya, and yellow fever. In the city of Tapachula, Mexico, mosquito control programs switched from pyrethroids to organophosphates for outdoor spatial spraying in 2013. Additionally, the spraying scheme switched from total coverage to focused control, prioritizing areas with higher entomological-virological risk. Five years after this strategy had been implemented, we evaluated the status and variability of insecticide resistance among Ae. aegypti collected at 26 sites in Tapachula. METHODOLOGY/PRINCIPAL
FINDINGS: We determined the lethal concentrations at 50% of the tested populations (LC50) using a bottle bioassay, and then, we calculated the resistance ratio (RR) relative to the susceptible New Orleans strain. Permethrin and deltamethrin (pyrethroids), chlorpyrifos and malathion (organophosphates), and bendiocarb (carbamate) were tested. The frequencies of the substitutions V1016I and F1534C, which are in the voltage-gated sodium channel and confer knockdown-resistance (kdr) to pyrethroid insecticides, were calculated. Despite 5 years having passed since the removal of pyrethroids from the control programs, Ae. aegypti remained highly resistant to permethrin and deltamethrin (RR > 10-fold). In addition, following 5 years of chlorpyrifos use, mosquitoes at 15 of 26 sites showed moderate resistance to chlorpyrifos (5- to 10-fold), and the mosquitoes from one site were highly resistant. All sites had low resistance to malathion (< 5-fold). Resistance to bendiocarb was low at 19 sites, moderate at five, and high at two. Frequencies of the V1016I ranged from 0.16-0.71, while C1534 approached fixation at 23 sites (0.8-1). Resistance profiles and kdr allele frequencies varied across Tapachula. The variability was not associated with a spatial pattern at the scale of the sampling. CONCLUSION/SIGNIFICANCE: Mosquito populations respond to selection pressure at a focal scale in the field. Spatial variation across sites highlights the importance of testing multiple sites within geographical regions.

Introduction

Aedes aegypti is the main vector of several arboviruses, including dengue, Zika, chikungunya, and yellow fever. The control of this mosquito species is challenging, mainly because it is highly adapted to urban and suburban areas and because it is widely dispersed in endemic regions [1]. Except for yellow fever, safe and effective treatments or vaccines for these diseases are still under study. Therefore, the suppression of Ae. aegypti remains the cornerstone to prevent transmission and control of outbreaks of these diseases [2].

Effective vector control involves several strategies, such as the elimination of potential breeding sites, application of chemical insecticides, and implementation of biological control. However, the application of chemical insecticides has become a common form of control because as a control is highly efficient and can be implemented promptly [3]. The most used insecticides in vector control have been the organophosphates temephos, used as a larvicide, and malathion, used as an adulticide by ultra-low volume application (ULV). Pyrethroids were introduced as adulticides in most Latin American countries in the 1990s [3]. In Mexico, according to the Mexican official policy for vector surveillance and control [4], the adulticide ULV formulation of permethrin, bioallethrin, and piperonyl butoxide (PBO) was used for more than 10 consecutive years (1999–2010). In the following 3–4 years, the pyrethroid d-phenothrin + PBO was introduced. Subsequently, the use of organophospates returned in 2013, with chlorpyrifos and malathion being used as adulticides, while carbamates were recommended for indoor residual application [5].

The prolonged use of pyrethroid insecticides resulted in the evolution of resistance to them in Ae. aegypti worldwide, including Mexico, where failures in dengue control strategies are due in part to resistance [5]. Given that resistance to insecticides has been reported in populations of Ae. aegypti globally, the World Health Organization (WHO) recommends testing to ensure an effective insecticide management program. Decisions based on evidence of the resistance and/or susceptibility of Ae. aegypti will ensure a better selection of insecticides in vector control programs [6].

Two mechanisms of resistance to insecticides have been identified: resistance due to the enhanced metabolism of insecticides and insensitivity at the target site of the insecticides. Both mechanisms are involved in resistance to pyrethroids [7]. Knockdown resistance (kdr) refers to a phenomenon in which insects are not knocked down immediately after exposure to pyrethroids. kdr is caused by specific mutations at the voltage-gated sodium channel (VGSC), which is the target site for pyrethroids and DDT [8]. The amino acid substitutions V1016I [9], F1534C [10], and V410L [11,12] frequently have been associated with resistance to pyrethroids. Once these mutations are fixed in a population, reversion to susceptibility is difficult to achieve [9]. Therefore, the detection and characterization of kdr mutations in mosquito populations before resistance fixation occurs is essential for insecticide management strategies.

In Mexico, Chiapas is one of the states with the highest rate of endemic dengue cases. In particular, the city of Tapachula reports the highest incidence of dengue in the state [13], which is attributed to the proliferation of vectors that transmit emerging and re-emerging diseases. Under the region’s tropical climate conditions, Ae. aegypti maintains high densities throughout the year. Consequently, dengue and other arboviruses transmitted by this vector have been prevalent in the region for a long time [14].

In the context of insecticide resistance management, we investigated the status of insecticide resistance to five insecticides, including two pyrethroids (permethrin and deltamethrin), two organophosphates (chlorpyrifos and malathion), and one carbamate (bendiocarb) and the spatial distribution of such resistance in populations of Ae. aegypti throughout the city of Tapachula. We expect that after 5 years of heavy use of organophosphates and the removal of pyrethroids from vector control campaigns, pyrethroid resistance will be lower, whereas organophosphate resistance will appear in focal points of the city. We tested 26 collection sites located in the city of Tapachula. Each collection site consisted of nine blocks and these were selected based on vehicle access for outdoor spraying. To minimize the effects of mosquito migration by flight range (50–150 m), sites were located at least 250 m apart. The spatial correlation between resistance and geographical distance was calculated for the 26 collection sites. In addition, since Tapachula’s vector control program uses a quadrant subdivision for spraying activities, we included a second analysis to test this source of variation by assigning sites to one of the four cardinal geographical quadrants (NE, NW, SE, and SW).

