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Table of Contents
Year : 2022  |  Volume : 59  |  Issue : 3  |  Page : 293-297

Novel pyriproxyfen based treatment for Aedes breeding control through a long-lasting formulation: Laboratory and field trials in Western Maharashtra, India

1 Dept of Community Medicine, Armed Forces Medical College, Pune, India
2 Indian Council of Medical Research, New Delhi, India

Date of Submission05-Jun-2021
Date of Acceptance31-May-2022
Date of Web Publication08-Dec-2022

Correspondence Address:
Dr. Reema Mukherjee
Indian Council of Medical Research, V. Ramalingaswami Bhawan, P.O. Box No. 4911, Ansari Nagar, New Delhi - 110029
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.353253

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Background & objectives: There is a need to evaluate novel techniques for dengue control in India. Several formulations of pyriproxyfen have been assessed for efficacy and duration of action. Pyriproxyfen is also used as a microencapsulated ready-to-use formulation against the Aedes vector. We evaluated a novel pyriproxyfen-based microencapsulated formulation. This slow-release, ready-to-use aqueous spray is a larvicidal formulation, and we assessed its efficacy and residual action through laboratory and semi-field trials against Aedes immature stages.
Methods: The study was carried out as per the guidelines for laboratory and field/small-scale field testing of mosquito larvicides by the World Health Organization. The evaluation was conducted in laboratory and semi-field conditions from August to December 2018. We tested the novel formulation on three materials (plastic, ceramic, and enamel) in the laboratory for its action as an antilarval. Four containers of each kind were sprayed with the formulation and kept as replicates. Four controls were used in the laboratory trials - 120 larvae (third instar) were introduced in the replicates and the controls each. Readings were taken daily till complete adult emergence or larval and pupal mortality. In the semi-field trials, we applied this formulation to the inside of desert coolers and observed larvicidal and pupicidal activity over five months. Data is presented in numbers and percentages, along with mean and standard deviation. Adult emergence and Emergence Inhibition was calculated.
Results: There was 100% adult emergence inhibition amongst the exposed larvae in the treated containers in the laboratory trials. In the untreated controls, adult emergence ranged from 80–95% in all types of containers. In the semifield trials, Inhibition Emergence was 100% in the treated desert coolers during the five months of the study period.
Interpretation & conclusion: This advancement in insecticide formulation technology promises to make dengue control more effective and efficient.

Keywords: Dengue; Aedes; Pyriproxyfen; novel microencapsulated formulation; paint technology

How to cite this article:
Tilak R, Wankhede U, Mukherjee R. Novel pyriproxyfen based treatment for Aedes breeding control through a long-lasting formulation: Laboratory and field trials in Western Maharashtra, India. J Vector Borne Dis 2022;59:293-7

How to cite this URL:
Tilak R, Wankhede U, Mukherjee R. Novel pyriproxyfen based treatment for Aedes breeding control through a long-lasting formulation: Laboratory and field trials in Western Maharashtra, India. J Vector Borne Dis [serial online] 2022 [cited 2023 Feb 2];59:293-7. Available from: http://www.jvbd.org//text.asp?2022/59/3/293/353253

  Introduction Top

Prevention of dengue depends on effective Aedes mosquitoes control. World Health Organization (WHO) and India’s National Vector Borne Diseases Control Programme (NVBDCP) advocate Integrated Vector Management (IVM) strategy[1],[2]. The multipronged IVM strategy emphasizes judicious chemical methods, environmental management, and vector control. IVM is an effective, efficient, and sustainable approach[3]. However, there are several limitations and challenges to its successful implementation; hence, chemical vector control remains the primary tool for dengue prevention in many countries.

In a study on preferential breeding sites of Aedes in Delhi, India, immature stages were found primarily in plastic water storage containers (35%), coolers (35%) followed by cement tanks (10%), other solid waste (8%), overhead tanks (5%), unused plastic containers (3%), earthen and planted pots (2%). Irrespective of the season: winter, pre-monsoon, monsoon, or post-monsoon, desert coolers were the preferred breeding site[4] outbreaks of dengue fever/dengue hemorrhagic fever (DF/DHF). Larvicides are widely used to treat Ae. aegypti larval habitats. There are several registered active ingredients used as larvicides. Organophosphates, insect growth regulators (IGRs), and bacterial insecticides are the most commonly used and recommended by the WHO Pesticide Evaluation Scheme (WHOPES)[5]. Temephos is the most widely used larvicide for Aedes mosquitoes, though certain reports claim inconclusive efficacy[6]· Insect growth regulator (IGR) is an increasingly used alternative in vector control across the globe. Juveniles like pyriproxyfen prevent reproduction, egg-hatching, and molting of insects, including mosquitoes, fleas, cockroaches, and houseflies[8],[9],[10],[11],[12]. Several studies have found pyriproxyfen to be effective against Ae. aegypti[8],[9],[10],[11] and within the WHO recommended larvicides, the juvenile hormone mimic-pyriproxyfen has been established as an effective and safe active ingredient for controlling its immature stages. The WHOPES working group has testified the efficacy of pyriproxyfen granules with an expected duration of residual efficacy (≥80% adult emergence inhibition) of 14–19 weeks for the solid slow-release granules in the control of Aedes spp[13].

