Resistance to insecticides and synergism by enzyme inhibitors in Aedes albopictus from Punjab, Pakistan

The widespread use of insecticides has ecological consequences such as emergence of insecticide resistance and environmental pollution. Aedes albopictus is a major vector of dengue virus in the Punjab province, Pakistan. Control of Ae. albopictus with insecticides along with source eradication is critical in the prevention and control of dengue fever but is threatened by the development of insecticide resistance. Here, field strains of Ae. albopictus from eight cities of Punjab were evaluated for resistance against temephos, deltamethrin and permethrin. For temephos, high resistance (RRLC50 > tenfold) was found in larvae of the Rawalpindi strain, moderate resistance (RRLC50 = five- to tenfold) in Multan, Faisalabad, Sialkot, Lahore and Sheikhupura strains, and low resistance (RRLC50 < fivefold) in Kasur and Sahiwal strains. In the case of deltamethrin, high resistance was seen in adults of the strain from Faisalabad, moderate resistance in the strains from Sialkot, Sheikhupura, Lahore and Kasur, and low resistance in Sahiwal, Multan and Rawalpindi strains. For permethrin, adults of all the field strains exhibited high levels of resistance. In synergism bioassays, toxicity of all the insecticides in the field strains significantly enhanced when tested in combination with piperonyl butoxide or S,S,S-tributylphosphorotrithioate, suggesting the probability of metabolic-based mechanisms of resistance. In conclusion, field strains of Ae. albopictus from Punjab exhibit resistance to temephos, deltamethrin and permethrin, which might be associated with metabolic mechanisms of resistance.


Scientific Reports
| (2020) 10:21034 | https://doi.org/10.1038/s41598-020-78226-0 www.nature.com/scientificreports/ temephos (an organophosphate) through larviciding, and deltamethrin and permethrin (pyrethroids) through space and indoor residual sprays have been in wide use in Aedes mosquitoes control programs. In this scenario, wide use of insecticides is fraught with the rapid development of insecticide resistance and environmental pollution 7,8 . Insecticidal bioassays in combination of enzyme inhibitors (e.g., S,S,S-tributylphosphorotrithioate (DEF or tribufos) and piperonyl butoxide (PBO)) to check their synergistic effect on insecticide toxicity is a rapid and inexpensive approach to provide clues regarding the possibility metabolic mechanisms of insecticide resistance 9,10 . Studies revealed that esterase-and/or oxidase-based mechanism of resistance could be present in insect species if toxicity of a particular insecticide is enhanced when used in combination with DEF, since DEF inhibits the activities of oxidases and esterases. Likewise, possibility of oxidase-based mechanism of resistance could be detected if the toxicity of an insecticide is synergized when used in combination with PBO, since it suppresses activities of oxidases 9,11,12 . Recently, development of insecticide resistance to organophosphate (temephos) and pyrethroid (deltamethrin and permethrin) insecticides have been reported in another important vector of dengue virus, Ae. aegypti (L.), from different areas of Punjab 5 ,and bioassays in combination of PBO and DEF suggested the probability of metabolic mechanism of resistance associated with resistance to the organophosphate. Likewise, there are chances of resistance development in Ae. albopictus that may reduce the efficacy of current insecticide-based control programs. Therefore, present study was planned to investigate the possibility of insecticide resistance development to commonly used insecticides (temephos, deltamethrin and permethrin) in Ae. albopictus collected from the urban areas of Punjab, Pakistan. Furthermore, synergism bioassays by using PBO and DEF in combination with insecticides were also performed to find the clues about the presence of metabolic mechanisms of resistance in the putatively resistant strains of Ae. albopictus.

Materials and methods
Aedes albopictus strains. The study was conducted on Ae. albopictus strains collected from eight cities of the Punjab province, Pakistan, during 2017-2018 (Table 1). The cities were selected on the basis of dengue fever outbreaks in recent years and wide use of insecticides application for control of Aedes mosquitoes. A reference strain (Ref-S) of Ae. albopictus was also collected from an area of very low chemical application and reared in the laboratory for 20 generations in insecticide-free environment. Although not truly susceptible, the susceptibility of the Ref-S strain to insecticides was quite higher as compared to the field strains (see results section) and hence can be used as baseline for resistance monitoring 13 . Immature Ae. albopictus were collected from different breeding sites and transported to the laboratory. About 500-700 immature Ae. albopictus were used to start each colony. In laboratory these strains were reared by maintaining 26 ± 1 °C, 65 ± 5% r.h. and photoperiod of 12:12 (L:D) h. In the laboratory, the diet was consisted of fish food for larvae and 20% sucrose solution for adult mosquitoes, while female Ae. albopictus were bloodfed from an anesthetized mouse thrice a week following in accordance with relevant guidelines and regulations. The field strains were reared up to F1 or F2 generations before starting bioassays. The study/bioassay protocols used against Ae. albopictus were performed according to the standard guidelines and regulations, and approved by the bioethics committee of Institute of Agricultural Sciences, University of the Punjab, Lahore.