Materials and methods

Collections

The study was conducted in the city of Tapachula, Chiapas, located in southern Mexico at 177 meters above sea level. The 26 collection sites located in four quadrants in the city: Northwest (NW), Northeast (NE), Southwest (SW), and Southeast (SE) are shown in Table 1. The biological material was collected from January to April 2018 using ovitraps of 1-L capacity [15]. Twelve ovitraps were installed at each collection site. Ovitraps were made by hand with transparent, inert, non-toxic polypropylene (PP) cups of a 1 L capacity, and painted black on the outside following the guidelines for Entomological Surveillance with Ovitraps of the Mexican Ministry of Health [15]. The interior of each ovitrap was lined with Whatman filter paper (No. 1) and filled with water to ¾ capacity; the paper was replaced weekly up to five times. The egg strips were transported to the insectary of the Regional Center for Research in Public Health/National Institute of Public Health (CRISP/INSP). The egg strips were submerged in 4 L of water in plastic containers (40 cm x 30 cm x 15 cm). On the third and sixth day, the hatched larvae were fed a diet of Harlan 5001 proteins, with 0.4 gr or 0.8 gr / 1.2 L for 1st-2nd stadium and 3rd-4th stadium at the 3rd and 6th day, respectively.

Table 1

Geographic location of 26 Aedes aegypti collection sites in Tapachula, Chiapas, Mexico, in 2018.

Quadrant	Site	Neighborhood	Abbreviation	Latitude	Longitude
Northeast
	NE-1	Colinas del Rey	Col	14°55’50.9”	92°14’50.2"
	NE-2	Galaxias	Gal	14°55’11.2”	92°15”06.5"
	NE-3	Barrio Nuevo	Bar	14°54’51.0”	92°15’05.3”
	NE-4	San Juan de los Lagos	SJL	14°54’26.3”	92°15’13.4”
	NE-5	Coapantes	Coa	14°54’23.0”	92°14’57.1”
	NE-6	Bonanza	Bon	14°54’02.8”	92°14’31.7”
	NE-7	Centro (Country-Club)	CCC	14°54‘22.7”	92°15‘32.8”
Southeast
	SE-1	Galeana	Gal	14°54’00.2”	92°15’56.0”
	SE-2	16 de Septiembre	16S	14°53’44.0”	92°15’42.1”
	SE-3	Calcáneo Beltrán	Cal	14°53’28.0”	92°15’43.4”
	SE-4	Benito Juárez 1	BJ1	14°53’21.8”	92°16’04.1”
	SE-5	Benito Juárez 2	BJ2	14°53’11.7”	92°16’10.3”
	SE-6	Emiliano Zapata	Zap	14°53’02.1”	92°16’14.2”
Southwest
	SW-1	Raymundo Enríquez	Ray	14°52’01.4”	92°18’48.8”
	SW-2	Pobres Unidos	Pob	14°53’14.0”	92°17’6.1”
	SW-3	Palmeiras	Pal	14°53’22.1”	92°18’06.4”
	SW-4	Nuevo Milenio	Nue	14°53’24.8”	92°17’59.4”
	SW-5	Primavera	Pri	14°53’39.3”	92°17’38.6”
	SW-6	Democracia	Dem	14°54’23.7”	92°16’33.5”
Northwest
	NW-1	5 de febrero	5Fe	14°55’33.7”	92°15’22.4”
	NW-2	Xochimilco 1	Xo1	14°55’48.9”	92°15’37.8”
	NW-3	Xochimilco 2	Xo2	14°56’02.2”	92°15’29.9”
	NW-4	Vergel 1	Ve1	14°56’21.2”	92°15’52.4”
	NW-5	Vergel 2	Ve2	14°56’32.9”	92°15’52.4”
	NW-6	Paraíso	Par	14°56’35.2”	92°15’19.7”
	NW-7	Centro (Nva. España)	CNE	14°54‘35.0”	92°15‘43.5”

Aedes aegypti mosquitoes were identified to species and placed in cages (30 cm3). Females were bloodfed from rabbit (under accepted procedures by the Ethical Commission of the Instituto Nacional de Salud Pública) to obtain the F1 generation. Environmental conditions consisted of a temperature of 27 ± 2°C, 70–80% humidity, and a 12:12 hour photoperiod. We used the insecticide-susceptible New Orleans reference strain of Ae. aegypti, provided by Dr. William Black and maintained in the CRISP/INSP insectary.

Bioassays

The F1 adults were exposed to the insecticides using a modified CDC bottle bioassay (Centers for Disease Control) [16]. Sigma brand technical grade insecticides were used to determine the lethal concentrations that killed 50% (LC50) at each site. The pyrethroids permethrin (Type I) and deltamethrin (Type II), the organophosphates malathion and chlorpyrifos, and the carbamate bendiocarb were used to represent the toxicological groups used by vector control programs in Mexico.

To determine the LC50, we tested five to six insecticide concentrations, which caused 10 to 90% mortality, in four replicates. Each insecticide LC50 required approximately 500 mosquitoes. Table 2 shows the insecticide concentrations (μg/bottle) used to coat 250 ml Wheaton bottles using acetone as the solvent. During the bioassay, 15 to 20 (2–3 day old) females were gently aspirated into each bottle. The knockdown effect was recorded every 10 minutes for 1 hour. After 1 hour of exposure, the mosquitoes were transferred to plastic containers and maintained in the insectary to observe the mortality at 24 hours. The LC50 of each insecticide was also determined for the susceptible New Orleans reference strain (NO) using a different set of insecticide concentrations (Table 2). Each insecticide LC50 was replicated at least five times during a 7-month period. As control, a bottle impregnated only with acetone was used each time a test with field or susceptible mosquitoes was run.

Table 2

Concentrations (μg/bottle) used to determine the LC50 of five different insecticides in the bottle bioassay for field Aedes aegypti and the susceptible reference strain.