Modern microencapsulation technology allows the creation of formulations in which the active ingredients (AIs) are released so that they exert a long-lasting effect. This technology formulation has been evaluated with organophosphates and pyrethroids against malaria vectors applied to bed nets[7] and walls[8]. Insecticide paints lead to a high residual effect in laboratory and field trials with tset-seflies[9], sandflies[10] and mosquitoes[11]. Unlike conventional liquid insecticides, the paints comprise of microencapsulated insecticides incorporated in a coating formulation. Larva IGR™ (Inesfly Corporation S.L., Paiporta, Spain) is a long-lasting liquid larvicide based on paint technology. It contains microencapsulated pyriproxyfen at 0.2% presented in a trigger spray packing which is ready to use. This aqueous spray dispersion can be directly applied on any surface or container. After a 24–48 h drying period, the containers can be filled with water, and pyriproxyfen is slowly released into the medium, causing larval and pupal mortality and preventing adult emergence. The product can be used in any container that holds non-potable water.

In this study, we evaluated the efficacy and residual action of the larvicidal formulation Larva IGR™ through laboratory and semi-field trials against Aedes immature stages applied on different containers and desert coolers.

  Material & Methods Top

The study was carried out per the guidelines for laboratory and field/small-scale field testing of mosquito larvicides by WHO[12]. The evaluation was conducted in laboratory and semi-field conditions from August to December 2018.

Laboratory trials

Three container materials, viz., enamel, plastic and ceramic, were considered for laboratory evaluation, being the most commonly used container types in Indian settings. Four insecticide-treated bowls constituted replicates, and four untreated bowls were controls. The replicates were sprayed with the recommended dosage (15 m2/L) of Larval IGRTM and allowed to dry for 24–48 h [Figure 1]. After drying, containers were filled with dechlorinated water [Figure 2]. Thirty laboratory-reared third larval instars were introduced into each bowl and kept at 25–28°C with 12:12 h photoperiod. Larval food was added to each bowl at two days intervals. All containers were covered with netting to prevent adult escape. Readings were taken daily till complete adult emergence or larval and pupal mortality and recorded in a format as advocated by WHO[12].
Figure 1: Spraying of the containers for lab trials.

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Figure 2: Treated replicates for lab trials.

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Small-scale field trials

Before starting the trial, study areas were surveyed for Aedes density by landing catches. Areas with similar landing rates were included in the trial. Four treated and two untreated desert coolers were used for the field trials. Desert coolers were cleaned and sprayed with the larvicide formulation at the recommended dosage. Only the inside surfaces of the desert coolers that were in contact with water were sprayed [Figure 3]. After 24–48 h drying, the coolers were filled with water and operated regularly. Larvicidal and pupicidal activity were assessed over five months, from August to December 2018. All pupae were picked up daily from both the treated and the control coolers Before starting the trial, study areas were surveyed for Aedes density by landing catches. Areas with similar and transferred to separate vessels (filled with water taken from the respective deser coolers) and kept in the laboratory, covered with gauze to monitor adult emergence inhibition.
Figure 3: Spraying of inner surface of desert coolers.

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Ethical statement: Not applicable

  Results Top

Lab trials and small-scale field trials were carried out to evaluate the efficacy and duration of action of a novel microencapsulated aqueous-based insecticide formulation against Aedes aegypti. The small-scale field trials were carried out from August to December 2018; the experiment was stopped after that, as the vector density declined. The results from the laboratory and the small-scale trials are described below.

