Stage-specific insecticide susceptibility and life-table analysis of the dengue vector mosquitoes

: Vector resistance to insecticides is a significant challenge in the control of mosquito-borne diseases such as dengue. Analysis of the growth rate and survivorship of the immature stages of larvae has been important in formulating effective vector control strategies. The present study aimed to assess insecticide resistance in both larvae and adults of Aedes aegypti and Ae. albopictus, and the life tables of their immature stages. Mosquito eggs collected from Kurunegala and Colombo districts of Sri Lanka from January to March 2023 were reared under laboratory conditions. Larval bioassays were carried out with temephos (0.0125 ppm, 0.0625ppm and 0.125ppm) and, the adult bioassays were with pyrethroids (0.03% deltamethrin, 0.05% and 0.08% lambdacyhalothrin, 0.04% permethrin) and malathion (1%and 5%). Biochemical assays were used to evaluate the activity of insecticide-detoxifying enzymes and the insensitivity of the organophosphate target site acetylcholinesterase. Survivorship of immature stages under laboratory conditions and semi-field conditions i.e., bamboo stumps, tyres and clay pots, were studied using Kaplan-Meier survival analysis. Larvae and adults of both species from Kurunegala were susceptible to all the insecticides tested, whereas Colombo populations were resistant. Acetylcholinesterases were largely sensitive in all vector populations. Higher activities of esterases, glutathione s-transferases and monooxygenases were found in Colombo populations. Results indicated that Ae. aegypti larvae were more resistant to insecticides than Ae. albopictus larvae. Also, the mechanisms of insecticide resistance were well developed in adults than in larvae. Aedes aegypti larval development time (5 days) was significantly shorter than that of Ae. albopictus (9 days) under laboratory conditions. Survival probabilities of them were 76-80%. Egg hatching to adult emergence period was 8 days for Ae. aegypti and 13 days for Ae. albopictus . This period, under semi-field conditions were; bamboo stumps 7-7.2 days, tyres 10-11.3 days, and clay pots 13.8-14 days with survival rates varied from 28.8 to 63% for both species. This study highlights stage specific response of dengue vectors to insecticides and survivorship of their immature stages in different habitats. This knowledge can be effectively used in future dengue vector control programmes.


INTRODUCTION
Dengue is a major mosquito-borne arboviral disease transmitted to humans by Aedes mosquitoes (WHO, 2005).More than 80% of the world's population lives in areas with the transmission of this disease.Vector control plays a key role in reducing the dengue disease burden in the absence of proper vaccines or treatments.Neuroinhibitory insecticides, delivered as larvicides or space spraying adulticides are widely used to control dengue vectors, especially during disease outbreaks.Organophosphates are used for larviciding and, both pyrethroids and organophosphates are used for space spraying.As a result of extensive exposure to insecticides, resistance to insecticides has emerged in vector mosquitoes and spread across all regions of the world (WHO, 2018a, Moyes et al., 2017, Karunaratne et al., 2018).
In Sri Lanka, reported cases of dengue have increased at an alarming rate recently (NDCU, 2022).Reduction in disease transmission is primarily attempted through the control of the vectors Aedes aegypti and Ae.albopictus.Although the elimination of vector breeding places is a major vector control strategy, lack of resources and poor community support hinder its success.Therefore, chemical control methods are used extensively for vector management (Sirisena et al., 2016).In Sri Lanka, health authorities use the organophosphate temephos (Abate®) as a larvicide and pyrethroids (PestGuard ® 161: d-tetramethrin 4% + cyphenothrin 12%, and Gokilaht 10MC: cyphenothrin 10.4%) as adulticides (Fernando et al., 2020).Organophosphate malathion was previously used as an adulticide until 2009 (NDCU, 2022, Fernando et al., 2020).Resistance has been reported to neuroinhibitory insecticides in both Ae.aegypti and Ae.albopictus from different regions of the country (Nugapola et al., 2020, Fernando et al., 2018, Fernando et al., 2020, Hegoda et al., 2017).
Insecticide resistance is achieved by mosquitoes mainly through two major mechanisms; target site insensitivity and increased detoxification (Karunaratne et al., 2018).Target sites become insensitive due to gene mutations which prevent the target site from binding to insecticides.Increased detoxification is achieved through increased activity of insect metabolic enzymes.Quantitative or qualitative changes of these enzymes in resistant mosquitoes cause insecticide detoxification at a much faster rate than done by their counterparts in susceptible mosquitoes.There are three groups of such enzymes; monooxygenases (also known as oxidases or cytochrome P450s), esterases and glutathione S-transferases (GSTs) (Hemingway et al., 2004, Karunaratne et al., 2018).
Vector resistance to insecticides and the prevalence of resistance mechanisms can vary markedly across vector populations in different regions, even within a country, reflecting the levels of selection pressure exerted by insecticide usage.Local vector populations can become resistant to several insecticides at any given time and they will not revert to susceptibility, at least not at a rate that is of immediate relevance to programmatic decision-making.
Understanding the type and level of resistance of local vector populations to insecticides in current use, or planned for use, is crucial to ensure the efficacy of the interventions (WHO, 2018b).
Survivorship analysis of the target species provides important data on the mortality, survival, and life expectancy of a vector species and has been important in formulating effective mosquito control strategies (Carey, 2001;Aida et al., 2011;Afrane et al., 2007;Carron et al., 2008).
The present study aimed to determine the survivorship of dengue vector immature stages and to compare insecticide resistance status between immature stages and adults collected from two different regions of Sri Lanka.Stagespecific expression of insecticide metabolizing enzymes and the presence of insensitive target site acetylcholinesterases were also examined in these two populations.

