MECHANISM S OF PYRETHRUM AND PYRETHROID REPELLENCY By Elizabeth Bandason A DISSERTATION Submitted to Michigan State University in partial fulfilment of the requirements for the degree of Entomology -Doctor of Philosophy 2018 ABSTRACT MECHANIS MS OF PYRETHRUM AND PYRETHROID REPELLENCY By Elizabeth Bandason Pyrethrum is a natural insecticide, extracted from the flower heads of Chrysanthemum cinerariifolium . Pyrethroids are synthetic compounds structurally derived from pyrethrins . Besides their insecticidal activity, it is well -documented from behavioral assays that pyrethrum and pyrethroids induce repellency. However, the molecular basis of pyrethrum/pyrethroid repellency is unknown. The aim of this study was to elucidate the mechanism of pyreth rum and pyreth roid repellency in mosquitoes and crop pests. To investigate repellency against mosqui toes, a previously established H and in cage assay was performed. We recorded mosquito landing rate on a hand in response to vapor from a mesh treated with p yrethrum or pyrethroids (transfluthrin , deltamethrin and permethrin). Three insecticide -susceptible Aedes aegypti strains, Waco, Orlando and Rock, two pyrethroid -resistant Ae. aegypti strains, Puerto Rico (PR) and Isokdr, an Ae. aegypti anosmic Orco (olfactory receptor co -receptor) mutant, and an insecticide -susceptible Anopheles gambiae strain, Kisumu were used. All Hand in cage experiments used serial dilutions of the test compounds to generate dose response curves. Different cohorts (50 per cohort ) of nulliparous 6 -8 day -old female mosquitoes were used per trial. Throughout the investigation, significant levels of repellency by pyrethrum and pyrethroids were observed in the insecticide -susceptible mosquito strains although the magnitude varied wi th test compounds. The repellent effects by pyrethrum and pyrethroids were reduced in the two resistant strains, PR and Isokdr, compared to the susceptible strains, Waco and Rock. Pretreatment of mosquitoes with piperonyl butoxide (PBO), a P450 inhibitor , enhanced transfluthrin repellency in both PR and Waco mosquitoes, but did not abolish the difference in repellency between them. Furthermore, repellency to DEET was not significantly different between susceptible and resistant mosquito strains. Repelle ncy against susceptible Aedes and Anopheles mosquitoes was also evaluated using two types of insecticide treated nets, PermaNet 2.0 and Olyset . Repellency of PermaNet2.0 on pyrethroid -susceptible Ae. aegypti Rock and Waco mosquitoes, and An. gambiae Kisumu mosquitoes was observed, but was reduced in pyrethroid -resistant Peurto Rico (PR) and Isokdr and anosmic orco mosquitoes. Repellency effect of the Olyset net on An. gambiae Kisumu mosquitoes was evident, but not on Ae. aegypti mosquitoes. Electroantennog raph (EAG) recordings were conducted from antenna of adult Ae. aegypti . Pyrethrum and pyrethroid s elicited EAG responses in Rock and Orlando mosquito strains. No EAG responses were recorded in Orco knockout mosquitoes. Studies on the diamondback moth and maize grain borer using T -maze assays and feeding choice tests revealed that pyrethroids also evoke repellency in the maize grain borer and diamondback moth. The larvae of diamondback moth preferred eating a leaf in an untreated arena than the treated one . Adults of maize grain borer preferred the untreated control arm in the T -maze assay. This study began to uncover the enigma of repellency of the one of the most important class of insecticides used globally to combat vector -borne human diseases. We conf irmed repellency by pyrethrum/pyrethroids in both disease vectors and crop pests, and demonstrated that Ae. aegypti mosquitoes can sense pyrethrum/pyrethroids via an authentic Orco -dependent olfactory pathway. The repellency is likely mediated by dual act ivation of both sodium channels and/or olfactory receptors. Collectively, our study provides a conceptual framework for understanding of the modes of action of pyrethrum/pyrethroids as an important group of insect repellents. iv ACKNOWLEDGEMENT S My appreci ation goes to my major professor Dr. Ke Dong who was so generous to dedicate her time and energy to supervise my work. She has been very supportive professionally and has helped me grow as a researcher. I am so thankful to my guidance committee: Dr. Edwar d Walker and Dr. Henry Chung at the Department of Entomology, Michigan State University (MSU) and Dr. Themba Mzilahowa at the Malaria Alert Center, College of Medicine (COM -UNIMA) for all their time and meaningful contributions to my research work. I also would like to thank late Dr Suzan Thiem for all her contributions, guidance and support. I would like to thank the Chair of Entomology department at MSU, Dr. Bill Ravlin for his support. Very special thanks to Heather Lenartson -Kluge for her assistance. I am thankful to all my lab mates for all kinds of assistance. I am grateful to Peng Xu, Qiang Wang and Feng Liu, postdoc associates in the Dong lab, for their help and collaboration to make this project work. My appreciation also goes to Dr Abel Sefasi (Bu nda-LUANAR) for his support. Thanks to Dr Kingdom Kwapata (Bunda -LUANAR) for providing space in his lab. Thanks to the Borlaug Higher Education for Agricultural Research and Development (BHEARD) team at MSU for their support. This material is based upon work supported by United States Agency for International Development as part of the Feed the Future initiative, under CGIAR Fund, award number BFS -G-11-00002, and the predecessor fund the Food Security and Crisis Mitigation II grant, award number EEM -G-00-04-00013, and a grant from NIH (R01GM115475). v TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................................... vii LIST OF FIGURES ....................................................................................................................................... viii CHAPTER 1 ...................................................................................................................................................... 1 INTRODUCTION ........................................................................................................................................... 1 Pyrethrins, pyrethroids and their use as insecticides ................................................................................ 2 Mode of action of pyrethroids ..................................................................................................................... 5 Pyrethroid resistance ..................................................................................................................................... 6 Insect olfaction and pyrethroid repellency ................................................................................................ 6 REFERENCES ................................................................................................................................................ 10 CHAPTER 2 .................................................................................................................................................... 16 REPELLENCY OF P YRETHRUM AGAINST Aedes aegypti AND Anopheles gambiae MOSQUITOES ............................................................................................................................................... 16 Abstract ......................................................................................................................................................... 17 Introduction .................................................................................................................................................. 18 Materials and methods ................................................................................................................................ 20 Mosquitoes ............................................................................................................................................... 20 Hand in cage behavior assay setup ........................................................................................................ 20 Experimental design, data acquisition and analysis ............................................................................ 23 Results ........................................................................................................................................................... 24 Repellency of pyrethrum and DEET on insecticide -susceptible Aedes and Anopheles mosquitoes ................................................................................................................................................ 24 Pyrethrum repellency is reduced in pyrethroid -resistant Aedes mosquitoes .................................... 24 Repellency effect of pyrethrum is Orco -dependent ............................................................................. 28 Discussion ..................................................................................................................................................... 30 Conclusion .................................................................................................................................................... 32 REFERENCES ................................................................................................................................................ 33 CHAPTER 3 .................................................................................................................................................... 37 TOXICODYNAMICS OF TRANSFLUTHRIN ON Aedes aegypti MOSQUITOES ......................... 37 Abstract ......................................................................................................................................................... 38 Introduction .................................................................................................................................................. 39 Materials and Methods ................................................................................................................................ 40 Mosquitoes ............................................................................................................................................... 40 Test compound and arm visits .............................................................................................................. 41 Hand in cage assay with cytochrome P450 inhibited mosquitoes using PBO ............................... 42 Hand in cage knockdown assay ............................................................................................................. 42 Results ........................................................................................................................................................... 43 Transfluthrin (TF) repellency in susceptible mosquito strains ......................................................... 43 Reduced transfluthrin repellency in resistant Aedes strains ................................................................ 46 Repellency effect of transfluthrin is Orco -dependent ........................................................................ 51 Pre-treatment of piperonyl butoxide (PBO) enhanced repellency elicited by transfluthrin ......... 52 vi Pre-treatment of PBO enhanced mosquito knockdown by transfluthrin. ...................................... 56 Discussion ..................................................................................................................................................... 60 Conclusion .................................................................................................................................................... 62 REFERENCES ................................................................................................................................................ 63 CHAPTER 4 .................................................................................................................................................... 67 REPELLENCY EFFECT OF PermaNet 2.0 AND Olyset NET ON Anop heles gambiae AND Aedes aegypti ........................................................................................................................................................ 67 Abstract ......................................................................................................................................................... 68 Introduction .................................................................................................................................................. 69 Materials and Methods ................................................................................................................................ 72 Mosquitoes ............................................................................................................................................... 72 Behavioral assays ..................................................................................................................................... 73 Results ........................................................................................................................................................... 74 Permethrin elicited repellency on pyrethroid -susceptible Ae. aegypti mosquitoes .......................... 74 PermaNet 2.0 repelled susceptible Aedes aeg ypti strains (Waco and Rock) and Anopheles gambiae (Kisumu) ................................................................................................................................................... 76 Permethrin and deltamethrin elicited electroantennogram (EAG) signals ..................................... 78 Discussion ..................................................................................................................................................... 81 Conclusion .................................................................................................................................................... 84 REFERENCES ................................................................................................................................................ 85 CHAPTER 5 .................................................................................................................................................... 90 TRANSFLUTHRIN AND PYRETHRUM REPELLENCY ON Plutella xylostela AND Sitophilus zeamais ................................................................................................................................................................ 90 Abstract ......................................................................................................................................................... 91 Introduction .................................................................................................................................................. 92 Materials and Methods ................................................................................................................................ 95 Insects ........................................................................................................................................................ 95 Test compounds ...................................................................................................................................... 95 Behavioral assays ..................................................................................................................................... 96 Data analysis .......................................................................................................................................... 100 Results ........................................................................................................................................................ 100 Repellency effect of pyrethrum and transfluthrin on Sitophilus zeamais (Lesser gain borer) ...... 100 Repellency effect of p yrethrum and transfluthrin on Plutella xylostella (Diamondback moth) ... 103 Pyrethrum and transfluthrin repel DBM larvae and reduce feeding on leaf discs in treated arena ........................................................................................................................................................ 105 Discussion .................................................................................................................................................. 108 Conclusion ................................................................................................................................................. 111 REFERENCES ............................................................................................................................................. 112 CHAPTER 6 ................................................................................................................................................. 116 CONCLUSIONS AND FUTURE DIRECTIONS ............................................................................... 116 REFERENCES ............................................................................................................................................. 120 vii LIST OF TABLES Table 2.1 Repellency effect of pyrethrum compared to DEET between strains of Aedes aegypti 1 and Anopheles gambiae (Kisumu) 1. ........................................................................................................... 27 Table 3.1 Repellency effect of TF compared between strains of Aedes aegypti 1 mosquitoes before and after PBO pre -treatment. ................................................................................................................ 48 Table 3.2 Repellency effect of DEET compared betw een strains of Aedes aegypti 1 mosquitoes before and after PBO pre -treatment .................................................................................................... 50 Table 3.3 Repellency effect of TF compared within and between strains before and after PBO pre -treatment of Aedes aegypti 1 mosquitoes ........................................................................................... 53 Table 3.4 Knockdown effect on Aedes aegypti 1 pretreated with PBO when exposed to 10 -3 TF dilution in hand in cage assay. ................................................................................................................ 58 Table 3.5 Knockdown effect on Aedes aegypti 1 pretreated with PBO when exposed to 10 -4 TF dilution in hand in cage assay. ................................................................................................................ 59 Table 4. 1 Repellency effect of permethrin o n Waco and PR Aedes aegypti 1 strains ................................ 75 Table 4.2 Repellency effect of PermaNet 2.0 and Olyset compared when tested on An. gambiae 1 and different Ae.aegypti 1 strains .............................................................................................................. 