Class	Mode of action	Insecticide	Concentration in μg/bottle
			Field colonies	New Orleans reference
PYRs	sodium channel activators	Permethrin	10, 20, 40, 80, 160	0.8, 1.2, 2.4, 3.2, 6
		Deltamethrin	1, 2, 4, 6, 8, 16	0.75, 0.1, 0.15,0.2, 0.4
OPs	cholinesterase inhibitors	Malathion	2, 3, 4, 6, 8	2, 3, 4, 6, 8
		Chlorpyrifos	2, 4, 6, 8, 12	0.2, 0.4, 0.8, 1.6, 3.2
CARBs		Bendiocarb	0.5, 0.75, 1, 1.5, 3	0.25, 0.3, 0.4, 0.6, 1.2

PYRs = pyrethroids, OPs = organophosphates, CARBs = carbamates.

The LC50, 95% confidence intervals, slope, intercept, and p values were determined using the binary logistic regression model with QCal software [17]. The null hypothesis (Ho) assumes the observed mortality curve adjusts to a binary logistic regression model. Thus, we expected p values higher than 0.05 to accept the Ho. When the Ho was rejected, the bioassay was repeated.

To estimate the level of resistance among sites, a resistance ratio (RR) was calculated by dividing the LC50 of the field sites by the LC50 of the NO strain. The RR criterion according to Mazzarri and Georghiou [18] classifies high resistance as RR values greater than 10, moderate resistance as RR values between 5 and 10, and low resistance as RR values less than 5.

Genotyping kdr-associated mutations

Genomic DNA was isolated from 50 F1 individual female mosquitoes from each collection site following the method of Black and DuTeau [19]. The DNA was resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8) and stored at -20°C. The V1016I and F1534C mutations were genotyped according to the protocols of Saavedra-Rodríguez et al. [9] and Yanola et al. [10], respectively. The genotype and allelic frequencies were tested for Hardy-Weinberg (HW) equilibrium. The null hypothesis is that equilibrium is present in the population, which was verified with a chi-square test (df = 1 and p value > 0.05).

We tested the spatial variation of the LC50s between the quadrants in the city using a linear model and ANOVA in R (car package). To test the hypothesis of resistance correlation with space, we created Moran’s I correlograms as implemented in PASSaGE 2.0 [20]. Mosquitoes from different collection sites were considered neighbors if the sites were within 250 meters of each other. We expected that the LC50s or kdr frequencies would be associated with geographical distance (i.e., that closer neighbor sites would show similar resistance levels, compared to those farther away). A second analysis to test the variation of the LC50s and kdr frequencies between and within quadrants using a linear regression model and ANOVA in R (car package) was conducted. Since the city is uniformly sprayed during a cycle, we did not expect variation between or within quadrants. Correlation between kdr frequencies and LC50s for permethrin and deltamethrin was tested using a Spearman test.

Results

The geographic distributions of the resistance ratios (RR) for each insecticide in the 26 sites in Tapachula are shown in Fig 1. The LC50 and confidence intervals for each of the five insecticides are shown in S1 Table. For the pyrethroids, we observed high levels of resistance widespread across sites. Fig 2A shows the permethrin RRs across Tapachula. High RRs were identified at 24 sites (RR from 11.4 to 43.1-fold). Only two sites—NE-3 and NW-2—showed moderate RRs (5.3 and 5.9-fold, respectively). The variation in RRs among quadrants was not significant (F = 0.56, df = 3, p value = 0.64). For deltamethrin, high RRs were determined in all 26 sites (10.6 to 101-fold). The variation among quadrants was not significant (F = 1.08, df = 3, p value = 0.37). Except for SW, all quadrants had at least one site with RR higher than 90-fold (Fig 2B).

Fig 1

Spatial distribution of insecticide resistance to five compounds in Aedes aegypti collected in Tapachula.

The number above each bar corresponds to the resistance ratio (RR). The RR was calculated relative to the susceptible New Orleans reference strain. Map obtained from the National Institute of Statistics and Geography (INEGI). Digital Map of Mexico. MDM: http://gaia.inegi.org.mx/mdm6.

Fig 2

Pyrethroids resistance ratios (RRs) of Aedes aegypti collected in 26 sites across Tapachula in 2018.

A) Permethrin and B) Deltamethrin. Dots represent the RR50 with 95% confidence intervals for each site. Horizontal lines indicate the threshold for low resistance (< 5-fold), moderate resistance (5- to 10-fold) and high resistance (> 10-fold).

The RRs for cholinesterase inhibitors (organophosphates and carbamates) are shown in Fig 3. For chlorpyrifos (Fig 3A), the RRs varied from low at 10 sites (0.68- to 4.9-fold) to moderate at 15 sites (5.2- to 7.2-fold) to high at one site (10.2-fold). No significant difference in RRs was found between quadrants (F = 1.08, df = 3, p value = 0.37). For malathion (Fig 3B), low resistance (0.86- to 4.5-fold) was identified at all 26 sites. However, a significant difference was observed between quadrants (F = 3.53, df = 3, p value = 0.03), with SE showing a mean RR of 2.6-fold (95% CI 1.9- to 3.2-fold). Resistance to bendiocarb was low (1.2- to 4.8-fold) at 19 sites, moderate (7.3- to 9.9-fold) at five sites, and high (10.3- to 11.2-fold) at two sites. No difference between quadrants was identified (F = 0.68. df = 3, p value = 0.57).

Fig 3

Cholinesterase inhibitors resistance ratios (RRs) of Aedes aegypti collected in 26 sites across Tapachula in 2018.

A) Chlorpyrifos (organophosphate), B) Malathion (organophosphate), and C) Bendiocarb (carbamate). Dots represent the RR50 with 95% confidence intervals for each site. Horizontal lines indicate the threshold for low resistance (< 5-fold), moderate resistance (5- to 10-fold) and high resistance (> 10-fold).