Mosquito collection
Aedes aegypti and Ae.albopictus eggs were collected weekly using 50 ovitraps, placed indoors in randomly selected 50 houses from Bandaranayakapura in Kurunegala district and from the Municipal Council area of Colombo district from January, 2023 to March, 2023 (Fig. 01).Study sites were residential areas where dengue incidence occurs year-round with monsoonal peaks.Larviciding using temephos sand granules, and fogging with pyrethroids have been in practice to control dengue transmission over the last 10 years in both these areas.However, exposure of mosquito populations to insecticides can differ probably due to the frequency of application, land-use pattens and other human activities.Herath et al.

Mosquito Rearing and survivorship experiments
Field-collected eggs were placed in separate white colour trays (32.5 x 26.5 x 6.5 cm) with 2 L of dechlorinated tap water under laboratory conditions (temperature = 25.6 o C ±1.9; relative humidity = 69.2±2.1; 12L:12D photoperiod).Once hatched, larvae were transferred into fresh trays with a 1.5 cm depth of water and fed with fish food (Prima fish food with 39% protein, Ceylon Grain Elevator, Sri Lanka) at a rate of approximately 0.1 g per 100 larvae per day.Pupae were transferred into adult emergence cages (30 cm × 30 cm × 30 cm), and 3-5-day-old adult female mosquitoes were used for experiments after species identification (WHO, 2022b).Daily observations were recorded from the larval emergence to pupal emergence.Larval life-table experiments were repeated three times for each species.For the pupal life-table tests, 50 newly emerged pupae were placed in mosquito cages and daily observations were made.Pupal experiments were also repeated three times per each species.
Tyres, clay pots and bamboo stumps were used as breeding habitats for semi-field experiments.They were placed in three separate sets, each set comprising one from each type.They were filled with dechlorinated water and left in the field for three days to simulate natural conditions.Laboratory-reared 1 st instar larvae from each species (50 larvae per container) were carefully placed in each habitat type and covered with a nylon mesh to prevent mosquito egg laying.All experimental settings were kept in semiopen locations under natural temperature and humidity, and half-shed lighting.Larval development was recorded daily and the larvae were not fed artificially.After the pupal emergence, observations were continued for the pupal study.

Larval Bioassays
To evaluate the efficacy of temephos (organophosphate), larval bioassays were carried out for three concentrations i.e., diagnostic dosage for temephos = 0.0125 ppm, x5 diagnostic dosage = 0.0625ppm, and x10 diagnostic dosage = 0.125ppm, following WHO recommendations and protocols (WHO, 2005;2016).Different concentrations were prepared by diluting the 3.125 ppm WHO stock solution (provided by the National Dengue Control Unit, Sri Lanka) with dechlorinated tap water.In each bioassay experiment, twenty 3 rd instar larvae were placed in 100 ml insecticide solution in 250 ml bioassay cups and each concentration was repeated five times.Control experiments were carried out using dechlorinated tap water alone.After a 1hr exposure period, dead larvae were counted and recorded.Mortality values were adjusted according to control mortalities using Abbott's formula (WHO, 2016;2022a).Test results were rejected if the control mortality exceeded 20%.