76 Table 5.1 Percent response 2 of Sitophilus zeamaise 1 when pyrethrum and transfluthrin were used in a T -maze choice trap assay ............................................................................................................. 102 Table 5. 2 Pe rcent response 2 of adult Plutella xylostella 1 when pyrethrum and transfluthrin were used in two choice test. ........................................................................................................................ 104 Table 5.3 Percent response 2 of Plutella xylostella 1 larvae when pyrethrum and transfluthrin were used in a larvae feeding preference assay .......................................................................................... 107 viii LIST OF FIGURES Figure 1.1 Chemical structures of selected pyrethroids and pyrethrins .................................................... 4 Figure 1.2 Schematic presentation of effect of pyrethroids on neuronal excitability ............................. 5 Figure 1.3 Peripheral olfactory system in mosquitoes .................................................................................. 7 Figure 2.1 Hand in cage assay setup .............................................................................................................. 22 Figure 2.2 No difference in the repellency effects of pyrethrum and DEET ....................................... 25 Figure 2.3 Difference in the repellency effects of pyrethrum and DEET on PuertoRico Ae.aegypti mosquitoes ............................................................................................................................... 26 Figure 2.4 Reduced repellency effect of pyrethrum in Aedes aegypti orco 5/16 mosquitoes ......................... 29 Figure 3.1 Transfluthrin repel susceptible Aedes mosquitoes .................................................................... 44 Figure 3.2 Transfluthrin repel susceptible Anopheles gambiae, Kisumu strain mosquitoes .................... 45 Figure 3.3 Transfluthrin repellency is reduced in re sistant mosquito strains. ........................................ 47 Figure 3.4 Repellency of DEET is not reduced in pyrethroid resistant mosquito strains .................... 49 Figure 3.5 Transfluthrin repellency is reduced in Aedes orco 5/16 and it is significantly high in Orlando Aedes aegypti strain ................................................................................................................ 51 Figure 3.6 Transfluthrin repellency is enhanced when Aedes aegypti mosquitoes are pretreated with PBO .................................................................................................................................................. 54 Figure 3.7 No difference in the landing response of PBO pretreated and untreated Aedes aegypti when expose d to untreated (control) .................................................................................................... 55 Figure 3.8 Transfluthrin knockdown effect is enhanced in mosquitoes pretreated with PBO ......... 57 Figure 4. 1 High permethrin repellency in pyrethroid susceptible Aedes aegypti Waco strain than pyrethroid resistant PR strain ................................................................................................................ 74 Figure 4.2 PermaNet2.0 repel pyrethroid susceptible Aedes aegypti strain s and Anopheles gambiae . ....... 77 Figure 4.3 Normalized EAG responses of Aedes aegypti to deltamethrin ................................................. 79 Figure 4.4 Normalized EAG resp onses of Aedes aegypti to permethrin ................................................... 80 Figure 5.1 T -Maze trap assay set up for Sitophilus zeamais (LGB) ............................................................. 96 ix Figure 5.2 Two choic e assay setup for adult diamondback moth insects .............................................. 97 Figure 5.3 Feeding preference assay set up for diamondback moth larvae ............................................. 99 Fi gure 5.4 Pyrethrum and transfluthrin repel Sitophilus zeamais (lesser grain borer) in a T-maze assay. ........................................................................................................................................ 101 Figure 5.5 Pyrethrum and transflu thrin repel Plutella xylostella (diamondback moth) in a choice assay. ................................................................................................................................... 103 Figure 5.6 Pyrethrum and transfluthrin repel and reduce feeding in Plutella xylostella (diamondback moth) larvae in a feeding choice assay. ................................................................... 105 Figure 5.7 Reduced feeding in pyrethrum and transfluthrin arenas in the choice feeding assay against DBM larvae .................................................................................................................... 106 1 CHAPTER 1 INTRODUCTION 2 Pyrethrins, pyrethroids and their use as insecticides Pyrethrins were originally derived from extracts of the flower heads of Chrysanthemum cinerariaefolium and they comprise six insec ticidal compounds namely pyrethrin I and II, cinerins I and II, jasmolins I and II (Anadón et al., 2009) . In their natural state, they are potent insecticides but are unstable when exposed to light, air and heat (Ensley, 2007) . Their use in form of crude extracts dates back to 400BC in persia (Ensley, 2007) . In the past decades, three developments have helped establish their main uses including delivering pyrethrins by incorporating pounded flowers with other ingredients into mosquito coils that repel, expel, knock down or kill mosquitoes (Casida, 1980). The second way is through aerosol can or comb which produces droplets belo w 30um in diameter, the third one is the use of additive synergists the piperonyl butoxide (PBO) which by itself is not toxic but increases the potency (Casida, 1980). Pyrethroids are synthetic analogs of pyrethrins with improved stability and greater insecticidal activity (Breckenridge et al., 2009; Davies et al., 2007; Ensley, 2007) With a few exceptions of more recently developed compound s, pyrethroids are typically esters of chrysanthemic acid (Fig. 1.1, Soderlund 2011). The first pyrethroid was allethrin which was identified in 1949 and has a basic cylopropane carboxylic ester structure as other type I pyrethroids, such as phenothrin and permethrin (Anadón et al., 2009) . The insecticidal activity of pyrethroids was enhanced by adding a cyno group to give alph a-cyano at the phenocybenzyl alcohol moiety of type II pyrethroids, such as deltamethrin, cyfluthrin and lamda -cyhalothrin (A nadón et al., 2009) . To date, pyrethroids remain important insecticides and have been used for more than thirty years to control insect vectors and crop pests. In other parts of the world they are also used as an active ingredient in many househol d insecticidal products (Sugiura et al., 2008) . Their use has extended to crop protection to minimize pre and postharvest losses. Cotton growing regions in the world , more especially in Africa , where 3 transgenic cotton is rarely used, pyrethroids have been used in field sprays to control insect pests (Christian et al., 2011; Symington et al., 2011; Yang et al., 2004) . Us e of pyrethroids in vegetable crops such as tomatoes, have also been documented (Haddi et al., 2012) . Dust formulated insecticides to control of storage pests in maize grains use a pyrethroid as an active ingredient (Kamanula, 2014) . Aside from agricultural and household uses, the use of pyrethroid insecticides has been largely documented in public health. To date, several pyrethroids, such as perme thrin and deltamethrin (Fig. 1.1), are used in insecticide treated nets (ITN) (Denham et al., 2015; Enayati and Hemingway, 2006; Stevenson et al., 2011; Takken, 2002) . To increase efficacy, some pyrethroid -bound nets are used together with piperoyl butoxide (PBO) as a synergist (Denham et al., 2015) . They also have been used in indoor residual sprays (IRS) and they are incorporated in mosquito coils. Recent research trends have documented the use of volatile pyrethroids such as transfluthrin (Fig. 1.1), inducing behavioral changes in mosquitoes due to sub -lethal exposure s. A study by Ogoma et al., (2014) extensively evaluated the effect of airborne pyrethroids on entomological parameters of malaria and gave a detailed account of deterrence, toxicity and blood feeding inhibition in mosquitoes due to exposure to air borne pyrethroids. Mosquitoe s that were captured in experimental huts did not feed or lay eggs. Details on several studies have revealed the use of pyrethroids in passive emanators, coils and transfluthrin impregnated hessian sacks to reduce outdoor mosquito bites (Govella et al., 2015; Ogoma et al., 2014, 2017; Ogoma, Ngonyani, et al., 2012; Ogoma et al., 2012 ) . 4 (A) (B) (C) (D) (E) Figure 1.1 Chemical structures of selected pyrethroids and pyrethrins . Indicated are: (A) Transfluthrin (B) Acetransfluthr in (ACTF), (C) deltamethrin and (D) permethrin. (E) Chemical structure of pyrethrin OOFFFFClClOOOClClOOFFFFOCNOOBrBr 5 Mode of action of pyr ethroids Voltage -gated sodium channels are integral transmembrane proteins that are critical for electrical signaling in most excitable cells. In response to membrane depolarization, sodium channels open (activate) and allow sodium ions to flow into the c ell, causing depolarization of the membrane potential. A few milliseconds after channel opening, the channel is inactivated (closed), i.e., fast inactivation which plays an important role in the termination of action potentials and prevents excessive depol arization of the resting membrane potential. Thus, sodium channels are essential components of cellular excitability. Pyrethroids inhibit channel inactivation and stabilize the open state of sodium channels, causing prolonged channel opening (Narahashi, 2000, 2000; Narahashi et al., 1995) . Type I pyrethroids cause repetitive discharges, whereas Type II pyrethroids cause membrane depolarizat ion accompanied by suppression of cellular excitability ( Narahashi 1986 , Fig. 1.2). Figure 1. 2 Schematic presentation of effect of pyrethroids on neuronal excitability . The diagram depicts the pyrethroid effects on individual channels on whole sodium currents and action potentials (Shafer et al., 2005 ). 6 Pyrethroid resistance Although pyrethroids remain reliable in insect control because of their low mammalian toxicity, their potency on insects has been affected by development of resistance. Some of the mechanisms through which insects develop resistance to pyrethroids include in creased metabolic detoxification, decreased sensitivity of the target site (sodium channels) to pyrethroids, reduced circular penetration or increased insecticide sequestration (Kasai et al., 2014; N. Liu, 2012; Nardini et al., 2012; Ranson et al., 2011; Toé et al., 2014) . A growing body of literature has presented two most common mechanisms of pyrethroid resistance: enhanced metabolic detoxification (mainly P -450 mediated) and knockdown resistance (kdr) due to mutations in the sodium channel gene (Ffrench -Constant et al., 2004; Liu et al. 2015; Matowo et al. 2014; Dong et al., 2014) . Insect olfaction and pyrethroid repellency Insects including Drosophila and mosquitoes rely on olfactory receptor neurons (ORNs) to sense odorants (Vos shal and Stocker, 2007; Carey and Carlson, 2011; Leal, 2013) . ORNs are housed in olfactory sensilla on antennae and maxillary palps (Fig.3. Odorants bind to specific olfactory receptors (ORs) in ORNs, which confer odor -specificity. An obligate OR co -recep tor ( Orco ) does not bind odorants by itself, but is necessary for the proper function of the OR/ Orco complex as ligand -gated cation channels (Benton et al., 2006; Larsson et al., 2004; Sato et al., 2008; Vosshall an d Hansson, 2011) . Host seeking in mosquitoes is mediated by this sensory modality (Bohbot et al., 2010). Others have demonstrated the effect on body odor affecting flight a nd landing in mosquitoes (Webster et al., 2015) . Detailed moment to moment flight maneuvers of the Aedes mosquitoes in response to human odor and carbon dioxide have been reported (Dekker and Carde, 2011) . Several authors have emphasized that behavioral expressions in insects are mediated by olfaction (T akken et al., 2001 ; Zwiebel and Takken,2004 ; Wang et al., 2010 , Takken a nd Verhulst, 2011) and olfactory 7 receptors which are mainl y found on the maxillary palpi and antenna of the insect are involved in the process (Zwiebel and Takken, 2004) . Figure 1.3 Peripheral olfactory system in m osquitoes . Different kinds of sensilla are located on different parts of the insect™s body including, the antennae, maxillary palps, labellum, tarsi and wing margin. For olfaction mediated behaviors, the odorant passes through openings in the sensillum to sensilla lymph from the air as shown in (B). When in the sensilla lymph it binds to olfactory binding proteins which interact with olfactory receptors in the neurons. (Bennett and Chopra, 1993) The olfactory sensilla can detect very lo w concentrations of air borne chemicals . For decades, DEET and other naturally derived insect repellents have been known to elicit behavior changes in mosquitoes through the olfactory pathway (Logan et al., 2010a; Masetti & Maini, 2006; McMahon et al., 2003; Syed and Leal, 2009) . Traditionally, most studies on pyrethroids have focused on contact toxicity and not the ability of the insects to detect very low concentrations which would elicit a change in their host seeking behavior. Details on the actual mechanisms underlying the resultant behavioral effects of exposure to pyrethroid sublethal effects are still elusive . Behavioral modifications due to insecticides with neurotoxic sub lethal effects such as pyrethroids, have been documented in a 8 review by (Haynes, 1988) . The review highlighted how permethrin and other insecticides affected mate locating behavior of males. Variations in the effect of the insecticides on the mate locating behavior were emphasized. A field study elucidated t he impact of metofluthrin impregnated slow release plastic cylinder against mosquitoes under indoor conditions, citing over 6 weeks of activity and demonstrated significant reducti on of mosquito indices after treatment (Kawada et al., 2006) . Another study has pointed towards insensitivity of mosquitoes carrying sodium channel mutations to transfluthrin repellency (Wagman et al., 2015) . They speculated that the repellency behavior evoked due to transfluthrin exposure in Aedes mosquitoes was m ediated by the neuroexcitation that affected mosquito locomotor behavior. Conflicting conce pts have been p resented in an attempt define a repellent when it comes to pyrethroids. Some studies have included the knockdown effect, mortality and deterrence in the term repellency (Adu -Acheampong et al., 2014 , Ogoma et at., 2012) and others emphasize that repellents are not supposed to cause mortality in insects, but have to reduce vector host contact and affect insect behavior at very low detectable limits (Maia et al., 2013) . Attempts qualify or define chemicals in terms of the behaviors they elicit in the insects has attracted an evolution of technical terms. Repellency has been defined focusing on insect locomotor behavior; as when an insect steers its course away from the source of stimuli by (Debboun et al., 2006). Others have defined repellency depending on w hether behavioral effects are observed after tarsal contact, and the resultant behavior has been referred to as fi contact repellencyfl or the behavioral effects observed when an insect does not make a tarsal contact with a source of stimuli by steering its c ourse away and this has been referred to as fi spatial repellencyfl (Achee et al., 2009; Debboun et al., 2006; Dusfour et al., 2009; Sathant riphop et al., 2014) . The terms spatial repellency and or contact repellency are still difficult to define as behavioral terms. A review by Miller et al.,(2009), emphasized on the terms to be desgnated to chemicals or insecticides in reference to th e locomotor 9 responses that they elicit in insects, updating terms by early behavioral scientists Diether and co investigators (1960). They e mphasized the definition of a fi repellent flas a chemical that causes a mover (an insect in this case) to make orient ed movements away from the source of stimuli and an fi attractant flas an oriented movement towards the source of stimuli. In the definition, Miller et al.,(2009) reinforced the assessment by Diether and coinvestigators, that these chemical s designated as attractants or repellents should act as odors. In the context of our study, we refer a repellent to a chemical that is causing a noncontact disengagement. Here, we hypothesize that olfactory receptor neurons are involved in the mechanisms of pyrethrum and pyrethroid repellency in mosquitoes and agricultural pests. We report different approaches to testing this hypothesis and these include; modified arm in cage assays (referred to as hand in cage assay hence forth) (Boyle et al., 2016; Kain et al., 2013; Logan et al., 2010; Masetti & Maini, 2006; Syed and Leal, 2009) . To te st involvement of the olfactory receptors, orco 5/16 mutant mosquitoes were used in the behavioral experiments. Aside hand in cage assay, other behavioral assays used to test the hypothesis include T -maze, two choice and feeding preference assays. We there fore report, repellency effect of pyrethrum and pyrethroids (transfluthrin and permethrin) in different Aedes and Anopheles mosquito strains. We also discuss the repellency effect of insecticide treated nets (PermaNet 2.0 and Olyset). Pyrethrum and transfl uthrin repellency on two agricultural pests Plutella xylostella and Sitophillus zeamais has also been reported. 10 REFERENCES 11 REFERENCES Achee, N. L., Sardelis, M. R., Dusfour, I., Kamlesh, R., & Grieco, J. P. (2009). Characterization of Spatial Repellent,Contact Irritant, and Toxicant Chemical Actions of Standard Vector Control Compounds. 