Project Title: Toxicity of Vapor Active Insecticides for ...€¦ · FDACS Project P0010729 Final Report: September 2017 1 1 Project Title: Toxicity of Vapor Active Insecticides for

FDACS Project P0010729 Final Report: September 2017

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Project Title: Toxicity of Vapor Active Insecticides for Multi-Vector Control 1

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Principle Investigator: Phillip E. Kaufman, PhD 3

Co-Principle Investigator: Christopher S. Bibbs, PhD Student 4

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Project Objectives: 6

1. Determine informative concentrations of active ingredient that include metofluthrin, 7

transfluthrin, prallethrin, and flumethrin to determine vapor toxicity against Aedes albopictus, an 8

initial screening species. 9

2. Utilize the informative concentration ranges determined in Objective 1 to replicate the vapor 10

activity bioassays using the four candidate insecticides against three additional vector-capable 11

mosquito species. 12

3. Replicate the vapor activity bioassays and analysis with a fifth candidate insecticide, 13

meperfluthrin, against all prior tested vector-capable mosquito species. 14

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ABSTRACT Objectives 1 and 2 were completed ahead of schedule, so an additional objective 16

was created in adding meperfluthrin to vapor bioassays. Volatile pyrethroid compounds are 17

among the tools commercially dubbed “spatial repellents.” Spatial repellents have been 18

advocated for urban vector management, and there is environmental overlap between mosquitoes 19

found in domestic settings and people that use spatial repellents for outdoor protection. Recent 20

research on several of these spatial repellents indicated considerable adulticidal action. With the 21

idea that these pyrethroid chemicals kill adult mosquitoes, metofluthrin, meperfluthrin, 22

transfluthrin, prallethrin, and flumethrin were evaluated against Aedes albopictus Skuse and 23

Aedes aegypti (L.), Culex pipiens quinquefasciatus Say, and Anopheles quadrimaculatus Say. 24

Dose response LC50 and LC90 data were obtained and analyzed for Ae. albopictus, Ae. aegypti, 25

Cx. quinquefasciatus, and Anopheles quadrimaculatus. It has been determined that transfluthrin 26

vapors had the highest overall toxicity against the four species. Meperfluthrin and metofluthrin 27

vapors demonstrated comparable toxicity. Prallethrin and metofluthrin vapors were similarly 28

toxic against Ae. albopictus, but prallethrin was less toxic than metofluthrin against the other 29

species. Flumethrin was the least toxic against all tested species. 30

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This project is applicable to the following three Florida Coordinating Council on Mosquito 32

Control research 2016 priorities (rank): 33

1. Pesticide- Efficacy/ Resistance (1) 34

2. Domestic Mosquito Control (3) 35

3. Application- Adulticides (8) 36

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INTRODUCTION 38

Domestic mosquito species, particularly Aedes albopictus Skuse and Aedes aegypti (L.), 39

provoke high levels of nuisance due to their cryptic oviposition that limits mosquito control 40

district treatment options. This is compounded by short flight ranges increasing the localized 41

human contact with these mosquitoes, and low resource demand for these species to reach high 42

numbers, as evidenced by breeding in shallow containers common across domestic properties. 43

These mosquitoes are also the associated vectors for dengue, chikungunya, and Zika viruses 44

(Derraik and Slaney 2015, Ngoagouni et al. 2015, Wilson and Chen 2015). This elevates the risk 45

of emerging pathogen establishment. Domestic risks also extend to Culex pipiens 46

quinquefasciatus Say, which is a ubiquitous urban vector of St. Louis encephalitis virus and 47