Bioassays. Bioassay methods have been described in detail elsewhere 5 . Briefly, toxicity of temephos was checked through larval bioassay in three replicates. A range of dilutions were prepared in acetone, while acetone alone was taken as a control. For Ref-S and field strains, the concentrations used were ranged between 0.01 to 0.32 µg/ml and 0.04 to 1.28 µg/ml, respectively.
Bottle-bioassay method developed by the Centre for Disease Control (CDC) was followed to check the toxicity of deltamethrin and permethrin against female adults of Ae. albopictus 9 . Deltamethrin and permethrin were dissolved in acetone to prepare concentrations. These concentrations were used to coat 250 ml glass bottles at the rate of 1 ml/bottle. Control bottles were coated with acetone alone. Range of concentrations used were as Synergism experiments. Synergism bioassays were performed as outlined in our previous report 5 . In brief, Ae. albopictus larvae (for temephos) or unfed females (for deltamethrin or permethrin) were exposed to PBO and DEF solutions for 1 h. After exposure to synergists, larvae or adults were then exposed to different concentrations (n = 25 per concentration per replicate) of insecticides via the insecticide solution (for larvae against temephos) or insecticide-treated bottles (for adults against deltamethrin or permethrin) as stated in the bioassay section. Additional details are provided in supplemental materials.
Data analyses. Data of the knockdown effect and mortality were subjected to Probit analysis using the software PoloPlus 14 . Median knockdown concentrations (KC 50s ) for deltamethrin and permethrin were calculated from the data after 1 h exposure. Median lethal concentrations (LC 50s ) for temephos, deltamethrin and permethrin were determined from the mortality data after 24 h exposure. KC 50s or LC 50s values of field strains were divided with those of the Ref-S strain to get resistance ratios (RR KC50 , RR LC50 ). The ratios were classified according to the following scale: RR < 5 folds (low resistance); RR ranged from 5 to 10 folds (moderate resistance); RR > 10 folds (high resistance) 5,15 . Simple linear regression was performed to find the association between LC 50 and KC 50 values for deltamethrin or permethrin in eight field strains. The resultant slope vales were analyzed following the criterion of Flores, et al. 16 : a slope value ≃ 1 indicates LC 50 ≃ KC 50 and most of the knockdown mosquitoes are dead after 24 h exposure; a slope value > 1 indicates the LC 50 value is greater than the KC 50 value most of the knockdown mosquitoes recovered after 24 h exposure and more insecticide concentration is needed to cause mortality of these mosquitoes 16 .

Results
The results of bottle-bioassays using adults of Ae. albopictus for estimating KC 50 and corresponding RR values for deltamethrin and permethrin are presented in Table 2. The results revealed the highest susceptibility of the Ref-S strain to deltamethrin and permethrin with KC 50 values 1.42 and 0.60 µg/ml, respectively. KC 50 values of different field strains ranged from 4.35 to 18.55 µg/ml for deltamethrin, and 6.85 to 15.47 µg/ml for permethrin. Field strains showed significant levels of RR values at KC 50 level when compared with the Ref-S strain, based on the 95% CIs of RR values did not include 1. In the case of deltamethrin, Multan, Rawalpindi and Sahiwal strains exhibited low resistance (RR 3.06, 3.39 and 3.50 fold, respectively), Sialkot, Kasur, Faisalabad and Lahore moderate resistance (RR 5.02, 6.02, 8.94 and 9.80 fold, respectively), and high resistance in the strain of Sheikhupura (13.06 fold) ( Table 2). www.nature.com/scientificreports/ Table 3 displays LC 50 values and corresponding RR values obtained for temephos (against larvae), deltamethrin and permethrin (against adults) in different strains of Ae. albopictus. All the insecticides showed the highest toxicity to the Ref-S strain with LC 50 values 0.05, 1.48 and 0.54 µg/ml for temephos, deltamethrin and permethrin, respectively. The LC 50 values for different field strains ranged from 0.17 to 0.64 µg/ml for temephos, 4.20 to 28.84 µg/ml for deltamethrin, and 6.44 to 37.14 µg/ml for permethrin. High resistance to temephos was found in the Rawalpindi strain (RR 12.80 fold), moderate resistance in Multan (RR 6.00 fold), Faisalabad (RR 6.00 fold), Sialkot (RR 7.80 fold), Lahore (RR 8.00 fold) and Sheikhupura (RR 8.20 fold) strains, and low resistance in Kasur (RR 3.20 fold) and Sahiwal (RR 3.40 fold) strains. In the case of deltamethrin, high resistance was seen in the strain of Faisalabad (RR 19.50 fold), moderate resistance in the strains of Sialkot (RR 6.44 fold), Sheikhupura (RR 7.51 fold), Lahore (RR 7.58 fold) and Kasur (RR 7.82 fold), and low resistance in Sahiwal (RR 2.84 fold), Multan (RR 3.51 fold) and Rawalpindi (RR 4.97 fold) strains. For permethrin, all the field strains exhibited high resistance with RR values ranged from 11.93 to 68.78 fold (Table 3).
