Insecticide resistance and underlying targets-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan

Insecticide resistant Aedes populations have recently been reported in Pakistan, imposing a threat to their control. We aimed to evaluate the susceptibility of Aedes aegypti and Aedes albopictus populations from Lahore to WHO-recommended insecticides and to investigate metabolic and target-site resistance mechanisms. For this purpose, we first carried out bioassays with the larvicides temephos and pyriproxyfen, and the adulticides malathion, permethrin, deltamethrin, alpha-cypermethrin, and etofenprox. We looked for Knockdown resistance mutations (kdr) by qPCR, High-Resolution Melt (HRM), and sequencing. In order to explore the role of detoxifying enzymes in resistance, we carried out synergist bioassay with both species and then checked the expression of CYP9M6, CYP9J10, CYP9J28, CYP6BB2, CCAe3a, and SAP2 genes in Ae. aegypti. Both species were susceptible to organophosphates and the insect growth regulator, however resistant to all pyrethroids. We are reporting the kdr haplotypes 1520Ile + 1534Cys and T1520 + 1534Cys in high frequencies in Ae. aegypti while Ae. albopictus only exhibited the alteration L882M. PBO increased the sensitivity to permethrin in Ae. aegypti, suggesting the participation of P450 genes in conferring resistance, and indeed, CYP928 was highly expressed. We presume that dengue vectors in Lahore city are resistant to pyrethroids, probably due to multiple mechanisms, such as kdr mutations and P450 overexpression.

Pyrethroids. Both species tested were resistant to the WHO diagnostic doses to the four pyrethroids, with the magnitude of resistance varying between species for different insecticides. As a whole, mortality rates were lower for permethrin and etofenprox compared to deltamethrin and alpha-cypermethrin (Fig. 2). Among pyrethroids, deltamethrin 0.03% caused the maximum mortality rates in both PAg (85.04%) and PAb (78.85%), while we observed minimum mortality rates with permethrin 0.25% for PAg (8.04%) and PAb (4.52%). In all experiments, control tubes showed no mortality, while Rock was 100% susceptible to all insecticides.
The pyrethroid with the longest estimated time for 50% of the exposed mosquitoes (KdT 50 ) to be knocked down was permethrin in PAg (145 min) and PAb (289 min), and the shortest KdT 50 was induced by deltamethrin in PAg (40.4 min) and PAb (46.8 min). Resistance ratios based on knockdown timings (KdRR 50 ) for the pyrethroids were higher for permethrin in PAb (8.4) and lower for deltamethrin in PAg (1.8) ( Table 2).

Mechanisms of resistance. Synergistic assay.
We tested if the synergist PBO would increase mortality to the pyrethroid permethrin to evaluate the possibility of metabolic resistance mechanisms involvement in permethrin resistance. Permethrin (0.25%) caused 22.3% mortality in PAg and 19.8% in PAb, in this assay, while for the pyrethroids permethrin and deltamethrin. The higher the SI, the less susceptible the population. Ae. aegypti Rockefeller strain (Rock) was used as a reference in all these larvicide bioassays. www.nature.com/scientificreports/ when pre-exposed to 4% PBO, the mortality increased to 100% and 50% in PAg and PAb, respectively. There was no mortality in the control and PBO-only conditions.