West Nile virus, and whose immatures develop in ditches and urban drain infrastructure (Noori 48

et al. 2015). Recently, this species has been suspected to have compatibility with Zika virus in a 49

laboratory study (Ayres 2016). Due to expansive residential development into swampland and 50

estuarine habitat in Florida, Anopheles spp. mosquitoes also are common species of interest in 51

such landscapes, with Anopheles quadrimaculatus Say being the most important U. S. species 52

tied to malaria transmission (Rutledge et al. 2005). Local malaria transmission occasionally 53

occurs, with Palm Beach County, FL being a Florida example (CDC 2003). 54

These examples demonstrate the importance of citizen awareness of risk and the 55

recognition of their employing personal protective solutions to supplement existing mosquito 56

control operations. Mosquito control programs recruit the citizen base as part of this 57

supplementation to be involved in mosquito habitat identification and source reduction (Marciel-58

de-Freitas and Lourenço-de-Oliveira 2011, Dowling et al. 2013, Fonesca et al. 2013). However, 59

the citizen base also can choose to supplement their vector prevention with over-the-counter 60

pesticides. Among those available tools, volatile pyrethroid compounds, or “spatial repellents,” 61


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have been advocated for urban vector management (Ritchie and Devine 2013). It is important to 62

evaluate the chemicals these consumers use for this supplemental effort. 63

Volatile pyrethroid compounds provide protection in an area well outside the source of 64

chemical dispersion (Achee et al. 2012, Kline and Strickman 2015) and are a marketing 65

alternative to topical repellents that garner favorable usage by citizens (Kline and Strickman 66

2015). By contacting target vectors in a gaseous state, as opposed to the liquid droplets employed 67

by the vast array of mosquito control operations, several different and beneficial properties are 68

achieved in their use. The marketing drive for their use revolves around repellency. Some 69

compounds exhibit repellency, such as metofluthrin repelling Ae. albopictus (Argueta et al. 70

2004), and prallethrin repelling Cx. quinquefasciatus and Culex tritaeniorhynchus Giles (Liu et 71

al. 2009). More consistently, these and similar compounds instigate a confusion or disorientation 72

in vectors including Ae. aegypti (Achee et al. 2009, Ritchie and Devine 2013). Some research 73

studies have reported mosquito mortality following use of these compounds, such as in Ae. 74

aegypti exposure to metofluthrin (Bibbs and Xue 2015, Ritchie and Devine 2013), Ae. albopictus 75

exposure to transfluthrin (Lee 2007), and Anopheles albimanus, Cx. quinquefasciatus, and Ae. 76

albopictus exposure to metofluthrin (Xue et al. 2012). 77

There is a need to measure the toxicity of volatile pyrethroids strictly in the vapor phase 78

to maximize the possibility of these chemicals preventing vector-borne pathogen transmission. 79

Being that volatile pyrethroids already have a pre-existing market in public-use products, a 80

systematic approach comparing the toxicity of candidate vapor active chemicals in a single study 81

may guide future use of these compounds and will provide valuable information to the end user. 82

This project will provide efficacy data on several available volatile pyrethroid compounds 83

against a variety of common domestic vector threats. This would provide an assessment on 84


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whether these compounds are detrimental or supplemental to the efforts of Florida mosquito 85

control programs, information that will be quite valuable as Florida continues to face the pending 86

arrival of vector-borne threats. 87

MATERIALS AND METHODS 88

Mosquitoes. Mosquito species used in this study were pyrethroid susceptible strains 89

acquired from the United States Department of Agriculture, Agricultural Research Service, 90

Center for Medical, Agricultural, and Veterinary Entomology (USDA-ARS-CMAVE) in 91

Gainesville, Florida. The strains used were the 1952 Orlando, FL, strain Ae. aegypti; 1998 92

Gainesville, FL, strain Ae. albopictus; 1952 Orlando, FL, strain Cx. quinquefasciatus; and 1952 93

Orlando, FL, strain An. quadrimaculatus. Mosquito strains were not exposed to insecticides prior 94

to evaluation and were not supplemented with wild-type introductions to the colonies. Rearing 95

conditions consisted of 26.6 °C, 85 ± 5% relative humidity (RH), with a photoperiod of 14:10 96

(L:D). Batches of 2,000 eggs were placed in larval pans containing 2,500 ml of reverse osmosis 97

(RO) water. Larvae were fed 1-3 g of liver and yeast mixture at a 3:2 ratio ad libitum in a 50-ml 98

suspension. Adult mosquitoes were kept in flight cages containing separate supplies of 10% 99

sucrose solution and reverse osmosis (RO) water. Subjects used in experiments were non-blood-100

fed, 5-7 day-old female mosquitoes. 101

Chemicals. Technical grade prallethrin (32917 Pestanal, Sigma-Aldrich Co. LLC, St. 102