Regression analysis between RR LC50 and RR KC50 for deltamethrin and permethrin is shown in Fig. 1. For deltamethrin, the slope value 0.73 indicates that majority of the knockdown mosquitoes failed to recover after 24 h. In contrast, the slope value 2.99 for permethrin revealed that most of the mosquitoes recovered after knockdown and a higher concentration of permethrin is required to eventually kill mosquitoes (Fig. 1).
The results of synergism bioassays against Ref-S and selected field strains are shown in Table 4. The data revealed that the toxicity of all the insecticides in the Ref-S strain did not change significantly when bioassayed in the presence of either PBO or DEF. The synergism ratios were non-significant based on 95% CIs of SRs include 1.
In the case of field strains, toxicity of all the insecticides enhanced significantly when bioassayed in the presence of PBO or DEF (based on synergism ratio test and non-overlapping 95% CIs of LC50 values), suggesting the possibility of metabolic mechanism of resistance. Toxicity of temephos against larvae of Ae. albopictus enhanced 2.25 and 2.66 fold in the presence of DEF and PBO, respectively. Similarly, toxicity of deltamethrin enhanced by 2.64 fold (+ DEF) and 2.07 fold (+ PBO), while toxicity of permethrin increased by 1.94 fold (+ DEF) and 2.62 fold (+ PBO) against adults of Ae. albopictus (Table 4).  22 reported the probability of resistance to deltamethrin and permethrin in Ae. albopictus from Rawalpindi. Similarly, Mohsin, et al. 21 reported probability of resistance to different insecticides in Ae. albopictus from the Lahore city of Punjab. However, the scope of both studies, in our opinion, was limited since mosquitoes were sampled from only one location in each study. Therefore, it was the need to explore other important areas of the province with dengue positive cases every year. In our study, we examined insecticide resistance in more detail by determining resistance ratios in www.nature.com/scientificreports/ Ae. albopictus from eight major cities with respect to dengue fever incidence, and also studied the possibility of metabolic mechanism of resistance. Pyrethroids such as deltamethrin and permethrin are dominant insecticides for the management of urban insect pests in residential environments. The results of the present study revealed low to high levels of resistance to deltamethrin and high levels of resistance to permethrin in different field strains of Ae. albopictus. In addition, regression analysis was performed to find the association between LC 50 and KC 50 values for deltamethrin or permethrin in different field strains. For deltamethrin, the slope value indicated that majority of the knockdown mosquitoes failed to recover after 24 h. In contrast, the slope value for permethrin revealed that most of the mosquitoes recovered after knockdown and a higher concentration of permethrin was required to eventually kill mosquitoes. These insecticides are widely used in Punjab to combat mosquitoes. The most probable reason for resistance Ae. albopictus could be the fact of usage of these insecticides in different forms such as fogging, indoor residual sprays, mosquito coils against Aedes mosquitoes in order to protect from dengue fever 5 . Furthermore, injudicious use of pyrethroid insecticides for different insect pests in urban settings 19 could also be responsible for resistance development in different strains of Ae. albopictus. Recently, we have reported resistance to permethrin and deltamethrin in Ae. aegypti from 12 cities (Faisalabad, Gujranwala, Lahore, Multan, Okara, Pattoki, Rawalpindi, Sahiwal, Sargodha, Sheikhupura, Sialkot and Kasur) of Punjab, Pakistan 5 . Except the Okara strain, all field strains exhibited high levels of resistance to permethrin. In case of deltamethrin, low levels of resistance were found in Multan, Okara and Sahiwal strains, moderate levels of resistance in Sialkot, Gujranwala and Sargodha strains, and high levels of resistance in Kasur, Pattoki, Lahore, Sheikhupura, Faisalabad and Rawalpindi. Previously, resistance to pyrethroid in Aedes mosquitoes has also been reported worldwide [28][29][30][31][32] .
