Development of Deltamethrin-Laced Attractive Toxic Sugar Bait to Control Aedes aegypti (Linnaeus) Population

Background The attractive toxic sugar bait (ATSB) is a promising strategy for controlling mosquitoes at the adult stage. The strategy is based on the use of a combination of fruit juice, sugar, and a toxin in order to attract and kill the adult mosquitoes. The selection of the components and optimization of their concentrations is significant for the formulation of an effective ATSB. Methods The present study formulated nine ATSBs and evaluated their efficacy against two laboratory strains (AND-Aedes aegypti and AND-Aedes aegypti-DL10) and two wildcaught colonized strains of Aedes aegypti (GVD-Delhi and SHD-Delhi). Initially, nine attractive sugar baits (ASBs) were prepared using a mixture of 100% fermented guava juice (attractant) with 10% sucrose solution (w/v) in 1 : 1 ratio. ATSBs were formulated by mixing each ASB with different concentrations of deltamethrin in the ratio of 9 : 1 to obtain final deltamethrin concentration of 0.003125–0.8 mg/10 mL ATSB. Cage bioassays were conducted with 50 mosquitoes for 24 h in order to evaluate the efficacy of each ATSB against the four strains of Ae. aegypti. The data were statistically analyzed using PASW software 19.0 program and 2-way ANOVA. Results The ATSB formulations registered 8.33–97.44% mortality against AND-Aedes aegypti and 5.15–96.91% mortality against AND-Aedes aegypti-DL10 strains of Ae. aegypti, while GVD-Delhi strain registered 2.04–95.83% mortality and SHD-Delhi strain showed 5.10–97.96% mortality. The administration of 0.8 mg of deltamethrin within 10 mL of attractive toxic sugar bait (ATSB) has led to the maximum mortality rate in adult mosquitoes. Conclusions The ATSBs formulated with guava juice-ASB and deltamethrin (9 : 1) showed toxin dose-dependent toxicity by all the four strains of Ae. aegypti. Most effective dosage was found as 0.8 mg deltamethrin/10 mL ATSB which imparted 96% to 98% mortality in adult mosquitoes. The investigations demonstrated the efficacy of deltamethrin-laced ATSB formulations against Ae. aegypti and highlighted the need for conduct of structured field trials and investigating the impact on disease vectors and nontarget organisms.


Introduction
Aedes aegypti (Linnaeus) and Ae.albopictus are vectors of global importance for transmission of arboviruses, such as dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV), while Ae.vittatus is reported from few SEAR countries [1].Lack of efective therapy and vaccination against these arboviruses, except for YFV, has increased the arboviral disease burden worldwide.In addition, emergence and re-emergence of these arboviruses has increased the disease prevalence.Among these, dengue has emerged as one of the fast-spreading diseases with approximately 100-400 million DENV infections/year [2].Te disease is reportedly endemic in more than 100 countries in the 5 WHO Regions with 3.5 billion people at risk of contracting dengue fever and 1.3 billion people living in dengue-endemic areas in 10 countries of South-East Asia Region (SEAR), except in DPR Korea.
In the absence of efective medication and vaccines, Aedes-borne diseases are presently managed by vector control [2].Of the diferent mechanical, biological, and chemical methods in use, Aedes control is still reliant on the chemical-based interventions [3].Unfortunately, the continued use of these chemicals over a long period of time has caused evolution of insecticide resistance in diferent mosquito species, including in Aedes spp.[4,5] that retard the disease control.
Use of attractive toxic sugar baits (ATSB) is a relatively new and efective strategy that consists of three components: sugar as a phago-stimulant, a toxin, and an odorant for alluring feeding [3,6,7].Te mosquitoes are attracted towards the bait because of the odour of fruit/fower juice and are killed on ingesting the toxin [8].ATSBs, initially formulated as toxic sugar baits (TSBs) without odorant, were unable to attract mosquitoes due to the absence of an attractant [6].Tus, fermented fruit/fower juices, with potential to lure mosquitoes by the production of attractive volatiles, were added in the formulations as attractants to formulate ATSBs [9,10].Use of ATSBs is contemplated as a reliable control method for both male and female mosquitoes as both quest for sugar sources outdoors.Various fruit juices have been assessed for their attractant potential against mosquitoes, such as guava, banana, mango, orange, tomato, watermelon, and papaya juice against Anopheles arabiensis [11], mango and guava juice against Ae.albopictus [12], guava juice against An.gambiae [13], and guava, mango, muskmelon, orange, papaya, pineapple, plum, sweet lemon, and watermelon juice against Ae.aegypti and An.stephensi [14,15].
ATSB formulations can be applied at the target site in a variety of forms, such as in traps, as toxin-incorporated phago-stimulants in bait stations, as baits at the entrance of storm drain system, or by spraying on plants [6,9,10,17].In addition, researchers have also demonstrated that use of ATSBs in wild has shown minimal harmful impact on nontarget arthropods including the benefcial ones [9,10,23,24].Tis novel vector control tool could be used efectively in attracting and killing a large number of mosquitoes, demonstrating its efectiveness in both indoor and outdoor environments.Despite its initial success, it is imperative to standardize the process to ensure its seamless integration and efectiveness at a larger scale.
Current study is a step in this direction with nine ATSB formulations containing cane sugar (10% sucrose solution in water as a phago-stimulant), fermented guava juice (attractant), and graded concentration of deltamethrin (toxin).In this study, guava juice-ASB was selected based on our laboratory studies that showed its superior attractancy than the other eight juices prepared from the locally available fruits [14].Te objective of the study was to assess the laboratory efcacy of nine ATSB formulations containing diferent concentrations of deltamethrin against two laboratory strains (AND-Aedes aegypti and AND-Aedes aegypti-DL10) and two wild-caught laboratory colonized strains (GVD-Delhi) and (SHD-Delhi) of Aedes aegypti to identify the most efective ATSB formulation.

