A novel non-contact bioassay method for quantitative evaluation of vapour phase toxicity of insecticides against mosquitoes

Accepted Manuscript A novel non-contact bioassay method for quantitative evaluation of vapour phase toxicity of insecticides against mosquitoes

Manas Sarkar, Ambadas Akulwad, Rajendra Kshirsagar PII: DOI: Reference:

S1226-8615(18)30423-0 doi:10.1016/j.aspen.2018.10.006 ASPEN 1265

To appear in:

Journal of Asia-Pacific Entomology

Received date: Revised date: Accepted date:

14 June 2018 27 September 2018 18 October 2018

Please cite this article as: Manas Sarkar, Ambadas Akulwad, Rajendra Kshirsagar , A novel non-contact bioassay method for quantitative evaluation of vapour phase toxicity of insecticides against mosquitoes. Aspen (2018), doi:10.1016/j.aspen.2018.10.006

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ACCEPTED MANUSCRIPT A novel non-contact bioassay method for quantitative evaluation of vapour phase toxicity of insecticides against mosquitoes Manas Sarkar*, Ambadas Akulwad, Rajendra Kshirsagar

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Eastern Express Highway, Vikhroli East, Mumbai 400079, India

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Research and Development Division, Godrej Consumer Products Ltd., Godrej ONE Building,

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Manas Sarkar, PhD Global Research and Development Division,

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Godrej Consumer Products Ltd.,

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Godrej ONE Building, Eastern Express Highway, Vikhroli East, Mumbai 400079, India Phone: +91-9920166889

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E-mail: [email protected]

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Running Title: A novel method for vapour toxicity

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*Corresponding author

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ACCEPTED MANUSCRIPT Abstract The recent advancement in new generation fluorinated pyrethroids (e.g., transfluthrin, metofluthrin etc.), the use of semi-volatile vapour phase insecticides for control of mosquitoes and other domestic pests rises. Enabling the examination of the vapour toxicity

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profiles of these molecules and many other similar new generation molecules will provide new avenues for researchers for understanding the bio-potency in the spatial killing of pests.

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Hence, it is critical to establish a well-controlled portable vapour-phase bioassay method that

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can provide the desired precision, accuracy, linearity and robustness. In this respect, we have designed a vapour-toxicity apparatus comprising glass assemblies and developed a novel

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bioassay method. We found that KT50 and percentage knockdown at 60 min reflect the

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concentration dependency. This validates and confirms that the method is sensitive enough to distinguish between concentrations and suitable for concentration-response experiments. We

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found that KT50 and percentage knockdown at 60 min at a given concentration does not differ significantly between experiments. Hence, the method has repeatability and precision.

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Percentage mortality and total KT50 against Culex quinquefasciatus shows that percentage mortality increases and KT50 decreases linearly with the increasing concentration. This

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method provides an easy to operate tool to test the vapour toxicity profiles of any vapour phase insecticide molecules against mosquitoes and flying insects.

Key Words Vapour

toxicity;

semi-volatile

insecticides;

transfluthrin; metofluthrin;

Introduction 2

non-contact

bioassay;

spatial

repellence;

ACCEPTED MANUSCRIPT Insect pests are major vectors of human infectious diseases that impose massive life losses and cost (Sarkar et al., 2015, 2009) . Chemical insecticides are the principal material of choice to control such pests. While insecticides are potent in the killing pests, long-term efficacy is often challenged by the evolution and spread of resistance to different types of insecticides (Kumar et al., 2011; Liu, 2015; Paine and Brooke, 2016). Innovative Vector

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Control Consortium (IVCC) is working together with several chemical companies to tackle

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the problem of insecticide resistance and develop new tools and insecticide molecules to

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ensure the efficacy of vector control. Some of these new generation insecticides require new, robust and simpler method for evaluation.