Louis, MO), flumethrin (N-13139, Chem Service, Inc., West Chester, PA), transfluthrin (N-103

13626, Chem Service, Inc., West Chester, PA), meperfluthrin (32065 Pestanal, Sigma-Aldrich 104

Co. LLC, St. Louis, MO), and metofluthrin were selected for this test. Metofluthrin was 105

extracted from OFF! Clip-on over-the-counter refill packs (31.2% metofluthrin, S. C. Johnson & 106

Son, Racine, WI) using pentane. Extracts were fractionated using automated flash 107


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chromatography (CombiFlash Rd 200i, Teledyne ISCO, Lincoln, NE) (Fig. 1) with simultaneous 108

electrospray ionization mass spectrometry (ESI-MS/MS) (Expressions CMS, Advion, Inc., 109

Ithaca, NY) (Fig. 2). Fractions were delivered using pentane as the non-polar solvent and ethyl 110

ether as the polar solvent at a 10 ml/min flow rate and a 5 ml peak runtime. Solvent was reduced 111

in a rotary evaporator and the resultant technical grade product was checked using gas 112

chromatography mass spectrometry (Supp. Fig. 1, Supp. Fig. 2.1 – 2.2). Each technical grade 113

pyrethroid was serially diluted in acetone to create screening concentrations of 5.00%, 1.00%, 114

0.50%, 0.10%, 0.05%, and 0.01% solutions by weight and stored in amber borosilicate vials (14-115

955-331, Thermo Fisher Scientific, Hampton, NH). Up to seven additional concentrations 116

(different for each chemical) were selected with respect to the initial six range-finding dilutions, 117

for a total of up to 13 concentrations, to collect sufficient data to determine LC50 and LC90 values 118

for each chemical with each mosquito species. 119

Fumigant Bioassays. Test cages consisted of single-use 473 ml clear polypropylene 120

snap-lid cups (MN16-0100, Dart Container Corp, Mason, MI) with the lid modified to have a 121

central 20-mm opening. Twenty female mosquitoes of a single species were aspirated into each 122

container. Filter paper strips (Grade 1 MFR# 28413934, Whatman PLC, Little Chalfont, UK) 123

were cut into 5-mm widths and 40-mm lengths and pleated every 5 mm before being treated with 124

40-µl of a chemical solution (Fig 3). Treated strips were allowed 6-min drying periods before 125

transfer into a mesh bag (Nylon Tulle No: 147356, Falk Industries, Inc., New York, NY) that 126

was suspended within the test cage through the hole in the modified lid (Fig 4). The hole was 127

then sealed to prevent vapor escape during testing. One concentration of a single chemical was 128

used in each treatment cage. Controls were strips treated with only acetone. Test cages were 129

stored in an incubator (Precision Mo: 818, Thermo Fisher Scientific, Hampton, NH) to maintain 130


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26.6 °C, 85 ± 5% RH, with a photoperiod of 14:10 (L:D) for the duration of data collection. 131

Mesh bags holding the filter paper strip were removed from cages after 2 hours and replaced 132

with a cotton ball soaked with a 10% sucrose solution. Contamination was limited through a 133

strict requirement that no cage materials were reused in subsequent tests. 134

Residual activity data were collected by allowing mosquitoes exposed to vapors to 135

remain in the original testing containers for the 24-hr test duration. Mosquitoes in a prone 136

position and suffering from ataxia that prevented proper upright resting, walking, and flight were 137

considered moribund. Mosquitoes in a prone position and rigidly immobilized were considered 138

dead. The mortality scored in a test cage was comprised of the total combined score of moribund 139

and dead mosquitoes. Mortality was scored at 2, 4, and 24 hours post exposure. 140

A second series of experiments following the above test cage construction and exposure 141

procedures was performed to assess recovery from vapor exposure. The holding conditions 142

deviate in that mosquitoes were transferred to untreated test cages after exposing them to vapors 143

for 2 hours. Mortality was scored by the same procedures. Mortality was recorded for these 144

insects only after being held for 24 hours. This was repeated for all mosquito species and 145

chemical concentrations to assess potential for metabolic recovery after treatment. 146