Rearing and Maintenance of Aedes aegypti Mosquitoes in
Insectary.Te cyclic colonies of Ae. aegypti mosquito strains were reared and cultured in the insectary in Insect Pest and Vector Control Laboratory at the Acharya Narendra Dev College, University of Delhi, India, since 2009.Te rearing conditions are maintained at regulated temperature (27 ± 2 °C), relative humidity (80 ± 10%), and photo-period regime (14L:10D).Adults are reared in cotton cloth fabric cages (45 × 40 × 40 cm) and fed on 10% sucrose solution soaked in a cotton swab kept on the cage roof.For egg maturation, female adults are provided with blood meals from an albino mouse.Laid eggs are collected in an ovicup placed in the cage.Te eggs are hatched and cultured in plastic trays half-flled with dechlorinated water and provided a mixture of fnely ground dog biscuits and yeast 3 : 2 (w/w) for larval nutrition.Te pupae are held in the mosquito cage for emergence to adults.

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Journal of Tropical Medicine

Strains of Aedes aegypti Employed in Study.
Two laboratory strains and two wild-caught colonized strains were used for the studies (Table 1).

Laboratory Strains
(a) Insecticide susceptible strain of Ae. aegypti (AND-Aedes aegypti): the strain was procured in 2009 from ICGEB (International Centre for Genetic Engineering and Biotechnology), New Delhi, India, and established in the laboratory.Since then, it is being maintained without any insecticide selection pressure.(b) Deltamethrin larval-selected strain of Ae. aegypti (AND-Aedes aegypti-DL10): the early fourth instars of the strain procured from ICGEB in 2009 were subjected to deltamethrin selection pressure at the LC 90 level for 10 successive generations.Te deltamethrin susceptibility status of F 10 was computed, and thereafter, each generation is being selected with the computed LC 90 value of deltamethrin in order to prevent variations in the deltamethrin susceptibility levels.Te adults of the strain, however, were susceptible to deltamethrin as they developed only 1.07fold deltamethrin resistance.1).Te LT 90 values and resistance ratios in these strains, when compared to susceptible AND-Aedes aegypti strain, decreased in the order of Aedes aegypti (GVD-Delhi) [1.8] > Aedes aegypti (SHD-Delhi) [1.68] > AND-Aedes aegypti-DL10 [1.12] > AND-Aedes aegypti [1.0] (Table 1).

Formulation of Attractive Toxic Sugar Baits (ATSBs).
Te guava juice-ASB was prepared by mixing fermented pure juice of guava and 10% sucrose solution (w/v) in 1 : 1 ratio [14].Deltamethrin was serially diluted in ethanol to obtain the concentrations in range of 0.03125 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 4.0 mg/mL, and 8.0 mg/mL.Te ATSB solutions were prepared by mixing 9 mL of guava juice-ASB with 1 mL of deltamethrin solution at a particular concentration (9 : 1 ratio), resulting in the nine ATSB formulations containing deltamethrin in the range of 0.003125-0.8mg/10 mL ATSB.