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Majority of the insecticides are nonvolatile organic molecule except fumigants. Volatile insecticides in the form of fumigant have long had applications in the protection of

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agricultural crops, stored products and commodities, as well as in the control and management of structural pests (William and Brown, 1951). But, with recent advancement in

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new generation fluorinated pyrethroids (polyfluorobenzyl insecticides), the use of these new

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generation semi-volatile insecticides for managing mosquitoes and other domestic pests is ever increasing especially in households (Denloye et al., 2017; Kuri-Morales et al., 2018; Mori, 2017). The most common semi-volatile polyfluorobenzyl insecticides are transfluthrin

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and metofluthrin. These molecules are frequently used in household insecticide products like liquid vaporizers, coils, aerosol and sprays. They are also described as vapour-phase insecticides (Killeen et al.,

2017).

The fast-acting effect of these polyfluorobenzyl

insecticides is predominantly due to the semi-volatile nature of the compounds, where the vapour of the compounds moves quickly through insect’s tracheae, resulting in a quick knockdown. This contrasts with the action of malathion or other non-volatile insecticides, where contact poisoning is the mode of entry. These vapour phase insecticides are also being

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ACCEPTED MANUSCRIPT tested for potential use for public health (Andrés et al., 2015; Horstmann et al., 2014; Killeen et al., 2017; Masalu et al., 2017; Ogoma et al., 2017). There are several guidelines and biological assay procedures for assessing the efficacy of different insecticide and spatial repellent molecules and products (Horstmann and Sonneck, 2016; Paramasivam, M.; Selvi, 2017; Scharf et al., 2007; WHO/Department of

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Communicable Disease Prevention, 1996; World Health Organization (WHO), 2009, 2006).

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Majority of these methods are based on either tarsal contact knockdown/mortality assay or

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spatial repellent assay. However, the mode of action of new generation polyfluorobenzyle insecticide is through vapour toxicity/spatial killing. Hence, it is important to effectively

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determine the non-contact (i.e., vapour) toxic potency of these semi-volatile insecticides against flying insects, both for development of new molecules as well as to test the efficacy

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of existing molecules. Most commonly, the spatial killing efficacy of these insecticides is evaluated by Peet Grady chamber as demonstrated by World Health Organization (WHO)

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(World Health Organization (WHO), 2009). However, Peet Grady is largely used to test

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formulated products, seldom for the active molecule. One needs a delivery format (product) to vaporize insecticide molecule in the Peet Grady chamber. There is another patented bioassay method (Scharf et al., 2007) is available for spatial killing or vapour phase bio-

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response, which is not much in use. There is no small-scale laboratory level bioassay, to measure vapour toxicity against flying insect, is available. Therefore, an efficient, reliable and easy to perform vapour-phase bioassay system could facilitate the development of novel insecticide molecules and enable screening of existing vapour-phase insecticide molecules. Here, in this report, we demonstrate a new comprehensive and controlled bio-toxicity assay system to evaluate the insect’s biological response to a volatile insecticide at vapour phase.

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ACCEPTED MANUSCRIPT Materials and Methods Insects and Chemicals Culex quinquefasciatus used in this bioassay were sampled from the permanent colony of Godrej Consumer Products Ltd., Mumbai. The mosquitoes were fed with 10% sugar solution

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from the day of emergence to the day of testing and reared at 27°C and 60±10% relative

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humidity.

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The technical grade transfluthrin (CAS No.118712-89-3) were purchased from Sigma-Aldrich Chemical Company and metofluthrin (CAS No. 240494-70-6) was purchased

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from Dr Ehrenstorfer GmbH.

Design of vapour-phase bioassay apparatus

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We conducted all bioassays using a vapour-phase toxicity test apparatus comprising of glass assemblies (Fig.1) conceptualized and designed. The engineering drawing was prepared using

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SolidWorks® Premium 2014 software. The glass apparatus consists of two parts Main vessel (Base part) (60mm height), upper lid part (75mm height), a wire mesh (87mm diameter and