Expression analyses. We assessed the levels of expression of five genes related to the metabolism of neurotoxic insecticides (CYP9M6, CYP9J10, CYP9J28, CYP6BB2, and CCEAe3a) and a gene of sensory appendage protein (SAP2) in the whole-body of PAg adult females, in comparison to the housekeeping gene Rps14. Comparing with the profile exhibited by Rock, the only gene with a significant relative fold change expression was the MFO P450 gene CYP9J28, 12.7 × more expressed in PAg (Supplementary Table S1). The SAP2 was nearly fourfold underexpressed in PAg than Rock. The fold change expression of these genes is found in Fig. 3, and more detailed data in Supplementary Table S1.   Figure S2). For the IIIS6 segment, however, 44 samples were kdr homozygous (1534 C/C), and one sample (that same #PAg_22) was heterozygote (1534 F/C), as obtained by TaqMan SNP assay for the F1534C SNP. In addition to this TaqMan assay, an HRM analysis in part of the exon 31 for this segment determined three variants (Fig. 4a), which when sequenced (see below), indicated variation in the 1520 (T1520I) in addition to the 1534 (F1534C) site. The genotypes were then determined as 1520 T/T + 1534 C/C (kdr homozygous at 1534) in 18.2% samples, 1520 I/I + 1534 C/C (kdr homozygous in both sites) in 27.3% samples, and 1520 T/I + 1534 C/C or 1520 T/T + 1534 F/C (heterozygous at 1520 or 1534 sites) in 54.5% samples (Fig. 4b). The exon 31 sequencing of the aforementioned #PAg_22 indicated its genotype as 1520 T/T + 1534 F/C. Summing up all of this information, the allelic frequencies in PAg were: 53.4% (IC), 45.5% (TC) and 1.1% (TF) (Fig. 4c). Three haplotypes were identified in the IIIS6 segment out of 13 sequenced samples (Supplementary Figure S2): the wild-type (PAg_3s6-1) without any nonsynonymous substitution, and two kdr haplotypes: one with the 1534C (PAg_3s6-2) and the other with both 1520I and 1534C kdr mutations (PAg_3s6-3). The PAg_3s6-1 haplotype was observed only under heterozygosis (overlapped peaks in the electropherogram) with PAg_3s6-2, confirming the #PAg_22 genotype (1520 T/T + 1534 F/C). We aligned these haplotypes with 69 other homologous sequences available on the NCBI GenBank and found that PAg_3s6-1 was similar to the wild-type haplotype described in Ae. aegypti populations from all continents. Likewise, PAg_3s6-2 was similar to the 1534C kdr sequences observed worldwide. PAg_3s6-3, with the double mutation 1520I + 1534C, was similar to sequences from Thai and Indian populations (Supplementary Figure S2).  Figure S2). The L882M substitution was also present in a sample from India (MF776970) 32 , although in a haplotype distinct to the Pakistani herein observed.
Concerning the exon 31, we obtained a 257 bp fragment from 13 samples, in which a total of six SNPs were identified, all synonymous substitutions. The sequences of eight haplotypes are available in (Supplementary Figure S2) (GenBank accession numbers: MT740758-MT740765). The haplotypes Pab_3s6-1 and Pab_3s6-2 were identical to sequences from Ae. albopictus from China and Malaysia, available on NCBI GenBank (Supplementary Figure S2).

Discussion
There are few insecticide resistance studies in Pakistani Aedes spp 12,13,15,16,[33][34][35][36][37][38][39][40] , in a scenario where vector control on its own has been one of the neglected aspects of arthropod-borne infections. There were interesting projects about IR status and mechanisms selected in Anopheles mosquitoes during the late 1970s and early 1980s 39,[41][42][43] . Nevertheless, the studies on this particular area were discontinued due to its geopolitical situation and other factors. Lahore, the second-most populous city in Pakistan, has faced various dengue outbreaks in the last decade. Before dengue vector control, several chemicals from pyrethroid, organophosphates, and organochlorine classes were employed in malaria vector control programs. They included deltamethrin, permethrin, malathion, DDT, among others. Here we evaluated the insecticide resistance status of Ae. aegypti and Ae. albopictus from Lahore to the larvicides temephos (organophosphate) and pyriproxyfen (IGR), as well to the adulticides permethrin, etofenprox, alpha-cypermethrin and deltamethrin (pyrethroids), and malathion (organophosphate). We evidenced that these species were still susceptible to both organophosphates and that the IGR is a suitable additional compound to be used in the region. On the other hand, Ae. aegypti and Ae. albopictus were resistant to all types of pyrethroids here evaluated. In addition to kdr mutations, an overexpressed P450 cyp gene might be playing a role in resistance to pyrethroids. Temephos has been employed in Punjab, Pakistan, against Aedes spp. since 2011-2012, and resistance to this larvicide was detected in Ae. aegypti from several cities of that district, including Lahore, collected in 2016 15 . Surprisingly, the LC 50 of temephos in Ae. aegypti from this same city was around 13X lower in our study than that samples collected 2 years earlier (0.08 15 against 0.006 µg/mL). A decrease in temephos resistance was indeed observed in laboratory lines maintained in the absence of selection pressure as well as in natural populations, some years later without the employment of temephos 44 (Rahman et al. 45 ), likely due to a substantial fitness cost associated with physiological changes selected for resistance 46 . However, this decrease can be relatively slight and requires several generations or years in the field without selection pressure, and to our knowledge, temephos is still applied in Lahore. Further studies with new collections are necessary in order to understand this phenomenon better. In the case of Ae. albopictus from Lahore, there were records of resistance to the organophosphates larvicides chlorpyrifos, profenofos, and triazophos 47 , while in our results Ae. albopictus presented a temephos LC 50 value similar to that of Ae. aegypti. Concerning the adulticide malathion, both Ae. aegypti and Ae. albopictus populations from Lahore were susceptible in collections performed in 2015 36 , and maintained this status as we observed here.