Cage Bioassays with ATSBs.
Te bioassay with each of the nine ATSB formulations was conducted in individual cloth cages (45 × 40 × 40 cm).A total of eighteen (nine controls and nine experimental) cotton discs (5 cm diameter, 1.5 cm thickness, 0.5 g) were prepared.Te cotton discs were soaked with 5 mL of 10% sucrose solution in water (w/v), and the experimental cotton discs were soaked with 5 mL of nine ( 9) diferent deltamethrin concentration ATSB formulations.
Te assay was conducted in cloth cages of the dimensions (45 × 40 × 40 cm).Earlier studies have shown the high attractant and non-toxic properties of guava juice-ASB [14], and the studies with ATSBs have shown their toxic efects against mosquitoes feeding upon them (unpublished data).To assess the efcacy of an ATSB in a controlled environment, one control disc and one experimental disc of given deltamethrin-ATSB concentration were placed on the two sides of a cage (Figure 1(a)).In each cage, 2-3 days old and unfed ffty Ae. aegypti adults, 25 females and 25 males, were released for 24 h (Figure 1(b)).After 24 h exposure, a number of mosquitoes, landed on ATSB and either knocked down (unable to fy but alive) or dead, were recorded and analysed.Four (4) replicates were tested for each concentration of ATSB.Tus, for each strain, a total of thirty-six cages were set-up for ATSB bioassays (9 doses × 4 replicates).Concurrently, control assays were held with attractive sugar baits (ASB) containing fermented pure juice of guava and 10% sucrose solution (w/v) in 1 : 1 ratio.

Statistical Analysis.
Te percent mortality in adults was calculated in each bioassay by using the following formula (equation ( 1)): percent mortality (%) � total number of dead adults total number of exposed adults × 100. (1)

Journal of Tropical Medicine
Te control bioassays resulting in >20% mortality were discarded, and experiments with control mortality in the range of 5-20% were corrected using Abbott's formula given in the following equation (2) [25]: where T is the percent mortality of Ae. aegypti on the guava juice-deltamethrin-ATSB and C is the percent control mortality of Ae. aegypti.Te mortality data were analyzed and interpreted using one-way ANOVA and Tukey's all pairwise multiple comparison test using Predictive Analytics Soft Ware (PASW) 19.0 program.

Results
Te cage bioassays were carried out with ATSB prepared with guava juice-ASB and deltamethrin insecticide (9 : 1) using nine doses of deltamethrin in the range of 0.003125-0.8mg/10 mL ATSB.Each strain showed a dosedependent mortality response to deltamethrin-ATSBs with respect to the deltamethrin dosage in the ATSB.After 24 hours of assay, the % mortality in AND-Aedes aegypti strain on the ATSBs ranged from 8.33% to 97.44%, while the % mortality in AND-Aedes aegypti-DL10 adults was in the range of 5.15% to 96.91% (Table 2).No mortality was recorded in the mosquitoes fed on ASB.
In comparison, relatively lower mortality was recorded in the adults of deltamethrin larval-selected AND-Aedes aegypti-DL10 strain (Table 2).Using 0.0625 mg/10 mL and 0.0125 mg/10 mL deltamethrin-ATSB as baits caused 10.53% and 17.71% adult deaths (P < 0.05) which enhanced to    2), with >80% mortality obtained with 0.8 mg deltamethrin/ 10 mL ATSB.Te attract and kill potential of nine ATSBs containing 0.003125 to 0.8 mg deltamethrin/10 mL ATSB was also investigated against two wild-caught strains of Ae. aegypti, the Aedes aegypti (GVD-Delhi), and Aedes aegypti (SHD-Delhi) strains which were colonized in the laboratory (Table 3).During 24 h exposure, the ATSB formulations induced 2.04% to 95.83% mortality in GVD-Delhi strain, while comparatively higher mortality of 5.10% to 97.96% was observed in the SHD-Delhi strain of Ae. aegypti.Te lowest adult mortality rates of 2.04% and 5.10% in Aedes aegypti (GVD-Delhi) and Aedes aegypti (SHD-Delhi), respectively, were observed with ATSB containing the lowest dose of deltamethrin (0.003125 mg/10 mL ATSB).Te increase in the concentrations of deltamethrin in the ATSB formulations increased the adult mortality in both the strains indicating a dose-mortality response correlation.
Te dose mortality response lines obtained on providing nine deltamethrin-ATSBs to AND-Aedes aegypti and AND-Aedes aegypti-DL10 showed R 2 values of 0.7983 and 0.8012, respectively (Figures 3(a