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0.2mm thickness and 210 micron mesh size) and a stopper (36 mm height) (Fig.2). Main vessel (base part) has a bottom diameter of 75mm and 98mm as open top diameter. There is a notch (shown as pointer no. 2, Fig.2), which is situated 17mm up from the bottom of the main vessel. The wire mesh (shown as pointer no. 4, Fig.2) is fitted into a notch that acts as the diaphragm of the system that separates the apparatus into two chambers – the bottom chamber (shown as pointer no. 1, Fig.2) and an upper chamber (shown as pointer no. 3, Fig.2). The floor of the main vessel is used for loading the insecticide to be tested and upper chamber is for insects to be exposed. Thus, the stainless steel wire mesh (No. 80 – 210

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ACCEPTED MANUSCRIPT micron) acts as a diaphragm that prevents insects from coming into direct contact with the insecticides under evaluation. This also ensures that knockdown happened due to the vapour of the insecticide molecule and not because of direct contact with the insecticide. The upper glass lid part (shown as pointer no. 5, Fig.2) of the vapour toxicity apparatus has an open basal end diameter of 87mm to fit tightly into the notch at the base. There is an inverted port

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(opening) of 14mm diameter (shown as pointer no. 6, Fig.2) on top of the lid to release the

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insects into the apparatus. We made the insect release port inverted for two reasons – (i) it

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prevents flying insects to be freed while releasing, (ii) potential to minimize vapour loss

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while releasing insects.

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Vapour Phase Bioassay Safety statement

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We have consulted the Material Safety Data Sheet (MSDS) for appropriate personal protective equipment before handling insecticide compounds. Specific laboratory training on

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bioassays.

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handling mosquitoes in laboratories was provided to personnel before attempting any

Preparing stock solutions The technical grade active insecticides were dissolved in acetone to prepare 0.1% master stock solution, which was used for the preparation of test concentrations. The master stock was stored at -4°C. For Cx quinquefasciatus, we have prepared 10, 2, 0.4, 0.08 and 0.02 µg/ml. For M. domestica Concentrations were 100, 20, 4, 0.8, 0.2 µg/ml. These concentrations were selected based on few range-finding pilot experiments (data not shown).

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Preparation of vapour-phase bioassay apparatus and systematic bioassay procedure We have used 12 numbers of vapour toxicity apparatus having the same specification for conducting the entire experiments. Before starting each experiment, we dismantled all parts

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of each vapour toxicity apparatus (Fig.2 and 3) and cleaned all parts. Cleaning was done with acetone, followed by an unscented soap solution in water followed by acetone and dried the

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apparatus in the oven (60°C) for 30 min to avoid any crossover residual contamination. After

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bringing the apparatus at room temperature, a blank test was run by releasing few 3-5 days old mosquitoes through the release port from top of the lid. The blank test was performed for

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half an hour to confirm no crossover contamination.

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After the satisfactory blank test for each test apparatus, we applied 1 ml of a respective concentration of pyrethroids solution on the bottom of the main vessel of the glass

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apparatus (picture 1 and 2, Fig.3). Then base was swirled on all sides in order to spread the insecticide solution across the inner surface of the base and then left open for half an hour in

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a fume hood at wind velocity of 1.0 m/second to evaporate the acetone completely. After 30 minutes, acetone evaporated completely and wire mesh was placed onto the notch of the main

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vessel (picture 5-7 in Fig.3) and closed the lid (picture 8 and 9 in Fig.3). The joint of the main vessel and the lid is covered with Teflon tape to prevent loss of insecticide vapours. Inverted insect release port is closed with stopper applied with grease for tight closure. The inner opening of the insect release port was also temporarily closed using a Teflon tape to minimize vapour loss during the release of insects (picture 3 and 4 in Fig.3). With these Teflon tapes, we ensured that there should not be any vapour loss from the apparatus during incubation and the exposure period. Here, we must acknowledge that insecticide vapour

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ACCEPTED MANUSCRIPT could lose during acetone evaporation and our experiment did not capture the quantum of this loss. Subsequently, the entire vapour toxicity apparatus was kept in an oven at 60°C for 15 min for incubation to help applied semi-volatile or volatile insecticides to vaporize. This ensures uniform vapour creation across the replicates. After incubation, all the test apparatus