Adult Bioassays
Adult bioassays were performed using three-to five-day old female mosquitoes of Ae. aegypti and Ae.albopictus, by tarsal contact exposure to insecticide-impregnated papers in standard test kits according to WHO guidelines (WHO, 2016;2022c).Insecticide-impregnated papers and control papers were obtained from the Vector Control Research Unit, University of Sains Malaysia.Susceptibility levels to 0.03% deltamethrin (discriminating dosage of deltamethrine for both species), 0.05% lambdacyhalothrin (discriminating dosage for Ae.aegypti), 0.08% lambdacyhalothrin (discriminating dosage for Ae.albopictus), 0.04% permethrine (discriminate dosage for both species) and 1% malathion (discriminating dosage for Ae.aegypti), 5% malathion (discriminating dosage for Ae.albopictus) were evaluated.For each bioassay, a batch of 20 mosquitoes was exposed to the insecticide-treated paper for one hour and the mortalities were recorded after a 24hour recovery period.Each experiment was repeated five times (n=100).Control experiments were carried out using solvent alone control papers without insecticides.Mortality values were adjusted according to control mortalities using Abbott's formula (WHO, 2016;2022c).Test results were rejected if the control mortality exceeded 20%.

Biochemical assays
All the biochemical experiments were carried out according to the procedures outlined by WHO (1998).From each study site, 50 healthy 3 rd instar larvae and 50 healthy adult mosquitoes from each species were individually subjected to total protein, esterase, GST, monooxygenase and acetylcholinesterase (AChE) assays.Each mosquito sample was homogenized in 200µl ice-cold distilled water.An aliquot of 100 µl was taken for AChE assay and the rest was centrifuged at 13,000 rpm for two minutes.The supernatant was used for esterase, GST, monooxygenase and protein assays.

Acetylcholinesterase (AChE) assay
Two, 25 μl replicates from each mosquito homogenate were placed in adjacent wells of a microtitre plate.The membrane-bound AChE in the mosquito homogenate was solubilized by adding 145 μl of Triton phosphate buffer [1% (v/v) Triton X-100 in 0.1 M phosphate buffer pH 7.8] to each.To one set of homogenates, 25 μl of 0.01 M acetylthiocholine iodide (ASChI) and 10 μl of 0.1 M propoxur solution (2.5 ml 0.1 M ASChI + 10 μl of 0.1 M propoxur in acetone) were added.To the other replicate, 25 μl of ASChI alone was added.The kinetics of the enzyme reaction were continuously monitored at 405 nm for five minutes using a microplate reader (BioTek-800 TS Microplate reader).Results were expressed as a percentage of remaining activity in the inhibited fraction compared to the control (uninhibited) activity.

Esterase assay
Ten (10) μl of each homogenate was mixed with 200 μl of 1 mM p-nitrophenyl acetate (pNPA) working solution (100 mM pNPA in acetonitrile: 50 mM sodium phosphate buffer pH 7.4, 1:99) in a microtitre plate well.The reaction was read immediately at 405 nm for two minutes as a kinetic assay at 21°C.An extinction co-efficient of 6.53 mM-1 (corrected for a path length of 0.6 cm) was used to convert the absorbance values to moles of product.Esterase specific activity per individual was reported as μmol product min-1 mg-1 protein.
Glutathione S-transferase assay Ten (10) μl of each homogenate was mixed with 200 μl of reduced glutathione (GSH)/1-chloro-2,4 dinitrobenzene (CDNB) working solution [95 parts of 10 mM reduced glutathione (GSH) in 100 mM phosphate buffer pH 6.5 + 5 parts of 63 mM CDNB diluted in methanol] in a microtitre plate well.The reaction was read at 340 nm immediately as a kinetic assay for 5 minutes.An extinction coefficient of 5.76 mM -1 (corrected for a path length of 0.6 cm) was used to convert absorbance values to moles of product.GST specific activity was reported as CDNB conjugated μmol product min-1 mg-1 protein.