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Pyrethrum extracts from the flower heads of Chrysanthemum ( Tanacetum cinerariifolium ) have been used as insect repellent against various biting arthropods for thousands of years and since 1902, pyrethrum has been i ncorporated as a key ingredient in commercial mosquito coils (Moore and Debboun, 2007). However, the mechanism of pyrethrum -mediated repellency remains unknown. This study reports repellency effect of pyrethrum when tested in Aedes aegypti Waco mosquit oes and Anopheles gambiae tested alongside DEET as a positive control. The magnitude of repellency elicited by pyrethrum was not significantly different from the one elicited by DEET in susceptible Aedes and Anopheles mosquitoes. However, repellency of p yrethrum was reduced in pyrethroid resistant PR strain. Experiments using orco 5/16 mosquitoes in Hand in cage assays revealed reduced repellency of pyrethrum in the highest concentration used, and in the low concentrations, repellency was abolished. These behavioral results were consistent with electroant enogram (EAG) recordings of Ae.aegypti where pyrethrum elicited robust EAG responses in Orlando mosquitoes and little EAG response in orco 5/16 mosquitoes 18 Introduction Pyrethrum is a botanical insect icide from the extracts of dry flowers of Tanacetum cinerariifolium ( also known as Chrysanthemum cinerariifolium) . T. cinerariifolium is grown commercially in many parts of the world, particularly in East Africa and Australia, for extraction of pyrethrum, which accumulates in the flower achenes ( Crombie, 1995; Greenhill, 2007 ). Pyrethrum is non -persistent in the environment and possesses low mammalian toxicity. Pyrethrum extract is a mixture of multiple chemical components with the pyrethrin (I and II) as the major component which are responsible for its insecticidal property. In addition to pyrethrins, other components, like sequiterpenes, flavonoids, triperpenols and sterols, are also found in pyrethrum extract (Casida and Quistad, 1995). Pyrethrum, and its synthetic analogs, the pyrethroids, are mostly known for their insecticidal activity (Anadón et al., 2009) . However, pyrethrum is also an insect repellent against various biting arthropods for thousands of years and since 1902, it has been incorporated as a key ingredient in commercial mosquito coils (Moore and Debboun, 2007). For example, its use in the form of a coil against Anopheles gambiae mosquitoes was reported more than five decades ago (Smith and Opudho, 1967). The burning of pyrethrum coils reduced the biting activity and caused mosquitoes to leave the experimental huts (Smith and Opudho, 1967). This study by Smi th and Opudho (1967) concluded that pyrethrum coils may greatly reduce the risk of malaria transmission and highlighted the mechanism of repellency was not known. The use of pyrethrum oil spray on flies attacking cattle has been reported to have repellent effect with variations depending on species of flies (Howell and Fenton., 1944) . Until recent decades, the use of pyrethrum as a repellent in micro doses is still evident (Glynne -Jones, 2001; Hoek et al., 2003) . Whether pyrethrum repellency is olfaction -based remains unknown. Perception of volatile chemicals by insects begins when the volatiles enter the lymph of olfactory sen silla (in antennae and also maxillary palps) via tiny pores and activate olfactory receptor 19 neurons (ORNs) ( Carey and Carlson et al., 2012; Leal, 2013 ). Odorant receptors (ORs) are located on the dendritic surface of olfactory receptor neurons (ORNs) that are housed in olfactory sensilla (Joseph and Carlson, 2015) . Individua l ORNs of basiconic and trichoid sensilla each expresses a single member of the OR family, which confers a characteristic odorant response profile of that neuron (Silbering & Benton, 2010) . Each OR is co -expressed with an obligate olfactory receptor co -receptor ( ORCO ) (Vosshall and Hansson, 2011) , which does not bind odorants by itself, but is essential for odorant perception (Benton et al., 2006; Larsson et al., 2004) . ORs and Orco form a complex functioning as a ligand -gated ion channel (Sato et al., 2008; Wicher et al., 2008; Smart et al., 2008). For decades, DEET has been a widely used repellent to minimize mosquito bites on humans, and it is known to evoke close range repellency through the olfactory system (Bohbot et al., 2011; Debboun et al., 2006; Naters and Carlson, 2006; Pickett et al., 2009; Stanczyk et al., 2013; Stanczyk et al., 2010; Syed and Leal, 2009) . Orco and the OR pathway are necessary for the olfactory effects of DEET on mosquitoes (DeGennaro et al., 2013). This study assessed repellency effects of pyrethrum in comparison with DEET repellency on an Ae. aegypti Orco mutant, and several pyrethroid -susceptible and pyrethroid resistant Aedes aegypti and Anopheles gambiae strains to evaluate the involvement of sodium channels and olfactory receptors in pyrethrum repellency. 20 Materials and methods Mosquitoes In this study, four Aedes aegypti mosquito strains were used; Waco, Puerto Rico (PR), Orlando and an Orco mutant ( orco 5/16 ). Waco is an insecticide -susceptible laboratory strain kindly provided by Dr. Zhiyong Xi at Michigan State University. Orlando is a wild -type Ae. aegypti strain kindly provided by Leslie Vosshall (Rockefeller University ). PR and two orco mutant lines ( orco 5 and orco 16 ) are from BEI Resources, NIAID, NIH. PR is a pyrethroid resistant strain possessing P450 -mediated pyrethroid resistance and three kdr mutations ( Reid et al., 2014 ; unpublished data from the Dong lab). The two orco lines were crossed to generate orco 5/16 mosquitoes for this study. The colonies of Ae .aegypti were reared at 27 oC, at least 60% humidity, at12h:12h light: dark photop eriod in growth chambers. Larvae were fed with liver powder and adults with 10% sucrose solution throughout the rearing period. Fifty females (4 -10 days old and mated) were used for behavioral experiments. Twenty four hours prior to the experiments, the mo squitoes were isolated into a clean cage and were given water only. Six hours before the experiments, the water was removed. Adults of An. gambiae , Kisumu strain were reared at Malaria Alert Center (MAC) insectary at University of Malawi, College of Medic ine (COM). They were reared on 10% sucrose solution and larvae on fish food. Colonies were maintained in growth chambers at approximately 28 0C and 70% relative humidity. Hand in cage behavior assay setup i. Test arena Behavioral assays were mostly carrie d out in summer. The temperature in the testing room ranged from 25 -280C and humidity 40 -70%. In colder weather, the room was conditioned by raising the temperature in the heating system and a humidifier was used to raise the humidity to at least 30%, 21 mak ing sure mosquitoes were active throughout the assay. The room was ventilated using a box fan to remove any background odors prior to the experiments. Test cages (30cmX30cmX30cm from Bio -quip) were cleaned in water with an odorless detergent and dried usi ng a box fan. One to two hours prior to the experiment, the cages were cleaned again with 99% ethanol using cotton wool. The cages were left to dry for another 30 minutes and a paper towel fitting the bottom of the cage was lined and held in place using odorless tape. ii. The glove The study adopted Hand in cage assay from Boyle et al., (2016) with slight modifications. A 5.8 cm by 5 cm window was made on an Ansell Tm sol -vex glove. To hold the window open, a magnet frame was glued to the glove. A piece of polyester mesh treated with test compounds was carefully cut and pla ced on top of the window (Fig 2.1A). On top of the treated mesh, five more magnet frames were stacked. An untreated mesh was place in between the fourth and the fifth magnet frames, which prevented the mosquitoes from getting in conta ct with the treated mesh (Fig 2.1 B -D). iii. Test compounds Pyrethrum from Sigma (Cat# N13151, 30.0% pyrethrin I and 19.9% pyrethrin II) was used. Pyrethrum was diluted volume by v olume using acetone as a carrier solvent. A range of dilutions from 10 -20 to 10 -2 were tested. The range varied by the mosquito species to be tested. In this study, Anopheles gambiae , Kisumu strain was very sensitive to insecticides as such lower concentra tions were used. Knockdown was not observed in Aedes strains, thereby; slightly higher concentrations were used to establish a dose dependent response. 22 Figure 2. 1 Hand in cage assay setup . (A -D) Assemblage of the glove to be used in the Hand in cage assay. (A) First layer of magnet frame that is glued to the glove, as a window frame and a first layer of mesh, which is usually treated with test compounds. It is placed on the top of the secured magnet frame. (B) Another magnet frame that holds the treated mesh in place (C) Extra stacks of four magnet frames and a second layer of mesh (untreated) between the fourth and the fifth magnet frames . (D) Complete set up of the glove, with clips that hold the stacked magnet frames and the treated and untreated mesh to the glove and (E) the complete setup of the Hand in cage assay. The camera on the top of the cage records landing activity of the mosquitoes during an arm visit. A C B D E 23 Experimental design, data acquis ition and analysis i. Experimental design Power analysis is important in determining the statistical power of an experiment. In this study, post -hoc power analysis was conducted using preliminary data in SAS 9.3 software and at least 3 replicates gave a p ower of more than 60%. To increase the power, most of the experiments were repeated at least 5 times, per compound per strain. Cohorts of 50 nulliparous mated and starved females were used as an experimental unit in one cage. ii. Data acquisition The assemb led test glove worn was inserted into the cage (referred to as arm visit he nce forth) for 5 minutes (Fig 2.1 E). For each arm visit, number of mosquitoes landing on the arm was counted starting from the second minute. The experiments recorded a cumulativ e number of mosquitoes that landed on the treatment window at time points 2, 3, 4, 5 minutes were recorded for each of the treatments for each replicate. Percent repellency for each treatment was calculated as; =1 100 Each test arena had 4 arm visits including the control arm. Mosquitoes were rested for at least 30 minutes before another concentration was tested. Each test was repeated for 5 times with different naïve mosquito cohorts. One cohort was used for each experiment and discarded af ter use. Experiments were completely randomized by day. iii. Data analysis Analysis of variance in SAS software (version 9.3) was used to compare mean percentage repellency. The repellency was compared within and between compounds and strains. A full model wa s used to establish the differences in the repellency of the mosquitoes on the treated window: model y=u+compound+ dose +compound*dose+e where y was the response: Percent repellency, u was overall 24 mean, compound was an effect due to compound, dose was an e ffect due to dose and compound*dose , was an interaction effect of dose and compound. Where necessary, separation of means was done using the Bonferroni posttests with alpha equal to 0.05. Results Repellency of pyrethrum and DEET on insecticide -susceptib le Aedes and Anopheles mosquitoes Hand in cage assay results revealed a significant repellent effect of pyrethrum when tested on Aedes aegypti Waco mosquitoes. The repellency increased with increasing concentrations of pyrethrum (Fig.2.2). Repellenc y was also observed from Anopheles mosquitoes, but at much low concentrations (Fig.2.2). For comparison, DEET was tested on Waco and Anopheles mosquitoes as a positive control. A comparison between pyrethrum and DEET repellency on Aedes mosquitoes reve aled no significant differences (Fig. 2.2). Similar results were observed when repellency of pyrethrum and DEET was compared on Anopheles mosquitoes (Fig. 2.2). When concentration effects of pyrethrum and DEET repellency were compared on Aedes and Anophel es mosquitoes; at each of the dilutions used, no significant differences were observed. Statistical comparisons of repellency effect between the two compounds when tested on Anopheles and Aedes mosquitoes are shown in table 2.1. Pyrethrum repellency is red uced in pyrethroid -resistant Aedes mosquitoes Further investigation using resistant Puerto Rico (PR) strain revealed that DEET was a more potent repellent on the resistant mosquitoes than pyrethrum. Statistical results revealed compound effect on the Pue rto Rico strain, with high mean repellency due to DEET (p<0.0001). Detailed comparisons of the test compounds by dos e are shown in Table 2.1, Fig 2.3 25 -4-3-2020406080100Pyrethrum DEET nsnsns(A) log[dilution] Percent repellency -20 -19 -18 020406080100DEET Pyrethrum ns ns nslog[dilution] Percent Repellency (B) Figure 2. 2 No dif ference in the repellency effects of pyrethrum and DEET . (A) Repellency of pyrethrum compared to DEET in Ae.aegypti Waco mosquitoes when tested at three different concentrations in Hand in cage assay. (B) Repellency of pyrethrum compared to DEET in Ano pheles gambiae Kisumu strain tested using Hand in cage assay at three different concentrations (Note Kisumu was tested at very low concentrations because it was too sensitive to pyrethrum and pyrethroids. Higher concentrations caused a knockdown effect). F emales were exclusively tested. Data analyzed using ANOVA. Bonferroni posttests 26 -4-3-2020406080100Pyrethrum DEET ****nslog[dilution] Percent repellecy Figure 2. 3 Difference in the repell ency effects of pyrethrum and DEET on PuertoRico Ae.aegypti mosquitoes. The Peurtorico(PR) strain, has two pyrethroid resistance mechanism (P450 -mediated and kdr mediated). Hand in cage assay was used to test the repellency effect at three different con centrations. Females were exclusively 27 Table 2. 1 Repellency effect of pyrethrum compared to DEET between strains of Aedes aegypti 1 and Anopheles gambiae (Kisumu) 1. Dose Pyrethrum Waco 2 (Mean repellency) DEET Waco 3 ( Mean repellency) Difference t-value P value -4 68.35 67.22 -1.130 0.2290 P > 0.05 -3 85.49 93.99 8.500 1.723 P > 0.05 -2 95.05 97.05 2.004 0.4061 P > 0.05 Dose Pyrethrum PR 4 (Mean repellency) DEET PR 5 (Mean repellency) Difference t-value P value -4 44.19 54.00 9.802 1.559 P > 0.05 -3 56.71 84.88 28.17 4.479 P<0.001 -2 74.90 92.18 17.28 2.747 P < 0.05 Dose Pyrethrum Kisumu 6 (Mean repellency) DEET Kisumu 7 (Mean repellency) Difference t-value P value -20 54.60 55.64 1.038 0.1594 >0.05 -19 76.48 76.10 0.625 0.0960 >0.05 -18 79.00 89.20 10.20 1.567 >0.05 1 4-10 days old, nulliparous female™s sugar sta rved 2 Aedes aegypti ; Waco strain exposed to pyrethrum 3 Aedes aegypti ; Waco strain exposed to DEET 4 Aedes aegypti ; Puerto -Rico strain exposed to Pyrethrum 5 Aedes aegypti ; Puerto -Rico strain exposed t o DEET 6 Anopheles gambiae ; Kisumu strain exposed to pyrethrum 7 Anopheles gambiae ; Kisumu strain exposed to pyrethrum 28 Repellency effect of pyrethrum is Orco -dependent An earlier study has reported lack of DEET repellency in anosmic orco 5/16 mosquitoes where the Orco gene was mutated (Degennaro et al., 2013) , confirming that DEET repellency is mediated by the OR pathway. To determine whether pyrethrum repellency is mediated by the OR pathway, we next ex amined the behavioral response of orco 5/16 mosquitoes to pyrethrum in the Hand in cage assay. At the low concentrations, pyrethrum repellency was abolished in orco 5/16 mosquitoes and a low level of repellency was detected at 10 -2 (Fig. 2.5), whereas both c ompounds elicited robust repellency in wild -type Orlando mosquitoes, from which the orco mutants were generated (Fig. 2.5). These results indicate that pyrethrum repellency is Orco -dependent. Dr. Feng Liu, a postdoc in the Dong lab, conducted electroanten nograph (EAG) recording of antenna of Ae. aegypti in response to pyrethrum. Pyrethrum elicited robust EAG responses in Orlando mosquitoes indicating that mosquitoes can sense the pyrethrum vapor and also suggesting that pyrethrum effectively activate olfac tory receptor neurons. Consistent with the behavioral results, little EAG response was detected in the antenna of orco 5/16 mosquitoes. 29 -4-3-2-20 020406080100log[Pyrethrum dilution] (A) Orco aabPercent repellency EAG Results Figure 2. 4 Reduced repellency effect of p yrethrum in Aedes aegypti orco 5/16 mosquitoes. (A) Showing repellency effect of pyrethrum in orco 5/16 mosquitoes tested at three different concentrations in Hand in cage assay. Repellency was abolished in the low concentrations slightly maintained in the h igh concentration. Data different from each other. (B) Showing Robust EAG response in Orlando strain and reduced response in orco 5/16 <0.05, Students T - (EAG recordings done by Feng Liu). (B) 30 Discussion It is well -established that pyrethrum and pyrethroids target sodium channels for their insecticidal activity (Corbel et al., 2004a; Dong et al., 2014; Du et al., 2011; Du et al., 2006; Li et al., 2012) . Pyrethrum and pyrethroids disrupt sodium channel function by prolonging sodium channel opening, increasing sodium i ons influx resulting in overstimulation of the insect nervous system which eventually leads to death. Although it has been well documented that pyrethrum repels mosquitoes and other insect pests, the mechanism of pyrethrum repellency has not been well unde rstood. The findings from this study established pyrethrum elicits repellency in Ae. aegypti and An. gambiae and indicated that pyrethrum repellency is olfaction -based. DEET is a well -known repellent which has been used for more than five decades (Stanczyk et al., 2010; Vi nauger et al., 2014) in many parts of the world. In this study, our results revealed that repellencies by DEET and pyrethrum are comparable denoting that pyrethrum is a potent repellent. Our study on pyrethroid susceptible Anopheles gambiae mosquitoes of the Kisumu strain confirmed repellency, indicating that pyrethrum as a repellent is not limited to one Aedes mosquito species. These results highlighted the need to explore the use of pyrethrum as a repellent to control mosquito bites. Pyrethrum has been cultivated in Africa, such as Kenya, and other parts of the world, implying access to crude extracts in the places where it is grown, can make a difference in reducing mosquito bites. The instability of insecticidal activity of pyrethrum under heat and l ight conditions have been documented (Glynne -Jones, 2001) . It would be interesting to discove r the length of time at which its repellency can last if it is to be used outdoors as a repellent. In places such as Africa, where pyrethrum is grown and mosquito borne diseases are endemic, pyrethrum crude extracts may be 31 easily accessible. Perhaps, mos t urgent studies should focus on how pyrethrum could be used as a repellent in semi field and field studies. While results in this study point towards pyrethrum being an efficient repellent, some important questions on its residual efficacy as a repellent need to be answered. Although pyrethrum might be readily available as a crude extract, since it is grown in some parts of Africa such as Kenya (Wandahwa et al., 1996), how its long -term use as a repellent might impact entomological parameters in semi field and field trials needs to be investigated further. Decreased repellency of pyrethrum compared to DEET when tested on pyrethroid resistant PR, indicate that pyrethr um potency as a repellent, is more effective against pyrethroid -susceptible mosquitoes. Knowledge on the resistan ce status of mosquito populations in the areas where pyrethrum is to be used as a repellent is vital. It should also be emphasized that not al l mosquito species may respond the same way to pyrethrum repellency. More studies on other mosquito species, to compare the minimum doses of pyrethrum as a repellent are vital. The current study observed a difference in the repellency due to pyrethrum when compared between two vector species, Anopheles gambiae and Aedes aegypti mosquitoes. While the differences in the sensitivity of the two vector species may be attributed to other factors such as behavioral plasticity, significant differences between speci es responses to pyrethrin based repellents has also been reported. A study by Sathantriphop et al. (2014) reported differences in the repe llency effect between vector species of Cx.quinquefasciatus, and An.minimus which exhibited a stronger behavioral response to pyrethroids as well as essential oils compared to Ae.abopictus and Ae.aegypti species. Reduced repellency in the orco mutan ts highlighted the importance of olfactory receptors in pyrethrum repellency, like DEET repellency (Degennaro et al., 2013) . However, detection of pyrethrum repellency in orco mutants at the high concentration suggested that the neurotoxic effect s 32 by pyrethrum on sodium channels could also evoke repellency. Reduced pyrethrum repellency in pyrethroid resistant mosquitoes further supports the involvement of sodium channels in pyrethrum repellency. Potential synergistic effects between these two mec hanisms are worth further investigation in future studies and should benefit the dev elopment of novel strategies in mosquito control, which will help reduce the risk of disease transmission. Conclusion Pyrethrum targets voltage -gated sodium channels which are critical for electrical signaling in the nervous system by prolonging the opening of sodium channels resulting in over -excitation the insect central nervous system (Dong et al, 2014). Here we found that pyrethrum activates olfactory receptor neurons i n mosquito antennae and evokes repellency in both Aedes and Anopheles mosquitoes. The comparable repellencies observed between pyrethrum and DEET in this study depicts pyrethrum as a potent repellent. Furthermore, we showed that pyrethrum repellency was reduced in Orco mutants and pyrethroid resistant mosquitoes, suggesting that pyrethrum repellency is olfaction -based and further enhanced by activation of sodium channels. Although pyrethrum might be readily available as a crude extract, since it is grown in some parts of Africa such as Kenya (Wandahwa et al., 1996) , how its long -term use as a repellent might impact entomological parameters in semi field and field trials remains to be investigated. 