Laboratory trials

There was 100% adult emergence inhibition amongst the exposed larvae in the treated containers [Table 1]. In the untreated controls, adult emergence ranged from 80–95% in all types of containers. Of the 120 instars introduced in the plastic bowls, 92.5% died in the larval stage, and the remaining 7.5% died in the pupal stage. While in the ceramic and the enamel bowls, the lethal effect was almost equal in both immature stages, with 45% larvae and 55% pupae dying in the ceramic bowls and 48.3% larvae and 51.7% pupae dying in the enamel bowls.
Table 1: Inhibition emergence of Aedes aegypti from treated bowls with Larva IGR

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Small-scale field trials

Breeding was noticed from 5th day onwards in the controls, whereas the treated coolers showed no breeding up to 22 days into the experiment [Table 2]. Pupae collected from the control coolers and kept in the laboratory showed 50%–70% adult emergence. Inhibition Emergence was 100% in the treated coolers during the entire five months of the study period [Figure 4]. After that the trial was discontinued because of a decline in the larval density in the control coolers.
Figure 4: Adult emergence (%) and Inhibition emergence (%) of Aedes in desert coolers on treatment with Larva IGR IE=inhibition emergence.

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Table 2: Average monthly larval density in treated and control coolers during period of observation in semi-field trials

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  Discussion Top

While various pyriproxyfen formulations have been evaluated in the laboratory and the field against Aedes mosquito[13], its formulation in a ready-to-use spray with long-lasting efficacy has not yet been tested against Ae, aegypti. This is one of the first trials to assess the residual efficacy of novel liquid insecticidal microencapsulated formulations of pyriproxyfen against Aedes mosquitoes in laboratory and semi-field conditions.

In our laboratory trials, we observed that the product was highly effective against Ae, aegypti larvae and pupae, with no adult emergence when used on different materials (plastic, ceramic and enamel). In our semi-field trials, we tested this product on desert coolers with a metallic water holding tank. This finding has important implications as most artificial water containers are either plastic, metal, or made with cement. The results showed that this larvicidal formulation would be effective when used on all these surfaces as a long-lasting treatment.

Our laboratory assays reflected higher larval mortality than other studies conducted with a granulated formulation of pyriproxyfen. The formulation’s inert ingredients probably were responsible. This observation is reinforced by the delay in mosquito breeding in the treated coolers (23 days) in comparison to control ones (five days). Reduced larval density in treated coolers also points to the larvicidal effect of the tested product. The product showed complete efficacy in preventing adult emergence from coolers for five months under standard field operating conditions. These findings are similar to other studies on the efficacy of pyriproxyfen in slow-release plastic discs impregnated formulation[14]. However, this is probably one of the first few studies on pyriproxyfen’s efficacy in a ready-to-use spraying formulation against Aedes. Current practices of biological larvicides applied on coolers report only efficacy for up to three weeks[6]. A long-lasting formulation of temephos used on water storage prevented breeding for up to two months. Other non-chemical methods like screening of water tanks in coolers were also found to be helpful in the prevention of breeding[15]. However, it is essential to note that eliminating oviposition sites may divert gravid females to other cryptic breeding sites, hindering the efficacy of other vector control activities. Adult emergence in the field trials was inhibited in all the treated coolers during the entire study period of five months. Over extended periods, a loss or reduction in efficacy could probably be observed due to the depletion of the active ingredient and sublethal exposure to the larvae. However, such exposure may still cause the emerged adult Aedes mosquitoes to exhibit reduced reproductive capacity[16].

Aedes control in urban areas, especially in desert coolers and non-potable water containers, is a challenging issue. The availability of a ready-to-use long-lasting antilarval spray product offers several distinct advantages.

  Conclusion Top

The long-lasting efficacy of the slow-release ready-to-use pyriproxyfen spray formulation indicates its feasibility for mosquito larvae control in private households and on a community level. This advancement in insecticide formulation technology makes larvae control more effective and efficient and consumes less manpower and resources. However, large scale cluster randomized trials are required to assess the impact of the novel larvicide at station levels on Aedes populations and disease transmission.

Our study is one of the first to test the efficacy and duration of action of a novel insecticidal formulation against Aedes. The results are promising; however, we could only carry out limited field trials in one area, and we acknowledge this as a limitation of our study.

Conflict of interest: None

  Acknowledgements Top

The authors would like to acknowledge the office of the Director General Armed Forces Medical Services’ office for the funds made available for this research and Dr Vikram Taneja for the help rendered.

Key message

A novel pyriproxyfen-based formulation was evaluated for its efficacy in preventing Aedes breeding both in the laboratory and semi-field settings. This advancement in insecticide formulation technology promises to make dengue control more effective and efficient.