The IGRs emerged as a prominent alternative to neurotoxic larvicides 48 . The chitin synthesis inhibitor compounds like diflubenzuron and buprofezin have been tested in Ae. aegypti from Lahore, with diflubenzuron causing higher mortality at the pupal stage while buprofezin resulting in more larval deaths 16 . Both Ae. aegypti and Ae. albopictus we evaluated here showed 100% of adult emergence inhibition against the analog of juvenile hormone IGR, pyriproxyfen, with maximum mortality at the pupal stage, as previously observed in 2015 40 . Also, as an alternative to chemicals, the biolarvicide Bti caused 100% mortality in the Aedes field populations from the same city 16,34 . Although resistance to pyriproxyfen and other IGRs is not common, there are reports of Brazilian and Malaysian populations resistant to pyriproxyfen and methoprene, respectively 49,50 . Therefore, although IGRs have emerged as a promising alternative to neurotoxicants, their effectiveness must be continuously monitored.
Although pyrethroids are not used as larvicides, we performed a rapid test with permethrin and deltamethrin, adopted as a preliminary method to indicate pyrethroid resistance in mosquito larvae 51 . Based on a similar assay, www.nature.com/scientificreports/ we did not evidence resistance to pyrethroid in Pakistani Ae. aegypti and Ae. albopictus larvae. However, both species were resistant to all tested pyrethroid adulticides. Levels of mortality were under 20% in adult bioassays with the pyrethroid type I permethrin, though around 80% to the type II pyrethroid deltamethrin. Resistance to permethrin and deltamethrin was previously recorded in Ae. aegypti from Lahore, collected in 2010 35 . In 2015, both Ae. aegypti and Ae. albopictus from four towns in the Lahore district were found to be resistant to permethrin, lambda-cyhalothrin, and deltamethrin, in addition to DDT 36 . Deltamethrin and permethrin resistant Ae. aegypti populations from Lahore showed a significant increase in mortality after being treated with synergist PBO 15 . Our study corroborated those findings by observing that a pre-exposure to 4% PBO increased mortality to permethrin (from 22 to 100% in Ae. aegypti and from 19.8% to 50% in Ae. albopictus), suggesting an influence of P450 detoxifying enzymes. Indicative of metabolic resistance in PY resistant Ae. aegypti from Lahore was previously shown by biochemical analysis, which indicated higher quantities of esterases, MFOs, GSTs and AchE 33 . Here we evaluated the expression of genes previously related to metabolic resistance, such as the P450 MFOs (CYP9J28, CYP9M6 and CYP6BB2) and the carboxylesterase CCE-Ae3a [52][53][54] . This trend has been identified in resistant Aedes population from Southeast Asia 29,54-56 , the Caribbean 57 , Central 58 , and South America 59 . Of them, we found CYP9J28 12-fold overexpressed in Ae. aegypti from Lahore. Its role has already been shown in vivo, when the transgenic expression of AaegCYP9J28 increased pyrethroidresistance in D. melanogaster 60 . As far as the overexpression of carboxylesterase genes in Ae. aegypti is concerned, they are more associated with OP resistance 61 . Indeed, as we did not observe resistance to the OPs temephos and malathion, it makes sense that the CCEAe3a gene was not overexpressed in our samples from Lahore.