33 REFERENCES 34 REFERENCES Anadón, a., Martínez -Larrañaga, M. R., & Martínez, M. a. (2009). Use and abuse of pyrethrins and synthetic pyrethroids in veterinary medicine. 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Acute Olfactory response of Culex mosquitoes to a human -and bird -derived attractant. Www.Pnas.Org/Cgi/Doi/10.1073pnas.0906932106 . Vinauger, C., Lutz, E. K., & Riffell, J. a. (2014). Olfactory learning and memory in the disease vector mosquito, Aedes aegypti. Journal of Experimental Biology , 2321Œ2330. https://doi.org/10.1242/jeb.101279 Wandahwa, F., Ranst, E. Van, & Damme, I. Van. (1996). Pyrethrum ( Chrysanthemum cinerariaefolium Vis .) cultivation in West Kenya : origin , ecological conditions and management, 6690 (96). 37 CHAPTER 3 TOXICODYNAMICS OF TRANSFLUTHRIN ON Aedes aegypti MOSQUITOES 38 Abstract Pyrethroid insecticides act on voltage -gated sodium channels for their insecticidal activity, and have played a cri tical role for effective control of insect pests and disease vectors. Besides their insecticidal activities, pyrethroids possess repellency against mosquitoes and other insects and have been widely used in bed nets, coils, emanators and vaporizer mats to c ombat mosquitoes. More recently, volatile pyrethroids, such as transfluthrin (TF), received more attention and exhibit great potential for global mosquito control (Govella et al., 2015; Ogoma et al., 2012, 2017) . However, details on the mechanism of action of transfluthrin are still elusive. This study reports olfaction - based repellency of transfluthrin in Aedes aegypti and Anopheles gambiae mosquitoes. The h and in cage assay was conducted to evaluate the action of TF on pyrethroid -susceptible and resistant mosquitoes. The main findings are 1) At lower concentrations, TF vapor elicited repellency in Ae aegypti and An gambiae ; 2) At higher concentrations, TF vapor induced knockdown (paralysis) of pyrethroid -susceptible mosquitoes, but not of pyrethroid -resistant mosquitoes carrying kdr mutations; 3) TF repellency was reduced in pyrethroid -resistant mosquitoes carrying kdr mutations; 4) Pretreatment of piperony l butoxide (PBO), an inhibitor of P450s, enhanc ed TF repellency in both susceptible and resistant mosquitoes; 5) TF repellency was significantly reduced in anosmic Orco mosquitoes. These findings demonstrated the complex toxicodynamics of transfluthrin ac tion on the mosquito nervous system: at high concentrations TF induces knockdown by targeting sodium channels, but induces repellency likely by activation of both olfactory receptors and sodium channels at sublethal concentrations. 39 Introduction Pyrethro ids have been highly featured in control of mosq uito borne diseases, either as aerosol sprays, indoor residual prays (IRS) and mosquito coils as repellents. It is well -established that pyrethroid insecticides act on voltage -gated sodium channels for thei r insecticidal activity. The success of pyrethroids in insect control, however, has been greatly affected by the development of pyrethroid resistance . Different mechanisms of resistance development, including target site resistance, kdr, and P450 -mediated resistanc e have been reported in several studies (Feyereisen, 1995; Hemingway et al., 2004; Liu, 2012; Reid et al., 2014; Salgado et al., 1983) . A number of studies have reported the implications of pyrethroid resistance in control o f insect vectors. (Matowo et al., 2014; Nardini et al., 2012; Strode et al., 2014) In transmission dynamics, the biting rate of a mosquito is key to disea se transmission among other important factors. For vectors, su ch as mosquitoes to locate a host for a blood meal, cues such as carbon dioxide and heat are important (Dekker and Carde, 2011 ; Lacey and Cardé, 2012;Webster et al., 2015) . Although pyrethroids have been traditionally understood as contact insecticides, in recent years, volatile pyrethroids, such as transfluthrin and metofluthrin, are main active ingredients of widely used insect repellent products including mosquito coil s, emanators and vaporizer mats. The repellency of pyrethroids is now being evaluated for human disease vector control and has the potential to become an important component of future malaria control programs in Africa (Hill et al., 2014 ; Kawada et al., 2005 ; Ogoma et al., 2014 ; Ogoma et al., 2012;2017; Reddy et al., 2011 ; Sugano and Ishiwatari, 2011 ). For example, TF has been reported to have a 90% protective effica cy when impregnated in hessian sacks over a period of 6 months in a semifield trial (Ogoma et al., 2012) . Reduction of out door bites with a 99% for Anopheles and 92% for Culex was evident when transfluthrin was used outdoors (Govella et al., 2015) . A recent behavioral stu dy reported reduced pyrethroid repellency in pyrethroid resistant Aedes mosquitoes 40 carrying kd r mutations (Wagman et al. , 2015). However, the me chanism(s) underlying the repell ency elicited by these compounds is largely unknown. In this study, we showed that transfluthrin evoked olfactory responses from the antennae of Ae. aegypti mosquitoes and elicited repellency. We further evaluated TF repe llency using two types of mutant mosquitoes: orco mutants and kdr mosquitoes. Our study established unique dual actions of TF on ORs and sodium channels as the underlying mechanism of TF repellency. We also showed that pretreatment of a P450 inhibitor pipe ronyl butoxide (PBO) enhanced both repellency and vapor toxicity of TF. Our study established a new paradigm for the understanding of the modes of action of volatile pyrethroids in mosquito control. Materials and Methods Mosquitoes In this study, six Aede s aegypti mosquito strains were used; Rockefeller (Rock), Isokdr, Waco, Puerto Rico (PR), Orlando and an Orco mutant ( orco 5/16 ). Rock and Isokdr were provided by Jeff Scott™s laboratory at Cornell University. Isokdr is highly resistant to pyrethroids poss essing two kdr mutations in the sodium channel and Rock is pyrethroid -susceptible; and they are isogenic (Smith et al., 2018). Waco is an insecticide -susceptible laboratory strain kindly provided by Dr. Zhiyong Xi at Michigan State University. Orlando is a wild -type Ae. aegypti strain kindly provided by Leslie Vosshall (Rockefeller University ). PR and two orco mutant lines ( orco 5 and orco 16 ) are from BEI Resources, NIAID, NIH. PR is a pyrethroid resistant strain possessing P450 -mediated pyrethroid resistan ce and three kdr mutations ( Reid et al., 2014 ; unpublishe d data from the Dong lab). The two orco lines were crossed to generate orco 5/16 mosquitoes for this study. The colonies of Ae .aegypti were reared at 27 oC, at least 60% humidity, at12h:12h light: dark photoperiod in growth chambers. Larvae were fed with li ver powder and adults with 10% sucrose 41 solution throughout the rearing period. Fifty females (4 -10 days old and mated) were used for behavioral experiments. Twenty four hours prior to the experiments, the mosquitoes were isolated into a clean cage and were given water only. Six hours before the experiments, the water was removed. Adults of An. gambiae , Kisumu strain were reared at Malaria Alert Center (MAC) insectary at University of Malawi, College of Medicine (COM). They were reared on 10% sucrose solut ion and larvae on fish food. Colonies were maintained in growth chambers at approximately 28 0C and 70% relative humidity. Test compound and arm visits Transfluthrin (95% purity) kindly provided by Dr. Kamal Chauhan (USDA), was used in this study . The compound was prepared volume by volume (v/v) with acetone as a carrier solvent. In a glass petri dish, 450µl was applied to white rectangular polyester netting on the treatment mesh (se e assembled glove design, Fig 2.1). Carrier solvent without the test compound was tested first, and the rest of the concentrations were tested from lowest to the highest concentration to avoid contamination. A range of concentrations from 10 -8 to 10 -2 was used for repellency assays using transfluthrin on PR and Waco. The assembled test glove worn was inserted into the arena (referred to as arm visit hence forth) for 5 minutes. Landing response of mosquitoes was recorded for 5 minutes. Each test arena had 4 arm visits including the control arm. Mosquitoes were rested for at least 30minutes before another concentration was tested. Each test was repeated for 5 times with different naïve mosquito cohorts. One cohort was used for each experiment and discarded after use. Experiments were completely randomized by day. 42 Hand in cage assay with cytochrome P450 inhibited mosquitoes using PBO Following slightly modified methods by (Reid et al., 2014) a nonlethal dose of 1ug was applied on the dorsal side of the thorax of the mosquitoes. Very minimum chilling was used in the mosquito preparation to avoid alte ring the behavior of the mosquitoes before the behavioral assay was conducted. Very active, ready to bite female mosquitoes starved on water were gently aspirated from the holding cage and chilled for 1 minute and then transferred to a 4 -degree Celsius gla ss petri dish for immobilization and 1ug of PBO was applied using a Hamilton syringe. Mosquitoes were treated in batches of 10 for efficient treatment and to reduce the cold treatment time. At least 50 PBO treated mosquitoes were transferred into a behav ioral assay arena (a 30x30x30 bio quip metal cage) and the cage was transferred into a behavioral experiment room, with at least 30% relative humidity and temperatures of 28 degrees Celsius. The mosquitoes were left in the behavioral test room for an hour to let the PBO to take effect as well as for the mosquitoes to acclimatize. The mosquitoes were then tested in hand in cage assay using transfluthrin and landing rates which were later transformed to percent repellency as previously illustrated in (Equa tion 1, chapter 2) were recorded. The procedure was repeated 5 times with di fferent cohorts of mosquitoes. Hand in cage knockdown assay Using hand in cage assay, mosquitoes were exposed to higher concentrations of transfluthrin to observe the knockdown e ffect. This assay used dilutions of 10 -5 to 10 -3, and recorded the number of mosquitoes knocked down over a period of 60 minutes. After 60 minutes the mosquitoes were provided with 10% sucrose and mortality was recorded after 24 hours. A similar procedure was followed with mosquitoes that were pretreated with PBO. 43 Results Transfluthrin (TF) repellency in susceptible mosquito strains To evaluate whether mosquitoes perceive TF through the olfactory system, we first conducted electroantennagram (EAG) reco rdings from the antennae of female Aedes mosquitoes. EAG signals were detected in response to TF vapor (Fig.3.1A), indicating that Aedes mosquitoes can sense the vapor of TF. We then conducted the hand in cage assays using susceptible Aedes mosquitoes, whi ch reveal ed transfluthrin (TF) elicited repellency effect in Waco and Rock mosquitoes (Fig. 3.1 A -B). For both mosquito strains, the repellency of TF increased with increasing concentration (p <0.001). To assess whether TF repellency is also in other mosq uitoes, the experiments were repeated using Anopheles gambiae mos quitoes of the Kisumu strain . Repellency of TF in Kisumu mosquitoes was observed at the concentration as low as 10 -20 (Fig.3.2). Unlike in Aedes mosquitoes where an overt dose response curv e was observed, in Anopheles mosquitoes, the dose response curve plateaued at a low concentration (10 -19 ). 44 Figure 3. 1 Transfluthrin repel susceptible Aedes mosquitoes . (A) Showing EAG signal from fema le mosquito antenna using TF and ACTF (Acetransflutrin), a TF -like structure shown in chapter 1 (Fig. 1.1). (B) Showing repellency of TF in Waco in tested in hand in cage assay at three different concentrations (C) Showing repellency of TF in Rock strain tested in hand in cage assay at three different concentrations. Female mosquitoes (4 -8 days) tested exclusively (EAG recordings done by Feng Liu ). (A) -8-7-6020406080100(B) log[TF dilution] Percent repellency -8-7-6020406080100(C) log[TFdilution] Percent repellency 45 -20 -19 -18 020406080100log[TFdilution] Percent repellency Figure 3. 2 Transfluthrin repel susceptible Anop heles gambiae, Kisumu strain mosquitoes. Female mosquitoes (4 -6 days old) tested exclusively in Hand in cage assay at three different concentrations. For Anopheles gambiae , TF was tested at very low concentrations because of its high sensitivity to pyreth roids. 46 Reduced transfluthrin repellency in resistant Aedes strains In the hand in cage assay, we observed that the landing of pyrethroid resistant PR and Isokdr mosquitoes when the second mesh close to the hand was not treated with an y chemicals, was not different from those of the two susceptible strains (Waco and Rock) indicating that the kdr mutations did not alter host -finding behavior. TF repellency was significantly reduced in both PR and Isokdr mosquitoes at all three concentr ation tested (Fig. 3.3). Hand in cage experiments using DEET, revealed no significant differences between the susceptible and resistance strains when compared at all the concentrations used (Fig. 3.4, Table 3.1). 47 -8-7-6020406080100Puertorico Waco ns***(A) log[TF dilution] Percent repellency -8-7-6020406080100Isokdr Rock ********(B) log[TFdilution] Percent repellency Figure 3. 3 Transfluthrin repellency is reduced in resistant mosquito strains . (A) Showing repellency effect of TF in PeurtoRico (PR) and Waco tested in Hand in cage assay at three different concentrations (B) Showing repellency effect of TF, in Isokdr and Rock mosquitoes tested in Hand in cage assay. Data analyzed using two way ANOVA. Bonferroni posttests ( 48 Table 3. 1 Repellency effect of TF compared between strains of Aedes aegypti 1 mosquitoes before and after PBO pre -treatment. Dose PRTF 2 (Mean repe llency) Waco TF 3 ( Mean repellency) Difference t-value P value 5 -8 4.0 18.6 -14.6 -1.73 P>0.05 -7 19.0 52.0 -33.0 -3.92 P<0.001 -6 41.8 68.8 -27.0 -3.21 P<0.001 Dose Isokdr 4 (Mean repellency) Rock 4 (Mean Repellency) Difference t-value P value -8 23.40 47.68 24.28 3.554 P<0.01 -7 43.60 77.07 33.47 4.898 P<0.001 -6 55.40 88.32 32.92 4.819 P<0.001 1 4-10 days old, nulliparous female™s sugar starved 2 3 sed to transfluthrin, without PBO pretreatment 4 5Analysis of Variance(ANOVA) P<0.01, 49 -4-3-2020406080100PuertoRico Waco nsnsnslog[DEET dilution] Percent repellency (A) -15 -12 -9-6-3-1020406080100Isokdr Rock nsnsnsnsnsnslog[DEET dilution] Percent repellecy (B) Figure 3. 4 Repellency of DEET is not reduced in pyrethroid resistant mosquito strains . (A) Showing repellency effect of DEET on PeurtoRico and Waco tested in Hand in cage assay at three different concentrations (B) Showing repellency effect of DEET, in Isokdr and Rock mosquitoes tested in Hand in cage assay at six different concentrations. Data analyzed using ANOVA. Bonferroni posttests ( ; 50 Table 3. 2 Repellency effect of DEET compared between strains of Aedes aegypti 1 mosquitoes before and after PBO pre -treatment Dose Rock DEET 2 (Mean repellency) Isokdr DEET 3 ( Mean repellency) Difference t-value P value 6 -15 43.39 41.79 -1.600 0.2947 P > 0.05 -12 54.18 54.58 0.4045 0.07451 P > 0.05 -9 65.31 70.27 4.961 0.9137 P > 0.05 -6 65.18 70.77 5.592 1.030 P > 0.05 -3 79.94 79.40 -0.5375 0.09901 P > 0.05 -1 86.13 82.89 -3.239 0.5966 P > 0.05 Dose Waco DEET 4 (Mean repelle ncy) PR DEET 5 ( Mean repellency) Difference t-value P value -4 54.00 67.22 13.22 2.568 P > 0.05 -3 84.88 93.99 9.112 1.770 P > 0.05 -2 92.18 97.05 4.874 0.9468 P > 0.05 1 4-10 days old, nulliparous female™s sugar starved 2 xposed to DEET 3 4 5 6 Analysis of Variance(ANOVA) 51 Repellency e ffect of transfluthrin is Orco -dependent To determine whether the OR pathway is involved in the olfactory response to TF in Ae. aegypti mosquitoes, we examined the response of orco 5/16 mutant mosquitoes from DeGannaro et al. (2013) and a wildtype strain , Orlando, which is isogenic to orco 5/16 in the hand -in-cage assay. As shown in Fig. 3.5, like in Waco and Rock, significant levels of TF repellency were observed in Orlando mosquitoes, but TF repellency was significantly reduced in orco 5/16 mutants. Abou t 30% of repellency was observed in orco 5/16 mosquitoes at the highest concentration of TF tested. -15 -12 -9-6020406080100Orco Orlando ************log[TF dilution] Percent repellecy Figure 3. 