  References Top

Integrated Vector Management (IVM). World Health Organization 2021. Available from: http://www.who.int/neglected_diseases/vector_ecology/ivm_concept/en/ (Accessed on January 08, 2021).  Back to cited text no. 1
MOHFW vector control. Available from: https://mohfw.gov.in/sites/default/files/79767166351454408152_0.pdf (Accessed on September 13, 2019).  Back to cited text no. 2
Arunachalam N, Tyagi BK, Samuel M, Krishnamoorthi R, Manavalan R, Tewari SC, et al. Community-based control of Aedes aegypti by adoption of eco-health methods in Chennai City, India. Pathog Glob Health 2012; 106(8): 488–496.  Back to cited text no. 3
Patel S, Singh P, Prakash V, Sharma AK, Dhan S, Singh R, et al. A Study on the influence of climatic factors on the preferential breeding places of Aedes, the Dengue vector, in Delhi, India. Int J Mosq Res 2020; 7(4). 95–104.  Back to cited text no. 4
WHO, WHO Pesticide Evaluation Scheme (WHOPES). Available from: https://www.who.int/water_sanitation_health/gdwqrevision/whopes.pdf (Accessed on January 19, 2021).  Back to cited text no. 5
George L, Lenhart A, Toledo J, Lazaro A, Han WW, Velayudhan R, et al. Community-Effectiveness of Temephos for Dengue Vector Control: A Systematic Literature Review. PLOS Neglected Tropical Diseases 2015; 9(9). e0004006.  Back to cited text no. 6
Sahu SS, Vijayakumar T, Kalyanasundaram M, Subramanian S, Jambulingam P. Impact of lambdacyhalothrin capsule suspension treated bed nets on malaria in tribal villages of Malkangiri district, Orissa, India. Indian J Med Res 2008; 128(3)262–270.  Back to cited text no. 7
Tchicaya ES, Nsanzabana C, Smith TA, Donzé J, de Hipsl ML, Tano Y, et al. Micro-encapsulated pirimiphos-methyl shows high insecticidal efficacy and long residual activity against pyrethroid-resistant malaria vectors in central Côte d’Ivoire. Malaria Journal 2014; 13(1): 332.  Back to cited text no. 8
Acapovi-Yao G, Kaba D, Allou K, Zoh DD, Tongué LK, N’Goran KE. Assessment of the Efficiency of Insecticide Paint and Impregnated Nets on Tsetse Populations. Preliminary Study in Forest Relics of Abidjan, Côte d’Ivoire. West African Journal of Applied Ecology 2014; 22(1). 17–25.  Back to cited text no. 9
Banjara MR, Das ML, Gurung CK, Singh VK, Joshi AB, Matlashewski G, et al. Integrating Case Detection of Visceral Leishmaniasis and Other Febrile Illness with Vector Control in the Post-Elimination Phase in Nepal. Am J Trop Med Hyg 2019; 100(1): 108–114.  Back to cited text no. 10
Mosqueira B, Soma DD, Namountougou M, Poda S, Diabaté A, Ali O, et al. Pilot study on the combination of an organophosphate-based insecticide paint and pyrethroid-treated long lasting nets against pyrethroid resistant malaria vectors in Burkina Faso. Acta Trop 2015; 148: 162–169.  Back to cited text no. 11
World Health Organization. (2005). Guidelines for laboratory and field testing of mosquito larvicides. World Health Organization. https://apps.who.int/iris/handle/10665/69101  Back to cited text no. 12
Vythilingam I, Hanni R, Bengi S. Laboratory and field evaluation of the insect growth regulator pyriproxyfen (sumilarv 0.5g) against dengue vectors. J Am Mosq Control Assoc 2005; 21(3): 296–300.  Back to cited text no. 13
Sai Zaw Min Oo, Sein Thaung, Yan Naung Maung Maung, Khin Myo Aye, Zar Zar Aung, Hlaing Myat Thu, et al. Effectiveness of a novel long-lasting pyriproxyfen larvicide (SumiLarv®2MR) against Aedes mosquitoes in schools in Yangon, Myanmar. Parasites & Vectors 2018.  Back to cited text no. 14
Singh A, Mukherjee R, Kotwal A, Rajiva R. Effectiveness of evaporative desert cooler mounted with fabricated metal mesh in preventing Aedes mosquito breeding in a North Indian City. Journal of Marine Medical Society 2018; 20: 34.  Back to cited text no. 15
Kamal HA, Khater EI. The biological effects of the insect growth regulators; pyriproxyfen and diflubenzuron on the mosquito Aedes aegypti. J Egypt Soc Parasitol 2010; 40(3): 565–74.  Back to cited text no. 16


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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