Knockdown resistance mutations in voltage-gated sodium channel (Na V ) alone or in combinations have been reported from several Ae. aegypti populations from Southeast and South Asian countries including Malaysia 62 23,74,75 . Mutations on these sites alone or in combination are expected in populations resistant to pyrethroids and DDT, as recently reviewed 21 . We did not detect these classical SNPs at IS6 (V1014L) and IIS6 (S989P and V1016G) Na V segments, nor any additional nonsynonymous substitutions in the IIS6 segment. Interestingly, the sequences of this segment in worldwide Ae. aegypti populations are divided into two clades, A and B, distinguished mostly by indels in the intron between exons 20 and 21. Besides, all IIS6 kdr mutations described so far are in haplotypes from clade A 23 . Here all IIS6 sequences of Ae. aegypti belonged to clade B. On the other hand, the 1534C kdr mutation in the IIIS6 segment was present in all Ae. aegypti samples from Lahore here evaluated, as indicated by TaqMan genotyping assay specific for the F1534C SNP and confirmed by sequencing. Moreover, HRM analyses displayed a distinct profile that verified the presence of the SNP T1520I. Summing up TaqMan, HRM, and sequencing analyses, we evidenced two kdr haplotypes: T1520 + 1534C (45.5%) and the double kdr 1520I + 1534C (53.4%). The haplotype without kdr T1520 + F1534 (1.1%) was present in only one sample. This partially explains the resistance observed in the Pakistani Ae. aegypti population to the pyrethroids. The F1534C kdr has been described in Ae. aegypti populations from several Asian countries 76 . In PY-resistant Indian Ae. aegypti populations, a PCR-RFLP analysis followed by sequencing showed the presence of three haplotypes [T1520 + F1534 (21%), T1520 + 1534C (66%), and 1520I + 1534C (13%)] as we have reported in our findings. It was also observed that the F1534C mutation always occurred independently while T1520I was always found in association with F1534C 14,23 .
In the case of Ae. albopictus, HRM analyses identified distinct variants in the analyses of both IIS6 and IIIS6 Na V segments. However, they all accounted for synonymous substitutions, as revealed by sequencing, except for one nonsynonymous in the IIS6 segment: L882M. Likewise, several mutations in PY-resistant Ae. albopictus populations from India were also reported 32 . Chinese Ae. albopictus presented a different mutation (F1534S) in pyrethroids resistant populations 70 . It is noteworthy that the diversity of haplotypes in Ae. albopictus was higher than Ae. aegypti, as expected since it is native to Asia while Ae. aegypti is an invasive species 77 . Consequently, lower diversity is expected in this species, compared to the autochthonous Asian tiger mosquito.

Conclusion
Ae. aegypti and Ae. albopictus from Lahore, Pakistan, were resistant to pyrethroids while susceptible to organophosphates and the IGR. Among compounds we tested herein, both larvicides temephos and pyriproxyfen, as well as the adulticide malathion, should be the most effective against both species. Synergist PBO increased mortality against permethrin, indicating the participation of metabolic resistance mechanisms. In fact, the P450 gene CYP9J28 was overexpressed in Ae. aegypti. Also, the kdr mutations T1520I and F1534C were present under high frequencies. Therefore, resistance to pyrethroids in Ae. aegypti from Lahore is likely related to multiple physiological mechanisms. These results' implications may be discussed with authorities responsible for Lahore's vector control actions, aiming to improve strategies against Aedes in Pakistan.

Methods
Sample collection and laboratory rearing. We collected larvae of Aedes aegypti and Aedes albopictus from different Union Councils (UCs)/neighborhoods of Mughulpura and airport areas of Lahore district with the help of sanitary agents and health workers recruited by the local health department for this purpose (Fig. 5).