Kdr-associated mutations

Genotype frequencies at the V1016I and F1534C loci in the voltage-gated sodium channel gene were determined in a sample of 45–50 individuals from each site (Table 3). The allele frequencies of the resistant allele I1016 fluctuated from 0.16–0.71. The lowest allele frequency (0.16) was scored for NE-3, whereas the highest frequency was from NW-6 (0.71). The remaining sites ranged from 0.2 to 0.5. Except for NE-2 and SW-4, the genotype frequencies at the V1016I loci were in HW equilibrium.

Table 3

Genotype counts and allele frequencies for two kdr-associated substitutions (V1016I and F1534C) from Aedes aegypti collected at 26 sites in Tapachula.

RR = homozygote resistant, RS = heterozygote, and SS = homozygote susceptible. * indicates a lack of Hardy-Weinberg equilibrium.

			V1016I genotypes				F1534C genotypes
Site	Abv	N	I/I	V/I	V/V	I1016 frequency	C/C	F/C	F/F	C1534 frequency
			RR	RS	SS		RR	RS	SS
NE-1	Col	48	4	23	21	0.32	47	1	0	0.99
NE-2	Gal	48	5	30	13	0.42*	39	8	1	0.90
NE-3	Bar	48	1	13	34	0.16	7	22	19	0.38
NE-4	SJL	48	6	22	20	0.35	47	1	0	0.99
NE-5	Coa	45	5	24	16	0.38	42	3	0	0.97
NE-6	Bon	48	8	22	18	0.4	43	5	0	0.95
NE-7	CCC	50	15	25	10	0.55	11	39	0	0.61*
	Subtotal	335	44	159	132	0.37	236	79	20	0.82*
SE-1	Gal	48	12	28	8	0.54	42	6	0	0.94
SE-2	16S	48	5	29	14	0.4	48	0	0	1*
SE-3	Cal	48	7	25	16	0.41	46	2	0	0.98
SE-4	BJ1	48	4	28	16	0.38	46	2	0	0.98
SE-5	BJ2	48	11	25	12	0.49	47	1	0	0.99
SE-6	Zap	48	10	25	13	0.47	34	14	0	0.85
	Subtotal	288	49	160	79	0.45	263	25	0	0.96
SW-1	Ray	48	5	28	15	0.39	48	0	0	1*
SW-2	Pob	48	14	21	13	0.51	47	1	0	0.99
SW-3	Pal	48	2	17	29	0.22	46	0	2	0.96*
SW-4	Nue	48	0	26	22	0.27*	48	0	0	1*
SW-5	Pri	48	8	22	18	0.39	47	1	0	0.99
SW-6	Dem	48	13	20	15	0.48	41	7	0	0.93
	Subtotal	288	42	134	112	0.38	277	9	2	0.98*
NW-1	5Fe	48	9	22	17	0.42	48	0	0	1*
NW-2	Xo1	48	10	29	9	0.51	48	0	0	1*
NW-3	Xo2	48	1	21	26	0.24	48	0	0	1*
NW-4	Ve1	48	11	23	14	0.47	48	0	0	1*
NW-5	Ve2	50	8	17	25	0.33	2	37	11	0.41*
NW-6	Par	48	24	20	4	0.71	48	0	0	1*
NW-7	CNE	50	4	26	20	0.34	0	38	12	0.38*
	Subtotal	340	67	158	115	0.43	242	75	23	0.82*
Total		1251	202	611	438	0.41	1018	188	45	0.89*

High allele frequencies of the resistant C1534 allele were determined at 22 of the 26 sites, ranging from 0.85 to 1.0. Lower frequencies (0.38–0.41) were found in NE-3, NW-5, and NW-7. While NE-7 was calculated with an intermediate value of 0.61. Most sites were in HW disequilibrium due to fixation of the resistant allele. We conducted a Spearman correlation test between the pyrethroid LC50s and the expected frequencies of resistant homozygous genotypes. We found significant correlation coefficients among permethrin LC50s, I1016/I1016 homozygotes (S = 2588, rho = 0.53, p value = 0.002), and C1534/C1534 homozygotes (S = 1966, rho = 0.515, p value = 0.004). Although it is known that C1534 shows protection only against permethrin (12), for deltamethrin a significant correlation was observed between the LC50 and I1016/I1016 homozygotes (S = 2643, rho = 0.467, p value = 0.008) and the C1534/C1534 homozygotes (S = 2945, rho = 0.507, p value = 0.002). However, the significance for both insecticides disappeared when observations from the New Orleans reference strain were removed.

To assess the correlation of LC50s with space, we generated Moran’s I correlograms for each of the five insecticides (Fig 4). The analysis included all 26 collection sites. We did not detect a discernable pattern in any of the tested insecticides. We expected a positive correlation (Moran’s I statistically > 0) between nearby sites, then as the distance increased (between the samples) the correlation would decrease, and later would turn negative (Moran’s I statistically < 0). However, this was not observed. Although, few of the distance classes were statistically different from zero (eg. bendiocarb at 3250 m, 3750 m, 4750 m, and 6250 m; malathion at 1500 m; and deltamethrin at 3750 m, and 4250 m), a caveat in our analysis is the possibility that there is autocorrelation at smaller distances than the ones we selected (x < 250 m). Our experimental design was not geared towards the detection of spatial correlation at smaller distances; there were a small number of samples below 250 meters.

Fig 4

Moran’s I correlograms as implemented in PASSaGE 2.0 assessing the correlation of LC50s with space for permethrin (pyrethroid), deltamethrin (pyrethroid), chlorpyrifos (organophosphate), malathion (organophosphate), and bendiocarb (carbamate).

The analysis included 26 collection sites in Tapachula, Chiapas, Mexico. Aedes aegypti mosquitoes from different collection sites were considered neighbors if the sites were within 250 meters of each other.