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were taken out from oven and were kept at room temperature for 10-15 min to cool down

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before releasing the insects. An appropriate number of 10-15 mosquitoes were released into

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each apparatus through the insect release port by puncturing the Teflon tape (this minimizes the loss of vapour from the upper exposure chamber). Once all the insects released into the

at every 10 minutes interval up to

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system, we closed the opening by the stopper and started recording the knockdown numbers 1 hour exposure period. After exposure, the mosquitoes

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were transferred to holding cages and provided with 10% sugar pad as food. Mortality was recorded the 24-hour post-exposure. All tests were performed at 25% ± 2 degrees and 60% -

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70% relative humidity. Each Concentration was tested in 6-9 replicates, repeated on three

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Data Analysis

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different days.

We calculated the 50% knockdown time (KT50 ) by Probit Analysis (DJ Finney, 1971). StatsDirect Statistical software v2.7.9 (07/09/2012) was used for data analysis. ANOVA was used to compare the means of KT50 and percentage knockdown during one-hour exposure (KD@60) of each pyrethroid. We have analysed the repeatability, intermediate precision (as experiments are done in different days), linearity and sensitivity parameter of the method from the knockdown data.

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Results Three-dimensional drawing of the vapour-phase toxicity test apparatus is shown in Fig. 1. An engineering drawing is presented in Fig. 2. For real-time photos of the vapour toxicity

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apparatus, please refer to Fig.3.

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The 50% knockdown time (KT50 ) data against mosquitoes are presented in Table 1

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(for transfluthrin) and Table 2 (for metofluthrin). The KT50 data shows that the method can clearly distinguish the efficacy of the two molecules against mosquitoes at even very low

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concentration levels. The results also represented graphically (Fig.4-5) to understand the linearity of the methods. The KT50 and percentage knockdown at 60th min (KD@60) for

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mosquitoes at different concentrations is presented in Table 3.

Discussion

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The vapour-phase bioassay developed and presented in this paper is based on the mechanism of non-contact toxicity of insecticides against insects. The insecticide vapour was generated

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by heating at 60°C for 15 min, which ensured that insects are only exposed to the vapour only. We have fixed the incubation temperature and time at 60°C for 15 min for operational feasibility.

It is critical to have the desired precision, accuracy, linearity and robustness in a wellcontrolled bioassay system. We tested the sensitivity of the method by analysing the variance between concentrations in a given experiment. We found that KT50 and percentage knockdown at 60th min (KD@60) at different concentrations in a given experiment defer significantly in mosquitoes (Table 3) – this pattern is consistent with both transfluthrin as 9

ACCEPTED MANUSCRIPT well as metofluthrin. This indicates that the method is sensitive enough to distinguish between concentrations and suitable for concentration-response experiments. We also tested the repeatability and precision of the method by analysing the variance of knockdown data (KT50 and KD@60) of a given concentration between different experiments in different days. We found that KT50 and percentage KD@60 for both transfluthrin and metofluthrin at a given

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concentration do not differ significantly between experiments (Table 3). This concludes that

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the method has repeatability and precision. We also tested the linearity of the method (see

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Fig.4-5). Percentage mortality and total KT50 (KT50 calculated using all the data from the experiments) for transfluthrin and metofluthrin against Cx quinquefasciatus shows that the

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percentage mortality and KT50 are inversely proportional.

With the enormous problem of vector-borne diseases and insecticide resistance, it

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becomes essential to develop newer strategies for vector and pest control. Vector control authorities are looking at two approaches – one approach is developing new insecticide

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molecules with a novel mode of action and other is the use of agricultural or household

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insecticide molecules for public health vector control. Under IVCC umbrella, many chemical companies are working to develop insecticide molecules with the newer mode of action. On the other hand vapour phase insecticide, transfluthrin has recently been reformulated into