We visited different domestic and industrial areas. Larvae mainly infested uncovered or partially covered water storage tanks, buckets, and small pots made from various materials, including plastic, steel, copper, and cement, which contained water and were located in shaded places. Larvae were brought to the insectary of the Department of Zoology, GC University Lahore, identified into species level with the help of morphological characters 78 , and provided with fish food (Tetra-Marine Granules, Tetra). Emerged pupae were shifted to clean and disinfected cages, and adults were provided with a 10% sugar solution. Adults were starved overnight but With the purpose of maximizing growth synchronization, we put the Aedes eggs to hatch in 1 L dechlorinated water, and after 3-4 h, 500 larvae were transferred to separate trays and grown to L3/L4 stage under standard lab conditions of temperature 26 °C ± 1 °C, light: dark 12 h: 12 h period and 70 ± 5% relative humidity 46 . The Ae. aegypti Rockefeller strain, the international reference for susceptibility to insecticides and vigor under laboratory conditions 79 , was reared in parallel and tested in all experiments as an internal control. We have referred to Ae. aegypti Rockefeller as Rock, and Ae. aegypti and Ae. albopictus Lahore populations as PAg and PAb, respectively. Bioassays with larvae. Temephos. We followed the dose-response WHO protocol to calculate the resistance ratios (RR) to temephos in the evaluated populations 80 . Temephos technical grade (Pestanal Sigma-Aldrich) was dissolved in ethanol to give a series of concentrations ranging from 0.002 to 0.018 mg/L in order to obtain mortality from 5 to 99%, as recommended for probit analysis. Each concentration and a negative control condition (containing 300 µL of ethanol only) was replicated four times, each containing 100 mL solution with ~ 20 late L3 or early L4 larvae, in a disposable plastic cup of 150 mL capacity. Mortality was calculated 24 h after initial exposure. The tests were performed three times with both PAg and PAb populations simultaneously, always in parallel to Rock as an internal control.
Pyriproxyfen. For the IGR pyriproxyfen, we performed dose-diagnostic assays. Each assay contained eight treatment replicas with pyriproxyfen (Sigma-Aldrich) at a diagnostic concentration of 0.3 μg/L. This dosage was previously obtained, as twice the CL 99 for Rock 49 . Besides, we included six control replicas (with 1 mL of the solvent instead of the IGR solution) in a 250 mL solution. A 300 mL disposable, transparent plastic cup was used for each replica, into which we added 10 L4 larvae. To nourish the larvae, we introduced 15 mg of fish food (Tetramarine Granules, Tetra) at the start of the assay and 10 mg on the fourth day of the experiment. All the cups were covered with gauze to avoid eventual adult escaping. Mortality and life stage transformation were cumulatively recorded bi-weekly. Parallel assays were done with Rock. Data was recorded until all individuals in the control condition emerged into adults. Assays were performed three independent times, under standard www.nature.com/scientificreports/ environmental conditions of temperature (26 °C ± 1 °C), light: dark regiments (12 h: 12 h), and relative humidity (75 ± 5%). Unlike neurotoxic insecticides, for IGRs, mortality itself is not the critical parameter to evaluate, but the index of Adult Emergence Inhibition (AEI). Populations are considered to be resistant when AEI is lower than 90%. In addition to AEI, we recorded the mortality observed at each developmental stage: larvae, pupae, and adult (adults that remained attached to the exuviae and died).
Susceptibility Index of Aedes larvae against pyrethroids. We adopted a simplified knockdown assay 51 to evaluate susceptibility to the knockdown effect to type I (permethrin) and type II (deltamethrin) pyrethroids in larvae. We prepared solutions with technical grades permethrin (Pestanal, Sigma-Aldrich) and deltamethrin (Pestanal, Sigma-Aldrich) by dissolving each in 1 mL acetone and then ethanol to make 250 ppm stock solutions. From this, we prepared a 20 mL solution for each pyrethroid, with 10 replicas for two concentrations (0.1 ppm and 0.4 ppm) in H 2 O. Two negative control cups with ethanol (32 µL) in 20 mL of H 2 O were run in parallel. We used one L4 larva per replica and registered the knockdown every 5 min, for half an hour. The KdT 50 , i.e., the time when 50% of the larvae were knocked down, was scored according to these categories: 1 (0-5 min), 2 (6-10 min), 3 (11-15 min), 4 (16-20 min), 5 (21-30 min) or 6 (> 30 min). The susceptibility index (SI) for each species against deltamethrin and permethrin was obtained by multiplicating the KdT 50 categories of both concentrations (0.1 and 0.4 ppm). Population with the lower SI is considered more sensitive. The obtained SI values were the mean of three independent assays, with Rock in parallel as an internal control in all of them.