5 Transfluthrin repellency is reduced in Aedes orco 5/16 and it is significantly high in Orlando Aedes aegypti strain. Repellency was tested at four different concentrations in Hand in cage P<0.001 52 Pre -treatment of piperonyl butoxide (PBO) enhanced repellency elicited by transfluthrin As shown earlier, both PR and Isokdr displayed a reduced repellency to transfluthrin compared to the pyrethroid -susceptible st rains (Fig.3.3). The Isokdr strain possesses only the kdr -mediated resistance and lacks P450 -mediated resistance mechanism. The reduced TF repellency in Isokdr is likely due to the kdr mutations in the sodium channel. However, the PR strain has both P450 mediated and kdr resistance mechanisms. To examine the role of P450 -mediated resistance in TF repellency, a topical application of 1µg of PBO was used to pretreat the mosquitoes before they were used in the hand in cage assay to inhibit the activity of P450 s. The PBO pretreatment resulted into enhanced repellency behavior in both Waco and PR mosquitoes. However, TF repellency maintained lower in PR mosquitoes pretreated with PBO compared to Waco mosquitoes (Fig 3.6, Table 3.3). These results suggest that both the P450 -mediated and kdr mechanisms contributed to reduced repellency in PR mosquitoes. To confirm that PBO itself did not affect the landing response of mosquitoes during an arm visit, untreated control was compared in PBO and non -PBO treated mosqui toes and no statistical d ifferences were observed (Fig 3.7) 53 Table 3. 3 Repellency effect of TF compared within and between strains before and after PBO pre -treatment of Aedes aegypti 1 mosquitoes Dose PRTF 2 (Mean repelle ncy) PR (TF+PBO) 3 ( Mean repellency) Difference t-value P value 5 -8 4.0 52.16 -19.5 -3.03 0.0058 -7 19.0 75.60 -14.5 -2.24 0.0343 -6 41.8 88.75 -29.8 -4.61 0.0001 Dose Waco TF 4 (Mean repellency) Waco(TF+PBO) 4 (Mean Repellency) Difference t-value P value -8 18.6 52.16 -33.6 -3.85 0.0008 -7 52.0 75.60 -23.6 -2.71 0.0123 -6 68.8 88.75 -19.9 -2.29 0.0312 Dose PR(TF+PBO )4 (Mean repellency) Waco(TF+PBO) 4 (Mean Repellency) Difference t-value P value -8 23.55 52.16 -28.60 -4.18 0.0194 -7 33.50 75.60 42.10 -6.15 0.0001 -6 71.59 88.75 17.16 -2.50 0.0003 1 4-10 days old, nulliparous female™s sugar starved 2 3 , without PBO pretreatment 4 5Analysis of Variance(ANOVA) P<0.01, 54 -8-7-6020406080100Puertorico PuertoricoPBO *ns***(A) log[TF dilution] Percent repellency -8-7-6020406080100WacoPBO PRPBO ******(B) log[TF dilution] Percent repellency Figure 3. 6 Transfluthrin repellency is enhanced when Aedes aegypti mosquitoes are pretreated with PBO . (A) Showing repellency effect of TF on PeurtoRico (PR) pretreated with PBO and without topical application of PBO tested in Hand in cage assay at 3 different concentrations (B) Repellency effect of TF, on Waco and PR mosquitoes when topically treated with PBO before the behavioral experiment. Least 55 PRNo PBO PBO Control 02040600.188 Landing response Waco No PBO PBO Control 02040600.562 Landing response Figure 3. 7 No difference in the landing response of PBO pretreated and untreated Aedes aegypti whe n exposed to untreated (control). Female mosquitoes exclusively tested; PBO -pretreatment conducted 60 minutes prior to experiment. Five cohorts of mosquitoes tested for each treatment, (unpaired two tailed t - 56 Pre -treatment of PBO enhanced mosquito knockdown by transfluthrin. Knockdown of Waco mosquitoes by TF was observed at the concentration of as low as 10 -5 in the hand in cage assay indicated T F vapor entered into mosquitoes interacting with sodium channels in the nervous system. At the highest concentration tested, 10-3, most of the 50 mosquitoes were knocked down but recovered within sixty minutes of observation (Fig 3.8). Therefore, the stud y sought to evaluate the effect of pretreatment with PBO on knockdown effect of TF on Waco mosquitoes. As shown in Fig. 3.8, pretreatment of Waco mosquitoes with PBO an hour before the hand -in-cage assay increased the knockdown effect by TF at 10 -4 and 10 -3. The knocked down mosquitoes did not recover at the end of the assay. The recovery from knockdown of the mosquitoes without PBO pretreatment (Fig. 3.8 A) is likely due to rapid metabolism of TF by P450s. 57 2345678910203040506001020304050-5TF -4TF -3TF (A) Exposure Time in minutes # of mosquitoes knocked down 2345678910203040506001020304050-4TF -4TFPBO (B) Exposure Time in minutes #of mosquitoes knocked down 2345678910203040506001020304050-3TF -3TF+PBO (C) Exposure Time in Minutes # of mosquitoes knockedown Figure 3. 8 Transfluthrin knockdown effect is enhanced in mosquitoes pretreated with PBO (A) Showing knockdown effect of TF when cohorts of 50 female Aedes aegypti Waco strain was exposed to TF in hand in cage assay for five minutes (shaded portion) to TF dilutions of 10 -5, 10 -4, 10 -3 comparison of Waco pretreated with PBO and without when exposed to 10 -4 TF. (C) Showing a comparison of Waco pretreated with PBO and without when exposed to 10 -3 TF. 58 Table 3. 4 Knockdown effect on Aedes aegypti 1 pretreated with PBO when exposed to 10 -3 TF dilution in hand in cage assay. + TF2 TF+PBO 3 Difference 95% CI of diff. t P value 4 Summary 2 8 22.8 14.8 2.509 to 27.09 3.584 P<0.01 ** 3 19 30.2 11.2 -1.091 to 23.49 2.712 P > 0.05 ns 4 30.6 39.4 8.8 -3.491 to 21.09 2.131 P > 0.05 ns 5 35.8 43.6 7.8 -4.491 to 20.09 1.889 P > 0.05 ns 6 41.6 44.6 3 -9.291 to 15.29 0.7265 P > 0.05 ns 7 42.6 45 2.4 -9.891 to 14. 69 0.5812 P > 0.05 ns 8 45 45.2 0.2 -12.09 to 12.49 0.04844 P > 0.05 ns 9 46 45 -1 -13.29 to 11.29 0.2422 P > 0.05 ns 10 44.8 44.8 0 -12.29 to 12.29 0 P > 0.05 ns 20 28.8 41.2 12.4 0.1089 to 24.69 3.003 P < 0.05 * 30 20.6 38.2 17.6 5.309 to 29.89 4.26 2 P<0.001 *** 40 15.4 35.4 20 7.709 to 32.29 4.844 P<0.001 *** 50 13.8 34.6 20.8 8.509 to 33.09 5.037 P<0.001 *** 60 11.2 34.6 23.4 11.11 to 35.69 5.667 P<0.001 *** 1 4-10 days old, nulliparous females sugar starved 2 luthrin, without PBO pretreatment 3 4 59 Table 3. 5 Knockdown effec t on Aedes aegypti 1 pretreated with PBO when exposed to 10 -4 TF dilution in hand in cage assay. Treatment 4TF TF+PBO Difference 95% CI of diff. t P value 4 Summary 2 5.4 9.2 3.8 -16.30 to 23.90 0.5628 P > 0.05 ns 3 10.6 16.4 5.8 -14.30 to 25.90 0.8591 P > 0.05 ns 4 14.6 22.8 8.2 -11.90 to 28.30 1.215 P > 0.05 ns 5 21 31.8 10.8 -9.297 to 30.90 1.6 P > 0.05 ns 6 22.4 33.8 11.4 -8.697 to 31.50 1.688 P > 0.05 ns 7 24.8 35.6 10.8 -9.297 to 30.90 1.6 P > 0.05 ns 8 23 35.2 12.2 -7.897 to 32.30 1.807 P > 0.05 ns 9 23 37.2 14.2 -5.897 to 34.30 2.103 P > 0.05 ns 10 21.2 33.8 12.6 -7.497 to 32.70 1.866 P > 0.05 ns 20 17 34 17 -3.097 to 37.10 2.518 P > 0.05 ns 30 10 35.2 25.2 5.103 to 45.30 3.732 P<0.01 ** 40 7.2 32.6 25.4 5.303 to 45.50 3.762 P<0.01 ** 50 5.6 33.4 27.8 7.703 to 47.90 4.118 P<0.01 ** 60 5.4 32.8 27.4 7.303 to 47.50 4.058 P<0.01 ** 1 4-10 days old, nulliparous females sugar starved 2 3 ansfluthrin after PBO pretreatment 4 0.01, 60 Discussion Pyrethroids are known to disrupt sodium channel function by prolonging its opening, increasing sodium ions in flux, which causes the knockdown effect, and may lead to death. Recently, studies (Xu et al., unpublished data) have confirmed the ability of pyrethroids to evoke olfactory response in Drosophila and mosquitoes. Evidence on transfluthrin™s ability to int erfere with mosquito biting behavior of different mosquito species in field and semi field trials have been reported (Govella et al., 2015; Ogoma et al., 2017; Ogoma, Lorenz, et al., 2014; Ogoma, Ngonyani, et al., 2014) Despite pyrethroid success, resistance in arthropods is a common phenomenon. Mutations in different arthropod species associated with pyrethroid resistance, have been reviewed in Dong et al. (2014) . Studies reporting P450 mediated pyrethroid resistance are not rare (Liu, 2012; Martin et al., 2 003; Ranson et al., 2011) . Insensitivity of insects to volatile repellent pyrethroid; transfluthrin, was recently reported in Aedes aegypti as a heritable trait (Wagman et al., 2015) . In this study we demonstrated the impact of both kdr and P450 mediated pyrethroid resistance on the toxicodynamics of transfluthrin action as a r epellent and as a knokdown agent in Aedes aegypti mosquitoes. Higher repellency levels in Waco and reduced repellency in resistant PR denote pyrethroid repellency efficacy is dependent on susceptibility of the mosquitoes to pyrethroids in general and these results concur with the finding from Wagman et al. (2015). Transfluthrin seem to have a duo action on sodium channels and olfactory receptors. It activates olfactory receptors to transduce action potentials, which are propagated and processed in the hig her brain centers to evoke repellency behavior in the mosquitoes. At the same time, in its vapor state, it diffuse s into an insect body and bind to the Na channels, through which action potentials may be propagated to the higher brain centers to induce kno ckdown effect. The duo action of a single compound, transfluthrin possibly leading to a synergistic propagation of action 61 potentials through the olfactory pathway and sodium channel prolonged opening leading to enhanced repellency behavior, seem to be dep endent on the susceptibility of mosquitoes to pyrethroids. Hence the observed low repellency levels in mosquitoes with kdr mutations when exposed to transfluthrin in the hand in cage assay could be attributed to the lack of synergy in between the signal t ransductions caused by the olfactory pathway and prolonged opening of sodium channels. Because of the mutations in the sodium channel, most likely, the signals that are propagated to the higher brain centers in the mosquito are only mediated through the ol factory pathway. Disruption of motor -neuron activity by transfluthrin has been reported (Wagman et al., 2015). This attests that resultant insect behavioral stimuli, termed as TF repellency is not exclusively due to the olfactory pathway. An increased re pellency in both strains after pretreatment with PBO, denote the important role that P450s may play in TF repellency. The P450s metabolize TF and therefore its availability is reduced for its action on sodium channels and olfactory receptors. The inhibitio n of P450 activity by PBO in the PR and Waco strains allowed us to elucidate the impact of P450 mediated pyrethroid resistance on transfluthrin repellency and toxicodynamics. Although contact bioassays using transfluthrin in the presence of PBO showed no e nhanced toxicity in mosquitoes with P450 mediated mechanisms (Horstmann and Sonneck, 2016) , here we report an enhanced vapor toxicity and repellency of TF on mosquitoes pretreated with PBO (see Fig 3.6). Enhanced repellency in PBO pretreated mosquitoe s could be explained by the inhibition of P450s in the insects body to increase the availability of TF for its interaction with sodium channels and` olfactory receptors which together with the olfactory responses elicited repellency behavior. Besides as an inhibitor of P450s, PBO has been suggested to enhance penetration of insecticides (Kasai et al., 2014) . Therefore, alternatively, enhanced TF repellency could be due to enhanced penetration of transfluthrin into the insect body (Kasai et al., 2014) . While, detailed studies by (Zhu et al., 2010) 62 have highlighted on a brain specific cytochrome P450 responsible for detoxification of deltamethrin in Tribolium insect species, others (Maïbèche -Coisne et al., 2 004; Pottier et al., 2012) have demonstrated a key role of P450s as olfactory degrading enzymes. Reported results attest the importance of P450s and kdr in TF toxicodynamics when used as a vapor and its implications in insect vector control. The increase in the knockdown effect of TF with increase in concentration, as well as repellency effect when used at sub lethal doses, denote the repel and kill effect that TF may have on mosquitoes. The repellency due to TF may decrease the mosquito biting rate. Biti ng rate is important as it somewhat relates to the mosquito densities which are key in vectoral capacity (Brady et al., 2016) . Although it was beyond the scope of this study to assess the vectoral capacity parameters that may be affected by the use of transfluthrin, these results certainly present a possibility of TF significantly affecting the transmission parameters of vector borne diseases. Conclusi on Collectively, this study reports repellency of TF against mosquitoes. TF repellency was almost abolished in Orco mutant mosquitoes, indicating olfaction -based TF repellency. 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Proceedings of the National Academy of Sciences of the United States of America , 107(19), 8557 Œ62. https://doi.org/10.1073/pnas.1000 059107 67 CHAPTER 4 REPELLENCY EFFECT OF PermaNet 2.0 AND Olyset NET ON Anopheles gambiae AND Aedes aegypti 68 Abstract The use of pyrethroid long lasting insecticidal nets (LLINs) remains one of the major ways in the control of mosquito borne diseases. The use of LLINs has registered tremendous success in reducing vector host contact although this has been limited due to development of resistance in some areas. Most studies on the mechanism of action of LLINs have reported excito -repellent effect and contact toxicity on mosquitoes. This present study reported olfactory based spatial repellency of deltamethrin treated net (PermaNet 2.0) and permethrin treated net ( Olyset) against mosquitoes. Specifically, in the Hand in cage assay, we observed 1) repellency effect of PermaNet 2.0 on pyrethroid -susceptible Ae. aegypti Rock and Waco mosquitoes, and An. gambiae Kisumu mosquitoes; 2) reduced repellency of PermaNet 2.0 against Ae. aegypti pyrethroid -resistant Puerto Rico (PR) and Isokdr and orco 5/16 mosquitoes; and 3) repellency effect of the Olyset net on An. gambiae Kisumu mosquitoes, but not on Ae. aegypti mosquitoes even though repellency was observed from permethrin -treated mesh. Furthermore, we found that permethrin and del tamethrin elicited electroantennogram (EAG) responses from Ae. aegypti mosquitoes, but not from anosmic orco 5/16 mosquitoes, providing the olfactory basis o f the repellency of LLIN s. 69 Introduction Among other known malaria vectors, Anopheles gamb iae is considered one of most important (Riabinina et al., 2016; Takken et al., 2001; World Health Organization(WHO), 2017) . It transmits a malaria parasite plasmodium falciparium which acco unts for 99% of all the malaria cases in the world. In the year 2016, 216 million cases of malaria occurred worldwide, causing estimated deaths of around 455000 (WHO, 2017) . Insecticide treated nets (LLINs) remain one of the most important tools in malaria control (Pennetier et al., 2013; Takken, 2002; Vantaux et al., 2014; World Health Organization, 2017) . Pyrethro ids are the only class of insecticides that have been approved to be used in LLINs (Ranson et al., 2011) . Most commonly used pyrethroids in LLINs include; -cypermethrin and deltamethrin (Mosha et al., 2008) . Commonly used LLINs include permethrin LLIN also referred to as Olyset Net and deltamethrin LLIN also referred to as PermaNet 2.0 (Guessan et al., 2001; Soleimani -Ahmadi et al., 2012) . The use of LLINs is popular but not limited to Anopheles mosquito control. They have also been used in the control of Aedes mosquitoes. For example, t he use of LLINs in the control of dengue vectors has been reported in Haiti (Lenhart et al., 2008) Although LLINs remain important in mosquito control, reduced efficacy in resistant mosquito populations has been reported (Enayati and Hemingway, 2006; Guessan et al., 2001; Thiam et al., 2012; Toé et al., 2014) . In the aforementioned studi es , there was reduced toxicity in resistant mosquitoes compared to pyrethroid susceptible ones. In addition, the need to increase the concentration of pyrethroids in insecticide treated nets to effectively reduce the host -vector contact in mosquitoes with kdr has been reported (Corbel et al., 2004 ). Moreover, the impact of agricultural insecticide use in cotton and rice growing areas has been emphasized as one of the causes in reduced 70 efficacy of the insecticide treated nets in agrarian societies (Bigoga e t al., 2012; Diabate et al., 2002; Fane et al., 2012; Hien et al., 2017) . To curb the reduced efficacy due to resistance development in mosquitoes, some LLINs have been incorporated with a synergist PBO. A study assessing the efficacy of PermaNet 2.0 (w ithout PBO) and PermaNet 3.0 (with PBO) revealed increased efficacy of PermaNet 3.0 on resistant mosquitoes (Koudou et al., 2011) . Similar findings were reported when PermaNet 2.0 and 3.0 were compared in an experimental hut trial in an earlier study (N™Guessan et al., 2010) . Similarly, reduced efficacy of Olyset n ets in mosquitoes with pyrethroid resistance and evidence on increased efficacy of the Olyset plus, a permethrin treated net with PBO incorporated has been reported (Guessan et al., 2001; Pennetie r et al., 2013) . Research on the mode of action of LLINs has mainly focused on contact toxicity of the nets (Ochomo et al., 2013) . Analysis of differ ential behavioral responses of Anopheles gambiae mosquitoes revealed a reduced frequency of contact of mosquitoes with pyrethroid treated nets but recorded an increased flying and sitting behavior (Siegert et al. , 2009). The observed behaviors were attributed t o the neurotoxic effect due to contact with the pyrethroid treated nets. Similar results have been reported by Kawada et al., (2014). A study, examining the leng th of time a mosquito spends in physical contact with the insecticide treated net and untreated one using infrared tracking system, revealed that mosquitoes spent less time on LLINs compared to untreated control ( Parker et al., 2015) . In assessing the interaction of the mosquitoes with the bed net overtime, Parker et al., (2015) observed less contact of the mosquitoes with the LLINs compare d to untreated control and concluded that the LLINs did not elicit repellency in the mosquitoes prior to physical contact, suggesting that LLINs mechanism of action is dependent on contact of the mosquito with the net . Different studies have attributed exi ting behavior, dete rrence and or contact irritancy (also referred to as contact disengagement) to use of permethrin and deltamethrin treated nets and 71 surfaces (Chareonviriyaphap et al., 2004; Ogoma, Lorenz, et al., 2014) . And, investigations on distribution of Anopheles gambiae mosquitoes due to the effect of permethrin treated nets, revealed a decrease in the mosquito densities with decreasing distan ce from the intervention areas (Gimnig et al., 2003) . Another study also r eported a spill over insecticide treated net effect to houses t hat were not using the bed nets in Haiti (Lenhart et al., 2008) . Although the study did n ot clearly evaluate the mechanism of the bednet action, the significant decrease in the dengue vectors was attributed to the deltamethrin treated net intervention . In addition to the reported spillover effect due to insecticide treated nets, exiting behavi or of mosquitoes from houses containing insecticide treated nets has been reported (Miller et al., 1991, Mosha et al., 2008). A study conducted in India, concur with earlier mentioned studies; that evaluation of the efficacy of Olyset plus (a permethrin treated net with PBO incorporated) showed a significant reduction in the house entry, a decline in the blood feeding rates in experimental hut and the deterrence effect of permethrin treated nets on Anophelesfluviatile was evident (Gunasekaran et al., 2016) . Moreover, the use of deltamethrin treated nets has been documented to significantly reduce host seeking by Culex mosquitoes when used around cattle enclosures (Maia et al., 2012) . The present study , we hypothesized that LLINs repellen cy ( non -contact disengagement) in different Anopheles and Aedes mosquito strains. In this study, we used the Hand in cage assay to examine potential repellency effect of Olyset and Permanet 2.0 on An.gambiae (the Kisumu strain) and Aedes aegypti (Waco, R ock, Isokdr and orco strains). 