Monooxygenase assay
Twenty (20) μl of homogenate was mixed with 80 μl of potassium phosphate buffer pH 7.2 + 200 μl of 6.3 mM tetramethyl benzidine (TMBZ) working solution [(0.01 g TMBZ dissolved in 5 ml methanol and then in 15 ml of sodium acetate buffer pH 5.0) + 25 μl of 3% (v/v) H2O2 solution] in a microtitre plate well.After a two-hour incubation at room temperature, the plate was read at 630 nm as an end-point assay.This assay does not measure the monooxygenase activity, but titrates the amount of bound haem in the mosquito homogenate.Since haem is present in the active site of monooxygenases, major changes in the amount of monooxygenases produce a measurable increase in haem.By using a standard curve of cytochrome C (which also contains bound haem) a crude estimate of the amount of the monooxygenases present was obtained and expressed as equivalent units of cytochrome P450 per mg protein.

Protein assay
To obtain specific activities of enzymes, protein concentrations of the individual homogenates were determined by Bio-Rad protein determination.In a microtitre plate well, 10 μl of each homogenate was mixed with 300 μl of BIO-RAD working solution (prepared according to the manufacturer's instructions) and absorbance was read at 630 nm after a five-minute incubation at room temperature.

Statistical analysis
Mortality levels were defined as susceptible (>97% mortality), possible resistance (90-97% mortality), and resistant (<90% mortality) according to the WHO susceptibility criteria (WHO, 2016).Significance in the spatial variations in corrected percentage mortalities of Ae. aegypti and Ae.albopictus larvae and adults was evaluated using one-way ANOVA by Tukey's test for mean separation.Enzyme activity/quantity values of Ae. aegypti and Ae.albopictus populations were compared using oneway ANOVA.Differences were considered significant for P < 0.05.Tukey's posteriori test was applied to determine significant differences between individual means.All statistical tests were performed, using Minitab 20.0 program.
In life table experiments, larval and pupal survivorship was evaluated using Kaplan--Meier survival analysis (Goel et al., 2010) Minitab 20 version.Log-rank test was used to compare the survival curves between different breeding habitats and the comparisons were made using one-way ANOVA and Tukey's pair-wise tests.

Larval Bioassay
Larvae of both Ae.aegypti and Ae.albopictus from Kurunegala were completely susceptible (100% mortality) to all tested temephos concentrations (Table 1).Temephos discriminating concentration 0.0125 ppm (WHO, 2005) could kill only 34% Ae. aegypti and 54% Ae. albopictus larvae of Colombo populations.Even the 10x discriminating concentration could only kill 84% of Ae. aegypti and 91% Ae. albopictus showing that Colombo dengue vector larvae are highly resistant to the organophosphate temephos (Table 01).Results also indicate that Ae. aegypti larvae are more resistant to temephos than Ae.albopictus larvae.

Adult Bioassays
Adult bioassays were conducted by exposing a total of 1800 adult female mosquitoes (Ae.aegypti = 900 and Ae.albopictus = 900) from each site to six insecticidediscriminating dosages along with control experiments.Figure 02 displays the resistance profiles of both vector species from the two study areas.Both Ae. aegypti and Ae.albopictus from Kurunegala showed >98% mortality for all the insecticides tested and therefore, susceptible to deltamethrin, lambdacyhalothrin, permethrin and malathion, according to WHO classification (WHO, 2022).However, both species from Colombo showed less than 90% mortality rate for all pyrethroids tested indicating that they are resistant to pyrethroids.Aedes aegypti adults from Colombo showed less than 90% mortality for both the 1% (Ae.aegypti diagnostic concentration) and 5% (Ae.albopictus diagnostic concentration) malathion (Fig: 02) showing that they are resistant to malathion.Aedes albopictus adults were possible resistance to the 5% malathion diagnostic dosage specified for it and resistant to the 1% Ae. aegypti discriminating dosage (Fig: 02).

Biochemical assays
Activity profiles of AChEs, esterases, GSTs and the amounts of monooxygenases of larvae and adult of both vector species from two different locations are shown in Fig. 03.
Propoxur inhibited larval and adult AChEs from both vector species showed remaining activities of less than 33% indicating that the target site AChEs are largely sensitive.However, insensitivity was emerging in both vectors from Colombo.Aedes aegypti larvae from Kurunegala showed a significantly higher sensitivity for inhibition (lower remaining activity) than that of adults (Fig. 03).
Esterase activities from Colombo Ae. aegypti were significantly higher than that of Kurunegala Ae. aegypti and, were even greater than the discriminating activity of 0.25 μmol/mg/min specified for resistant Anopheles mosquitoes (Perera et al., 2008).Aedes albopictus adults from Colombo showed a significantly higher (p=0.021)esterase activity than that of their larvae (fig.03).Adults of both species had significantly higher GST activities than that of their larvae and these values were greater than the GST discriminating value of 0.40 μmol/mg/min given for resistant anophelines (Perera et al., 2008).Significantly higher amounts of monooxygenases were observed in Colombo Ae. aegypti and these values were above the monooxygenase discriminating amount of 0.35 equivalent units of cytochrome P450 per mg protein specified for resistant anophelines (Perera et al., 2008).Also, the adult monooxygenase levels were significantly higher than that of larvae in this population.