Adulticides. Pyrethroids. We followed the WHO-like tube tests procedure, with modifications 81,82 for the tarsal contact tests with insecticides-impregnated filter papers. Papers (Whatman grade 1) were impregnated with diagnostic concentrations of four pyrethroids following WHO standard protocol and dosages: deltamethrin 0.03%, permethrin 0.25%, etofenprox 0.5% and alpha-cypermethrin 0.03% 81 . Solutions were prepared from technical grade insecticides (Pestanal Sigma-Aldrich) in acetone and silicone oil (used as a carrier) and evenly applied to a filter paper (Whatman grade 1). Papers were allowed to air dry for 72 h before use. Papers impregnated with just solvent were used as the control. In each assay, we put 15-20 female mosquitoes, 3-5 days old, non-blood-fed, in a resting tube (tube without insecticide) to acclimatize for 30 min. After that, they were gently blown into tubes having insecticides-impregnated papers, and knockdown was checked every 5 min for 2 h. This differed from the WHO protocol 81 , which recommends exposure time of 60 min. In the case of Rock, mortality was checked every two minutes. After they were exposed to insecticides, mosquitoes were shifted back to resting tubes, provided 10% sugar water, and mortality evaluated after 24 h.
Organophosphate. For the OP malathion bioassays, we used CDC bottle tests 83 , impregnating 250 mL glass bottles (Wheaton) with 20 µg/mL malathion (Cheminova Brasil Ltda, São Paulo) dissolved in acetone. For impregnating the bottles, 1 mL of the insecticide solution was distributed evenly to all parts of the bottle, including its cap, and left to air dry for at least 24 h before the test. In each bottle, 20-25 female mosquitoes, three to five days old, non-blood fed, were left for one hour, and mortality was recorded. Bottles impregnated with 1 mL acetone were used as control. This experiment was done in four replicates, three separate times. Standard conditions of temperature, humidity, and light-dark periods were maintained throughout the assays, as described earlier.
Synergist assay. In order to evaluate the occurrence of metabolic resistance mechanisms to the pyrethroid type I permethrin, we carried out WHO bioassays with the synergist PBO (Piperonyl Butoxide) 84 . This substance inhibits the action of Multi-Function Oxidases (MFOs) and can revert resistance 85 . For this purpose, papers were impregnated with technical grade PBO (Endura) and permethrin (Pestanal, Sigma-Aldrich), as described earlier. The test was composed of four conditions: (i) exposure to 4% PBO, (ii) a pre-exposure to 4% PBO and then to 0.25% permethrin, (iii) exposure to 0.25% permethrin only, and (iv) exposure only to solvent control. The susceptible strain, Rock, was carried out parallelly. Each assay consisted of four tubes, and each tube contained 20-25 female mosquitoes, 4-6 days old, non-blood-fed. All exposures lasted 1 h, after which mortality was checked in each assay. Mosquitoes were then transferred to resting tubes and provided 10% sugar solution. The number of dead mosquitoes was counted after 24 h, according to the standard criteria of WHO 81 .
Calculations. In the case of both pyrethroids and organophosphates bioassays, we expected 100% mortality of the reference strain and no (zero) mortality in the control. If mortality in the control exceeded 20%, the test should be repeated. If less, Abbott's correction formula would be applied 86 . Temephos Lethal Concentrations (LC) were obtained by log x probit transformations followed by linear regression analyses 87 with the sum of the values from the three assays. We calculated the resistance ratios (RR) by dividing the LC of both PAg and PAb by the LC of Rock, in the absence of a reference lab strain for Ae. albopictus at that time.
To estimate knockdown time to pyrethroids (KdT 50 ), readings were likewise submitted to probit analysis. Accordingly, knockdown-time Resistant Ratios (KdT-RR 50 ) were calculated by dividing KdT 50 of PAg and PAb by the KdT 50 of Rock.