72 Materials and Methods Mosquitoes In this study, five Aedes aegypti mosquito strains were used; Rockefeller (Rock), I sokdr, Waco, Puerto Rico (PR), and an Orco mutant ( orco 5/16 ). Rock and Isokdr were provided by Jeff Scott™s laboratory at Cornell University. Isokdr is highly resistant to pyrethroids possessing two kdr mutations in the sodium channel and Rock is pyrethroid -susceptible; and they are isogenic (Smith et al., 2018). Waco is an insecticide -susceptible labora tory strain kindly provided by Dr. Zhiyong Xi at Michigan State University. Orlando is a wild -type Ae. aegypti strain kindly provided by Leslie Vosshall (Rockefeller University ). PR and two orco mutant lines ( orco 5 and orco 16 ) are from BEI Resources, NI AID, NIH. PR is a pyrethroid resistant strain possessing P450 -mediated pyrethroid resistance and three kdr mutations ( Reid et al., 2014 ; unpublished data from the Dong lab). The two orco lines were crossed to generate orco 5/16 mosquitoes for this study. The colonies of Ae. aegypti were reared at 27 oC, at least 60% humidity , at12h:12h light: dark photoperiod in growth chambers. Larvae were fed with liver powder and adults with 10% sucrose solution throughout the rearing period. Fifty females (4 -10 days old and mated) were used for behavioral experiments. Twenty four hours pr ior to the experiments, the mosquitoes were isolated into a clean cage and were given water only. Six hours before the experiments, the water was removed. Adults of An. gambiae , Kisumu strain were reared at Malaria Alert Center (MAC) insectary at Universit y of Malawi, College of Medicine (COM). They were reared on 10% sucrose solution and larvae on fish food. Colonies were maintained in growth chambers at approximately 28 0C and 70% relative humidity. 73 Behavioral assays i. Repellency effect of permethrin on Aed es aegypti This study was conducted to test the repellency of permethrin itself as a compound before testing the permethrin treated insecticide treated nets. Unlike other pyrethroids, permethrin is less volatile yet it is used in insecticide treated nets as a contact irritant. This study was conducted to establish non-contact repellency of permethrin. The permethrin was of 99% purity, from Sigma Aldrich. Fifty milligrams of the compound was weighed and diluted in acetone as a carrier solvent to be used in Hand in cage assay. Hand in cage assays were carried out follo wing methods explained in Fig 2.1. The tests were conducted on both resistant (PR) and susceptible (Waco) mosquito strains. Females were tested exclusively. ii. Repellency effect of Olyset and PermaNet 2.0 on Ae.aegypti and An.gambiae . The test nets were provided by Malaria Alert Centre in Malawi. Olyset net is impregnated with 1000mg per meter squared of the treated net (Siegert et al., 2009) . For the 5cmx5.8cm net pieces used in this study, they contained approximately 3 mg. PermaNet 2.0 mosquito nets have been reported t o have 55mg of deltamethrin per meter square (Koudou et al., 2011) , converting to 0.159mg on the Hand in cage net piece used. To assess the repellency effect of insecticide treated nets, Hand in cage assays were carried out follo wing methods explained in Fig 2.1 except that, an untreated insecticide treated net was used as control. For the other treatments; 5cmx5.8cm net pieces were cut and used in the behavio ral assays. At least 5 different cohorts of mosquitoes were tested on each of the treatments. Untreated net was tested first. 74 Results Permethrin elicited repellency on pyrethroid -susceptible Ae. aegypti mosquitoes We first examine d the potential perme thrin repellency in the Hand in cage assay using permethrin -treated mesh. Repellency by permethrin was detected on Waco mosquitoes and there was an increase in the repellency effect with an increase in the concentratio n of the compound used (Fig. 4 .1). There were no significant differences in the repellency effect of permethrin between the two lowest concentrations used (10 -4 and 10 -3 in Fig 4.1 ). A significant difference in the repellency effect of permethrin due to concentration was only observed between the lowest (10 -4) and the highest concentration (10 -2 equivalent to 5mg of permethrin). However, the repellency on the PR strain was abolished (Fig. 4.4). T able 4.1 is an excerpt of statistical multiple comparisons comparing the PR and Waco strai n at specific doses . -4-3-2020406080100Waco PRns***Percent repellency Figure 4. 1 High permethrin repellency in pyrethroid susceptible Aedes aegypti Waco strain than pyrethroid resistant PR strain tested at three different concentrations in Hand in c age assay. Repellency was abolished in pyrethroid resistant PR strain and was maintained in susceptible Waco strain. Data analyzed P<0.001 75 Table 4. 1 Repellency effect of permethrin on Waco and PR Aedes aegypti 1 strains Dose Waco 2 (Mean repellency) PR3 ( Mean repellency) Difference t-value P value 4 -4 9.708 13.54 3.830 0.3195 P > 0.05 -3 45.18 8.793 -36.38 3.035 P < 0.0 5 -2 62.59 13.35 -49.24 4.107 P<0.01 1 4-10 days old, nulliparous female™s sugar starved 2 Waco strain exposed to permethrin in Hand in cage assay 3 4Analysis of Variance(ANOVA) Bonf P<0.01, 76 PermaNet 2.0 repelled susceptible Aedes aegypti strains (Waco and Rock) and Anopheles gambiae (Kisumu) We later carried out the non Œcontact Hand in cage assay to assess potenti al repellency of the deltamethrin treated net (PermaNet 2.0) and permethrin treated net (Olyset). Indeed, significant levels of repellency of PermaNet 2.0 was found in the pyrethroid susceptible Ae. aegypti mosquitoes: Waco, Rock, and the An. gambiae Kisum u strain. However, repellency of the Olyset net was observed in the An. gambiae mosquitoes, surprisingly not in the Aedes mosquitoes (Fig. 4.3). In all the tests, repellency effect on Kisumu was significantly higher than that on Aedes aegypti strains (Wac o and Rock). However, repellency of PermaNet 2.0 was abolished in Isokdr and PR strains and orco mosquitoes. Detailed multiple comparisons of repellency effect on mosquito strains due to insecticide treated net e ffect are presented in table 4.2 . Table 4.2 Repellency effect of PermaNet 2.0 and Olyset compared when tested on An. gambiae 1 and different Ae.aegypti 1 strains Strain/species PermaNet 2.0 2 Olyset 3 Difference t-value P-value 9 Waco 5 54.01 12.39 -41.62 5.450 P<0.001 Rock 5 66.97 18.31 -48.66 6.373 P<0.001 Isokdr 6 4.486 -3.064 -7.551 0.7659 P > 0.05 PR7 -1.780 -6.354 -4.574 0.5990 P > 0.05 Orco 8 -6.150 -3.768 2.382 0.3119 P > 0.05 Kisumu 4 85.96 60.54 -25.42 2.978 P < 0.05 1 4-10 days old, nulliparous female™s sugar starved 2 3 4 Kisu Anopheles gambiae mosquito of Kisumu strain 5 Aedes aegypti ; pyrethroid susceptible laboratory strains 6 Aedes aegypti strain with Kdr mediated resistance 7 Aedes aegypti strain with both Kdr and P450 mediated resistance 8 Orco Aedes aegypti strain, with Orco -coreceptor knocked out 9 Analysis of Variance(ANOVA) 77 Waco Rock Isokdr PROrco Kisumu -20 020406080100 abbcccPercent repellency (A) Waco Rock Isokdr PROrco Kisumu -20 020406080100abccbdbdbPercent repellency (B) Figure4. 2 PermaNet2.0 re pel pyrethroid susceptible Aedes aegypti strains and Anopheles gambiae . (A) Showing repellency effect of PermaNet2.0 tested using net fabric in Hand in cage assay. (B) Showing reduced repellency effect of olyset net fabric on susceptible Aedes aegypti mo squito strains and increased repellency in Anopheles gambiae . Both nets are manufactured by Sumitomo Company; PermaNet 2.0 has 50mg/m 2 of deltamethrin and Olyset 1000mg/ m 2 of the net. 78 Permethrin and deltamethrin elicited electroantennogram (EAG) sign als In EAG recording experiments conducted by Feng Liu a postdoc associate in Dong Lab, deltamethrin and permethrin evoked olfactory responses from the mosquito antenna. Both deltamethrin and permethrin, when heated up, following methods by Slone et al., (2017) , evoke d EAG signals from the antenna of Rock mosquitoes in a d ose dependent manner (Fig 4.3A and 4.3 A). The EAG recording experiments were repeated using Orlando and Orco mosquitoes. Similarly, EAG signal was detected from mosquitoes of another susceptible strain, Orlando, in response to both deltame thrin and permethrin (Fig 4.3A and 4.4 A ). However, no EAG response was observed from Orco mosquitoes (Fig 4.3B and 4.4 B), indicating EAG responses by deltamethrin and permethrin are Orco -dependent. 79 Figure 4. 3 Normalized EAG responses of Aedes aegypti to deltameth rin . (A) Showing EAG responses in females Aedes aegypti Rock strain. (B) Showing a comparison of EAG responses in Orlando and Orco Aedes aegypti EAG recording done by Feng Liu) 01 234567-2-10Normalized EAG response Log [Deltamethrin dilution] (A) (B) 80 Figure 4. 4 Normalized EAG responses of Aedes aegypti to permethrin. (A) Showing EAG responses recorded in Aedes aegypti Rock strain. (B) Showing a comparison of EAG responses in Orlando and Orco Aedes aegypti mosquitoes. Fem EAG recording done by Feng Liu) 01234 561.0 2.0 3.0 Normailized EAG response Log [Permethrin dilution] (A) (B) 81 Discussion The use of pyrethroid trea ted insecticide bed nets remains important in the control of mosquito borne diseases, more especially malaria. The bed nets act as a b arrier from mosquito bites for people sleeping inside them. Research has demonstrated excito -repellency effects, others mortality in mosquitoes due to contact with the pyrethroid insecticide treated nets and surfaces (Corbel et al., 2004; Denham et al., 2015; Ritthison et al., 2014) . The current study has demonstrated an olfactory basis of action of the pyrethroids, deltamethrin and permethrin, which are used in LLINs. Results on behavioral assays using permethrin compound on Aedes aegypti mosquitoes demonstrated a dose dependent noncontact repellency in the pyrethroid susceptible strain Waco and not in the resistant strain PR, depicting that pyrethroid repellency is not very much restricted to very volatile pyrethroids, such a s transfluthrin and metofluthrin. The positive impact of insecticide treated nets on houses that are close to intervention areas compared to those that were far, supporting the hypothesis that bed nets may have a community effects in controlling mosqu ito vectors has been documented (Gimnig et al., 2003) . The actual mechanism underlying the community effects of pyrethroid treated nets is still debatable. The current study has demonstrated the ability of permethrin and deltamethrin to evoke olfactory response in EAG recordings in the antenna of the mosquito. The lack of EAG responses in mosquitoes with Orco co -receptor knocked out, support the olfactory basis of action of these test compounds, which somewhat might point towards the underlying mechanism of the community effects of the nets among other factors. Behavioral experiments evaluating the repellency effect of PermaNet 2.0 and Olyse t net adopted the Hand in cage assay, importantly because the assay was designed in such a way that insects do not come into contact with the treated net, avoiding the 82 excito repellent effects of insecticide treated nets that have been reported by a grow ing body of literature including (Kawada et al., 2014; Killeen and Smith, 2007) . The lack of LLIN repellency against pyrethroid -resistant mosquitoes (PR) observed in this study echoes a challenge of the use of deltamethrin treated bednets in areas with mosquito populations that are resistant. The efficacy of pyre throid treated bednets when mosquitoes that have both kdr -type and metabolic resistance mechanisms was questioned (Enayati and Hemingway, 2006b) . In th eir study, they highlighted significant differences between entry rates of pyrethroid and susceptible and resistant Anopheles mosquitoes in an exposure arena containing permethrin treated bed net. Results in the current study, support their findings. Simil ar findings have been reported (Ochomo et al., 2013 , Toé et al., 2014) . This study has presented evidence on the repellency effect of net bound permethrin and deltamethrin on different strains of Ae.aegypti . Different mosquito populations may develop resistance against the insecticides or change their behavior by avoiding contact with treated surfaces (Takken and Verhulst, 2011) . In this study, the noncontact repellency effect of the del tamethrin treated net, PermaNet 2.0 evident in susceptible Ae.aegypti mosquito strains; Waco and Rock and the reduced repellency in the resistant strain, Isokdr, signify the impact of resistance development on the efficacy of the insecticide treated nets i n areas with and without resistant mosquito populations. A study by Kawada et al., (2014) revealed that L1014S KDR Anopheles mosquito field populations wit h reduced frequency takeoffs from a permethrin treated nets and those lacking kdr mutations maintaining high levels of contact repellency irrespective of their metabolic factors to pyrethroids. Our study has demonstrated that non -contact repellency due t o Olyset and PermaNet 2.0 exposure may be affected by mosquito metabolic factors. Repellency effect of both nets was abolished in PR Aedes strain which has both P450 and kdr mediated resistance, unlike in Isokdr which only has kdr mediated resistance. Wh ile this study does not offset the excito repellency 83 effect of pyrethroid treated nets, the abolished repellency in Orco due to non -contact exposure of the mosquitoes through Hand in cage assay confirm the role that olfactory receptors play in the repellen cy effect of the Olyset and PermaNet 2.0 insecticide treated nets. Further, increased repellency effect of the nets on An.gambiae compared to Ae.aegypti mosquito strains, signify the differences in the sensitivity of different mosquito species to pyret hroids as repellents. Repellency effect observed in this study could be a resultant behavior of olfactory processing of odors. To dissect the differences in the sensitivity of the mosquito species to repellency effect of the pyrethroids, studies exploring in the actual signal propagation and neural circuits in the nervous system of the mosquito species resulting into the repellency behavior could help in elucidating the underlying cause of the sensitivity differences In addition, this study has reported g reater repellency effect of Permanent 2.0 compared to Olyset net in all the mosquito strains of Aedes and Anopheles tested. Other studies have also reported varying results on insecticide treated nets (Mosha et al., 2008; Spitzen et al., 2014) . While Mosha et al. (2008) reported high protective efficacy of permethrin treated net over deltamethrin insecticide treated net , Spitzen et al., (2014) reporte d absence of close range repellency of deltamethrin treated net against Anopheles mosquitoes. In the present study, non -contact hand in cage assays using permethrin showed repellency in susceptible Waco mosquitoes yet there was no repellency of the permet hrin treated net (Olyset). The differences in the repellency effect of the nets used in this study could be attributed to the fabric properties of the nets and the concentrations used . 84 Conclusion In summary, pyrethroid repellency may not be limited to very volatile pyrethroids. Permethri n and deltamethrin also elicit repellency behavior i n mosquitoes through the olfactory pathway as observed in the EAG recordings and behavioral assays. Lack of EAG response in Orco mosquitoes when exposed to permethrin and deltamethrin compounds, yet overt responses in mosquitoes with their Orco co -receptor intact, confirm the involvement of the olfactory receptors in the action of permethrin and deltamethrin compounds. As regards to PermaNet 2.0 and Olyset, the finding s of this study point towards a non -contact repellency mechanism of action of the nets apart from the well -known contact repellency. Abolished repellency observed in behavioral assays in resistant mosquitoes indicate that the nets may be more efficient to ols in reducing mosquito bites in areas with susceptible mosquito populations compared to areas with resistant mosquito populations. It is important to note that the conclusions presented here are from the evaluation of PermaNet 2.0 and Olyset. Furthe r studies may consider using other nets that are available on the market from different manufacturers to evaluate the potency of their repellent activities on different mosquito species and mosquitoes with differ ent resistance status. 85 REFERENCES 86 REFERENCES Bigoga, J. D., Ndangoh, D. N., Awono -Ambene, P. H., Patchoke, S., Fondjo, E., & Leke, R. G. F. (2012). 