FDACS Project P0010729 Final Report: September 2017

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

3 Principle Investigator: Phillip E. Kaufman, PhD

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

6 Project Objectives:

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

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

9 initial screening species.

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

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

12 mosquito species.

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

33 Control research 2016 priorities (rank):

34 1. Pesticide- Efficacy/ Resistance (1)

35 2. Domestic Mosquito Control (3)

36 3. Application- Adulticides (8)

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

87 arrival of vector-borne threats.

88 MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

119 for each chemical with each mosquito species.

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

159 measures ANOVA.

160 RESULTS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

178 Repeated measures ANOVA indicated a statistically significant increase in mortality

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

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

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

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

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

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

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

186 DISCUSSION

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

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

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

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

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

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

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

194 of the rearing facility.

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

209 considered completed.

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

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

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

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

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

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

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

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

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

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

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

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

242 examine in future study.

FDACS Project P0010729 Final Report: September 2017

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

265 insecticides.

FDACS Project P0010729 Final Report: September 2017

266 ACKNOWLEDGEMENTS

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

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

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

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

271 facilitating completion of the work to date.

272 REFERENCES CITED 273 Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J Econ

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289 Bibbs, C.S. and R.D. Xue. 2015. OFF! Clip-on repellant device with metofluthrin tested on

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FDACS Project P0010729 Final Report: September 2017

338 Tables

Table 1. Comparative LC50 of four volatile pyrethroids, delivered as a vapor, to susceptible strains of four vector a mosquito species 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 3 Values are LC50 with 95% fiduciary limits (lower FL, upper FL) shown in ppm (µg/cm 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. 339 340

FDACS Project P0010729 Final Report: September 2017

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

2 hours Transfluthrin 0.017 (0.011 – 0.026) 0.023 (0.016 – 0.032) Meperfluthrin 0.024 (0.016 – 0.036) 0.015 (0.010 – 0.024) Metofluthrin 0.384 (0.256 – 0.576) 0.040 (0.030 – 0.055) Prallethrin 0.211 (0.137 – 0.326) 0.369 (0.255 – 0.533) Flumethrin 3.382 (1.626 – 7.037) 69.153 (29.375 – 162.797)

4 hours Transfluthrin 0.011 (0.008 – 0.017) 0.016 (0.011 – 0.022) Meperfluthrin 0.021 (0.014 – 0.031) 0.012 (0.007 – 0.018) Metofluthrin 0.227 (0.151 – 0.341) 0.032 (0.023 – 0.045) Prallethrin 0.150 (0.100 – 0.225) 0.290 (0.199 – 0.422) Flumethrin 2.631 (1.247 – 5.552) 44.988 (18.870 – 107.258)

24 hours Transfluthrin 0.008 (0.005 – 0.014) 0.011 (0.007 – 0.015) Meperfluthrin 0.011 (0.007 – 0.018) 0.009 (0.006 – 0.014) Metofluthrin 0.142 (0.092 – 0.219) 0.023 (0.017 – 0.030) Prallethrin 0.091 (0.057 – 0.145) 0.202 (0.134 – 0.305) Flumethrin 2.135 (0.996 – 4.573) 22.282 (9.987 – 49.715)

2 hours, Transfluthrin 0.020 (0.014 – 0.028) 0.043 (0.032 – 0.058) transferred Meperfluthrin 0.036 (0.025 – 0.050) 0.020 (0.013 – 0.030) Metofluthrin 0.729 (0.510 – 1.041) 0.033 (0.025 – 0.043) Prallethrin 0.277 (0.201 – 0.382) 0.463 (0.315 – 0.680) Flumethrin 5.421 (2.567 – 11.448) 56.002 (22.710 – 138.102)

FDACS Project P0010729 Final Report: September 2017

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

2 hours Transfluthrin 0.229 (0.167 – 0.315) 0.350 (0.247 – 0.496) Meperfluthrin 0.238 (0.166 – 0.342) 0.133 (0.095 – 0.184) Metofluthrin 2.342 (1.702 – 3.224) 2.570 (1.750 – 3.775) Prallethrin 4.424 (3.121 – 6.272) 4.149 (2.900 – 5.936) Flumethrin 135.870 (84.450 – 218.600) 2727.086 (957.076 – 7770.547)