In the present study, low to moderate levels of resistance to temephos were found in seven field strains, while the Rawalpindi strain exhibited high resistance to temephos. In Punjab, temephos is the most widely used as larvicide to manage larvae of Aedes mosquitoes that might be responsible for resistance development to temephos in Ae. albopictus. Temephos resistance in Ae. aegypti has also been reported from different cities (stated above) of Punjab 5 . Of these, high levels of resistance were found in Gujranwala, Lahore, Kasur, Rawalpindi and Faisalabad strains, moderate levels of resistance in Okara, Sahiwal, Pattoki, Sialkot and Sheikhupura, and low levels of resistance in Sargodha and Multan. There are variable reports of resistance development to temephos in Ae. albopictus from different areas of the world. For instance, Ponlawat, et al. 32 reported low levels of resistance to temephos in Ae. albopictus from different localities of Thailand. Larvae of Ae. albopictus from Selangor, Malaysia were found to be highly resistant to temephos 33 . In contrast, larvae of Ae. albopictus from Central Africa were found to be susceptible to temephos 34 . The different expression of resistance might be due to differences in geographic origin of Ae. albopictus strains, insecticide exposure histories and/or different environmental conditions.
Insecticidal bioassays in combination of enzyme inhibitors (e.g., PBO or DEF) to check their synergistic effect on insecticide toxicity is a rapid and inexpensive approach to provide clues regarding the possibility metabolic mechanisms of insecticide resistance 9,10 . Studies revealed that esterase-and/or oxidase-based mechanism of resistance could be present in insect species if toxicity of a particular insecticide is enhanced when used in Table 4. Toxicity of temephos, deltamethrin, and permethrin with or without synergist in Aedes albopictus strains. £SR, synergism ratio = (LC 50 of temephos, deltamethrin or permethrin alone) ÷ (LC 50 of temephos, deltamethrin or permethrin plus PBO or DEF). ns, non-significant (p > 0.05) based on overlapping 95% CI of LC 50 values of insecticides plus PBO or DEF when compared with that of the LC 50 of insecticide alone. **Significant SR, 95% CIs of SR did not include 1 39 . www.nature.com/scientificreports/ combination with DEF, since DEF inhibits the activities of oxidases and esterases. Likewise, possibility of oxidasebased mechanism of resistance could be detected if the toxicity of an insecticide is synergized when used in combination with PBO, since it suppresses activities of oxidases 9,11,12  These results indicate the possibility of esterase-and oxidase-based mechanisms of resistance linked with insecticide resistance in field strains. Both of these enzyme inhibitors have also been reported to enhance toxicity of temephos in Ae. aegypti from Cucuta 35 and in different field strains of Ae. aegypti from Punjab, Pakistan 5 . Resistance to pyrethroid insecticides in insect pests can be due to the enhanced activities of metabolic enzymes and/or modification to the target site of these insecticides. Sayyed, et al. 12 reported the synergistic effect of PBO and DEF on the toxicity of deltamethrin in Chrysoperla carnea (Stephens). In contrast to the present study, toxicity of deltamethrin and permethrin did not synergize by enzyme inhibitors in field strains of Ae. aegypti 5 .In the present study, the Sialkot strain showed about 69 fold RR at LC 50 level, and its toxicity was enhanced by < threefold in the presence of PBO or DEF. This high level of resistance to permethrin indicates the possibility of altered target site mechanism responsible for resistance to permethrin. Smith, et al. 36 reported metabolic detoxification and altered target site as the major mechanisms of resistance to pyrethroid insecticides in Ae. albopictus and Ae. aegypti. However, altered target site mechanism alone was found to be the major mechanism of resistance to pyrethroid in Ae. aegypti in Puerto Rico 37 . Multiple mechanisms could be present at the same time in resistant mosquitoes 38 , depending upon the history and geographical origin of insect strains. Future studies may be planned at molecular level to further confirm the mechanisms of resistance in Ae. albopictus.
Insecticide resistance is a major hindrance in the management of Aedes mosquitoes that ultimately lead to seasonal outbreaks of dengue fever in different areas of Pakistan. An important strategy could be to lessen the use of insecticides by adopting integrated vector management (IVM) tool such as mosquito-breeding source reduction, the use of mosquito nets, and management of rainwater collecting bodies. Aedes albopictus from different cities of Punjab, Pakistan, have shown resistance development to insecticides used for mosquito control, which may result in severe outbreaks of dengue fever in the future. To avoid this situation, regular insecticide resistance monitoring along with the use of alternative measures could be effective tools for managing Ae. albopictus.