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ambient emanator formats for potential public health use (Andrés et al., 2015; Killeen et al., 2017; Masalu et al., 2017; Ogoma et al., 2017). For every phase of insecticide molecule or product development, bioassay method with high specificity and ability to unambiguously evaluate the bio-response of the insecticide of interest is vital. This non-contact vapour-phase bioassay described here is best suited for assessing the volatile and semi-volatile insecticide molecules. This method may not be appropriate or relevant for non-volatile molecules. The method is practical and easy to operate with simple standardization on the incubation temperature and time (we have used 60°C for 15min) based 10

ACCEPTED MANUSCRIPT on the concentration, nature of insecticide molecule, type of insects used and operational feasibility. This artificial heating ensures uniform vapour creation across the replicates; otherwise, the experiments can be done at a consistent room temperature above 25°C. Increasing the incubation temperature and/or time may accelerate the knockdown and killing of insects at a lower concentration due to a higher concentration of vapour; in such a

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situation, one may need to reduce the exposure concentration to have a dose-response effect.

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Insect release port may also be used for collection of air sample from the vessel for testing

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the concentration over time inside the vessel to confirm if the any insecticide vapour is lost from the vessel. However, we have not performed this experiment.

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Information on the potential efficacy of vapour phase insecticides can be obtained using this bioassay. This method can be used to screen potential vapour phase active

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molecules that can be formulated for use as pesticides. The new bioassay system can also serve as a system for studying concentration response of new generation volatile insecticide

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candidates. To the best of our knowledge, this is the first comprehensive portable non-contact

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method for evaluation of the efficacy of vapour phase insecticides. We would further recommend that the WHO should consider evaluating our vapour phase toxicity bioassay

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method described here with an aim to incorporate in WHO pesticide evaluation guidelines.

Acknowledgments For the rearing and supply of the test insects, we thank Uday Chugle and Sanjay Gamre. Authors also acknowledge the technical help of Mr Dipon Sarkar in conducting few experiments. We are grateful for the support and scientific advice of Dr Sunder N Mahadevan.

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ACCEPTED MANUSCRIPT References Andrés, M., Lorenz, L.M., Mbeleya, E., Moore, S.J., Andres, M., Lorenz, L.M., Mbeleya, E., Moore, S.J., 2015. Modified mosquito landing boxes dispensing transfluthrin provide effective protection against Anopheles arabiensis mosquitoes under simulated outdoor

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conditions in a semi-field system. Malar J 14, 255. doi:10.1186/s12936-015-0762-8

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Denloye, A.A., Ajelara, K.O., Alafia, A.O., Babalola, O.O., Bajulaiye, A.A., Okoh, H., Dike, P.I., 2017. Evaluation of unorthodox preparations and their comparative efficacy relative

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to orthodox insecticides against household insects in Lagos. Int. J. Entomol. Res. 5, 43–

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DJ Finney, 1971. Probit analysis by Finney, D. J.: University Press - Better World Books, 3rd

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Tetrafluorobenzyl Pyrethroids against Target- Site and Metabolic Resistant Mosquitoes.

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PLoS One 11, e0149738. doi:10.1371/journal.pone.0149738 Horstmann, S., Sonneck, R., Velten, R., Werner, S., 2014. Use of a compound comprising a

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polyfluorobenzyl moiety against insecticide-resistant pests. WO2014079928A1. Killeen, G.F., Tatarsky, A., Diabate, A., Chaccour, C.J., Marshall, J.M., Okumu, F.O., Brunner, S., Newby, G., Williams, Y.A., Malone, D., Tusting, L.S., 2017. Developing an expanded vector control toolbox for malaria elimination. BMJ Glob. Heal. 2, e000211. doi:10.1136/ Kumar, K., Sharma, A.K., Kumar, S., Patel, S., Sarkar, M., Chauhan, L.S., 2011. Multiple insecticide resistance/susceptibility status of Culex quinquefasciatus, principal vector of bancroftian filariasis from filaria endemic areas of northern India. Asian Pac. J. Trop.