4 hours Transfluthrin 0.153 (0.112 – 0.209) 0.278 (0.187 – 0.412) Meperfluthrin 0.202 (0.138 – 0.295) 0.125 (0.087 – 0.179) Metofluthrin 1.518 (1.202 – 1.918) 1.424 (0.940 – 2.160) Prallethrin 3.388 (2.129 – 5.393) 3.362 (2.286 – 4.944) Flumethrin 72.330 (48.290 – 108.339) 294.724 (120.486 – 720.935)

24 hours Transfluthrin 0.076 (0.059 – 0.097) 0.058 (0.024 – 0.141) Meperfluthrin 0.132 (0.092 – 0.191) 0.101 (0.069 – 0.146) Metofluthrin 1.298 (1.005 – 1.677) 0.487 (0.323 – 0.733) Prallethrin 2.162 (1.337 – 3.495) 1.810 (1.210 – 2.706) Flumethrin 36.296 (25.884 – 50.895) 205.738 (85.927 – 492.609)

2 hours, Transfluthrin 0.160 (0.121 – 0.210) 0.108 (0.089 – 0.132) transferred Meperfluthrin 0.277 (0.197 – 0.392) 0.175 (0.125 – 0.245) Metofluthrin 2.899 (2.260 – 3.719) 0.847 (0.546 – 1.314) Prallethrin 1.675 (1.286 – 2.180) 33.801 (17.766 – 64.308) Flumethrin 73.544 (48.567 – 111.366) 5527.478 (1732.754 – 17632.628)

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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

2 hours Transfluthrin 0.195 (0.124 – 0.306) 0.151 (0.107 – 0.215) Meperfluthrin 0.186 (0.126 – 0.274) 0.165 (0.104 – 0.261) Metofluthrin 3.666 (2.446 – 5.494) 0.211 (0.155 – 0.287) Prallethrin 2.397 (1.553 – 3.699) 3.301 (2.283 – 4.771) Flumethrin 156.367 (75.157 – 325.327) 3306.676 (1679.380 – 9307.141)

4 hours Transfluthrin 0.082 (0.055 – 0.123) 0.097 (0.068 – 0.138) Meperfluthrin 0.161 (0.109 – 0.237) 0.117 (0.074 – 0.184) Metofluthrin 2.280 (1.517 – 3.426) 0.209 (0.149 – 0.293) Prallethrin 1.439 (0.957 – 2.163) 2.610 (1.793 – 3.801) Flumethrin 134.516 (63.752 – 283.827) 2246.582 (942.308 – 5356.138)

24 hours Transfluthrin 0.111 (0.066 – 0.186) 0.065 (0.044 – 0.095) Meperfluthrin 0.121 (0.076 – 0.193) 0.066 (0.044 – 0.101) Metofluthrin 1.510 (0.981 – 2.325) 0.102 (0.077 – 0.137) Prallethrin 1.061 (0.665 – 1.693) 2.161 (1.432 – 3.260) Flumethrin 120.487 (56.241 – 258.121) 1076.406 (482.436 – 2401.667)

2 hours, Transfluthrin 0.136 (0.095 – 0.196) 0.191 (0.143 – 0.256) transferred Meperfluthrin 0.202 (0.144 – 0.283) 0.191 (0.123 – 0.296) Metofluthrin 11.858 (8.299 – 16.943) 0.146 (0.112 – 0.191) Prallethrin 1.811 (1.313 – 2.496) 4.836 (3.291 – 7.107) Flumethrin 263.637 (124.844 – 556.728) 3141.073 (1273.753 – 7745.880)

FDACS Project P0010729 Final Report: September 2017

347 Figures

348

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

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

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

352 runtime.

353

FDACS Project P0010729 Final Report: September 2017

354

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

356 metofluthrin fractions during automated flash chromatography.

357

FDACS Project P0010729 Final Report: September 2017

358

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

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

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

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

363

364

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365

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

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

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

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

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

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

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

373 collection.

374

FDACS Project P0010729 Final Report: September 2017

375 Supplemental Figures

376

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

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

FDACS Project P0010729 Final Report: September 2017

379

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

381 chemical ionization. Fingerprinting of three out of four fraction cycles shown. 26

FDACS Project P0010729 Final Report: September 2017

382

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

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

385 tabulated output for all four chromatograms.

FDACS Project P0010729 Final Report: September 2017

386

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

388 tandem with mass spectrometry.