Discussion
Te ATSB is a mixture of three components; fruit juice, toxin, and sugar solution; to attract for feeding and kill the adults by toxin feed.It is based on the fact that mosquitoes require a sugar diet throughout their life for energy, growth, development, mating, and egg production [8,26].Since mosquitoes search for sugar sources in the environment, the ATSB with table sugar competes with the available sources of plant sugar and provides nourishment for survival [6].
Te frst toxic sugar bait (TSB) was developed against Ae.aegypti using malathion and 20% sucrose solution combination [16].Malathion was added to sucrose in diferent concentrations (1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, and 0.1 mg/mL) resulting in up to 85.2% adult mortality.However, TSBs, though efective in laboratory evaluation, could not register comparable mortalities in the feld due to the presence of the competing environment's natural sugar sources and attractants.Consequently, addition of odour attractants, in the form of fruit juices, fower nectar, or bug honeydew, resulted in the formulation of ATSBs [9,10,27].Te laboratory or feld trials with diferent ATSBs have showed varied efcacy which may be not only because of the toxin used but also due to the attractant used, type and prevalence of mosquito species, level of resistance in the mosquitoes to the toxin, and ecological factors.Tereafter, several toxins have been used in the ATSB formulations such as deltamethrin, boric acid, dinotefuran and spinosad
Earlier studies with nine ASBs prepared by combining nine diferent fermented pure fruits juices with 10% sucrose solution in water in 1 : 1 ratio revealed guava juice-ASB as the most efective attractant for Ae.aegypti [14].Te present study is to validate these laboratory results on wild-caught laboratory colonized Ae. aegypti strains with nine ATSBs formulated by adding 9 parts of guava juice-ASB to 1 part of various dosages of a contact pyrethroid insecticide, deltamethrin, in the range of 0.003125 to 0.8 mg/10 mL ATSB.Tese ATSBs were evaluated for their toxic potential against the two laboratory strains (AND-Aedes aegypti and AND-Aedes aegypti-DL10) and two wild-caught laboratory colonized strains (Aedes aegypti (GVD-Delhi) and Aedes aegypti (SHD-Delhi)) of Ae. aegypti.Te studies revealed a dosedependent mortality response in adult Ae. aegypti of ATSBs after 24 h exposure.Te recorded mortality in AND-Aedes aegypti and AND-Aedes aegypti-DL10 ranged from 8.33 to 97.44% and 5.15-96.91%,respectively, whereas these formulations induced relatively less mortality in the laboratory colonized wild-caught deltamethrin susceptible GVD-Delhi strain (2.04-95.83%)and SHD-Delhi strain (5.10-97.96%).Te formulations with 0.4 mg deltamethrin/10 mL ATSB caused >80% mortality in the adults of AND-Aedes aegypti, while rest of the three strains registered >80% mortality in the adults with 0.8 mg deltamethrin/10 mL ATSB.
Te observed mortality in cage bioassays was found to be correlated with the LT 50 values of deltamethrin obtained against these strains.Increased adult mortality was observed in the AND-Aedes aegypti strain (LT 50 = 4.431 min) followed by AND-Aedes aegypti-DL10 (LT 50 = 4.766 min) and Aedes aegypti (SHD-Delhi) (LT 50 = 8.382 min), while Aedes aegypti (GVD-Delhi) (LT 50 = 8.787 min) showed lowest mortality relative to the other strains.Tese results are encouraging and need to be validated with the feld studies as to date, most of the research in the feld of ATSB has been carried out with oral toxins: dinotefuran, spinosad, chlorfenapyr, and boric acid.Contact insecticides-TSBs, though investigated, have been in limited focus.
A few studies have assessed ATSBs formulated using diferent pyrethroids such as deltamethrin, permethrin, cyfuthrin, and bifenthrin and found them efective against diferent species of mosquitoes, Cx. quinquefasciatus, An. quadrimaculatus, An. stephensi, Ae. aegypti, and Ae.taeniorhynchus [15,[19][20][21].It was reported that formulations were generally more efective against pyrethroid-susceptible populations than the pyrethroid-resistant populations [21].Tus, ATSBs containing insecticides with modes of action diferent from that of pyrethroids were formulated and found efective against pyrethroid-resistant mosquitoes [13].It has been thus recommended that use of insecticides with an alternate mode of action to the existing insecticide in use should be preferred as a strategy for efective vector management [1].
Current laboratory investigations revealed the efcacy of deltamethrin as a toxin component in the ATSB to control Ae. aegypti population.Te dosage of 0.8 mg deltamethrin in 10 mL ATSB was found to be highly efective resulting in 96% to 98% mortality in adult mosquitoes.However, reports have suggested the reduced efcacy of ATSBs in the felds in comparison to the laboratory assays probably due to the development of resistant strains [13,35].Moreover, it can be due to the availability of natural sugar sources in the natural environment which compete with bait stations.
Based on the encouraging results from this study, it is pertinent to assess the efcacy of the developed deltamethrin-ATSB formulation for use in the feld against wild mosquitoes.Further, supplementary studies are recommended on the impact of ATSBs on the environment and non-target organisms, which would help in ascertaining the safe use of ATSBs.