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ACCEPTED MANUSCRIPT Med. doi:10.1016/S1995-7645(11)60119-3 Kuri-Morales, P.A., Correa-Morales, F., González-Acosta, C., Moreno-Garcia, M., DávalosBecerril, E., Benitez-Alva, J.I., Peralta-Rodriguez, J., Salazar-Bueyes, V., GonzálezRoldán, J.F., 2018. Efficacy of 13 Commercial Household Aerosol Insecticides Against Aedes aegypti (Diptera: Culicidae) From Morelos, Mexico. J. Med. Entomol. 55, 417–

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Liu, N., 2015. Insecticide Resistance in Mosquitoes: Impact, Mechanisms, and Research

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Directions. Annu. Rev. Entomol. 60, 537–559. doi:10.1146/annurev-ento-010814-

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Ogoma, S.B., 2017. Efficacy and user acceptability of transfluthrin-treated sisal and hessian decorations for protecting against mosquito bites in outdoor bars. Parasit.

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Vectors 10, 197. doi:10.1186/s13071-017-2132-6 Mori, T., 2017. Recent findings of new synthetic pyrethroids. Acta Hortic. 1169, 47–52.

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doi:10.17660/ActaHortic.2017.1169.8 Ogoma, S.B., Mmando, A.S., Swai, J.K., Horstmann, S., Malone, D., Killeen, G.F., 2017. A

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low technology emanator treated with the volatile pyrethroid transfluthrin confers long term protection against outdoor biting vectors of lymphatic filariasis, arboviruses and malaria. PLoS Negl. Trop. Dis. 11, e0005455. doi:10.1371/journal.pntd.0005455 Paine, M.J.I., Brooke, B., 2016. Insecticide Resistance and Its Impact on Vector Control, in: Advances in Insect Control and Resistance Management. Springer International Publishing, Cham, pp. 287–312. doi:10.1007/978-3-319-31800-4_15 Paramasivam, M.; Selvi, C., 2017. Laboratory bioassay methods to assess the insecticide toxicity against insect pests-A review. J. Entomol. Zool. Stud. JEZS 5, 1441–1445. 13

ACCEPTED MANUSCRIPT Sarkar, M., Borkotoki, A., Baruah, I., Bhattacharyya, I.K., Srivastava, R.B., 2009. Molecular analysis of knock down resistance (kdr) mutation and distribution of kdr genotypes in a wild population of Culex quinquefasciatus from India. Trop. Med. Int. Heal. 14, 1097– 1104. doi:10.1111/j.1365-3156.2009.02323.x Sarkar, M., Kumar, K., Sharma, A., Gupta, A.K., K, G.A., 2015. Ento-epidemiological

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characterization of Dengue in Uttarakhand (India). J. Mosq. Res. J. Mosq. Res. J. Mosq.

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Res. 555, 1–10. doi:10.5376/jmr.2015.05.0017

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Scharf, M.E., Nguyen, S.N., Song, C., Koehler, P.G., 2007. Bioassay for volatile low molecular weight insecticides and methods of use cross-reference. US 2007/0154393

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A1. doi:US 2010/0311130 Al

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WHO/Department of Communicable Disease Prevention, C. and E., 1996. Report of the WHO Informal Consultation On Evaluation and testing of insecticides. Geneva,

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Switzerland .

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William, A., Brown, A., 1951. Insect Control by Chemicals. John Wiley & Sons, New York. World Health Organization (WHO), 2009. Guidelines for efficacy testing of household insecticide products control of neglected tropical diseases WHO Pesticide Evaluation

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Scheme. Geneva, Switzerland.

World Health Organization (WHO), 2006. Guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mosquito nets control of neglected tropical diseases WHO Pesticide Evaluation Scheme. Geneva, Switzerland.

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ACCEPTED MANUSCRIPT Figure Legends Fig.1: 3D drawing of Vapour-phase toxicity apparatus comprising of glass apparatus. Fig. 2: Engineering drawing of the glass apparatus. Main vessel (base part) (60mm height), bottom diameter of 75mm and top diameter 98mm having (1) Bottom chamber, (2) Notch

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situated 17mm up from the bottom, (3) Upper chamber; (4) Wire mesh fitted into the notch; (5) Upper glass lid part with open basal end diameter of 87mm to fit tightly into the notch at

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the base, (6) Inverted port (opening) of 14mm diameter to release the insects into the

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apparatus; (7) Glass stopper of 36 mm height.