Life table analysis of dengue vector species
Survivorship of larval and pupal immature stages of both vector species under laboratory and semi-field conditions, obtained using Kaplan-Meier survival analysis, are shown in the Fig. 04.Aedes aegypti larval development time (5 ± 0.14 days) was significantly shorter than that of Ae. albopictus (9 ± 0.31 days) under laboratory conditions.Their respective survival probabilities were 80% and 76%.
Although the larval periods were significantly different among the different semi-field breeding habitats for Ae.aegypti (F=11.16,p=0.023) and Ae.albopictus (F=10.6,p=0.03), both species had comparable development periods in each semi-field type.Shortest larval periods were observed in bamboo stumps (5-5.2 days) and the highest were observed in clay pots (9.8-10 days) for both species.
Pupae to adult mean development times for both vector species were 3 days under laboratory conditions.It was significantly different (p=0.03)from 4 days observed for Ae.aegypti, under semi-field conditions.Aedes albopictus pupal development times were significantly different (p=0.023)among semi-field habitats i.e., bamboo stumps -2 days, tyres -3 days and clay pots -4 days.Pupal survival probabilities were 72% under laboratory conditions for both species.Under semi-field conditions, Ae. aegypti pupae had a 90% survival probability in clay pots, 68.4% in tyres and 45.2% in bamboo stumps whereas Ae. albopictus pupae had a survival probability of 90% for all three different habitats (Fig. 04).
The period from the first instar larva (egg hatching) to adult emergence is 8 days for Ae.aegypti and 13 days for Ae.albopictus under laboratory conditions.These figures for semi-field habitats were; bamboo stumps 7 and 7.2 days, tyres 10 and 11.3 days, and clay pots 14 and 13.8 days respectively for Ae.aegypti and Ae.albopictus.The survival rate from egg hatch to adult was 57.6% for Ae.aegypti and 54.72% for Ae.albopictus under laboratory conditions.These values were 32% for bamboo stumps, 38.4% and 28.8% for tyres, and 63% and 57.6% for clay pots respectively, for the two species.