Discussion

Efforts to control Ae. aegypti populations are hindered by widespread insecticide resistance worldwide. Local insecticide resistance monitoring is necessary for the design of specific and successful resistance management programs [21]. In Latin America, pyrethroids have been used for adult mosquito control since the 1990s. The switch to pyrethroids was based on environmental concerns that led to the use of less toxic classes of insecticides [22]. In Mexico, vector control programs implemented the use of permethrin in 1999 and continued their use until 2010. Local selection pressure caused a rapid evolution of pyrethroid resistance in Ae. aegypti across Mexico [9,23-27], resulting in policy modifications that recommended the use of insecticides with different toxicological modes of action.

In Tapachula, vector control programs replaced the use of permethrin with a different Type I pyrethroid (d-phenothrin + piperonyl butoxide) from 2010 to 2013. In 2013, pyrethroids were replaced by the organophosphate chlorpyrifos, and in 2017, by malathion. This study reveals the current status and response of local Ae. aegypti populations to these insecticide shifts. Despite the switch to organophosphates in the last 5 years, we observed that high levels of pyrethroid resistance remain widespread in Tapachula. An assumption in insecticide resistance management is that insecticide resistance has negative fitness costs. Therefore, when insecticide pressure is removed, populations are expected to reverse to susceptibility [28,29]. Currently, we are conducting a study to determine the degree of loss of resistance to pyrethroids from 2016 to 2020 in this study area, which will demonstrate whether mosquito populations in Tapachula are undergoing a process of decreasing resistance that will take several years. Another explanation is that pyrethroid resistance is maintained in Ae. aegypti populations by the domestic use of pyrethroids [30]. Surveys in Merida, Mexico, indicate that 85% of households took action to kill pests, and 89% exclusively targeted mosquitoes. Interestingly most of the aerosol spray cans contained pyrethroid insecticides [31].

Interestingly, RRs for deltamethrin—a Type II pyrethroid—were higher than permethrin RRs across sites. Deltamethrin was authorized by CENAPRECE for indoor residual use in 2009 for control of the malaria vector, but its use was restricted to rural areas. Therefore, direct selection pressure from the use of deltamethrin in public health is unlikely to be responsible for the high RRs in Ae. aegypti from Tapachula. Although all pyrethroids act at the same target site, the variability of resistance to their different types is attributed to different binding sites for Type I and Type II pyrethroids at the voltage-gated sodium channel. Additionally, the presence of enzymes that have a greater affinity to metabolize specific molecules within the same toxicological group might explain this variability [32].

Knockdown resistance (kdr) is a major mechanism of pyrethroid resistance in Ae. aegypti from Mexico. In this study, we measured the frequency of this mechanism by molecular tests that identify mutations that confer changes to amino acids in the VGSC. The allele frequencies of the resistant allele I1016 ranged from 0.4 to 0.7, and for the resistant allele C1534, from 0.85 to 1.0 (except for three sites that had ~0.4). Historical data of kdr mutations indicated that C1534 confers low level of resistance on its own, and that resistance increased dramatically when I1016 evolved from the V1,016/C1,534 haplotype in field mosquito collected in different places from Mexico [33]. Those results demonstrated that I1016 was unlikely to have evolved independently, and that both mutations need coexist in the same mosquito in order to confer higher levels of resistance. Moderate correlations were significant between the resistant allele frequencies and the RRs for permethrin and deltamethrin only when including the New Orleans datapoints. This significance might be explained by most of the allele frequencies being distributed within a small range of variability.

This study was conducted after chlorpyrifos had been used for 5 years in outdoor spraying by vector control programs. Our results provide evidence of the response of Ae. aegypti populations to chlorpyrifos pressure. Ten sites showed low RRs, 15 sites showed moderate resistance, and one site was highly resistant. Interestingly, Ae. aegypti from all 26 sites were susceptible or had low RRs to malathion, thereby indicating that resistance to chlorpyrifos does not predict the lack of effectivity of malathion. Additionally, the RRs to bendiocarb were variable: mosquitoes from 19 sites had low RRs, those from three were moderate, and those from two were highly resistant. Only a few sites showed moderate to high resistance to both chlorpyrifos and bendiocarb (NE-5, NW-6, and SE-4). The lack of cross-resistance between organophosphates and carbamates suggests that the resistance mechanisms are not due to the insensitivity of their target site (the acetylcholinesterase) [34] and, in fact, no mutations have been found in ace-1 gene in Aedes aegypti [35].

A survey in Veracruz, Mexico, identified high RRs to chlorpyrifos in Cosoleacaque (RR = 13.9), moderate RRs in Poza Rica (RR = 7.9), and low RRs in five sites in Veracruz [36]. By using a discriminating dose of 50 μg/bottle and 85 μg/bottle for 30 minutes, two additional studies were able to identify chlorpyrifos resistance in Mexico [26,37]. Since neither of these studies found a history of chlorpyrifos use in vector control programs, the resistance might be explained by indirect exposure to chlorpyrifos through the extensive use of this insecticide to control agricultural pests [36].

During vector control programs, the city of Tapachula is uniformly sprayed, using the same insecticide, frequency and intensity. More yet, we selected sites based in their accessibility to spraying-vehicles. Assuming that no spatial heterogeneity in frequency and intensity of spraying, we did not expect the high levels of variation in resistance profiles across the city. For example, significant heterogeneity in the frequency of kdr haplotypes was detected in Ae. aegypti collected between city blocks in a town of Yucatan, suggesting that selection for these haplotypes occurs at a fine spatial scale (37). However, in contrast to our study, insecticide application was highly variable in space and time, creating a mosaic of selection pressures. In our study, some sources of heterogeneity could occur from mosquito migration from untreated sites due to vehicle inaccessibility, including parks, cemeteries, steep and unpaved streets. A second source of insecticide pressure is by use of household aerosol insecticides. For example, in a previous study from Merida, Mexico approximately 87% of households used commercially available pyrethroid products to control mosquitoes in their homes (31). Future studies should include an assessment of this source of selection pressure in Tapachula.