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The Lancet Infectious Diseases , 12(7), 512 Œ513. https://doi.org/10.1016/S1473 -3099(12)70107 -X Toé, K. H., Jones, C. M., N™Fale, S., Ismail, H. M., Dabiré, R. K., & Ranson, H. (2014). Increased pyrethroid resistance in malaria vectors and decreased bed net effectiveness, Burkina Faso. Emerging Infectious Diseases , 20(10), 1691 Œ6. https://doi.org/10.3201/eid2010.140619 Vantaux, A., Lefèvre, T., Dabiré, K. R., & Cohuet, A. (2014). Individual experience affects host ch oice in malaria vector mosquitoes. Parasites & Vectors , 7, 249. https://doi.org/10.1186/1756 -3305-7-249 World Health Organization. (2017). World Malaria Report 2017 . World Malaria Report 2017 . https://doi.org/10.1071/EC12504 90 CHAPTER 5 TRANSFLU THRIN AND PYRETHRUM REPELLENCY ON Plutella xylostela AND Sitophilus zeamais 91 Abstract Pyrethroids are also important in the control of different agricultural pests. In their use in agricultural field pest control, they are sprayed on the crops to reduce damage by insects. In grain storage, they are mainly incorporated as active ingredients in pesticide dusts. The current use of pyrethroids in the control of agricultural insects attracts repeated sprays and pesticide applications which may be very costly for small holder farmers. The use of pyrethroids as repellents in agriculture is very rare yet it has potential to reduce pesticide load on food and the environment. This study reports the repellency effect of transfluthrin and pyrethrum on Plutel la xylostela and Sitophilus zeamais . It illustrates 1) avoidance of pyrethrum and transfluthrin treated arenas in adult Plutella xylostela and Sitophilus zeamais insects 2) feeding preference of Plutella xylostela larvae in pyrethrum and transfluthrin trea ted arenas 3) reduced feeding in Plutella xylostella larvae in pyrethrum and transfluthrin treated arenas 92 Introduction Insect pests rely on chemosensation to locate their plant hosts, to find oviposition sites, suitable mates and food. Reviews (Naters and Carlson, 2006; Tricoire -Leignel et al., 2012) emphasized that insect olfaction cues as crucial in agricultural production. Olfaction measures long range attraction (Naters and Carlson, 2006) or repellency and some studies have proved its importance in diamondback moth in d istinguishing suitable plant hosts from unsuitable ones (Henniges -Janssen et al., 2011; Liu et al., 2005) . Diamondback moth has been reported to avoid its natural enemies using olfactory cues (Reddy et al., 2002) . Similar results have also been reported in Drosophila ; to avoid natural enemies and harmful chemical substances in oviposition sites (Ebrahim et al., 2015; Stensmyr et al., 2012) . These studi es confirm the key role that olfaction plays for insects in crop production. In fact, the economically important damage that insect pests cause on crops is largely driven by olfactory cues which they use to find oviposition sites; which are mostly the plan t host where the larvae hatch and cause direct damage. Insects such as diamondback moth ( Plutella xylostella ) are a leading cause of damage to cruciferous crops and lesser grain borer ( Sitophilus zeamais ) cause significant post -harvest loss in maize crop in Africa and other parts of the world. Like other insect pests, effective control still relies on insecticides. Earlier, before restrictions were placed on some major insecticides, control of most agricultural pests in Africa relied on the inexpensive or ganochlorides and methyl carbamates which are not only toxic to insects but also humans (Casida, 1980) . Currently, even with restrictions, use of less expensi ve yet toxic insecticides such as carbamates is still common for profit oriented small holder farmers. In some cases organophosphates and pyrethroids such as cypermethrin are used to reduce preharvest losses, more especially in Malawi. On the other hand, pesticide dusts with deltamethrin incorporated are used in reducing postharvest losses in maize yield to control maize 93 weevils and other beetles such as larger grain borer. In some cases, multiple applications of these pesticides are required, to maintain yield quality. This may pose a health risk. Studies have confirmed health effects of overuse of insecticides in some parts of Africa (Naidoo etal., 2013) . Unfortunately, the use of insecticide sprays also select for resista nce in Anopheles gambiae mosquitoes (Hien et al ., 2017) complicating the control of malaria. Unlike carbamates and organochlorides, pyrethroids are less toxic to humans and other mammals (Casida, 1980; Dong et al., 2014; Ensley, 2007; Soderlund et al., 2002) . Despite their favorable chemical properties, the use of pyrethroids in agriculture and public health has been affected by development of insecticide resistance requiring frequent reapplications to protect crops exceeding required recommendations, risking food toxicities and making agricultural production for small holder farmers in Africa, very costly. Recent progress in insect chemosensation studies, offers opportunities for developing novel ways of insect pest control. Olfaction, in conjunction with the existing insect control strategies may enhance crop protection in Agriculture. Olfacti on in agricultural insect pest control, is essential in trap designs (Dendy et al., 1989) . Although the potential in using repellents to push insects away from crops has not been largely tapped, Arnold et al., (2015) emphasize d on how in -depth knowledge on insects orient ation to stimuli may play a vital role in designing mass trapping of insects for control. Their study demonstrated the importance of odor and color on the ab ility of lesser grain borer (LGB) in locating the maize grain. What these studies lacked was an exploration of chemical cues that would push the insects away from the storage facility and pulled into a trap, to enhance mass trapping. Majority of studies taking advantage of the insect olfaction cues for mass trapping to reduce crop damage, have maximized on the host odor and color cues. Yet, recent studies by have presented evidence on the ability of the pyrethroid; transfluthrin to prevent outdoor 94 mosqui to bites through repellency when used and reused in low cost impregnated hessian sacks (Govella et al., 2015; Ogoma et al., 2012, 2017) . Although the studies mainly focused on repellency in mosquitoes, their results warranted further exploration of simila r mechanisms in other insects, more especially agricultural pests. Pyrethroids feature highly in the control of insect pests of crops like cotton, horticultural crops and are also incorporated in pesticide dusts for control of storage pests such as LGB in Malawi. They are also important in the control of livestock pests such as tsetse fly. Currently, pyrethroid blanket sprays are common in the control of insect pests in the field. Establishing repellency effect of pyrethroids might be helpful in control of insect pests in the field as a push strategy at minimal insecticide amounts. This study sought to establish repellent effect of pyrethrum and transfluthrin on two agricultural insect pests; Plutella xylostella (diamondback moth) and Sitophilus zeamais (lesser grain borer) in Malawi. 95 Materials and Methods Insects Sitophilus zeamais A laboratory colony of Sitophilus zeamais (lesser grain borer), also referred to as LGB was obtained from Lilongwe University of Agriculture and Natural Resource s (LUANAR), Bunda campus and used for the experiments. The lesser grain borer was reared on untreated maize grain to avoid pre -exposure to pesticides before the insects were used in a behavioral assay. Both males and females were used in the assay. Plute lla xylostella A colony of Plutella xylostella (Diamondback moth) was established by collecting larvae from Dedza, Bembeke area central Malawi. Dedza is an area where cabbage is commonly grown and the diamondback moth problems are not scarce. The lar vae were collected from a farmer™s field and reared at Bunda College campus biotechnology laboratory on fresh cabbage plants until pupation. Insects were collected from Dedza bi weekly to sustain the colony. Adults were fed 10% sugar solution and were rea red in 30x30x30 bioquip plastic cages. Test compounds Technical grade transfluthrin and pyrethrum compounds with at least 95% purity were used for the study. The test compounds were diluted in acetone as a carrier solvent. In preliminary behavioral studi es, transfluthrin caused knockdown in the behavioral assays, as such it was used at a slightly lower concentration (volume/volume) 10 -3 as opposed to pyrethrum which was diluted at a higher concentration 10 -2. These concentrations were maintained throughou t the whole study. 96 Behavioral assays i. T-Maze trap assay T-Maze assays for Drosophila melanogaster (Stensmyr et al., 2012) and (Ebrahim et al., 2015) were modified to examine repellency of pyrethrum and transfluthrin on lesser grain borer (Fig 5.1). Ins ects were gently released into the set up through the delivery tube (A) into the decision tube (B). Once all the insects were released into the decision tube they were left for 60 minutes to make a choice. The assay was designed in such a way that once th e insect makes a choice and goes into either of the holding cups (C) it has limited opportunity to walk back into the decision tube. The holding cups contained filter papers (D1 or D2) which were treated with either 100 µl of acetone or a test compound. Tw enty beetles were used per assay. Each assay was repeated atleast five times with different cohorts of insects. The experiments were conducted in a room with temperature of 25 oC and 60% relative humidity. Figure 5. 1 T-Maze tra p assay set up for Sitophilus zeamais (LGB). (A) Showing a d elivery tube, both males and females were used in the assay. (B) Showing a d ecision tube; insects walk freely into the decision chamber to make a choice, insects are left to make a choice for a t least 60 minutes, after which any insects remaining in the decision tube are scored as unde cided. (C) Showing a holding cup and (D1-D2) showing filter papers (3x4cm) loaded with either 100 µl of acetone or test compound. 97 ii. Two -choice repellency assay The two choice repellency assay was set using three world health organization (WHO) bioassay test tubes. The three tubes were connected together to make; a decision tube (A in Fig. 5.2), and choice tubes o n each side (B1 and B2 in Fig 5.2). Diamondback mo th adults (5males and 5 females) were released into the decision tube and left in the behavioral test room (25 0C; 60% relative humidity) for 30 minutes to acclimatize. Later the choice tubes with filter papers loaded with acetone (for control) or test compound as treatment were connected. Soon after the choice tubes were connected, doors (C in Fig. 5.2) on both sides of the decision tube were opened to allow the insects move and make their choice. After 30 minutes, once the choices were made, the doors were closed and insects that remained in the decision tube were considered undecided. Figure 5. 2 Two choice assay setup for adult diamondback moth insects . (A) Showing a d ecision tube which holds both male and female d iamondback moths. (B1 -B2) Showing c hoice tubes containing filter papers either loaded with acetone (as control) or test compound (pyrethrum or transfluthrin) diluted in acetone . (C) Showing decision tube doors and (D) side view of a filter paper loaded w ith acetone on B2, the decision tube B1 also contains a filter paper loaded with a test compound pyrethrum. 98 iii. Larvae feeding preference assay Third instar larvae of diamondback moths, starved for 12 hours, were used in the assay. Two plastic sample bo ttles, 6cm high and 4cm in diameter on the base were used in the assay as feeding arenas (Fig. 5.3) The bottles (used as a feeding arena) into which the leaves were placed were cleaned to certainty first with water then with 95% alcohol and left to dry. A filter paper, once loaded with either acetone or a test compound, was placed inside the cover of the bottle to hold it and the bottle (the feeding arena) with the leaf and filter paper were placed in a feeding chamber (Fig. 5.3). Discs (4cm in diameter) w ere cut from a freshly harvested leaves and kept in distilled water to maintain freshness until they were used for experiments. Then, 12 hour starved, 3 rd instar larvae were carefully released into the middle of the feeding chamber using a camel brush. The feeding chamber was placed in an experimental room with at least 25 0C and 60% relative humidity for 24 hours. Results were observed the next day. Number of larvae in each of the feeding arena was counted; those that remained in the feeding chamber we re considered as undecided. 99 Figure 5. 3 Feeding preference assay set up for diamondback moth larvae . The assay was carried out in a feeding chamber (30cmx15cmx10cm). (A) Showing a filter paper treated (3x4cm) with either acetone (for control) or test compound (transfluthrin or pyrethrum) as treatment. (B) Showing a leaf disc of a cabbage plant (4cm in diameter). The leaf was cleaned with clean water and had no contact with the test compounds. (C) Showing an arena window f or entrance of larvae that has made a choice and (D) Diamondback moth larvae; 3 rd 100 Data analysis The response index/repellency index (RI) from all the behavioral assays was calcul ated following modified methods by (Stensmyr et al., 2012) . In brief the indices were calculated as (Ta -Tb)/T, where Ta is the number of insects in the treatment and Tb number of insects in the control(acetone), and T is the number of insects that parti cipated in the trial (Ta+Tb). The index ranged from -1 to +1, where the latter signified complete avoidance (repellency) and the former, attraction. The RI were analyzed using Students™t - assay, further ana lysis of the images of the leaf discs remaining after 24 hours of feeding in the feeding arenas, were captured and processed in Image J software to calculate the leaf area left. The area of the leaf discs remaining either in control arena or treated arena were calculated and subjected to Student™s t - Results Repellency effect of pyrethrum and transfluthrin on Sitophilus zeamais (Lesser gain borer) To assay the behavioral effects of exposing lesser grain borer (LGB) to pyrethrum (10 -2 dilution), a T-Maze trap assay was used (Fig 5.1). Adult LGB, were used in cohorts of 20 re leased into the decision tube . Notably, when the beetles were released, they oriented towards the untreated arena of the maze, steering their course away from the tr eated one. Similar experiments were repeated using a 10 -3 transfluthrin dilution. Exposed LGB, displayed a similar pattern orienting towards the untreated arena, depicting a negative response from transfluthrin. Although transfluthrin was used at a lower concentration than pyrethrum, results still revealed significantly high percent response towards the control arena denoting that in both cases, pyrethrum and transfluthrin are effectively repelling the LGB. 101 Pyrethrum Acetone -1.0 -0.5 0.0 0.5 1.0 (A) Repellency Index Transfluthrin Acetone -1.0 -0.5 0.0 0.5 1.0 (B) Repellency Index Figure 5. 4 Pyrethrum and transfluthrin repel Sitophilus zeamais (lesser grain borer ) in a T -maze assay. (A) Showing repellency index due to pyrethrum in lesser grain borer ( LGB ). Negative repellency index denote avoidance/ or an orientation away from the arena and positive index denote attraction to the arena. (B) Showing repellency of transfluthrin against LGB. Both males and females tested in the T -maze assay, 20 insects released in the decision tube (A in Fig 5.2) . Experiments replicated 5 ti mes, with 4 experimental units erent from each other (P<0.0001), Student™s T - 102 Table 5. 1 Percent response 2 of Sitophilus zeamaise 1 when pyrethrum and transfluthrin were used in a T-maze choice trap assay Treatment Difference 3 t-value 95% CI of diff P value Summary 3 Acetone vs pyrethrum 87.50 41.23 82.26 to 92.74 P<0.0001 *** Acetone vs undecided 89.25 42.05 84.01 to94.49 P<0.0001 *** pyrethrum vs undecided 1.750 0.8246 -3.485 to 6.985 P>0.05 ns Treatment Difference 2 t-value 95% CI of diff P value Acetone vs transfluthrin 85.25 26.15 77.21 to 93.29 P<0.0001 *** Acetone vs undecided 91.50 28.07 83.46 to 99.54 P<0.0001 *** transfluthrin vs undecided 6.250 1.917 -1.791 to 14.29 P>0.05 ns 1 Sitophilus zeamais (Lesser grain borer) adults 2 Percent response denotes a proportion of insects steering a course towards an arena. 3Difference (mean percent response) compares the number of insects that oriented to each of the arenas. Th e control attracted more insects than the treatment, hence the positive differences. 4 ANOVA Bonferroni posttests ( 103 Repellency effect of pyrethrum and transfluthrin on Plutella xylostella (Diamondback moth) Pyrethrum and transfluthrin elicited a repellency behavior when tested on diamondback moth tw o choice set up (Fig 5.2). Diamondback moths oriented towards the untreated arm than the treated arm containing a filter paper loaded with the test compound. For transfluthrin and pyrethrum, insects preferred the untreated side, hence the negative respon se index for both compounds (see table 5.3). In this experiment, transfluthrin was used at a lower dose (10 -3) compared to pyrethrum (10 -2) to keep the flying insects from knockdown effect. Insects that did not make a choice after 60 minutes of the exper iment were considered undecided. Pyrethrum Acetone -1.0 -0.5 0.0 0.5 1.0 (A) Repellency Index Transfluthrin Acetone -1.0 -0.5 0.0 0.5 1.0 (B) Repellency Index Figure 5. 5 Pyrethrum and transfluthrin repel Plutella xylostella (diamondback moth) in a choice assay . (A) Showing repellency index in pyrethrum against diamondback mo th (DBM) in a choice assay using WHO bioassay test tubes. Negative index denote avoidance in the insect, from the arena and positive index denote attraction to the specific arena. (B) Showing repellency index in transfluthrin against DBM . Five males and f emales tested in the choice assay, 10 insects released in the decision chamber. Experiments replicated 5 different from each other (P<0.0001), St udent™s T - 104 Table 5. 2 Percent response 2 of adult Plutella xylostella 1 when pyrethrum and transfluthrin were used in two choice test. Treatment Difference 3 t-value 95% CI of diff P value Summary 4 Cont rol vs pyrethrum 66.67 10.35 49.32 to 84.02 P <0.0001 *** Control vs undecided 83.33 12.94 65.98 to 100.7 P <0.0001 *** pyrethrum vs undecided 16.67 2.588 -0.6828 to 34.02 P >0.05 ns Treatment Difference 2 t-value 95% CI of diff P value Summary 3 Control vs transfluthrin 70.00 11.74 53.94 to 86.06 P <0.0001 *** Control vs undecided 80.00 13.42 63.94 to 96.06 P <0.0001 *** transfluthrin vs undecided 10.00 1.677 -6.062 to 26.06 P >0.05 ns 1 Plutella xylostella (Diam ondback moth) adults (5 males and 5 females) 2 Percent response denotes a proportion of insects steering a course towards an arena. 3Difference (mean percent response) compares the number of insects that oriented to each of the arenas. The control attr acted more insects than the treatment, hence the positive differences. 4 ANOVA Bonferroni posttests ( 105 Pyrethrum and transfluthrin repel DBM larvae and reduce feeding on leaf discs in treated arena Having assayed the repellency effect of pyrethrum and transfluthrin on adult DBM, the foll owing studies sought to explore whether the test compounds would have repellent activity on the larvae and affect their feeding behavior. Results in this study confirmed significant differences in the percent response of the larvae, when released in the f eeding chamber. Calculated response indices for both compounds revealed a negative response for the treated arenas co mpared to untreated ones (Fig 4.5 A-B). Calculated leaf areas using image J, revealed larger leaf areas remained in treated arenas co mpa red to untreated ones(Fig 4.5 C -D and Fig 4.6) Transfluthrin Acetone -1.0 -0.5 0.0 0.5 1.0 (A) Repellency Index Pyrethrum Acetone -1.0 -0.5 0.0 0.5 1.0 (B)Repellency Index Acetone Transfluthrin 01234567p=0.031 Leaf area in cm (C) Acetone Pyrethrum 0123456p=0.005 Leaf area in cm (D) Figure 5. 6 Pyrethrum and transfluthrin repel and reduce feeding in Plutella xylostella (diamondback moth) larvae in a feedin g choice assay . (A -B) Showing repellency index of transfluthrin and pyrethrum against diamondback moth larvae. (C -D) Showing the remaining leaf area in the feeding arenas, after 24hrs of the feeding trial. Experiments replicated 5 times, with 4 experimen tal units a day for each of the compounds. Six- - 106 (A) (B) (C) (D) Figure5. 