DISCUSSION
Vector-borne diseases are a major cause of sickness worldwide and their prevention heavily depends on vector control (WHO, 2022a).Insecticides are the widely used effective tool in insect vector control and as a result of frequent and indiscriminate use of insecticides, resistance to insecticides has emerged among insect populations creating a major threat to insect control programmes worldwide (WHO, 2022a).Understanding the status of resistance in local vector populations to commonly used insecticides is crucial to ensure the success of insect vector control programmes.In Sri Lanka, organochlorine DDT was introduced in malaria control programmes in the late 1940s.Due to resistance development in mosquito vectors and environmental concerns, DDT was replaced with organophosphates for mosquito control in the health sector and with carbamates for insect pest control in the agricultural sector in 1975/77.Pyrethroids were introduced to mosquito control programmes in 1994 (Karunaratne, 1999).Pyrethroids and organophosphates are still widely used in mosquito control programmes (NDCU, 2022).
Both the bioassay and biochemical assays conducted during the present study revealed that Kurunegala dengue vector populations are susceptible to all tested pyrethroids and organophosphates.Colombo vector populations are resistant to both these groups of insecticides and have increased activities of esterases, GSTs and monooxygenases for insecticide detoxification.All these three enzyme groups are capable of detoxifying pyrethroids and organophosphates effectively (Karunaratne et al., 2018).Elevation of the activities of these enzymes above the discriminating levels given for Anopheles mosquitoes (Perera et al. 2008) It is clear from our study that the activities of esterases, GSTs and monooxygenases found in adults of both species were significantly higher than those found in their larvae.
To our knowledge, this has not been reported in mosquitoes previously.However, a similar scenario has been reported from the tea defoliator Hyposidra talaca (Geometridae: Lepidoptera).Expression of the same three detoxifying enzymes varied greatly in all five larval stages of Hyposidra talaca and increased with each successive stage suggesting that these enzymes are serving as a defensive mechanism to increasing insecticidal stress (Roy et al. 2021).When dengue vectors are considered, insecticidal pressure is higher on adults than on larvae, since insecticides are the major tool used for adult control.Larval control is largely attempted by clearing breeding habitats.Although temephos has been recommended as a larvicide for larval control, practically it is impossible to apply larvicides to all possible larval breeding habitats and the insecticidal pressure on larvae is comparatively low.Also, there can be an evolutionary significance as adults are more vulnerable to xenobiotic exposure since, they feed on plant juice and animal blood.
Development time and survivorship of various stages of mosquitoes under different environmental conditions are of particular importance, as they affect the vectorial capacity, which is tightly linked to mosquito-borne disease transmission (Garrett-Jone et al., 1964).In this study, we observed that the micro-environmental conditions had a significant effect on the larval growth period and survival rate of Ae. albopictus and Ae.aegypti.Under laboratory conditions, the larva to adult development period was 8 days, with a survival rate of 57% for Ae.aegypti and 13 days with 54% for Ae.albopictus indicating that, Ae. aegypti development is significantly faster.Similar to our results, Lopes et al. (2014) and Agnew et al. (2002) have shown that Ae. aegypti larval development period under laboratory conditions is 7 days.However, shorter periods (five days) of development had been achieved by rearing larvae individually in the laboratory, and longer periods (up to 10 days) had been observed with the larvae collectively reared outside the laboratory, a situation similar to our observation (Lopes et al., 2014;Almério et al., 1995;Yang et al., 2020).Our results show that the development period of dengue larvae from egg hatching to adult is 7 -7.2 days for bamboo stumps, 10 -11.3 days for tyres, and 13.8 -14 days for clay pots.Survival rates were 32% for bamboo stumps, 28.8% -38.4% for tyres, and 57.6% -63% for clay pots.Almério et al. (1995) have shown longer periods for Ae.albopictus egg hatching to adult emergence as 19.6, 27.3 and 37.5 days, respectively for a tree hole, a bamboo stump and an auto tyre.Variation in the rate of larval growth of Ae. aegypti has occurred according to the food types given to them (Hotchnink, 1985).The faster growth rate in the laboratory may be due to the more nutritious larval food they received under laboratory conditions.Differences in the rate of development and survivorship under semi-field conditions can be due to the availability of food and various other biotic and abiotic factors.

CONCLUSIONS
Dengue vector mosquitoes from Kurunegala were susceptible to all insecticides tested, whereas those from Colombo were resistant to the tested insecticides.Resistance of the Colombo population was mainly due to increased activities of insecticide metabolising enzymes.Results indicated that, Ae. aegypti larvae were more resistant to insecticides than Ae.albopictus larvae.A lso, adults were more resistant than larvae with well-developed resistant mechanisms.Development of Ae. aegypti is faster and their survival rate is also better than that of Ae. albopictus.

Figure 1 :
Figure 1: Map of Sri Lanka showing the study sites

Figure 4 .
Figure 4. Survivorship of larval and pupal immature stages of Aedes aegypti and Ae.albopictus in laboratory and semi field condition.A-Larval survivorship curves under laboratory conditions, B-Pupal survivorship curves under laboratory conditions, C-Larval survivorship curves for different containers, D -Pupal survivorship curves for different containers.

Table 01 :
Percentage mortality of Aedes aegypti and Ae.albopictus larvae exposed to different concentrations of temephos.Five batches of 20 third instar larvae of each species from each location were tested for each concentration.
Dalpadado et al. (2021)ficant contribution to organophosphates and pyrethroids.It is evident from our study that altered target site mechanism, with insensitive organophosphate target site AChE, is emerging in both Ae.aegypti and Ae.albopictus populations from Colombo.Fernando et al. (2018),Nugapola et al. (2021)andDalpadado et al. (2021)have reported the presence of kdr-type mutations in the voltage-gated sodium channel genes, producing insensitive target sites for pyrethroids.During the present study, it was noted that both the larvae and adults of Ae. aegypti populations are more resistant to insecticides than those of Ae. albopictus both in Kurunegala and Colombo.Therefore, the reason behind recommending higher discriminating dosages for Ae.albopictus than those for Ae.aegypti by the WHO (2016; 2022c) is questionable.