The spatial variability in insecticide resistance observed across the 26 sites in Tapachula is likely associated with the presence or appearance of “hot spots or dengue foci,” which contribute to the persistent transmission of the diseases and therefore to focal areas with greater spray intervention [38]. In addition, the spatial variability of resistance highlights the importance of evaluating resistance in multiple sites within a defined geographic area for the application of appropriate vector control decisions. Although no geographical correlation/association/pattern between resistance was found in Tapachula, more specific and finer environmental characteristics must not be discarded in future studies. A previous study used mitochondrial ND4 haplotypes to determine gene flow patterns among 38 Ae. aegypti coastal collections in Mexico [39]. Three genetic clusters were identified, the Northeast, Pacific, and Yucatan peninsula. For all sites, genetic distances remained small below geographic distances of 90 km and became large at distances >150 km. The Pacific cluster had the highest gene flow and diversity. A second study in the Yucatan Peninsula showed high gene flow occurring across 27 Ae. aegypti collection sites located up to 150 km of distance. Single nucleotide polymorphism (SNPs) at eleven loci did not vary across sites, suggesting high levels of gene flow. In contrast, insecticide resistance loci, including kdr alleles (I1016 and C1534) were highly variable across sites, indicating that insecticide resistance offsets the homogenizing effects of gene flow [40]. In this study, we assume complete gene flow among collection sites because: 1) Tapachula belongs to the Pacific cluster, 2) Ae. aegypti is well established throughout the year and, 3) collection sites are within 10 km of distance. However, this remains to be tested.

Conclusion

Despite more than 5 years having passed since the removal of pyrethroids from vector control programs in Tapachula, high levels of pyrethroid resistance and kdr-associated alleles persist in Ae. aegypti populations. Future resistance surveys will reveal if pyrethroid resistance is maintained in mosquito populations. We observed that, after 5 years of chlorpyrifos use in vector control programs, more than 50% of the sites have moderate to high chlorpyrifos resistance but complete susceptibility to malathion. Since malathion was introduced later in 2017, future studies to evaluate the selection of malathion resistance in the field are needed. Two different analyses were conducted 1) the spatial analysis included all 26 sites and, 2) the quadrant analysis to identify operational sources of heterogeneity. The quadrant analysis doesn’t include a geographical component and has limitations. Insecticide resistance varied spatially, most likely as a consequence of the pattern of insecticide use combined with environmental factors. Based on the results of our study, we suggest that both of the studied organophosphates and the carbamate remain viable options for use in the control strategy for this vector. The return to a pyrethroid (at least permethrin and deltamethrin) for outdoor spraying is recommended when the levels of resistance have decreased to RR less than 10-fold and once mechanisms other than kdr have been elucidated for pyrethroid resistance.

Lethal concentration to kill 50% (LC50) for five insecticides at 26 Aedes aegypti sites in Tapachula, Chiapas, Mexico.

The LC50 is in micrograms (μg) of active ingredient per bottle. The 95% confidence intervals around the LC50 are enclosed in parentheses. p values higher than 0.05 indicate the observed data fit the binary logistic regression model.

(XLSX)

Click here for additional data file.

Genotype for two kdr-associated substitutions (V1016I and F1534C) per individual Aedes aegypti mosquito (n = 47 to 50) at 26 sites in Tapachula, Chiapas, Mexico.

For V1016I: AA = homozygote for Ile1016, resistant; GG = homozygote for Val1016, susceptible; AG = heterozygote Ile1016/Val1016. For F1534C: TT homozygote for Phe1534, susceptible; GG = homozygote for Cys1534, resistant; TG = heterozygote Phe1534/Cys1534.

(XLSX)

Click here for additional data file.

8 Apr 2021

Dear Dr. Penilla-Navarro,

Thank you very much for submitting your manuscript "Insecticide resistance in Aedes aegypti from Tapachula, Mexico: spatial variation and response to historical insecticide use" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Pattamaporn Kittayapong, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Amy Morrison, Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Study Objectives: Yes, clearly stated – to test for resistance to five different insecticides of three different chemical classes, and to perform spatial mapping of resistant mosquitos.

Hypothesis: Yes, clearly stated – pyrethroid resistance is predicted to be lower, while organophosphate resistance is predicted to increase at specific focal points in Tapachula.

Study Population: Yes, clearly described and appropriate. The study is of Aedes aegypti populations in Tapachula, the city with the highest incidence of dengue in Chiapas state, making the results of this study of potentially high public health impact.

Study Design

Critiques:

- Line 153: “Females were bloodfed to obtain the F1 generation.” Please specify the animal species on which the females were bloodfed.

- Lines 198-199: The authors describe the use of city quadrants as the basis for some of their spatial variation analysis, however this seems like a somewhat arbitrary geographical division on which to perform spatial analysis. For instance, two collection sites may be very close in linear distance, but are located in different quadrants, leading to underestimation of spatial effects between quadrants. While it is okay to keep the quadrant spatial analysis in the paper, I recommend they discuss the limitations of this approach in more detail in the Discussion section.

- Lines 200-207: The authors describe a correlation analysis (using Moran’s correlograms) testing the hypothesis that mosquitos from neighboring collection sites will have more similar LC50s than mosquitos from distant sites, however I do not see the results of this analysis in the Results or Discussion section. Please include this data in the revised manuscript (even if the results are negative or placed in Supplemental Data), as they provide more robust and meaningful spatial information than the quadrant-based approach.

Strengths:

- A major strength of the study is the integration of spatial, phenotypic (insecticide resistance, as measured by bottle bioassay), and genotypic data.

- The geographic locations of the Aedes collection sites are precisely and thoroughly described.

Statistics: Yes, statistical approach is sound overall with sufficient sample sizes.

Ethical and Regulatory: No concerns.

Reviewer #2: Methods are written clearly, sample sizes are adequate, analyses are appropriate overall.