Data analysis. Probit analyses were performed in PoloPlus (Version 1.0, LeOra Software 147

LLC, Cape Girardeau, MO) to determine descriptive statistics and predictive dose responses of 148

prallethrin, flumethrin, transfluthrin, metofluthrin, and meperfluthrin for Ae. aegypti, Ae. 149

albopictus, Cx. quinquefasciatus, and An. quadrimaculatus at each repeated measure of time. A 150

minimum of four replications with at least 2,080 individuals for each mosquito species were used 151

per chemical to generate LC50 and LC90 values with a 95% confidence limits (CL), expressed in 152

m/v (mass/volume or g/100 ml). Data was discarded if control mortality in excess of 10% 153


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occurred within a replicate. Probit analysis included correction for control mortality using 154

Abbott’s formula (Abbott 1925). If the lower CL and upper CL of two LC values did not 155

overlap, either within one chemical but across species or with any chemical but within species, 156

then the difference was considered significant (p < 0.05). Variation between chemicals and 157

across time points was analyzed in JMP 13.1.0 (SAS Institute, Inc., Cary, NC) using repeated 158

measures ANOVA. 159

RESULTS 160

As identified in our grant objectives, we completed testing of Ae. albopictus, Ae. aegypti, 161

Cx. quinquefasciatus, and An. quadrimaculatus with all four original candidate chemicals as well 162

as one additional chemical, meperfluthrin. Objective 1 and 2 has been completed. The LC50 and 163

LC90 values representing vapor toxicity of each pyrethroid against Ae. albopictus, Ae. aegypti, 164

Cx. quinquefasciatus, and An. quadrimaculatus are listed in Table 1 and Table 2, respectively. 165

Toxicity responses for many of the selected chemistries were mixed when evaluated 166

across the tested mosquito species. Confidence limit comparisons with LC50 values indicated 167

meperfluthrin had the highest vapor toxicity against Ae. albopictus, Ae. aegypti, and An. 168

quadrimaculatus. Transfluthrin had the second highest toxicity, followed by metofluthrin, 169

prallethrin, and then flumethrin. In contrast, transfluthrin demonstrated the highest toxicity in Cx. 170

quinquefasciatus, followed by meperfluthrin, prallethrin, metofluthrin, and flumethrin. 171

Flumethrin vapors were much less toxic than the differences observed between any other 172

comparisons of tested pyrethroids, regardless of species. When taken with LC90 comparisons, 173

transfluthrin and meperfluthrin were not significantly different in responses generated against Ae. 174

albopictus, Cx. quinquefaciatus, and An. quadrimaculatus. When evaluating the confidence 175


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limits at LC90 of sets where mosquitoes were exposed for 2-hr then transferred for metabolic 176

recovery, transfluthrin was more toxic than meperfluthrin against all four species. 177

Repeated measures ANOVA indicated a statistically significant increase in mortality 178

between 2-hr, 4-hr, and 24-hr time points within each chemical against Ae. albopictus (F = 0.97, 179

df = 2, p < 0.0001), Ae. aegypti (F = 0.95, df = 2, p < 0.0001), Cx. quinquefasciatus (F = 175.80, 180

df = 2, p < 0.0001) and An. quadrimaculatus (F = 163.84, df = 2, p < 0.0001). Differences in the 181

relative toxicity between chemicals was observed as exposure time increased against Ae. 182

albopictus (F = 15.86, df = 4, p < 0.0001), Ae. aegypti (F = 9.10, df = 4, p < 0.0001), Cx. 183

quinquefasciatus (F = 7.72, df = 4, p < 0.0001) and An. quadrimaculatus (F = 29.41, df = 4, p < 184

0.0001), with the slopes being different between chemicals. 185

DISCUSSION 186

Mosquitoes. Susceptible mosquito strains were acquired through Joyce Urban, the 187

USDA-ARS-CMAVE collaborator included in the submission of the grant. Joyce Urban, Haze 188