Conclusions
Te study was conducted using nine ATSB formulations with fermented guava juice (100%), sucrose solution (10% w/v), and nine doses of pyrethroid deltamethrin (0.003125-0.8mg/10 mL ATSB) to assess their toxic potential against two laboratory strains and two wild-caught colonized strains of Ae. aegypti.Te studies revealed a deltamethrin dose-dependent impact of ATSBs on the mortality in adult mosquitoes.Te recorded mortality in laboratory strains, AND-Aedes aegypti and adults derived from AND-Aedes aegypti-DL10 strain selected with deltamethrin at the larval stage for 10 generations, ranged from 8.33-97.44% to 5.15-96.91%,respectively, whereas these formulations induced 2.04-95.83%and 5.10-97.96%mortality in laboratory colonized wild-caught GVD-Delhi and SHD-Delhi strains of Ae. aegypti, respectively.Te investigations indicating a positive correlation between the % mortality in the adults and the deltamethrin susceptibility demonstrated the efcacy of these ATSB formulations against Ae.aegypti with deltamethrin.Tis study highlighted the need to conduct structured feld trials and investigation of the impact on non-target organisms.
Aedes aegypti strains are presented in Table 1.Te adult strains were completely susceptible to deltamethrin.Te LT 50 values were in the range of 4.43 to 8.78 min and LT 90 values in the range of 8.85 to 15.99 min (Table Colonized Strains.Te wild strains have been maintained in the laboratory since May-June 2021.Since then, approximately 42 generations of these wild strains have been passed.(a)Govindpuri strain of Ae. aegypti (GVD-Delhi): larvae were collected in June, 2021, from the Govindpuri locality of the Southeast Delhi, India, (28.534 °N, 77.265 °E) and maintained at Acharya Narendra Dev College, India, without insecticide selection pressure.(b)Shahdara strain of Ae. aegypti (SHD-Delhi): larvae were collected in May, 2021, from the Shahdara locality of the East Delhi, India, (28.689 °N, 77.290 °E) and maintained at Acharya Narendra Dev College, India, without insecticide selection pressure.Te adult susceptibility data to deltamethrin of the four

Table 1 :
Deltamethrin susceptibility status of the four strains of Aedes aegypti: laboratory strains (AND-Aedes aegypti and AND-Aedes aegypti-DL10) and wild-caught colonized strains (GVD-Delhi and SHD-Delhi) of Aedes aegypti after 24 h exposure to 0.05% deltamethrin-impregnated papers.

Table 2 :
Number of adults of laboratory strain (AND-Aedes aegypti and AND-Aedes aegypti-DL10) of Aedes aegypti attracted and killed in ATSB cage bioassays.Four replicates each with n � 50, 25 males and 25 females (24 h), total n � 200.* * Corrected percent mortality; values in the table represent the number of mosquitoes dead; ATSBs with diferent letters (column-wise) are signifcantly diferent (P < 0.05) computed by one-way ANOVA followed by Tukey's all pair wise multiple comparison test. *

Table 3 :
Number of adults of wild-caught colonized population of Aedes aegypti (GVD-Delhi) and Aedes aegypti (SHD-Delhi) strains of Aedes aegypti attracted and killed towards ATSB formulation during ATSB cage bioassays.ATSB (guava juice-ASB + mg deltamethrin/10 mL)No. of dead adults ± SE (mortality) Aedes aegypti (GVD-Delhi) * Four replicates each with n � 50, 25 males and 25 females (24 h), total n � 200.* * Corrected percent mortality; values in the table represent the number of mosquitoes dead; ATSBs with diferent letters (column-wise) are signifcantly diferent (P < 0.05) computed by one-way ANOVA followed by Tukey's all pair wise multiple comparison test. *