Reviewer #3: Yes, the objectives were clearly articulated with clear hypothesis stated. The study design, population description and choice, sample size, and statistical analysis were appropriate for this type of study. I have no reservations about ethical or regulatory issues.

I had three specific critiques of the methods:

-Line140: What was the material and color of the ovitraps used? Where they a specific brand or homemade?

-Line 146: At what approximate concentration (grams of diet per pan, etc.) and at what frequency were the larvae fed?

-Lines 165-175: What was the control treatment used for the LC50 testing? Was acetone alone used? Were the control mosquitoes untreated? Both? Please clarify.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis presented does not match the stated analysis plan, as I do not see any results for correlating LC50 profile similarity with distance to other collection sites (Lines 200 to 207), as discussed above.

The results are otherwise clearly and completely represented. The figures are of sufficient visual quality and clarity.

Reviewer #2: Line 260. Table 3. It would be useful to describe the mutation status per individual mosquito (raw data as supplement) and a summary in the results for the two mutation sites. It is not likely that the mutations are evolving or acting independently.

Line 269 - What is known about these particular mutations and insensitivity towards Type I vs Type II pyrethroids?

Line 275 - The significance is said to have disappeared, but the sentence is ambiguous and the reader cannot tell whether this is just for the deltamethrin analysis or the permethrin analysis as well. This is clarified in the Discussion, but it would be better to clarify at this first instance where it is mentioned.

Line 337 - Target-site mutations (acetylcholinesterase) are known to be unlikely in Aedes aegypti, so this is not an unexpected result. A reference about this should be included here.

Line 352 - No geographical correlation with resistance patterns were found. Is anything known about the genetic population structure or history of colonisation by Ae. aegypti in Mexico? Resistance alleles can be brought in with mosquitoes invading from other places. Is this a likely scenario in Tapachula? In some cases it might explain the presence of resistance where there is no local history of use of a particular insecticide in the local area.

Reviewer #3: The analyses, results, tables, and figures were appropriate and clearly presented the data.

I have a specific critique of Figure 1:

-Please clarify the location of ALL the sites by placing a dot indicating each site. Some sites (e.g. Raymundo Enriquez, Paraiso, etc.) have the resistance ratios placed adjacent to the site, but the site location is not clear.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Please address the limitations of the quadrant-based spatial analysis, as detailed above.

Yes, the conclusions are supported by the presented data. The authors do a good job discussing how the data advance the field and explicitly state how their findings impact Tapachula and mosquito control more generally.

Minor points to clarify:

- Discuss the possible contribution of regional/spatial heterogeneity in the intensity/frequency or type of insecticide used in Tapachula that can explain the spatial variation in resistance profiles. It sounds like insecticides were used uniformly throughout the city, adhering to national guidelines, but it would be helpful for the authors to include these potential contributing factors in the Discussion.

- Discuss how much population mixing you would expect between Aedes in neighboring collection sites. How frequently and how far does Aedes tend to migrate between different areas in urban habitats? How does that inform how you interpret the spatial variation you observe in your phenotypic and genotypic data?

Reviewer #2: It would be useful to discuss the VGSC results in light of the hypotheses of co-evolution of Ile1016 and Cys1534 in Mexico put forward by Vera-Maloof et al. 2015 (PlosNTD)

Reviewer #3: The conclusions appear to be correctly founded on the literature and supported by the data presented. The limitations and recommendations for future action are clearly described and appropriate. The relevance to the advancement of the field and to public health are addressed.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: None.

Reviewer #2: Zika should be written with a capital Z as it is named after Zika forest.

Page 7 - Line 128 - remove apostrophe from OP's. It is better written as OPs (no possessive required).

Reviewer #3: My editorial suggestions:

Minor edits in the Introduction section:

-Lines 110-111: Knockdown resistance is defined, however, I think it would be useful to also indicate that knockdown is a characteristic effect of the pyrethroid insecticide class.

-Line 128: Remove the apostrophe for OPs (should be OPs, not OP's)

Minor edits in the Methods section:

-Line140: What was the material and color of the ovitraps used? Where they a specific brand or homemade?

-Line 146: At what approximate concentration (grams of diet per pan, etc.) and at what frequency were the larvae fed?

-Lines 165-175: What was the control treatment used for the LC50 testing? Was acetone alone used? Were the control mosquitoes untreated? Both? Please clarify.

-Line 184: The second H null here is written as 'Ho', please correct to H null similar to elsewhere in the paper.

Minor edit to the Results section:

-Line 234: Please correct 'RR ... is shown' to 'RRs ... are shown'.

Minor edit to the Discussion section:

-Line 282: Please correct '[2014]' to '[21]'.

Minor edit to Figure 1:

-Please clarify the location of ALL the sites by placing a dot indicating each site. Some sites (e.g. Raymundo Enriquez, Paraiso, etc.) have the resistance ratios placed adjacent to the site, but the site location is not clear.

Minor

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This manuscript by Solis-Santoyo, et al. describes a study of spatial variation in phenotypic and genotypic insecticide resistance in Aedes aegypti in the city of Tapachula, which has one of the highest incidences of dengue in Mexico. The authors report the discovery of significant spatial variation in insecticide resistance, including widespread, high-level pyrethroid resistance despite the discontinuation of pyrethroid use in the city 5 years prior to sample collection. Furthermore, one of the alleles that confers knockdown resistance to pyrethroids has nearly achieved fixation throughout Tapachula. The strengths of the study include the clarity of writing, detailed study design, and interesting findings that can inform the strategy of other mosquito control campaigns. The main weakness in the study is the absence of determining the relationship between distance of neighboring collection sites and insecticide resistance profile similarity, i.e. if neighboring mosquitos have more similar resistance phenotypes/genotypes than spatially distant non-neighbors. Related to this criticism is the use of a quadrant-based system to determine spatial effects, which has methodological limitations. Overall, however, this is a well-designed study with interesting findings, and I recommend minor revisions before it can be accepted for publication.