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Fig.3: Real-time photos of the vapour toxicity apparatus. (1-2): insecticide application area, (3-4): Temporarily closing of the inner opening of the insect release port using a Teflon tape

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to minimize vapour loss during the release of insects, (5-7): wire mesh placed onto the notch of the main vessel and (8-9): Lid closed.

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Fig.4: Graph shows concentration-dependent percentage mortality (post 24hrs post exposure) and concentration-dependent time to get knockdown against mosquitoes using transfluthrin.

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Percentage mortality increases and total KT50 decreases linearly with the increasing concentration of transfluthrin against Culex quinquefasciatus.

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Fig.5: Graph shows Concentration-dependent percentage mortality (post 24hrs post exposure) and Concentration-dependent time to get knockdown against mosquitoes using metofluthrin. Percentage mortality increases and total KT50 decreases linearly with the increasing concentration of metofluthrin against Culex quinquefasciatus.

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ACCEPTED MANUSCRIPT Conflict of Interest: There is no conflict of interest. The authors of the present study are employees of Godrej

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Consumer Products Ltd. (a Godrej Group company) at the time laboratory studies were conducted. Godrej Consumer Products Ltd. acts as an employer and provided support in the form of salaries for the authors, but did not have any additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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ACCEPTED MANUSCRIPT Highlight Portable non-contact bioassay method for evaluation of vapour phase insecticides

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Enable evaluation of the vapour toxicity profiles of all semi-volatile insecticides

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Enable evaluation of spatial killing ability of household insecticide actives

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Method can be used to screen potential new vapour phase insecticide molecules

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ACCEPTED MANUSCRIPT Table 1. Probit analysis results of Transfluthrin against Culex quinquefasciatus KT50

Dose (µg)

(min)

0.02

286.53

118 - 1000

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1.913

1.548

0.08

39.146

33.644 – 47.192

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2.443

1.444

0.4

35.46

29.60 – 44.32

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2.01

3.399

2

22.90

18.642 – 28.11

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Chi-square

Exposure

Number

1.65

1.965

10

11.08

8.318 – 13.71

1.87

1.963

Confidence Interval

Slope (n)

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(df = 6)

ACCEPTED MANUSCRIPT Table 2. Probit analysis results of metofluthrin against Culex quinquefasciatus Exposure

KT50 (min)

Confidence

Number

Interval

(n)

Dose (µg)

Slope

Chi-square (df = 6)

73.4

53.87 – 127.34

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1.625

0.771

0.08

28.35

24.198 – 33.603

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2.249

4.151

0.4

20.53

18.027 – 23.17

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3.144

4.709

2

10.43

8.725 – 12.07

36

10

9.00

7.47 – 10.45

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0.02

3.276

5.293

3.24

3.953

ACCEPTED MANUSCRIPT Table 3. ANOVA of KT50 and percentage knockdown at 60 min exposure (KD@60) for transfluthrin and metofluthrin against Culex quinquefasciatus mosquito. Transfluthrin

Metofluthrin

Knockdown (60

Knockdown (60

KT50 of

KT50 of min) of

min) of individual

Parameter

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individual individual experiments

F

P-value

P-value

F

P-value

value

Variance in

P-value

0.0013* 13.00 < 0.0001* 56.60 < 0.0001* 219 in a given

value

< 0.0001* 50.80

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experiment

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Variance in

0.1111

2.67

0.4042

1.05

0.1366

2.24

0.7923

0.347

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experiments

F

value

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different dose

dose between

experiments

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value

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F

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experiments

same given

individual

experiments

Note: * represent the difference is significant. (1) KT50 and KD@60 are significantly different between doses in a given experiment; it proves that method is sensitive enough to distinguish between doses. (2) Variance in a given dose between experiments is not significantly different. Therefore, repeatability and intermediate precision of the experiment is established.

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