Brown, and Greg Knue assisted in the training of an insectary technician for the grant and 189

delivered a starting egg clutch to begin the experiment colonies. As with Ae. albopictus, 190

pyrethroid susceptible strain colonies of Aedes aegypti, Culex quinquefasciatus, and An. 191

quadrimaculatus were established from eggs provided by the USDA-ARS-CMAVE. These 192

colonies were maintained in the same abiotic conditions as Ae. albopictus, but in different rooms 193

of the rearing facility. 194

Chemicals. Metofluthrin was difficult to acquire. Through a different collaborative 195

agreement offered at the time of the grant’s submission, Dr. Uli Bernier and associates with the 196

USDA-ARS-CMAVE team assisted with extraction of metofluthrin from commercially-available 197


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products in order to circumvent supply barriers (Fig. 1, Fig. 2, Supp. Fig. 1, Supp. Fig. 2.1 – 2.2). 198

The metofluthrin extracted was sufficient to test in the four intended mosquito species. 199

Fumigant Bioassays. The original testing design was inefficient with vapor dispersal. A 200

borosilicate glass housing was used to exclude mosquitoes from physical contact during testing 201

(Fig 4). This design resulted in limited and inconsistent mortality data, perhaps due to vapors 202

being unable to disperse upwards and out of the vial in a consistent manner. Preliminary data 203

generated by the old design were set aside. A new data set was collected using the modified 204

design reported in the Materials and Methods section. We have generated LC50 and LC90 data, 205

with results tabulated in this report (Table 1, Table 2). With the data acquired from the adapted 206

experimental design the LC50 and LC90 values for these three species were successfully 207

completed along with the addition of meperfluthrin (Table 1, Table 2). Objectives 1 and 2 are 208

considered completed. 209

Upon conclusion of study, some residual contamination and degradation of resources was 210

accounted for among rearing supplies, bioassay tools and materials, and technical grade chemical 211

stores. Replacement of contaminated, damaged, or otherwise compromised resources utilized 212

within the duration of this study was exacted from the leftover supply funding in the grant. The 213

allocation of funding and supplies was sufficient to address the expected needs. 214

Importance to Florida Mosquito Control. Transfluthrin, meperfluthrin, and 215

metofluthrin are type-I pyrethroids containing polyfluorinated alcohols in their structure. These 216

chemistries generated higher mortality than either prallethrin, a non-fluorinated type-pyrethroid 217

already used as a mosquito control adulticide, and flumethrin, a type-II pyrethroid with a 218

monofluorinated alcohol. Volatile pyrethroids containing polyfluorinated alcohols appear to be 219

better development targets based on the results of this dose response study. Very poor 220


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performance was observed with flumethrin across all species, with the lowest activity observed 221

in Ae. albopictus. It is suspected that limited vaporization pressure with flumethrin is the cause 222

of the reduced efficacy. After reviewing literature again and discussing actions with 223

collaborating toxicologists, appropriate formulation could circumvent this drawback. We are 224

investigating possible avenues to improve the delivery of flumethrin with future work. 225

Because of the consistently low activity of flumethrin, meperfluthrin was added to the 226

treatment assays. Meperfluthrin generally performed as well or better than transfluthrin when 227

comparing LC50 values. However, when comparing LC90 values and the sets in which mosquitoes 228

were transferred to clean containment for recovery, transfluthrin and meperfluthrin generally 229

performed equivalently, with transfluthrin outperforming meperfluthrin against Ae. albopictus, 230

Ae. aegypti, and Cx. quinquefasciatus. Interestingly, treatments in which mosquitoes were 231

transferred to clean containment after exposure to enable metabolic recovery resulted in 232

transfluthrin yielding the highest toxicity against all four mosquito species. The LC50 is generally 233

a metric with fewer errors when comparing chemistries, and by this metric meperfluthrin was the 234

highest performing compound against all species. However, the LC90 is more relevant to future 235

interest because of the need to attain a minimum of 80% effect for target chemistries to move 236

forward in product development. The cohorts in which mosquitoes were allowed to recover from 237

exposure in clean containment are also more realistic to application environments, because it is 238

unlikely that mosquitoes will have sustained contact for long periods of time. By the LC90 239

metrics, particularly when evaluating the recovery group, transfluthrin significantly 240

outperformed meperfluthrin. This suggests that transfluthrin is a more pragmatic chemistry to 241

examine in future study. 242


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To date, transfluthrin and meperfluthrin are not components in any EPA labeled products 243

in the United States. These two chemistries could be good candidates to move forward with 244

product development given the success of metofluthrin, an active ingredient in several EPA 245

registered products. Furthermore, the potency of all three of these pyrethroids with 246

polyfluorinated alcohols warrants study in other areas. Spatial repellents are only one delivery 247

mechanism for these chemistries, and it is still unclear what sub-lethal impacts might occur in 248

vectors when the targets are exposed to pyrethroid vapors. How vapor active compounds, 249

especially volatile pyrethroids, interface with resistance management issues is also an 250

unanswered dilemma. However, there are benefits to working with vapors. 251

Traditional adulticiding, in which pyrethroids are a critical chemical class for public 252

health pest control, relies on dispersing find droplets through the air to deliver active ingredients 253

to the mosquito. The chemistry must impinge upon and penetrate into the cuticle of the target. A 254

weak, but common and non-selective, resistance mechanism is to have resistance to penetration. 255

Volatile pyrethroids are vapors. This promotes a different route of entry that potentially bypasses 256

penetration resistance due to inhaling the toxin instead of absorbing it through the outer cuticle. 257

Furthermore, the higher performing chemistries evaluated in this study, pyrethroids with 258

polyfluorinated alcohols, are intended to be resilient to detoxification enzymes due to the 259

fluorine molecules occluding target sites for cytochrome p450. Metabolic resistance is a strong 260

mechanism for reducing the efficacy of public health treatment efforts. In addition, resistance 261

mechanisms tend to have multiplicative interaction with each other. Therefore, compounds with 262

qualities that mitigate more than one pathway of resistance are practical topics of study. We 263

believe transfluthrin, meperfluthrin, and metofluthrin are strong candidates as vapor active 264

insecticides. 265


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ACKNOWLEDGEMENTS 266

Funding was provided by the Florida Department of Agriculture and Consumer Services 267

project P0010729. Gratitude is extended to Maia Tsikolia, Nurhayat Tabanca, and Ulrich Bernier 268

from the USDA-ARS-CMAVE for sharing lab space, equipment, and their expertise in the 269

chemical extraction of metofluthrin. Jeff Bloomquist’s logistical consultation is appreciated for 270

facilitating completion of the work to date. 271

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Tables 338

339

340

Table 1. Comparative LC50 of four volatile pyrethroids, delivered as a vapor, to susceptible strains of four vector mosquito species a Exposure Reading b Pyrethroid Ae. albopictus Ae. aegypti

2 hours Transfluthrin 0.052 (0.038 – 0.072) 0.055 (0.039 – 0.078) Meperfluthrin 0.035 (0.025 – 0.051) 0.025 (0.018 – 0.034) Metofluthrin 0.588 (0.427 – 0.809) 0.269 (0.183 – 0.396) Prallethrin 0.936 (0.660 – 1.327) 0.673 (0.471 – 0.963) Flumethrin 16.222 (10.083 – 26.100) 8.956 (3.143 – 25.519) 4 hours Transfluthrin 0.034 (0.025 – 0.047) 0.034 (0.023 – 0.050) Meperfluthrin 0.026 (0.018 – 0.038) 0.019 (0.014 – 0.028) Metofluthrin 0.444 (0.351 – 0.561) 0.159 (0.105 – 0.241) Prallethrin 0.335 (0.210 – 0.533) 0.445 (0.302 – 0.654) Flumethrin 11.390 (7.604 – 17.060) 2.215 (0.906 – 5.419) 24 hours Transfluthrin 0.029 (0.023 – 0.037) 0.002 (0.001 – 0.006) Meperfluthrin 0.019 (0.013 – 0.028) 0.015 (0.011 – 0.022) Metofluthrin 0.318 (0.246 – 0.410) 0.053 (0.035 – 0.080) Prallethrin 0.222 (0.137 – 0.359) 0.280 (0.187 – 0.419) Flumethrin 7.662 (5.464 – 10.744) 1.842 (0.769 – 4.409) 2 hours, Transfluthrin 0.041 (0.031 – 0.054) 0.043 (0.036 – 0.053) transferred Meperfluthrin 0.048 (0.034 – 0.067) 0.030 (0.022 – 0.043) Metofluthrin 0.848 (0.661 – 1.088) 0.077 (0.050 – 0.120) Prallethrin 0.543 (0.417 – 0.706) 1.139 (0.599 – 2.167) Flumethrin 11.508 (7.600 – 17.426) 11.114 (3.484 – 35.455) a Values are LC50 with 95% fiduciary limits (lower FL, upper FL) shown in ppm (µg/cm3 or µg/ml). Based on serial dilutions of compounds applied to paper strips in a 473.18 ml air space. b Mosquitoes exposed without removal from original test container (2 hr, 4 hr, and 24 hr) and mortality recorded or exposed for 2 hr and transferred to clean containers with mortality recorded 24 hr after initial exposure.


18

Table 1, Continued. Comparative LC50 of four volatile pyrethroids, delivered as a vapor, to susceptible strains of four vector mosquito species a Exposure Reading b Pyrethroid Cx. quinquefasciatus An. quadrimaculatus


341


19

342

343

Table 2. Comparative LC90 of four volatile pyrethroids, delivered as a vapor, to susceptible strains of four vector mosquito species a Exposure Reading b Pyrethroid Ae. albopictus Ae. aegypti



20

344

345

346

Table 2, Continued. Comparative LC90 of four volatile pyrethroids, delivered as a vapor, to susceptible strains of four vector mosquito species a Exposure Reading b Pyrethroid Cx. quinquefasciatus An. quadrimaculatus



21

Figures 347

348

Figure 1. Metofluthrin extracts were fractionated using automated flash chromatography 349

(CombiFlash Rd 200i, Teledyne ISCO, Lincoln, NE). Fractions were delivered using pentane as 350

the a-polar solvent and ethyl ether as the polar solvent at a 10ml/min flow rate and a 5ml peak 351

runtime. 352

353


22

354

Figure 2. Simultaneous mass spectrometry (Expressions CMS, Advion, Inc., Ithaca, NY) of 355

metofluthrin fractions during automated flash chromatography. 356

357


23

358

Figure 3. Chemical solution being applied to Whatman No.1 filter paper, which was cut into 359

strips with dimensions of 5 mm x 40 mm, pleated every 5 mm in length. Applications were made 360

by using a 20-µl pipette fitted with a filter tip to administer 40 µl of solution in two passes. 361

Aliquots were kept in amber borosilicate vials to protect the chemical integrity. 362

363

364


24

365

Figure 4. Clear polyethylene test containers with a volume of 473 ml. Snap-lids were modified 366

with a 20-mm opening to allow admission of 20 female, non-blood-fed, 5-7 day-old mosquitoes. 367

Left: Treated filter paper strips were contained within a 4 ml borosilicate vial to allow passage of 368

vapors while excluding direct contact. This method failed to allow consistent vapor dispersal. 369

Right: Cages were modified to a design where treated filter paper strips were contained within a 370

mesh bag suspended from the opening to allow passage of vapors while excluding direct contact. 371

Container openings were sealed during testing. This modification allowed consistent data 372

collection. 373

374


25

Supplemental Figures 375

376

Supplemental Figure 1. Automated flash chromatography (CombiFlash Rd 200i, Teledyne 377

ISCO, Lincoln, NE) inputs/outputs for metofluthrin fraction separation. 378


26

379

Supplemental Figure 2.1. Total ion chromatogram generated with atmospheric-pressure 380

chemical ionization. Fingerprinting of three out of four fraction cycles shown. 381


27

382

Supplemental Figure 2.2. Total ion chromatogram generated with atmospheric-pressure 383

chemical ionization. Fingerprinting of the fourth of four fraction cycles shown. Followed by 384

tabulated output for all four chromatograms. 385


28

386

Supplemental Figure 2.3. Positive and negative electrospray ionization responses, run in 387

tandem with mass spectrometry. 388

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