7 Reduced feeding in pyrethrum and transfluthrin arenas in the choice feeding assay against DBM larvae . (A -B) Showing the leaf area left (dark shaded spots) in untreated arenas without pyrethrum and transfluthrin. (C -D) Showing the leaf area left in pyr ethrum and transfluthrin treated arena .Ten 3 rd instar larvae released into the feeding chamber. Faded regions of the leaf disc image depict the areas eaten by the larvae. Leaf image analyzed using ImageJ software. 107 Table 5. 3 Percent response 2 of Plutella xylostella 1 larvae when pyrethrum and transfluthrin were used in a larvae feeding preference assay Treatment Difference 3 t-value 95% CI of diff P value Summary 4 Control vs pyrethrum 41.00 7.712 27.89 to 54.11 P <0.0001 *** Control vs undecided 61.50 11.57 48.39 to 74.61 P <0.0001 *** pyrethrum vs undecided 20.50 3.856 7.386 to 33.61 P <0.0001 *** Treatment Difference 2 t-value 95% CI of diff P value Summary 3 Control vs transfluthrin 27.50 4.881 13.60 to 41.40 P <0.0001 *** Control vs undecided 54.50 9.674 40.60 to 68.40 P <0.0001 *** transfluthrin vs undecided 27.00 4.793 13.10 to 40.90 P <0.0001 *** 1 Plutella xylostella (Diamondback moth) larvae 2Percent res ponse denotes a proportion of insects steering a course towards an arena. 3Difference (mean percent response) compares the number of insects that oriented to each of the arenas. The control attracted more insects than the treatment, hence the positive dif ferences. 4 ANOVA Bonferroni posttests ( 108 Discussion In most parts of Africa, insecticide use remain important in pre - and post -harvest crop protection, although a number of studies hav e reported some occupational hazards due to overuse and misuse of pesticides in some parts of the continent (Naidoo et al., 2013) . Repellency studies on insects of agricultural importance, due to sub lethal compound exposures are rare. This study was inspired by the efficiency of a low cost technology that uses transflut hrin hessian sacks to control outdoor biting mosquitoes (Govella, Ogoma, Paliga, Chaki, & Killeen, 2015; Ogoma et al., 2012, 2017) . The observed behavioral responses of LGB when exposed to pyrethrum were consistent with respon ses to transfluthrin. More insects oriented towards the untreated arm than the treated one reinforcing the notion that pyrethrum and transfluthrin evoked repellency behavior in the LGB. A closely related study focused on the use of permethrin treated n ets to protect maize in storage (Anaclerio et al., 2 015) targeting the contact toxicity of the permethrin treated net, to reduce the penetration effect of insects from one infected storage bag to uninfected one. Concurring with a recent study (Barbosa et al., 2017) which demonst rated the LGB avoided surfaces treated by deltamethrin more than spinosyns. They attributed contact irritancy to be the underlining cause of the avoidance behavior. Contrary to the findings of this study, our study has demonstrated the ability of pyrethru m and transfluthrin to repel LGB without getting in contact with treated arenas. Although our study did not look at the specific olfactory receptors responsible and did not repeat the experiments on LGB insects with their Orco co -receptors knocked out; ear lier studies have demonstrated reduced repellency in Orco mosquitoes. Thus, in the current study, the possibility of olfaction mediated repellency behavior in LGB may not be overruled. In addition to suggesting the olfactory mediated avoidance behavior, be cause of the volatile nature of pyrethrum and transfluthrin, neuro -physiological excitation of the LGB due to sodium 109 channel activation when exposed to the sublethal vapors could also influence the resultant avoidance behavior of insects from the treated a rm and orientation to the untreated arm. When extended to a field setup, this study presents an opportunity of decreasing pesticide load in maize storage by decreasing LGB infestations in storage facility not only through contact toxicity, but also the abi lity of the compounds to steer insects away from the storage facilities without contact. This may enhance push and trap of LGB in the storage facilities. Apart from the LGB the current study further explored repellency due to pyrethrum and transfluthrin on diamondback moth adults in absence of attractant odors from a host plant. The increased response to the untreated arm of the test tube confirmed the ability of the transfluthrin and pyrethrum to elicit avoidance behavior in diamondback moth. Studies hav e investigated the roles of olfaction on diamondback moth host finding (Couty et al., 2006) . Others have focused the role of chemo sensation on diamondback moth res ponses to natural enemies (Reddy et al ., 2002) yet studies on olfactory mediated behaviors due to pyrethroid exposure are rare. Our laboratory trial on repellency and transfluthrin and pyrethrum, has demonstrated the ability of the test compounds to elicit repellency behavior on diamondback moth, suggesting the need to further explore the repellency effect in semi -field and field trials. In addition to the adult insects, our observation on repellency effect of pyrethrum and transfluthrin on diamondback moth larvae in a feeding choice assay, confirm how repellency may not only be important to adult diamondback moth, but also in larval foraging behavior. The orientation of the larvae to the untreated arena more than the treated one in the feeding preference assay, depicts chemo sensation is not exclusively used in the adult diamondback moth insects. The effect of non -plant host odors such as deterrents on diamondback moth larvae using leaf discs that have been dipped in a compound of interest was demonstrated by (Guangli et al.,2011) . Earlier 110 review by (Ramaswamy, 1988) , gave a detailed description of sensory modalities and behaviors in moths, diamondback moth included; highlighting findings by Diether (1982) that; host recognition and prefe rence of moths could be complex. It may involve the complex neural and metabolic processes. Thus studies using feeding preference experiments with the substrate treated with a test compound, may be complex to interpret in such a way that it may be difficu lt to deduce the cause of the released behavior of the larvae since the larvae may have to come into contact with the treated substrate and the resultant behavior could either be acceptance, rejection and or deterrence not necessarily repellency. The res ults observed in the feeding preference experiments in this current study, were exclusively due to odor cues and not contact of the insects with the treated surface. The increased response to the leaf disc in the untreated arena than the one treated with e ither pyrethrum or transfluthrin; depict the importance of olfactory cues in the choices of food sources for the larvae diamondback moth. The leaf area analysis revealing a small leaf area remaining in the untreated arena, compared to the treated ones fo r both compounds; pyrethrum and transfluthrin, depict that the larvae were not only repelled by the test compounds, but they also accepted the leaf in the untreated arena more than the one in the treated arena. These results confirmed, the repellency eli cited by the test compounds steered the insects more into the untreated arena suggesting that repellency may have an important role in controlling crop damage, not just by steering the insects away but also reducing the crop damage itself. Although this cl aim warrants further exploration of the actual larvae olfactory receptors involved, it echoes a promising role repellents may play in the future control of crop pests to reduce pesticide overload in the environment. 111 Conclusion These results have emphas ized the importance of repellents in reducing food losses. Although repellents have mainly featured in public health, from this study, it seems they may be important in reducing insect -host contact, and reduce direct damage on crops. This study has shown that pyrethrum and transfluthrin can elicit repellency in Plutella xylostella and Sitophilus zeamais . Normally, prevention of food losses in most parts of the world, Africa specifically, involve repeated application of pesticides directly on the food pr oducts. These practice results into pesticide overload on food crops and may cause health hazards. The steering away of the test insects from treated arenas in this study presents a promising opportunity of reducing pesticides application on food. Additi onally, the reduced feeding in diamond back moth larvae in arenas treated with pyrethrum and transfluthrin suggest that the repellency mechanism is not limited to adult insects only but also larvae. Further studies will test the extent to which these repe llents can reduce food losses in semifield studies. Studies to test more pyrethroids and to identify olfactory receptors responsible for this repellency behavior in Sitophilus and Plutella xylostella , might be also be important. 112 REFERENC ES 113 REFERENCES Anaclerio.M, Pellizzoni.M, Todeschini, T. . and B. . (2015). 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RESEARCH ARTICLE A low technology emanator treated with the volatile pyrethroid transfluthrin confers long term protection against outdoor biting vectors of lymphatic filariasis , arboviruses and malaria. Ogoma, S. B., Ngonyani, H., S imfukwe, E. T., Mseka, A., Moore, J., & Killeen, G. F. (2012). Spatial repellency of transfluthrin -treated hessian strips against laboratory -reared Anopheles arabiensis mosquitoes in a semi -field tunnel cage, 1 Œ5. Ramaswamy, S. B. (1988). Host Finding By Moths : Sensory And Behaviours , 34(3), 235 Œ249. Reddy, G. V. P., Holopainen, J. K., & Guerrero, A. (2002). Olfactory responses of Plutella xylostella natural enemies to host pheromone, larval frass, and green leaf cabbage volatiles. Journal of Chemical Ec ology , 28(1), 131 Œ143. https://doi.org/10.1023/A:1013519003944 Soderlund, D. M., Clark, J. M., Sheets, L. P., Mullin, L. S., Piccirillo, V. J., Sargent, D., – Weiner, M. L. (2002). Mechanisms of pyrethroid neurotoxicity: Implications for cumulative risk assessment. Toxicology , 171, 3Œ59. https://doi.org/10.1016/S0300 -483X(01)00569 -8 Stensmyr, M. C., Dweck, H. K. M., Farhan, A., Ibba, I., Strutz, A., Mukunda, L., – Hansson, B. S. (2012). A conserved dedicated olfactory circuit for detecting harmful microbe s in drosophila. Cell , 151(6), 1345 Œ1357. https://doi.org/10.1016/j.cell.2012.09.046 115 Tricoire -Leignel, H., Thany, S. H., Gadenne, C., & Anton, S. (2012). Pest insect olfaction in an insecticide -contaminated environment: Info -disruption or hormesis effect . Frontiers in Physiology . https://doi.org/10.3389/fphys.2012.00058 Wang, G., Huang, X., Wei, H., & Fadamiro, H. Y. (2011). Sublethal effects of larval exposure to indoxacarb on reproductive activities of the diamondback moth, Plutella xylostella (L.) (Le pidoptera: Plutellidae). Pesticide Biochemistry and Physiology , 101(3), 227 Œ231. https://doi.org/10.1016/j.pestbp.2011.09.010 116 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS 117 Pyrethroids are compounds that are structu rally derived from pyrethrins and they make up almost 17% of the insecticides that are sold on the market (Sparks, 2013) . Pyrethrum and pyrethroids are well known for their insecticidal activity upon contact with insects through their action on the voltage gated sodium channel (Davies et al., 2007; Dong et al., 2014; Du et al., 2011) . A growing body of literature has documented from behavioral assays that pyrethrum and pyrethroids induce repellency (Bibbs & Kaufman, 2018; Bowman et al., 2018; Chareonviriyaphap et al., 2004; Govella et al., 2015; Kawada et al., 2006; Ogoma et al., 2014; Sathantriphop et al., 2014) . This study was conducted to elucidate the mechanism of pyrethrum and pyrethroid repellency in mosquitoes and agricultural pests. In an attempt to find out the mechanism of repellency, we utilized behavioral assays and electroantennogram re cordings. We used different strains of Aedes aegypti mosquitoes that are pyrethroid susceptible and pyrethroid resistant (kdr and P450 mediated mechanisms). We also tested repellency on Anopheles gambiae , Kisumu strain. An orco mutant Aedes aegypti mosqui to made from Orlando strain was used to verify the involvement of olfactory receptors. Our study used DEET as a positive control since it is a well -known mosquito repellent. We further explored repellency effect of pyrethrum and transfluthrin on agricultur al insect pests, Sitophilus zeamais and Plutella xylostella . The data in our study suggest that pyrethrum and pyrethroids evoke repellency behavior in mosquitoes. Our Hand in cage behavioral assay was designed in such a way that the mosquitoes do not ge t in contact with the treated surfaces so that we could exclusively test involvement of olfactory receptors in the resultant behavioral stimuli. Landing frequency transformed into repellency percentage was compared between compounds and mosquito strains. U nexpected, our study showed enhanced repellency due to pyrethrum and pyrethroids in pyrethroid -susceptible mosquito strains than in resistant ones. This difference between strains was not observed when DEET was tested. It was expected that if olfactory re ceptors were exclusively involved in the repellency effect 118 of pyrethrum and pyrethroids, there would not be overt differences in the repellency levels between resistant and susceptible strains. This suggests that sodium channel activation also plays a ro le in the repellency. Interestingly the abolished and/or reduced repellency due to pyrethrum and pyrethroids in pyrethroid susceptible orco mutants when compared to a susceptible isogenic strain in the behavioral assays still signified the importance of o lfactory receptors in the repellency mechanism. Our electroantennogram studies in the mosquito antenna showed that olfactory receptors were activated by pyrethrum and pyrethroids. These findings echo the importance of both olfactory receptors and sodium channel action in the repellency due to pyrethrum and pyrethroids. The details on the extent to which olfactory processing and sodium channel activation contribute to pyrethroid repellency in mosquitoes remain to be investigated. In addit ion, our study has shown enhanced repellency and knock down effect of transfluthrin when mosquitoes are pretreated with piperonyl butixide (PBO). We have shown that inhibiting P450 activity in mosquitoes enhances transfluthrin repellency and toxicity. Thes e studies have laid out a foundation for further investigation of the effect of P450 activity inhibition on transfluthrin repellency. It should be noted that these results cannot be generalized for all pyrethroids, they may vary. Future studies will con sider testing this effect of P450 inhibition on repellency using different kinds of pyrethroids. One of the ways through which pyrethroids have been used to combat vector borne diseases, is their incorporation in the insecticide treated nets. Their mech anism of action is still debatable. Our study has shown that the deltamethrin treated net (PermaNet2.0) and permethrin treated net (Olyset) repel mosquitoes and the magnitude of repellency may vary from one species to another. It should be emphasized tha t based on our findings, the repellency of the bed net may also vary from one net type to another. It would seem unlikely that the pyrethroid treated nets, especially 119 the permethrin and deltamethrin treated nets would elicit repellency in mosquitoes becau se permethrin and deltamethrin are not very volatile. Our study has shown that the repellency effect is not limited to very volatile pyrethroids. Infact, our behavioral experiments showed repellency due to permethrin in corroboration with electroantennogr am studies that showed a robust response. Why permethrin treated net showed reduced repellency than deltamethrin treated net, yet behavioral assay and electroantennogram recordings showed overt responses would depend on several factors, including; how th e compound is incorporated in the nets as well as the material of the net used. Thus it should be noted that these results cannot be generalized on all bed nets and all mosquito species. Based on the observed variations in these results, further studies wi ll test repellency of several pyrethroid treated net types that are being used. Further, our study has revealed the potential of pyrethroid repellency in reducing food losses in laboratory experiments. Normally, insecticides such as pyrethroids are used in sprays and not as repellents in the control of insect crop pests. In our study, both pyrethrum and transfluthrin have shown the ability to steer insects away from the treated arenas without physical contact. Future studies on pyrethroid and pyrethrum r epellency on agricultural insect pests, will attempt replicating the laboratory studies under field and semi field studies. The studies will also examine the impact of pyrethrum and pyrethroids on oviposition behavior of agricultural pests, more especially the diamond back moth. Overall, our studies on mosquitoes and agricultural pests present a great step forward in exploring the use of pyrethrum and pyrethroids as repellents in the control of insect pests and vectors without overloading the environment w ith pesticides. 120 REFERENCES 121 REFERENCES Bibbs, C. S., & Kaufman, P. E. (2018). Volatile Pyrethroids as a Potential Mosquito Abatement Tool : A Review of Pyrethroid -Containing Spatial R epellents, 8(June). https://doi.org/10.1093/jipm/pmx016 Bowman, N. M., Akialis, K., Cave, G., Barrera, R., Apperson, S., & Meshnick, S. R. (2018). Pyrethroid insecticides maintain repellent effect on knock -down resistant populations of Aedes aegypti mosqu itoes, 1 Œ14. Chareonviriyaphap, T., Prabaripai, A., & Bangs, M. J. (2004). Excito -repellency of deltamethrin on the malaria vectors, Anopheles minimus, Anopheles dirus, Anopheles swadiwongporni, and Anopheles maculatus, in Thailand. Journal of the America n Mosquito Control Association , 20(1), 45 Œ54. Davies, T. 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Impregnating hessian strips with the volatile pyrethroid transfluthrin prevents outdoor exposure to vectors of malaria and lymphatic filariasis in ur ban Dar es Salaam , Tanzania. Parasites & Vectors , 8Œ12. https://doi.org/10.1186/s13071 -015-0937-8 Kawada, H., Iwasaki, T., Luu, L. L., Tran, K. T., Mai, N. T. N., Shono, Y., – Takagi, M. (2006). Field evaluation of spatial repellency of metofluthrin -impr egnated latticework plastic strips against Aedes aegypti (L.) and analysis of environmental factors affecting its efficacy in My Tho City, Tien Giang, Vietnam. American Journal of Tropical Medicine and Hygiene , 75(6), 1153 Œ1157. https://doi.org/75/6/1153 [ pii] Ogoma, S. B., Ngonyani, H., Simfukwe, E. T., Mseka, A., Moore, J., Maia, M. F., – Lorenz, L. M. (2014). The Mode of Action of Spatial Repellents and Their Impact on Vectorial Capacity of Anopheles gambiae sensu stricto. PLoS ONE , 9, e110433. https:// doi.org/10.1371/journal.pone.0110433 Sathantriphop, S., White, S. 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