Exploration of knockdown resistance (kdr) mutations. We evaluated the nucleotide diversity in the genomic region corresponding to IS6, IIS6, and IIIS6 Na V segments to investigate kdr mutations classically found in Ae. aegypti pyrethroid-resistant populations. Genomic DNA was isolated from male mosquitoes (n = 45) of both PAg and PAb with a column-based DNA extraction kit (NuleoSpin, Macherey-Nagel Laboratories), according to manufacturer's instructions. We did not use females to avoid eventual amplification of DNA inside their  (Fig. 6). We focused on these exons because the known kdr sites 989, 1016, and 1534 are respectively placed in the exons 20, 21, and 31 of the Ae. aegypti NaV gene. Reactions were performed with 1X MeltDoctor  -TCG TTC TAC CTT GTA AAT  T-NFQ  Leu: FAM-TTC GTT CTA CCT TTT AAA  TT-NFQ   989, IIS6  AH21C3C  TCT/CCT  (Ser/Pro)   for: TGA TCG TGT TCC GGG TAT TATGC  rev: CCA TCA CTA CGG TGG   Expression analysis of genes related to detoxification of insecticides. We evaluated the expression profiles of genes previously associated with resistance in Aedes populations from South-east Asia in PAg populations: three were from the CYP9 family (CYP9M6, CYP9J10, and CYP9J28) and one from CYP6 (CYP6BB2). The expression profile of one carboxylesterase (CCEAe3a) and one sensory appendage protein (SAP2) was also investigated 31,91,92 . The expression levels were relative to the housekeeping gene of the ribosomal protein S14 (RpS14) 93 . Five-day-old female mosquitos were pooled in a tube. For each population, we used four pools as biological replicates per population. In order to isolate RNA, these mosquitoes were macerated in 300 µL TRIzol (Invitrogen, CA, USA) and then homogenized with glass beads in TissueLyser II (Qiagen, Venlo, Netherlands). RNA was precipitated with TRIzol (Invitrogen, CA, USA) and chloroform and then washed with ethanol to remove any debris of DNA and protein, according to the manufacturer's protocol. The pellet obtained was air-dried and eluted with RNase-free water. The isolated RNA was quantified with Qubit RNA HS Assay kit (Invitrogen), and cDNA was then synthesized in an RT-PCR reaction with SuperScript Vilo MasterMix (Invitrogen), using 5 µL RNA and molecular grade water qi 20 µL. The reagents were incubated at room temperature for 10 min, 42 °C for 1 h, and 85 °C for 10 min. We used the Qubit dsDNA HS Assay kit (Invitrogen) to quantify this cDNA and diluted it to 4 ng/µL for qPCR use.
The qPCR reactions were performed in a 96-Well MicroAmp reaction (Invitrogen) plate, with the kit KAPA SYBR FAST qPCR Master Mix (1x) and ROX LOW (KAPA Biosystems) kit, 0.2 µM of each forward and reversed primers (Table 5), 4 ng/µL cDNA and molecular grade water q.s. 10 µL. Each pool (biological replicate) was divided into four technical replicates. Thermocycling conditions in a QuantStudio 6 Flex Real-Time PCR system (ThermoFisher Scientific) consisted of enzyme activation and initial denaturation at 50 °C and 95 °C for 2 and 3 min, respectively, followed by 40 cycles of denaturation (95 °C for 3 s) and annealing/extension of primers (60 °C for 1 min), with an additional standard melting curve analysis step.
Analysis of gene expression. We obtained the Ct values of four replicates for each gene and calculated their means. Replicates presenting different Ct from Grubbs values (outliers) 98 or with an unexpected peak in the melting analysis were excluded. Relative quantification analyses were performed using the ΔΔ Ct method 99 , where the RpS14 was taken as the reference gene and Rock, reared alongside test population, as the reference strain. For all genes, the Ct threshold was set at 0.2. The 2 (−ΔΔCt) equation was applied to define the relative fold-change Table 4. Primers for HRM analyses in Aedes aegypti and Aedes albopictus. All primer sequences are in the 5′-3′orientation.