Reviewer #2: The paper is clearly written and the experimental approach seems rigorous. A little more attention to the published literature at the points mentioned above would improve the manuscript.

Reviewer #3: Overall, this was a well executed and analyzed study that will be useful not only to Mexico, but to other areas facing insecticide resistance issues with a limited complement of chemical control tools. The difference in the resistance to chlorpyrifos vs. malathion was intriguing and warrants a follow up study in a few years time.

--------------------

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Reviewer #1: Yes: Joshua R. Lacsina

Reviewer #2: No

Reviewer #3: Yes: Natasha M. Agramonte, PhD

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

9 Jul 2021

Submitted filename: Responses to review comments_1.docx

Click here for additional data file.

19 Aug 2021

Dear Dr. Penilla-Navarro,

We are pleased to inform you that your manuscript 'Insecticide resistance in Aedes aegypti from Tapachula, Mexico: spatial variation and response to historical insecticide use' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Pattamaporn Kittayapong, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Amy Morrison, Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: In the revised manuscript, the authors did an excellent job in clarifying the distinction between the spatial analysis performed using the 26 collection sites and the quadrant analysis to look for operational sources of variation in spraying activities. This makes much more sense, and strengthens the manuscript. I have no methodological concerns.

Reviewer #2: More information is needed about the blood-feeding of mosquitoes on rabbits. Were rabbits anaesthetised.? How long was each feeding event? Are there any ethical standards that you need to report to about this activity?

Reviewer #3: The objectives were clearly stated with an appropriately designed study to test the stated objectives. The population was appropriate and detailed well. The sample sizes were sufficient for the rigor of the statistical analyses used to support the conclusions. I discerned no ethical or regulatory concerns.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Yes, by including the Moran's I correlogram in the revised manuscript, the analysis now clearly matches the analytic plan. Particularly with the addition of Figure 4, it is evident that the null hypothesis for correlating LC50s with space cannot be rejected, making this part of the analysis much clearer for the reader. I have no concerns about the results or analysis.

Reviewer #2: (No Response)

Reviewer #3: The analysis presented matched the methods stated. The results were clearly presented and the figures were sufficiently clear to elucidate the data described.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes, the conclusions are supported by the data and the limitations of the analysis are clearly addressed. The impact and public health relevance of their discoveries are thoroughly discussed.

Reviewer #2: (No Response)

Reviewer #3: The conclusions appear to be supported by the data presented and the limitations noted are appropriate for this study. The authors discussed both the public health relevance and the usefulness of the study in advancing the field.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: No modifications recommended.

Reviewer #2: Line 61. Any use of insecticides may contribute to selection for insecticide resistance. Mismanagement of their use can have worse effects, but even with rational use, resistance can still develop.

Line 87: change to ‘As a control,’

Line 192: there is an unnecessary full stop after aegypti

Line 298: no apostrophe needed in LC50s.

Line 304: change to ‘were statistically significant’. This sentence is incomplete, but makes sense if it is joined to the next sentence with a comma …425 m), a caveat….

Line 309: change to ‘there is a very small number’

Line 363: change ‘resistant’ to ‘resistance’

Line 378: change ‘effectivity’ to ‘efficacy’

Line 384: change to ‘and, in fact, no mutations’

Line 395: does ‘More yet’ mean ‘Furthermore’?

Line 396: change to ‘based on their accessibility’ and ‘Assuming no spatial heterogeneity’

Line 398: change ‘de city’ to ‘the city’

Line 399: correct spelling of ‘aegypti’

Line 401: correct in-text reference

Line 404: change to ‘migration from sites that were untreated due to vehicle'

Line 408-9: This study needs a reference. This information has been stated earlier in the Discussion. Remove the repetition.

Reviewer #3: Accept

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This revised manuscript by Solis-Santoyo, et al. describes a study of spatial variation in phenotypic and genotypic insecticide resistance in Aedes aegypti in the city of Tapachula, which has one of the highest incidences of dengue in Mexico. The authors report the discovery of significant spatial variation in insecticide resistance, including widespread, high-level pyrethroid resistance despite the discontinuation of pyrethroid use in the city 5 years prior to sample collection. Furthermore, one of the alleles that confers knockdown resistance to pyrethroids has nearly achieved fixation throughout Tapachula. Importantly and contrary to the authors’ initial hypothesis, there was no correlation between LC50 and distance between collection sites despite largely uniform application of insecticide throughout the city. These results are both unexpected and intriguing as to the source of such high spatial variability. The revised manuscript is much improved due to (1) clarifying the reasons for and distinction between the spatial analysis and the quadrant analysis, and (2) the addition of the Moran’s I correlogram analysis. The authors have addressed all my critiques, and I recommend the manuscript be accepted for publication.

Reviewer #2: Most points identified in the first review have been changed appropriately.

The convention is a capital letter for Zika – Surely you are referring to Zika, the disease. Why are you being informal in a scientific paper? The journal editor can decide what is standard for this journal, but a brief perusal of articles shows the use of a capital letter by other authors in PloS NTD.

Reviewer #3: The clarity and rigor of the manuscript is much improved. Nominal minor edits include:

-Line 331, 353-354: Type 1 and/or Type 2 typically precede the pyrethroid (e.g. Type 1 pyrethroid)

-please italicize 'kdr' throughout the manuscript

-Line 401: this appears to be a reference formatting typo and (37 GROSSMAN) should be changed to [37]

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Joshua R. Lacsina

Reviewer #2: No

Reviewer #3: Yes: Natasha Marie Agramonte

Submitted filename: PNTD Review - 20-02240R1.pdf

Click here for additional data file.

6 Sep 2021

Dear Dr. Penilla-Navarro,

We are delighted to inform you that your manuscript, "Insecticide resistance in Aedes aegypti from Tapachula, Mexico: spatial variation and response to historical insecticide use," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases