Mosquitocidal Activity and Mode of Action of the Isoxazoline Fluralaner

International Journal of Environmental Research and Public Health

Article Mosquitocidal Activity and Mode of Action of the Isoxazoline Fluralaner

Shiyao Jiang 1, Maia Tsikolia 1, Ulrich R. Bernier 2 and Jeffrey R. Bloomquist 1,*

1 Emerging Pathogens Institute, Department of Entomology and Nematology, University of Florida, Gainesville, FL 32610, USA; shiyao.jiang@uﬂ.edu (S.J.); [email protected] (M.T.) 2 USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL 32608, USA; [email protected] * Correspondence: [email protected]ﬂ.edu; Tel.: +1-352-294-5166

Academic Editor: Paul B. Tchounwou Received: 22 December 2016; Accepted: 31 January 2017; Published: 6 February 2017

Abstract: Mosquitoes, such as Aedes aegypti and Anopheles gambiae, are important vectors of human diseases. Fluralaner, a recently introduced parasiticide, was evaluated as a mosquitocide in this study. On Ae. aegypti and An. gambiae fourth-instar larvae, fluralaner had 24-h LC50 (lethal concentration for 50% mortality) values of 1.8 ppb and 0.4 ppb, respectively. Following topical application to adult Ae. aegypti, fluralaner toxicity reached a plateau in about 3 days, with 1- and 3-day LD50 (lethal dose for 50% mortality) values of 1.3 ng/mg and 0.26 ng/mg, suggesting a slowly developing toxicity. Fipronil outperformed fluralaner by up to 100-fold in adult topical, glass contact, and feeding assays on Ae. aegypti. These data show that fluralaner does not have exceptional toxicity to mosquitoes in typical exposure paradigms. In electrophysiological recordings on Drosophila melanogaster larval central nervous system, the effectiveness of fluralaner for restoring nerve firing after gamma-aminobutyric acid (GABA) treatment, a measure of GABA antagonism, was similar in susceptible Oregon-R and cyclodiene-resistant rdl-1675 strains, with EC50 (half maximal effective concentration) values of 0.34 µM and 0.29 µM. Although this finding suggests low cross resistance in the presence of rdl, the moderate potency, low contact activity, and slow action of fluralaner argue against its use as an adult mosquitocide for vector control.

Keywords: Aedes aegypti; Anopheles gambiae; Drosophila melanogaster; ﬁpronil; GABA receptor

1. Introduction Gamma-aminobutyric acid (GABA) is present in both mammals and invertebrates and is an important inhibitory neurotransmitter [1]. Among the different types of GABA receptors, the GABA receptor-chloride channel complex is an important target site for insecticides, such as lindane and fipronil [2]. However, target site mutations have significantly reduced the utility of conventional GABAergic insecticides. Mutation A301S [3,4], which was originally described as A302S [5] and studied in cultured neurons from Drosophila melanogaster, confers ca. 100-fold and ca. 1000-fold resistance to picrotoxinin and lindane, respectively [6]. Therefore, development of insecticides that work on novel binding sites of this receptor could help avoid cross resistance problems and contribute to effective control of pest insects. Isoxazolines and meta-diamides have emerged as second-generation GABAergic compounds in the search for novel insecticides [7]. Two representative isoxazolines, fluralaner (Figure1) and afoxolaner, were developed by scientists from Nissan Chemical Industries in Japan and DuPont in the U.S., respectively [8,9]. Both compounds have been approved by the U.S. Food and Drug Administration (FDA) to be commercialized as parasiticides. Fluralaner shows no cross-resistance in both in vivo and in vitro studies against various insects species compared to other classic GABAergic

Int. J. Environ. Res. Public Health 2017, 14, 154; doi:10.3390/ijerph14020154 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2017, 14, 154 2 of 17 Int. J. Environ. Res. Public Health 2017, 14, 154 2 of 17 compounds [10,11], and demonstrates good selectivity towards mammals vs. invertebrate pests [10,12]. compounds [10,11], and demonstrates good selectivity towards mammals vs. invertebrate pests [10,12]. According to Gassel et al. [11], fluralaner has toxicity greater than fipronil against six insect and According to Gassel et al. [11], fluralaner has toxicity greater than fipronil against six insect and parasite species, and blocks homo-oligomeric GABA receptors expressed in cell lines with high parasite species, and blocks homo-oligomeric GABA receptors expressed in cell lines with high potency. The only available data on mosquito toxicity of fluralaner is that a low concentration of potency. The only available data on mosquito toxicity of fluralaner is that a low concentration of 1.2 ppt (parts per trillion, 10−12 g/mL) killed more than 90% of first-instar Ae. aegypti larvae, which is 1.2 ppt (parts per trillion, 10−12 g/mL) killed more than 90% of first-instar Ae. aegypti larvae, which is ca. 16,000-fold more potent than fipronil [11]. The goal of the present study is to investigate fluralaner ca. 16,000-fold more potent than fipronil [11]. The goal of the present study is to investigate fluralaner toxicity on Ae. aegypti and An. gambiae via different exposure routes, and its activity on native GABA toxicity on Ae. aegypti and An. gambiae via different exposure routes, and its activity on native GABA receptor responses of CNS preparations of susceptible and resistant D. melanogaster strains. receptor responses of CNS preparations of susceptible and resistant D. melanogaster strains.

Figure 1. ChemicalChemical structures structures of fipronil ﬁpronil and fluralaner. ﬂuralaner.

2. Materials and Methods

2.1. Chemicals

Samples of dieldrin and L-aspartic-aspartic acid acid were were purchased from from Sigma-Aldrich Chemical Chemical Co. Co. (St. Louis,Louis, MO, USA).USA). FipronilFipronil (Figure(Figure1 )1) was was donated donated by by Rhône-Poulenc Rhône-Poulenc Ag Ag Co. Co. (now(now BayerBayer CropScience, Research Triangle Park, NC, USA), and Triton X-100 was acquired from Thermo Fisher ScientificScientific Inc. (Waltham, (Waltham, MA, MA, USA). Diethyl maleate (DEM) was purchased from Sigma-AldrichSigma-Aldrich Chemical Co., and piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) were obtained from Chem Service Inc. (West (West Chester, PA, USA). Rapeseed-oil methyl ester (RME) was purchased from UCY Energy (Alfter, Germany), and silicon oil was acquired acquired from from Dow Dow Corning Corning Co. Co. (Auburn, MI, USA). Ethanol, acetone and dimethyl su sulfoxidelfoxide (DMSO) used as solvents were obtained from Sigma-Aldrich Chemical Co. Fluralaner was extracted and and purified purified from from a a commercial commercial canine canine formulation formulation of of BRAVECTO™ BRAVECTO™ Chews (for large dogs, 44–88 lbs, each containing 1 g of fluralaner), fluralaner), manufactured by Merck Animal Health. Isolation of the the compound compound was was performed performed wi withth a a Teledyne Teledyne Isco flash flash chromatography chromatography system (Lincoln, NE, USA) usingusing hexanes/ethylhexanes/ethyl acetate acetate as as eluent eluent system. Solvents, Solvents, hexanes and ethyl acetate were obtained from Acros Organics (Morris Plains,Plains, NJ, USA). Melting point was determined on a hot-stage apparatusapparatus and and is uncorrected.is uncorrected. Nuclear Nuclear magnetic magnetic resonance resonance (NMR) analyses(NMR) wereanalyses performed were performedat the Nucleic at the Magnetic Nucleic Resonance Magnetic FacilityResonance of the Facility University of the ofUniversity Florida. NMR of Florida. spectra NMR were spectra recorded were in 1 recordedCDCl3 with in CDCl tetramethylsilane3 with tetramethylsilane as the internal as the standard internal for standardH (500 for MHz) 1H (500 and MHz) CDCl and3 as CDCl the internal3 as the 13 internalstandard standard for C (125 for 13 MHz).C (125 MHz). One half of a Bravecto Chew (3.70 g) was placed in a 250-mL separatory funnel, water (80 mL) was added and the funnel was shaken well to obtainobtain a homogenous suspension. Then, ethyl acetate × (80 mLmL × 3) was usedused forfor extraction.extraction. Organic phasesphases werewere combined,combined, washedwashed withwith aq.aq. NaNa22COCO33 solution, aq. 1 N HCl solution, brine and then drieddried over anhydrous sodium sulfate. Any remaining solvent was distilled off under reduced pressure and the resultant residue was purifiedpurified by flashflash chromatography usingusing hexanes/ethyl hexanes/ethyl acetate acetate nonlinear nonlinea gradientr gradient to obtain to obtain 0.48 g0.48 of the g targetof the product target product4-[(5RS)-5-(3,5-dichlorophenyl)-4,5-dihydro-5-(trifluoromethyl)-1,2-oxazol-3-yl]- 4-[(5RS)-5-(3,5-dichlorophenyl)-4,5-dihydro-5-(trifluoromethyl)-1,2-oxazol-3-yl]-N-[2-oxo-2-(2,2,2-N-[2-oxo-2- (2,2,2-trifluoroethylamino)ethyl]-trifluoroethylamino)ethyl]-o-toluamide.o-toluamide. Product Product identity identity and purityand purity (99%) (99%) were were confirmed confirmed with with thin layer chromatography (TLC) and NMR analysis. It was a colorless solid after recrystallization

Int. J. Environ. Res. Public Health 2017, 14, 154 3 of 17 thin layer chromatography (TLC) and NMR analysis. It was a colorless solid after recrystallization ◦ 1 from ethanol; m.p. 171.0–172.0 C; H-NMR (CDCl3) δ 7.54–7.41 (m, 6H), 7.29 (t, J = 6.5 Hz, 1H), 6.92 (t, J = 6.9 Hz, 1H), 4.21 (d, J = 5.1 Hz, 2H), 4.10–4.06 (m, 1H), 3.97–3.88 (m, 2H), 3.72–3.68 (m, 1H), 13 2.44 (s, 3H). C-NMR (CDCl3) δ 169.8, 169.4, 155.4, 138.8, 137.4, 137.1, 135.6, 129.8, 129.5, 129.4, 127.7, 123.4 (q, J = 278.9 Hz), 123.7 (q, J = 285.4 Hz), 87.3 (q, J = 30.5 Hz), 43.9, 43.6, 34.9 (q, J = 40.7 Hz), 19.7.

2.2. Insects Fourth-instar Ae. aegypti larvae were kindly provided by the Center for Medical, Agricultural & Veterinary Entomology (CMAVE), U.S. Department of Agriculture-Agriculture Research Service, Gainesville, FL, USA. The larvae were fed a mixture of ground liver and yeast, maintained under 75% relative humidity and 28 ◦C, with a 12 h:12 h dark:light cycle, and reared to adulthood for bioassays. Eggs of An. gambiae were provided by BEI Resources under the CDC-MR4 program. The emerged larvae were fed with fish flakes (Tetra, Blacksburg, VA, USA), maintained under 75% relative humidity and 28 ◦C, with a 12 h:12 h dark:light cycle, and reared to adulthood for bioassays. Bioassays were performed on susceptible G3 (MRA-112) strain [13]. Susceptible (Oregon-R) and cyclodiene-resistant (rdl-1675) strains of D. melanogaster were used in bioassays and electrophysiology experiments. The Oregon-R strain was originally provided by Dr. Doug Knipple from Cornell University, Ithaca, NY, USA, and maintained in culture at the University of Florida since 2009. The rdl-1675 strain was purchased from the Bloomington Drosophila Stock Center at Indiana University, Bloomington, IN, USA. Both strains were reared at 21 ◦C and provided with artificial media purchased from Carolina Biological Supply, Burlington, NC, USA.

2.3. Larval Mosquito Bioassays Compounds were dissolved in ethyl alcohol, followed by serial dilution to generate an appropriate number of concentrations. A 5 µL aliquot (or more depending on compound solubility) of this solution was added to 5 mL of tap water containing 10 fourth-instar larvae of each treatment group. Tap water treated with the corresponding amount of ethanol was used as the control group. Larvae were held at room temperature (21 ◦C) and observed periodically for behavioral effects, such as convulsions and paralysis, for the first 4 h after treatment. Larval mortality was recorded after 24 h, 48 h and 72 h, or until toxicity reached a stable plateau. In these assays, the plateau was defined as the treatment day where toxicity was not significantly different from the value observed 2 days later. Larvae were fed during the experiment with a few grains of ground fish flakes added to the petri dish every day. A Triton X-100 solution of 10 ppm was also tested to see whether it would increase the toxicity of fluralaner. Paralytic activity of compounds to headless larvae was assessed as described by Islam and Bloomquist [14], with slight modification. Heads of fourth-instar larvae were detached with forceps and treated as described above, except in physiological saline instead of tap water. The saline was composed of (mM): NaCl (154), KCl (2.7), CaCl2 (1.4), HEPES (4) at pH 7.2. Mosquito saline treated with 0.5% ethanol and 100 ppm L-aspartic acid were used as negative and positive controls, respectively. An insect pin was used to probe larvae every hour to observe swimming behavioral responses. Compared to the control group, larvae showing irregular behaviors such as consistent convulsion, twitching or only slight movement were counted as paralyzed. Each concentration was repeated on at least three different batches of mosquitoes. LC50 (lethal concentration for 50% mortality) and PC50 (concentration for 50% paralysis) values were calculated as described in the statistics section.

2.4. Adult Mosquito Bioassays For topical toxicity assays [15], 5–7 days old adult female mosquitoes (non-blood fed, n = 10) were chilled on ice for 2 min, during which time 0.2 µL of insecticide solution (in ethanol) was applied to the thorax by a hand-held microdispenser (Hamilton, Reno, NV, USA). A solvent-only treatment was included in each experiment as a negative control. Following treatment, mosquitoes were transferred Int. J. Environ. Res. Public Health 2017, 14, 154 4 of 17 to paper cups covered with netting. A 10% sugar solution in tap water was supplied via a cotton ball placed on the netting and changed every day. Paper cups were held at room temperature (21 ◦C), and the mosquitoes were observed for behavioral effects such as convulsion or paralysis and the onset of toxicity for the first 4 h. To test for synergistic effects, synergists were applied topically to the abdomen of mosquitoes 4 h before treatments (PBO, 500 ng per mosquito; DEF, 200 ng per mosquito; or DEM, 1 µg per mosquito) at amounts that generally produced little or no mortality. Mortality was recorded after 24 h, 48 h, and 72 h, or until toxicity reached a stable plateau. Each dose was repeated on at least three different batches of mosquitoes. The LD50 (lethal dose for 50% mortality) without synergist and LD50 with synergist were calculated, as described in the statistics section. The synergist ratio was determined by the equation: LD50 of insecticide alone/LD50 of insecticide + synergist. Mosquito injection bioassays [16] were performed under a microscope with a manual microsyringe pump (World Precision Instruments, Sarasota, FL, USA) and a fine glass pipette (TW100-4, World Precision Instruments) fabricated by a pipette puller (Sutter Instrument, Novato, CA, USA), and broken at the tip (ca. 20 µm). Compound solutions were prepared in mosquito saline (described in headless larvae assays) with 5% ethanol as a vehicle. A 5% ethanol solution in saline was used as control. Non-blood fed female mosquitoes (5–7 days post-emergence, n = 10) were chilled on ice and injected with 0.2 µL compound solution into the side of their thorax. Treated mosquitoes were then placed into paper cups and maintained as described above for topical assays. Each dose series was repeated on at least three different batches of mosquitoes, with LD50 values calculated as described in the statistics section. For feeding assays [17], female mosquitoes (5–7 days of age and non-blood fed) were starved for 6 h and chilled on ice for 2 min, then transferred to glass test tubes (n = 10). Compound solutions were prepared in 10% sugar water with 0.5% ethanol used as vehicle. The control group was assigned 10% sugar water with 0.5% ethanol alone. Cotton balls were treated with 1 mL of prepared compound solution and inserted at the top of the test tubes. Test tubes were maintained at room temperature (21 ◦C), with the cotton ball changed and solution reapplied every day. To assess synergist effects on feeding assays, synergists (compounds and doses as given above) were topically applied 4 h before treatment. Mortality data was recorded after 24 h, 48 h, and 72 h, or until toxicity reached a stable plateau, with each experiment repeated at least three times. LC50 values were calculated as described in the statistics section. A contact filter paper toxicity assay was conducted according to WHO protocol [18]. Adult female mosquitoes (n = 10) were 5–7 days of age and non-blood fed at the time of experimentation. Serial dilutions of each insecticide dissolved in ethanol were prepared prior to treatment and 2 mL of each concentration was applied to a 180 cm2 (12 cm × 15 cm) filter paper (Chromatography Paper, Fisher Scientific Pittsburgh, PA, USA). Papers were left to dry for 24 h prior to use. Mosquitoes were chilled for 2 min on ice, after which they were transferred to a WHO cylindrical plastic holding chamber and held for 1 h to acclimatize. The mosquitoes were then gently transferred into the treatment chamber, which contained the treated paper, and exposed for 1 h in the vertical tube. After the 1-h exposure time, the mosquitoes were transferred to the holding chamber as described above, with 10% sugar solution supplied on the netting and changed every day. Holding chambers were left at room temperature (21 ◦C) after the treatment, and mortality was recorded after 24 h, 48 h, and 72 h, or until toxicity reached a stable plateau. Each experiment was repeated on at least three different batches of mosquitoes. LC50 values with and without synergist, as well as synergist ratios were calculated, as described in the statistics section. Additional surface contact assays were conducted in borosilicate glass test tubes (20 mm × 150 mm) with an inner surface area of ca. 85 cm2 (Fisher Brand 14-961-33). Compounds were dissolved in acetone and 250 µL was added to each tube, with 250 µL of acetone as the control group. The test tube was carefully rotated to allow the acetone to evaporate and to distribute the compound evenly inside the tube. Adult female mosquitoes (n = 10) 5–7 days from emergence (non-blood fed) were chilled on ice for 2 min and transferred into compound-coated tubes. To ensure parallel comparison Int. J. Environ. Res. Public Health 2017, 14, 154 5 of 17 to WHO paper assay, in another set of experiments mosquitoes were transferred to clean tubes after 1-h exposure to insecticide-coated glass tubes. A cotton ball with 10% sugar water solution was used to seal test tubes and changed every day to ensure food supply for mosquitoes. Test tubes were maintainedInt. J. Environ. at room Res. temperaturePublic Health 2017, 14 (21, 154◦ C) and mortality data was collected after 24 h, 485 h, of and17 72 h, or untilto toxicity seal test reached tubes and a stablechanged plateau. every day Each to ensure experiment food supply was for repeated mosquitoes. on at Test least tubes three were different batchesmaintained of mosquitoes. at room LC temperature50 values were(21 °C) calculated and mortalit asy describeddata was collected in the statisticsafter 24 h, section.48 h, and 72 h, or until toxicity reached a stable plateau. Each experiment was repeated on at least three different 2.5. Adultbatches D. melanogaster of mosquitoes. Bioassays LC50 values were calculated as described in the statistics section.

Adult2.5. Adult females D. melanogaster (n = 10) from Bioassays both strains (CS-OR and rdl-1675) that were 5–7 days of age were tested in feeding assays and glass contact assays, and the resistance ratio (LC rdl-1675/LC CS-OR) Adult females (n = 10) from both strains (CS-OR and rdl-1675) that were 5–750 days of age were50 was assessedtested viain feeding both routes assays ofand exposure. glass contact Procedures assays, and for the both resistance assays ratio were (LC the50 rdl same-1675/LC as for50 CS-OR) the mosquito bioassays,was except assessed that viaD. both melanogaster routes of exposure.was anesthetized Procedures by for a constantboth assays ﬂow were of the CO same2 for as several for the seconds. mosquito bioassays, except that D. melanogaster was anesthetized by a constant flow of CO2 for several 2.6. Electrophysiologicalseconds. Recording on D. melanogaster Larval CNS

Electrophysiological2.6. Electrophysiological recordings Recording on from D. melanogasterD. melanogaster Larval CNSthird-instar larval CNS were performed as described previously [19], with slight modification. The CNS was dissected in physiological saline Electrophysiological recordings from D. melanogaster third-instar larval CNS were performed as containingdescribed (mM) previously NaCl (157), [19], KCl with (3), slight CaCl modification.2 (2), HEPES The (4), CNS at pH was 7.2, dissected and either in physiological left intact orsaline transected posteriorcontaining to the cerebral (mM) NaCl lobes (157), to eliminate KCl (3), CaCl the blood-brain2 (2), HEPES (4), barrier at pH (BBB) 7.2, and and either facilitate left intact penetration or of chemicalstransected into the posterior central to synapses. the cerebral A lobes recording to elim suctioninate the electrode blood-brain was barrier pulled (BBB) with and a facilitate pipette puller (Sutter Instrument,penetration of Novato, chemicals CA, into USA)the central and synapses broken. atA therecording tip (ca. suction 40 µ m)electrode by fine was forceps pulled with before a being filled withpipette saline. puller Several (Sutter Instrument, peripheral No nervevato, CA, trunks USA) wereand broken drawn at the into tip the(ca. 40 electrode µm) by fine to forceps record nerve before being filled with saline. Several peripheral nerve trunks were drawn into the electrode to activity descending from the CNS. record nerve activity descending from the CNS. ElectricalElectrical signals signals were were amplified, amplified, digitized, digitized, andand transmitted to tothe theanalysis analysis software, software, wherein wherein spikes werespikes converted were converted to a rateto a rate (spikes/s (spikes/s or or Hz) Hz) byby a PowerLab PowerLab analog analog to digital to digital converter converter hardware hardware and LabChartand LabChart 7 software 7 software (ADInstruments, (ADInstruments, Colorado Colorado Springs, Springs, CO, CO, USA). USA). To eliminate To eliminate background background noise, spikesnoise, werespikes onlywere talliedonly tallied if they if they exceeded exceeded a a fixedfixed threshold, threshold, which which was was set when set when no peripheral no peripheral nerves werenerves attached were attached to the to suctionthe suction electrode. electrode. After After baseline frequency frequency was was established, established, 1 mM 1GABA mM GABA was added to the saline bath to inhibit nerve activity, and allowed to incubate for 5 min (Figure 2). was added to the saline bath to inhibit nerve activity, and allowed to incubate for 5 min (Figure2).

Figure 2.FigureD. melanogaster 2. D. melanogasterlarval larval CNS CNS recordings recordings and and samplingsampling for for statistical statistical analysis. analysis. (A) Nerve (A) Nervefiring firing displayeddisplayed as a frequency as a frequency of spikes/s of spikes/s (Hz). (Hz). For For statisticalstatistical analysis, analysis, baseline baseline firing firing frequency frequency and nerve and nerve discharge inhibited by 1 mM GABA were calculated by averaging over 3-min periods as shown in the discharge inhibited by 1 mM GABA were calculated by averaging over 3-min periods as shown in the figure. The drug-induced nerve firing rate was averaged over 3-min periods after treatment; (B) Nerve firing of the same preparation displayed as a voltage recording. Int. J. Environ. Res. Public Health 2017, 14, 154 6 of 17

Experimental compounds (e.g., fluralaner) were then added to the saline bath (1 µL of DMSO solution in a 1 mL bath volume) and mixed by gentle pipetting to assess their ability to reverse the inhibitory effect of GABA. The baseline (pre-treatment) and GABA inhibited nerve firing rates were each averaged over a 3 min period. The drug-induced nerve firing rate was averaged over 3 min periods after treatment (Figure2). Fipronil and dieldrin at a concentration of 10 µM were used as positive controls. If, after GABA treatment, the nerve firing rate recovered to at least 25% of the original pretreatment level, the toxicant was considered to have induced a positive response. Drug effects on larval CNS with intact BBB also were assessed, with experimental compounds added to the bath after establishing the baseline frequency, and each drug concentration was replicated 3–9 times. Fipronil at 10 µM was used as positive control. Recording data obtained within 36 min after addition of experimental compounds were included in the analysis.

2.7. Test of GABAergic Compounds on Mammalian GABAA Receptors

Effects of fluralaner, fipronil, dieldrin, and picrotoxin were tested on GABA α1β3γ2 ion channels expressed in HEK293 cells by ChanTest Corporation, Cleveland, OH, USA. Briefly, HEK293 cells were transfected with cDNA of GABA α1β3γ2 ion channels and maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 with addition of 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 µg/mL streptomycin sulfate, and 500 µg/mL G418. Extracellular buffer containing (mM): NaCl (137), KCl (4), CaCl2 (1.8), MgCl2 (1), HEPES (10), Glucose (10), at pH 7.4 was loaded into planar patch clamp (PPC) plate wells (11 µL per well). Cell suspension was then pipetted into the intracellular compartment (9 µL per well) of the PPC planar electrode. With whole-cell recording, effects of compounds on transfected HEK293 cells were detected in agonist mode (application of test compounds only) and in antagonist mode (application of GABA with test compound), with solution added 10 µL/s for 2 s. GABA and picrotoxin were used as agonist positive control and antagonist positive control, respectively. The agonist effect of the test compounds and GABA (positive control) was calculated as: % activation = (ITA/IMax) × 100%, in which ITA was the compound-induced current at various concentrations, and IMax was the mean current induced with 300 µM GABA. Inhibitory effect of the test compounds and picrotoxin on the channel was calculated as: % Inhibition = (ITA/IEC80) × 100%, where ITA was the GABA EC80-induced current in the presence of various concentrations of the test compound and IEC80 was the mean current elicited with GABA EC80. GABA EC80 values were selected based on ChanTest historical data: 60 µM for α1β3γ2 GABA ion channels. Inhibitory concentration-response data were fitted to an equation of the form: % Inhibition = N % VC + {(% PC − % VC)/[1 + ([Test]/IC50) ]}, where [Test] was the concentration of the compound, IC50 was the concentration of the compound producing 50% inhibition, N was the Hill coefficient, % VC was the mean current at the passive control (picrotoxin EC50), % VC was the mean current at the vehicle control (GABA EC50) and % inhibition was the percentage of ion channel current inhibited at each concentration of the test compound. Nonlinear least squares fits were solved with the XLfit add-in for Excel software (Microsoft, Redmond, WA, USA).

2.8. Statistical Analysis

Control mortality was corrected by Abbott’s formula and LC50, LD50, and PC50 values were calculated by SAS software (Proc Probit, SAS 9.4, SAS Institute Inc., Cary, NC, USA). EC50 and IC50 values, and concentration-response curves of drugs in Drosophila larval CNS recordings were obtained by nonlinear regression to a four-parameter logistic equation in GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA, USA). The two-tailed Student’s t-test, one-way ANOVA with Bonferroni or Tukey’s post-test used to analyze results between treatment group means, with a p < 0.05 considered to be statistically signiﬁcant. Int. J. Environ. Res. Public Health 2017, 14, 154 7 of 17

3. Results

3.1. Larval Mosquito Bioassays

The 24-h LC50 values of fluralaner on fourth-instar Ae. aegypti and An. gambiae larvae were 1.8 ppb and 0.4 ppb, respectively; a statistically significant difference (p < 0.05) by a factor of 4.5 (Table1). The toxicity of fluralaner on Ae. aegypti larvae also increased significantly (p < 0.05) by a factor of 3.6 after 48 h of exposure (Table1).

Table 1. Larval bioassays of ﬂuralaner on fourth-instar Ae. aegypti and An. gambiae larvae.

Species Bioassay LC50 or PC50, ppb; (95% CI) 24 h LC 1.8 (1.4–2.2) Intact larval toxicity in water 50 48 h LC50 0.5 (0.3–0.6)

Ae. aegypti Intact larval toxicity in saline 24 h LC50 2.9 (2.2–4.0) Intact larval toxicity in water + Triton 24 h LC50 2.7 (2.1–3.6) Headless larval paralysis in saline 5 h PC50 3.0 (1.1–6.0) Intact larval paralysis in saline 5 h PC50 30 (26–35)

An. gambiae Intact larval toxicity in water 24 h LC50 0.4 (0.3–0.5)

Aedes larval assays performed in mosquito physiological saline and in 10 ppm Triton X-100 solution displayed a small, but statistically-significant increase (p < 0.05) of LC50 values to 2.9 ppb and 2.7 ppb, respectively, compared to those performed in tap water (Table1). The 24 and 48 h LC 50 values of fipronil on fourth-instar Ae. aegypti larvae were 23 ppb and 5.1 ppb, respectively, and showed 10- to 13-fold less potency compared to the toxicity of fluralaner in the same assay. The LC90 values at the 48-h time point of fluralaner and fipronil on fourth-instar Ae. aegypti larvae were 2.2 ppb (1.4–6.0 ppb) and 11 ppb (8–21 ppb), respectively. Paralysis studies with fluralaner on headless and intact Ae. aegypti larvae yielded a significant (p < 0.05) 10-fold difference in PC50 values (Table1). Intoxication signs of fluralaner in mosquito larvae were spontaneous asynchronous contractions and convulsions, similar to signs of fipronil exposure.

3.2. Adult Mosquito Bioassays In adult mosquito bioassays, intoxication signs included an inability to stand upright, and flying along the bottom of holding container while on their backs. In topical assays, the 24 h LD50 values of fluralaner on Ae. aegypti and An. gambiae were 1.3 ng/mg and 0.29 ng/mg, respectively (Table2), a species difference of 4-fold. In time course studies with Ae. aegypti, the toxicity of fluralaner on mosquitoes increased progressively (Figure3), and the LD 50 value following a single topical application decreased significantly (p < 0.05; one-way ANOVA, Tukey’s test) on a daily basis until day 3 (Table2). At day 3, the LD50 value was 0.26 ng/mg (Table2), five-fold lower than that observed on day 1. At observation days 4, 5 and 6, the toxicity did not increase further (p > 0.05) compared to the LD50 on day 3 (Figure3), and at the two lowest doses tested did not reach 100% mortality, even after 7 days of observation (Figure3). For An. gambiae, fluralaner toxicity reached its plateau at day 2, with an LD50 value of 0.21 ng/mg (Table2), a difference of only 1.4-fold compared to 24 h. For comparison, topical assays of fipronil on Ae. aegypti found an LD50 value after 24 h of 0.062 ng/mg (Table2). Toxicity of fipronil in Ae. aegypti topical assay stopped increasing significantly at day 2, with a LD50 value of 0.021 ng/mg (Table2). Fipronil was more active than fluralaner on Ae. aegypti in topical assays by 21-fold at 1 day post-exposure and 17-fold at day 2. Int. J. Environ. Res. Public Health 2017, 14, 154 8 of 17 Int. J. Environ. Res. Public Health 2017, 14, 154 8 of 17

Table 2. Topical bioassays of ﬂuralaner on Ae. aegypti and An. gambiae adult females. Table 2. Topical bioassays of fluralaner on Ae. aegypti and An. gambiae adult females.

CompoundsCompounds SpeciesSpecies Time Time (Day) (Day) LD50, ng/mg; LD50 (95%, ng/mg; CI) (95% CI) 1 11.3 (1.0–2.3) 1.3 (1.0–2.3) 2 2 0.50 (0.38–0.65) 0.50 (0.38–0.65) * * 3 3 0.26 (0.22–0.31) 0.26 (0.22–0.31) * * Ae.Ae. aegypti aegypti 4 40.21 (0.17–0.31) 0.21 (0.17–0.31) FluralanerFluralaner 5 50.15 (0.12–0.18) 0.15 (0.12–0.18) 6 0.14 (0.12–0.17) 6 0.14 (0.12–0.17) 1 10.29 (0.25–0.35) 0.29 (0.25–0.35) An.An. gambiae gambiae 2 2 0.21 (0.19–0.24) 0.21 (0.19–0.24) * * 3 0.20 (0.18–0.23) 3 0.20 (0.18–0.23) 1 10.062 (0.050–0.076) 0.062 (0.050–0.076) FipronilFipronil Ae.Ae. aegypti aegypti 2 2 0.029 (0.022–0.042) 0.029 (0.022–0.042) * * 3 0.019 (0.015–0.023) 3 0.019 (0.015–0.023) * Signiﬁcant difference at p < 0.05, compared to the LD50 values of the preceding day. * Significant difference at p < 0.05, compared to the LD50 values of the preceding day.

FigureFigure 3. MortalityMortality data for adult topicaltopical assayassay showingshowing the the increase increase of of toxicity toxicity over over time time at at a rangea range of ofdoses doses labeled labeled to to the the right right of of the the symbols symbols and and expressed expressed as as ng/mosquito. ng/mosquito. No No mortality mortality was was observed observed in incontrols controls over over six six days days when when treated treated with with solvent solvent vehicle. vehicle.

ForFor injection bioassays of fluralanerfluralaner on Ae.Ae. aegypti aegypti adultadult females, the 24-h LD50 valuevalue was was 0.60.6 ng/mg ng/mg (Table (Table 3),3), with with no no increase increase of of toxici toxicityty at at the the 48-h 48-h and and 72-h 72-h time time point. point. This This LD50 LD value50 value was lesswas than less thanthat in that 24-h in topical 24-h topical assays assays by a factor by a factorof 2. The of 2.24-h The LC 24-h50 values LC50 forvalues glass for contact glass and contact feeding and assaysfeeding were assays 13 ng/cm were 132 and ng/cm 49 ppm,2 and respectively 49 ppm, respectively (Table 3). Toxicity (Table3). of Toxicity fluralaner of fluralaner by feeding by and feeding glass contactand glass methods contact increased methods increasedby factors by of factors 2- to of4-fo 2-ld to after 4-fold 48 after h, compared 48 h, compared to 24-h to exposures. 24-h exposures. For 2 2 2 2 comparison,For comparison, fipronil fipronil had hada glass a glass contact contact LC50 LC value50 value of 10 of ng/cm 10 ng/cm after after24 h and 24 h 0.7 and ng/cm 0.7 ng/cm after 48after h, which48 h, which is a 14-fold is a 14-fold increase increase in toxicity in toxicity (Table (Table 3). 3). InIn feedingfeeding assays,assays, fipronil fipronil had had LC 50LCvalues50 values of 0.47 of ppm0.47 andppm 0.12 and ppm 0.12 for ppm 24 h andfor 4824 h, h respectively, and 48 h, respectively,and the toxicity and did the not toxicity increase did significantly not increase after significantly 48 h (Table 3after). Compared 48 h (Table to fluralaner, 3). Compared fipronil to fluralaner,had ca. 8-fold fipronil greater had toxicityca. 8-fold in greater glass contact toxicity assays in glass at contact the 48-h assays time pointat the and48-h ca. time 100-fold point and higher ca. 100-foldtoxicity inhigher feeding toxicity assays in at feeding 24-h and assays 48-h timeat 24-h points. and 48-h At the time 24-h points. time point At the in 24-h the glass time contact point in assay, the glassthe difference contact assay, in toxicity the difference of fluralaner in toxicity and fipronil of fluralaner was not and statistically fipronil was significant. not statistically significant. InIn WHO WHO paper paper assays, fluralaner fluralaner was dissolved inin ethanol,ethanol, withwith 3.63.6 mg/cmmg/cm²2 ofof either either silicon silicon oil oil oror RME RME added added to to the the solvent. solvent. These These formulations formulations at 2 at mg/paper 2 mg/paper (ca. 11,000 (ca. 11,000 ng/cm ng/cm2) of fluralaner2) of fluralaner killed atkilled most at 12% most at 12% the at 48-h the 48-htime timepoint. point. In glass In glass cont contactact assays assays with with exposure exposure time time of of 1 1h hto to match match the the 22 WHO paper assay, fluralanerfluralaner displayeddisplayed aa LCLC5050 value of 504 (152–1338) ng/cmng/cm , ,which which is is significantly significantly moremore toxic than that in WHO paper assays by a factor of at least 22-fold.

Int. J. Environ. Res. Public Health 2017, 14, 154 9 of 17

Table 3. Bioassays of ﬂuralaner and ﬁpronil on Ae. aegypti adult females.

Compounds Bioassay Time (h) LD50 or LC50; (95% CI) Injection 24 0.6 (0.4–0.9) ng/mg 24 13 (9–19) ng/cm2 Glass Contact 2 Fluralaner 48 6 (5–9) ng/cm * 24 49 (34–84) ppm Feeding 48 12 (6–20) ppm * 72 5 (3–6) ppm 24 10 (7–17) ng/cm2 Glass Contact 48 0.7 (0.4–1.1) ng/cm2 * Fipronil 24 0.47 (0.36–0.42) ppm Feeding 48 0.12 (0.09–0.15) ppm *

* Signiﬁcant difference at p < 0.05, compared to the LD50 or LC50 values of the preceding day.

Synergist assays, in which PBO, DEF, or DEM was topically applied to adult female Ae. aegypti 4 h before treatment, were performed in topical and feeding assay formats (Tables4 and5). PBO displayed little effect on fluralaner toxicity in either assay, with synergist ratios of at most 2.2-fold. The synergist DEM decreased toxicity to fluralaner in the feeding assay (Table4), but increased toxicity up to about 2-fold in topical treatments (Table5). In contrast, DEF displayed strong synergism of fluralaner in feeding assays (Table4), but weak synergism in topical assays (Table5).

Table 4. Effects of synergists in feeding assays of ﬂuralaner against Ae. aegypti adult females.

1 Synergist Time (Day) LC50, ppm; (95% CI) SR 1 39 (29–58) 1.3 PBO 2 5.5 (3.9–7.5) 2.2 3 4.6 (2.8–8.7) 1.1 1 13 (8–24) * 3.8 DEF 2 1.5 (1.1–2.0) * 8.0 3 0.8 (0.5–1.2) * 6.3 1 60 (40–104) 0.8 DEM 2 31 (21–48) 0.4 3 12 (7–20) 0.4 1 Synergist ratio = LC50 of fluralaner (from Table3)/LC 50 of fluralaner + synergist; * Significant difference at p < 0.05, compared to data for fluralaner alone on the corresponding day (Table3).

Table 5. Effects of synergists in topical assays of ﬂuralaner against Ae. aegypti adult females.

1 Synergist Time (Day) LD50, ng/mg; (95% CI) SR 1 0.72 (0.50–1.41) 1.8 2 0.29 (0.22–0.37) 1.7 PBO 3 0.19 (0.14–0.24) 1.4 4 0.12 (0.06–0.15) * 1.8 5 0.12 (0.06–0.15) 1.3 1 0.97 (0.77–1.38) 1.3 2 0.36 (0.26–0.45) 1.4 DEF 3 0.30 (0.25–0.35) 0.9 4 0.17 (0.10–0.24) 1.2 5 0.17 (0.10–0.24) 0.9 1 0.69 (0.55–0.97) * 1.9 2 0.38 (0.27–0.47) 1.3 DEM 3 0.31 (0.24–0.39) 0.8 4 0.22 (0.15–0.29) 1.0 5 0.22 (0.15–0.29) 0.7 1 Synergist ratio = LD50 of fluralaner (from Table2)/LD 50 of fluralaner + synergist; * Significant difference at p < 0.05, compared to the LC50 values of the preceding day. Int. J. Environ. Res. Public Health 2017, 14, 154 10 of 17

3.3. Adult D. melanogaster Bioassays To confirm resistance in the rdl-1675 strain, dieldrin feeding assays were performed. In these studies,Int. the J. Environ. susceptible Res. Public strain Health 2017 (CS-OR), 14, 154 had a LC50 value of 2.5 ppm (95% CI = 2.1–3.0 ppm)10 toof 17 dieldrin, whereas the resistant strain (rdl-1675) had no mortality when fed 100 ppm dieldrin, indicating a 3.3. Adult D. melanogaster Bioassays resistance ratio greater than 40-fold. In both glass contact and feeding assays, fluralaner displayed To confirm resistance in the rdl-1675 strain, dieldrin feeding assays were performed. In these equivalent toxicity to susceptible and resistant strains of D. melanogaster (Table6). The LC 50 values of studies, the susceptible strain (CS-OR) had a LC50 value of 2.5 ppm (95% CI = 2.1–3.0 ppm) to dieldrin, both D. melanogaster strains were not significantly different from each other at the 24-h and 48-h time whereas the resistant strain (rdl-1675) had no mortality when fed 100 ppm dieldrin, indicating a point (Tableresistance6). Inratio glass greater contact than assays,40-fold. In LC both50 valuesglass contact of fluralaner and feeding on assays,Ae. aegypti fluralanerand displayedD. melanogaster were notequivalent significantly toxicity different to suscep fromtible and each resistant other atstrains recorded of D. melanogaster time points (Table (Tables 6). The3 and LC650). values However, of the toxicityboth of fluralaner D. melanogaster in feeding strains were assays not against significantlyD. melanogaster different fromwas each greater other at than the 24-h that and against 48-h timeAe. aegypti by a factorpoint of (Table ca. seven 6). In (Tablesglass contact3 and assays,6). As LC was50 values observed of fluralaner for mosquitoes, on Ae. aegypti mortality and D. inmelanogasterD. melanogaster increasedwere significantly not significantly at 48 different h compared from each to 24other h (Table at recorded6). time points (Tables 3 and 6). However, the toxicity of fluralaner in feeding assays against D. melanogaster was greater than that against Ae. aegypti by a factor of ca. seven (Tables 3 and 6). As was observed for mosquitoes, mortality in Table 6. Glass contact and feeding bioassays of fluralaner against susceptible and resistant adult D. melanogaster increased significantly at 48 h compared to 24 h (Table 6). D. melanogaster strains. Table 6. Glass contact and feeding bioassays of fluralaner against susceptible and resistant adult 1 D. melanogaster Assaystrains. Strain Time (h) LC50; (95% CI) Ratio 24 18 (13–27) Assay StrainCS-OR Time (h) LC50; (95% CI)5.8 Ratio 1 Glass Contact 4824 3.1 (2.0–5.3)18 (13–27) * 2 CS-OR 5.8 Glass(ng/cm Contact) 2448 13 3.1 (9–21) (2.0–5.3) * (ng/cm2) rdl-1675 24 13 (9–21) 3.9 rdl-1675 48 3.3 (1.7–5.3) * 3.9 48 3.3 (1.7–5.3) * 24 6.5 (3.4–12.3) 24 6.5 (3.4–12.3) CS-ORCS-OR 3.43.4 4848 1.9 (0.6–3.1) 1.9 (0.6–3.1) * * FeedingFeeding (ppm) (ppm) 2424 7.0 (5.2–10.4)7.0 (5.2–10.4) rdlrdl-1675-1675 3.93.9 4848 1.8 (1.0–2.7) 1.8 (1.0–2.7) * * 1 1 LD50 at 24 h/LD50 at 48 h; * Significant difference at p < 0.05, compared to the LC50 values of the LD50 at 24 h/LD50 at 48 h; * Significant difference at p < 0.05, compared to the LC50 values of the preceding day. preceding day.

3.4. CNS3.4. Recordings CNS Recordings on D. on melanogasterD. melanogaster InitialInitial experiments experiments compared compared the the solvent solvent responsesresponses of of intact intact and and severed severed (BBB (BBBdisrupted) disrupted) larval larval CNS preparations.CNS preparations. Intact Intact and and severed severed susceptiblesusceptible (CS-OR) (CS-OR) CNS CNS treated treated with withDMSO DMSO showed showed a a consistentconsistent nerve nerve ﬁring firing pattern, pattern, which which is is cycliccyclic oscillations (Figure (Figure 4A–C),4A–C), but butoverall overall spike spikerates rates declineddeclined over theover 30-min the 30-min observation observation period period (Figure(Figure 44D).D).

B 0.1% DMSO A 0.1% DMSO 40 Hz 40 Hz

3min 3min

0Hz------

0.1% DMSO D C 40 Hz

3min

0Hz------

FigureFigure 4. DMSO 4. DMSO effects effects on D. on melanogaster D. melanogasterlarval larval CNS. CNS. Ordinate Ordinate in all in tracesall traces is spike is spike rate rate in Hzin Hz (spikes/s) vs. time(spikes/s) (abscissa). vs. time (A) Recording(abscissa). ( ofA) intactRecording CS-OR of intact larval CS-OR CNS treatedlarval CNS with treated DMSO; with (B )DMSO; Recording of severed(B CS-OR) Recording larval of severed CNS treated CS-OR withlarval DMSO; CNS treated (C) Recordingwith DMSO; of (C intact) Recordingrdl-1675 of intact larval rdl CNS-1675 treated larval CNS treated with DMSO; (D) Summary plot of DMSO effects on D. melanogaster larval CNS. with DMSO; (D) Summary plot of DMSO effects on D. melanogaster larval CNS. Each data point was Each data point was averaged over at least three replicates and error bars represent ± SEM. Dashed averaged over at least three replicates and error bars represent ± SEM. Dashed line in A–C is the zero Hz baseline in this and subsequent ﬁgures displaying nerve ﬁring measurements. Int. J. Environ. Res. Public Health 2017, 14, 154 11 of 17

line in A–C is the zero Hz baseline in this and subsequent figures displaying nerve firing Int. J. Environ.measurements. Res. Public Health 2017, 14, 154 11 of 17

On severed CS-OR larval CNS, discharge rates dropped to 72% ± 11% of baseline firing rate 30 minOn after severed the addition CS-OR larval of DMSO. CNS, Firing discharge frequencies rates dropped of intact to CS-OR 72% ± and11% rdl of-1675 baseline (resistant) firing CNS rate 30at min30 min after after the DMSO addition addition of DMSO. were Firing 85% frequencies± 3% and 89% of ± intact 18%, CS-ORrespectively, and rdl which-1675 (resistant)were greater CNS than at 30that min of aftersevered DMSO CS-OR addition CNS (Figure were 85% 4D).± This3% anddiffere 89%nce,± 18%,however, respectively, was not whichstatistically were significant. greater than that ofOn severed CS-OR CS-OR larval CNS preparations, (Figure4D). This 10 µM difference, fluralaner however, showed was neuroexcitatory not statistically effects significant. within five minutesOn CS-ORof addition larval to CNSthe bath preparations, (Figure 5A,B). 10 µ AfterM fluralaner the treatment showed of 10 neuroexcitatory µM fluralaner, effectsfiring patterns within fiveon larval minutes CNS of additiondisplayed to more the bath rapid (Figure cyclic5 A,B).oscillations After theon treatmenttop of a greater of 10 µtonicM fluralaner, discharge firing rate, patternsconsistent on with larval a CNSdisruption displayed of normal more rapidpattern cyclic generation. oscillations Blocking on top of of nerve a greater discharge tonic discharge was observed rate, consistentfollowing withthe aneuro-excitation, disruption of normal and patternshowed generation. some evidence Blocking of of greater nerve discharge effect in was the observed severed followingpreparations; the neuro-excitation, viz., there is more and showedresidual somefiring evidence evident of in greater the intact effect preparations. in the severed The preparations; effects of viz.,10 µM there fipronil is more on residualintact and firing severed evident CN-OR in the larval intact CNS preparations. were similar The to effectsthat of of10 10µMµM fluralaner; fipronil on an intactexcitatory and severedaction followed CN-OR by larval inhibition CNS were of nerve similar firing to that(Figure of 10 5C,D).µM fluralaner; an excitatory action followed by inhibition of nerve firing (Figure5C,D).

Intact Severed A 10 μMFluralaner B 10 μM Fluralaner

40 Hz 40 Hz

2 min 2 min

0 Hz------CD 10 μMFipronil 10 μMFipronil 40 Hz 40 Hz 30 s

2 min

0Hz------

Figure 5. Electrophysiological recordings on intact or severed CS-OR D. melanogaster larval CNS. (AFigure) Intact 5. CNS Electrophysiological treated with 10 µ Mrecordings fluralaner; on (B )intact Severed or CNSsevered treated CS-OR with D. 10 melanogasterµM fluralaner; larval (C)Intact CNS. CNS(A) Intact treated CNS with treated 10 µM fipronil;with 10 (DµM) Severed fluralaner; CNS ( treatedB) Severed with CNS 10 µM treated fipronil. with 10 µM fluralaner; (C) Intact CNS treated with 10 µM fipronil; (D) Severed CNS treated with 10 µM fipronil. Excitation and nerve blocking effects of fluralaner were concentration-dependent, and lower Excitation and nerve blocking effects of fluralaner were concentration-dependent, and lower concentrations required longer incubation times to cause either effect. This relationship is evident in concentrations required longer incubation times to cause either effect. This relationship is evident in electrophysiological traces (Figure6), and explored in measurements of time to 50% block of nerve electrophysiological traces (Figure 6), and explored in measurements of time to 50% block of nerve firing (Figure7). In both susceptible and resistant CNS preparations, reduction in firing occurs more firing (Figure 7). In both susceptible and resistant CNS preparations, reduction in firing occurs more quickly and is more extensive at higher concentrations (Figure6). Similar results were obtained on quickly and is more extensive at higher concentrations (Figure 6). Similar results were obtained on intact rdl-1675 larval CNS, where 10 µM fluralaner and fipronil blocked nerve discharge significantly intact rdl-1675 larval CNS, where 10 µM fluralaner and fipronil blocked nerve discharge significantly faster than 0.1 µM fluralaner (Figures6 and7). Time to block of nerve firing was used to quantify faster than 0.1 µM fluralaner (Figures 6 and 7). Time to block of nerve firing was used to quantify effects on the CNS. However, there was not a statistically significant difference in time to 50% nerve effects on the CNS. However, there was not a statistically significant difference in time to 50% nerve block for fluralaner between intact and severed CNS preparations from the susceptible Oregon-R strain block for fluralaner between intact and severed CNS preparations from the susceptible Oregon-R (Figure7). Given the variability inherent to spontaneous nerve discharge recordings, no other analyses strain (Figure 7). Given the variability inherent to spontaneous nerve discharge recordings, no other were attempted. analyses were attempted. For comparison, dieldrin, fipronil, and fluralaner were applied to severed susceptible and resistant CNS preparations pretreated with 1 mM GABA to inhibit nerve discharge. Dieldrin (10 µM) stimulated recovery of firing in CS-OR CNS (Figure8A), but on rdl-1675 CNS yielded virtually no recovery of nerve activity (Figure8B). Application of 0.1% DMSO had no effect on GABA-treated CNS preparations (data not shown). At 3 µM, all preparations from both strains showed a positive response (recovery to at least 25% of baseline firing frequency within a 30 min observation period) to fluralaner (Figure8C,D). Similarly, fipronil at 10 µM showed similar recovery of firing on severed larval CNS in both susceptible and resistant strains (Figure8E,F). There was no response to 30 nM fluralaner for either Drosophila Int. J. Environ. Res. Public Health 2017, 14, 154 12 of 17 strain, but higher concentrations were effective (Figure9). For both susceptible and cyclodiene-resistant CNS preparations, 100 nM fluralaner was the lowest concentration that reversed, even partially, the GABA inhibitory effect (Figure9). The EC 50 values for fluralaner on CS-OR and rdl-1675 severed CNS Int. J. Environ. Res. Public Health 2017, 14, 154 12 of 17 preparations were 0.34 µM (95% CI: 0.06–1.8 µM) and 0.29 µM (95% CI: 0.09–0.92 µM); not significantly Int. J. Environ. Res. Public Health 2017, 14, 154 12 of 17 different from each other. CS-OR rdl-1675 CS-OR rdl-1675 A 10 μMFluralaner B 10 μMFluralaner 40 Hz A 10 μMFluralaner 40 Hz B 10 μMFluralaner 40 Hz 40 Hz 2 min 2 min

2 min 2 min

0Hz------0Hz------1 μM Fluralaner 1 μM Fluralaner C D 40 Hz 1 μM Fluralaner 50 Hz 1 μM Fluralaner 40 Hz C 50 Hz 2 min D 2 min

2 min 2 min

0Hz------0Hz------E F 0.1 μMFluralaner 40 Hz 0.1 μMFluralaner E F 40 Hz 0.1 μMFluralaner 40 Hz 2 min 0.1 μMFluralaner 40 Hz 2 min 2 min 2 min

0Hz------0Hz------

Figure 6. Electrophysiological recordings on CS-OR or rdl-1675 D. melanogaster intact larval CNS. Figure 6. Electrophysiological recordings on CS-OR or rdl-1675 D. melanogaster intact larval CNS. Figure(A) CS-OR 6. Electrophysiological CNS treated with recordings10 µM fluralaner; on CS-OR (B )or rdl rdl-1675-1675 CNS D. melanogastertreated with intact 10 µM larval fluralaner; CNS. (A) CS-OR CNS treated with 10 µM fluralaner; (B) rdl-1675 CNS treated with 10 µM fluralaner; (A(C) )CS-OR CS-OR CNS treatedtreated with with 1 µM10 µM fluralaner; fluralaner; (D) rdl(B-1675) rdl-1675 CNS treatedCNS treated with 1 withµM fluralaner; 10 µM fluralaner; (E) CS-OR (C) CS-OR CNS treated with 1 µM fluralaner; (D) rdl-1675 CNS treated with 1 µM fluralaner; (E) CS-OR (CCNS) CS-OR treated CNS with treated 0.1 µM with fluralaner; 1 µM fluralaner; (F) rdl-1675 (D) rdl CNS-1675 treated CNS treated with 0.1 with µM 1 fluralaner. µM fluralaner; (E) CS-OR CNS treated with 0.1 µM fluralaner; (F) rdl-1675 CNS treated with 0.1 µM fluralaner. CNS treated with 0.1 µM fluralaner; (F) rdl-1675 CNS treated with 0.1 µM fluralaner.

30 a 0.1 µM fluralaner a 30 a 0.11 µM µM fluralaner fluralaner a 1 µM fluralaner ab 10 µM fluralaner 10 µM fluralaner 20 ab 10 µM fipronil 20 ab 10 µMa fipronil ab a ab a ab a 10 b b a 10 b b b a a Time to 50% block, min block, to 50% Time b a Time to 50% block, min block, to 50% Time 0 0 IntactIntact CS-ORIntact Intact rdl-1675 Severed Severed CS-OR IntactIntactCS-OR CS-ORIntact rdl-1675Intact rdl-1675 Severed Severed CS-OR CS-OR CS-OR rdl-1675 CS-OR

FigureFigure 7. 7.Time Time to to 50% 50% reduction reduction of of firing firing rate rate after after chemical chemical treatments treatments of ofD. D. melanogaster melanogasterlarval larval CNS CNS ± Figurepreparations.preparations. 7. Time Each toEach 50% bar bar reduction is is the the average average of firing of of atrate at least least after three th chemicalree replicates replicates treatments and and error error of D. bars ba melanogasterrs show show ±SEM. SEM. larval Letters Letters CNS preparations.indicateindicate statistical statistical Each significance barsignificance is the average at at the the p ofp= =at0.05 0.05 leastlevel level threeusing using replicatesa a two-way two-way and error ANOVA ANOVA bars calculated calculatedshow ± SEM. with with Letters Prism Prism p indicatesoftware.software. statistical Comparisons Comparisons significance were were made made at the across across p = 0.05 different differe levelnt treatmentsusing treatments a two-way within within ANOVA a a nerve nerve preparation preparationcalculated with ( (p< < 0.0001) Prism0.0001) as well as for a given treatment across the different CNS dissections (p = 0.1263). Bars not labeled by software.as well as Comparisons for a given treatment were made across across the differe differentnt treatments CNS dissections within a ( pnerve = 0.1263). preparation Bars not (p labeled < 0.0001) by the same letter are significantly different. asthe well same as forletter a given are significantly treatment across different. the different CNS dissections (p = 0.1263). Bars not labeled by the same letter are significantly different. For comparison, dieldrin, fipronil, and fluralaner were applied to severed susceptible and resistantFor comparison, CNS preparations dieldrin, pretreated fipronil, with and 1 mMfluralan GABAer wereto inhibit applied nerve to discharge. severed susceptibleDieldrin (10 and µM) resistantstimulated CNS recovery preparations of firing pretreated in CS-OR with CNS 1 mM (Figure GABA 8A), to inhibitbut on nerverdl-1675 discharge. CNS yielded Dieldrin virtually (10 µM) no stimulatedrecovery of recovery nerve activity of firing (Figure in CS-OR 8B). CNSApplication (Figure of8A), 0.1% but DMSO on rdl -1675had noCNS effect yielded on GABA-treated virtually no recoveryCNS preparations of nerve activity (data not (Figure shown). 8B). At Application 3 µM, all preparations of 0.1% DMSO from had both no strainseffect onshowed GABA-treated a positive CNS preparations (data not shown). At 3 µM, all preparations from both strains showed a positive

Int. J. Environ. Res. Public Health 2017, 14, 154 13 of 17 Int. J. Environ. Res. Public Health 2017, 14, 154 13 of 17 response (recovery to at least 25% of baseline firing frequency within a 30 min observation period) to fluralanerresponse (recovery (Figure 8C,D). to at least Similarly, 25% of fipronil baseline at firing 10 µM frequency showed within similar a recovery30 min observation of firing on period) severed to larvalfluralaner CNS (Figure in both 8C,D). susceptible Similarly, and resistantfipronil at strain 10 µMs (Figure showed 8E,F). similar There recovery was no of response firing on to severed 30 nM fluralanerlarval CNS for in eitherboth susceptible Drosophila strain,and resistant but higher strain concentrationss (Figure 8E,F). were There effective was no (Figure response 9). toFor 30 both nM susceptiblefluralaner for and either cyclodiene-resistant Drosophila strain, butCNS higher preparations, concentrations 100 werenM effectivefluralaner (Figure was 9).the For lowest both concentrationsusceptible and that cyclodiene-resistantreversed, even partially, CNS th e preparations,GABA inhibitory 100 effect nM (Figurefluralaner 9). The was EC 50the values lowest for fluralanerconcentration on CS-OR that reversed, and rdl-1675 even severedpartially, CNS the preparationsGABA inhibitory were effect 0.34 µM(Figure (95% 9). CI: The 0.06–1.8 EC50 values µM) and for 0.29fluralaner µM (95% on CS-OR CI: 0.09–0.92 and rdl µM);-1675 not severed significantly CNS preparations different from were each 0.34 other. µM (95% CI: 0.06–1.8 µM) and 0.29Int. J. µM Environ. (95% Res. CI: Public 0.09–0.92 Health 2017 µM);, 14 ,not 154 significantly different from each other. 13 of 17 CS-OR rdl-1675

A CS-OR B 1 mM GABA 1 mM GABA rdl-1675 50 Hz 40 Hz A B 1 mM GABA10 μM Dieldrin 1 mM GABA10 μMDieldrin 2 min 2 min 50 Hz 40 Hz 10 μMDieldrin 10 μM Dieldrin 2 min 2 min

0Hz------

C 0Hz------D 3 μM Fluralaner ------1 mM GABA 40 Hz 40 Hz

C 3min D1 mM GABA3 μM Fluralaner 2 min 1 mM GABA3 μMFluralaner 40 Hz 40 Hz 1 mM GABA 3 μMFluralaner 3min 2 min

0Hz------

E 0Hz------F 1 mM GABA ------40 Hz 1 mM GABA 40 Hz 1 mM GABA 1 min E 10 μM Fipronil 1 min F 10 μM Fipronil 40 Hz 1 mM GABA 40 Hz 1 min 10 μM Fipronil 1 min 10 μM Fipronil

0Hz------

Figure 8. Electrophysiological recordings on severed D. melanogaster larval CNS, with GABA Figure 8. Electrophysiological recordings on severed D. melanogaster larval CNS, with GABA inhibition. inhibition. Test compounds were added after 5-min incubation with 1 mM GABA. (A) CS-OR CNS FigureTest compounds 8. Electrophysiological were added after recordings 5-min incubation on severed with D. 1 mMmelanogaster GABA. (A larval) CS-OR CNS, CNS with treated GABA with treated with 10 µM dieldrin; (B) rdl-1675 CNS treated with 10 µM dieldrin; (C) CS-OR CNS treated inhibition.10 µM dieldrin; Test compounds (B) rdl-1675 were CNS added treated after with 5-min 10 µ Mincubation dieldrin; with (C) CS-OR1 mM GABA. CNS treated (A) CS-OR with 3CNSµM with 3 µM fluralaner; (D) rdl-1675 CNS treated with 3 µM fluralaner; (E) CS-OR CNS treated with treatedfluralaner; with (D 10) rdlµM-1675 dieldrin; CNS treated(B) rdl-1675 with 3CNSµM fluralaner;treated with (E )10 CS-OR µM dieldrin; CNS treated (C) CS-OR with 10 CNSµM fipronil;treated 10 µM fipronil; (F) rdl-1675 CNS treated with 10 µM fipronil. with(F) rdl 3-1675 µM fluralaner; CNS treated (D with) rdl-1675 10 µM CNS fipronil. treated with 3 µM fluralaner; (E) CS-OR CNS treated with 10 µM fipronil; (F) rdl-1675 CNS treated with 10 µM fipronil.

100 100 80 80 60 60 40 rdl-1675 % Response,

increased firing increased 40 20 CS-ORrdl-1675 % Response, increased firing increased 20 CS-OR 0 -8 -7 -6 -5 0 log [Fluralaner], M -8 -7 -6 -5 log [Fluralaner], M FigureFigure 9. 9. ConcentrationConcentration response response curve curve (% (% responding responding preparations) preparations) of of fluralaner ﬂuralaner on on resistant resistant and and susceptible strains of D. melanogaster, in transected larval CNS recordings. Fluralaner was added after susceptibleFigure 9. Concentration strains of D. melanogaster response curve, in transected (% responding larval preparations)CNS recordings. of fluralanerFluralaner onwas resistant added after and a 5-min incubation of 1 mM GABA to inhibit nerve activity. Solid line and dotted line represent curves asusceptible 5-min incubation strains of D.1 mM melanogaster GABA to, inhibitin transected nerve activity.larval CNS Solid record line ings.and dotted Fluralaner line represent was added curves after for rdl-1675 and CS-OR preparations, respectively. fora 5-min rdl-1675 incubation and CS-OR of 1 mMpreparations, GABA to inhibitrespectively. nerve activity. Solid line and dotted line represent curves for rdl-1675 and CS-OR preparations, respectively. 3.5. Potency on Mammalian GABA Receptors

Fluralaner was screened on a mammalian GABAA receptor construct under contact by TM ChanTest . Only GABA displayed agonist activity among the compounds tested, and it had an EC50 value of 11 µM on mammalian GABAA α1β3γ2 receptors. Fluralaner and dieldrin showed low potency for block of these receptors, with IC50 values above 30 µM. For comparison, ﬁpronil and picrotoxin had IC50 values of 4.9 µM and 3.9 µM, respectively. Int. J. Environ. Res. Public Health 2017, 14, 154 14 of 17

4. Discussion Gassel et al. [11] performed toxicity comparisons of fluralaner with other commercialized compounds, including fipronil. On Ctenocephalides felis (cat flea), Ae. aegypti, Lucilia cuprina Meigen (sheep blowfly), and Stomoxys calcitrans Linnaeus (stable fly), fluralaner outperformed dieldrin and imidacloprid, as well as deltamethrin, except on S. calcitrans. Ozoe et al. [10] also reported that fipronil outperformed fluralaner on C. felis by a factor of five in dry film contact assays. For mosquitoes, Gassel et al. [11] reported a single finding that fluralaner (48 h LC90 value = 1.2 ppt) was ca. 16,000-fold more potent than fipronil on Ae. aegypti first-instar larvae (48 h LC90 value = 20 ppb). In the present study, fluralaner gave a 48 h LC90 value of 2.2 ppb on fourth-instar Ae. aegypti larvae, differing from the data on first-instar larvae by over 1800-fold. However, fipronil showed a 48-h LC90 value of 11 ppb, similar to toxicity observed by Gassel et al. [11] for first instars (20 ppb for >90% mortality). In addition, on fourth-instar Ae. aegypti larvae, fipronil was 10- to 13-fold less potent than fluralaner. These data suggest that fluralaner might be an excellent larvicide, although the life stage of the mosquito larvae has a large impact on chemical sensitivity. On adult Ae. aegypti, fipronil had higher toxicity than fluralaner in topical (7- to 21-fold), feeding (ca. 100-fold), and glass contact assays (8-fold at 48 h). These findings, plus the greater speed of action, suggest fipronil would be a better overall adult mosquitocide, at least in the absence of resistance. Bioassays and CNS recordings provide some insight into the slow toxicity of fluralaner. It took 3 days for fluralaner toxicity to plateau, and its toxicity was not enhanced much by injection (about two-fold compared to topical application). In contrast, the LD50 value by injection can be reduced by more than 10-fold compared to topical treatments for carbamates such as propoxur [16], another compound that must reach central synapses to exert its effects. Cuticle thickness is known to have a significant and positive correlation with the time to knock down by permethrin, suggesting that thicker cuticle led to a slower rate of insecticide penetration [20]. For larval assays of Ae. aegypti, the concentration leading to 50% paralysis on intact larvae was 10-fold higher than that for headless larvae. Thus, the cuticle of fourth-instar Ae. aegypti proved to be a more important factor influencing fluralaner toxicity than in adults. The large size (molecular weight = 556) and high lipophilicity (log p = 5.0) could influence fluralaner penetration of barriers and contribute to its slowly developing toxicity. These barriers would include the blood-brain-barrier, and although there was no significant difference between speed of nerve discharge block in severed vs. intact D. melanogaster larval CNS, the blood brain barrier in adult mosquitoes may have a different permeability to fluralaner. At present, there is no published information on the metabolism of fluralaner in insects. A decrease in toxicity was observed with DEM in adult sugar feeding assays (Table5), but the overall effect was small, as was the potentiation of toxicity by PBO. Both findings argue against significant glutathione-S-transferase and P450 monooxygenase metabolism. DEF is a potent carboxylesterase inhibitor [21] and in this study had the most significant positive synergist ratios in feeding assays with Ae. aegypti adults. However, little synergism was noted in topical applications of fluralaner, suggesting that the factor responsible resides in the alimentary system. No ester linkage is present in the fluralaner molecule, but it does have two adjacent amide groups. Carboxylamidases have been identified in different insect species, including Lepidoptera, Orthoptera, and Dictyoptera, which can use p-nitroacetanilide as a model substrate, and were found most abundantly in the midgut [22,23]. The only purified carboxylamidase studied from insects was insensitive to DEF [22]; however, a carboxylamidase might be present in adult Ae. aegypti that metabolizes fluralaner in a DEF-sensitive fashion. Alternatively, DEF might have other mechanisms for potentiating the oral toxicity of fluralaner. All these possibilities await further investigation. Fluralaner showed no target site cross resistance with other GABA receptor-directed compounds. Ozoe et al. [10] reported fluralaner to be equally effective in topical assays on dieldrin-resistant and susceptible strains of houseflies, with LD50 values of 1.01 ng/mg and 0.85 ng/mg, respectively. These values were similar on a per mg insect weight basis to the topical toxicity found for fluralaner on Ae. aegypti adults (1.3 ng/mg at 24 h). Additionally, Asahi et al. [24] showed that fluralaner had similar Int. J. Environ. Res. Public Health 2017, 14, 154 15 of 17 potent toxicity on fipronil-resistant and susceptible strains of Laodelphax striatellus Fallén, whereas fipronil showed a resistance ratio of about 1700. These findings are in agreement with results of both feeding and glass contact assays on dieldrin-resistant and susceptible strains of D. melanogaster reported here, where fluralaner showed similar activity at all recorded time points. On homo-oligomeric RDL (resistant to dieldrin) GABA receptors, fluralaner showed no cross-resistance to classical GABA non-competitive antagonists. Gassel et al. [11] reported fluralaner to be a potent blocker of dieldrin-resistant D. melanogaster and C. felis RDL recombinant, homomultimeric receptors in cell-based fluorescence dye assays, with IC50 values as low as 2.8 nM and 1.7 nM, respectively. In two-electrode voltage clamp (TEVC) recordings of oocytes expressing M. domestica RDL receptors, fluralaner had similar IC50 values of 2.8 nM on rdl-type receptors with A299S mutation and 5.3 nM on wild-type receptors [10]. Fluralaner also had an IC50 value of 12 nM on fipronil-resistant RDL receptors from a plant-feeding mite, Tetranychus urticae Koch, in TEVC experiments, whereas 30 µM of fipronil only blocked 27% of GABA-induced current [24]. In the present study, fluralaner was tested on native D. melanogaster GABA receptors, in situ, instead of heterologously expressed RDL receptors. In line with data on expressed RDL receptors, fluralaner showed similar potent activity on native dieldrin-resistant and -susceptible GABA receptors of D. melanogaster larval CNS, with EC50 values of 0.29 µM and 0.34 µM, respectively. The lower potency in CNS preparations can be attributed to the need for penetration into the neuropile, as well as any differences between native and recombinant homomultimeric receptors. These results further document that fluralaner has potent activity on insect strains that are resistant to classical GABAergic compounds. Gassel et al. [11] also reported 18-fold resistance to fipronil in D. melanogaster homo-oligomeric RDL GABA receptors (Ser isoform). In the present study, no resistance to fipronil was observed in RDL larval CNS preparations, at least when tested at 10 µM (Figure8). We would note that this concentration is over 10-fold greater than the IC50 for blocking the Ala isoform in homo-oligomeric receptors, reported to be 663 nM [11]. So, the resistance may be largely circumvented at this concentration. Good selectivity of fluralaner between mammals and invertebrates also has been demonstrated in both in vivo and in vitro studies. Currently commercialized as a parasiticide, fluralaner was reported to be safe to dogs at or above recommended treatments [25,26]. In radioligand binding studies on rat brain membranes, 10 µM of fluralaner showed around 40% inhibition of radiolabeled 4-ethynyl-4-N-propylbicycloorthobenzoate (EBOB) binding, which is more than 2000-fold less sensitive than its binding to housefly GABA receptors [10,12]. Additionally, fluralaner had low activities on either recombinant β3 homopentamers or α1β2γ2 heteropentamers [10,12]. In the present study, mammalian GABAA α1β3γ2 receptors were tested against fluralaner and fipronil. In line with previous reports, the IC50 value of fluralaner was higher than 30 µM. Moreover, the IC50 value of fipronil was 4.9 µM, suggesting fluralaner has a lower toxicity to mammals than fipronil. The mode of action and toxicology of isoxazolines such as fluralaner are similar to another new insecticide class, the meta-diamides, which also work on invertebrate GABA receptors. According to Nakao et al. [27], meta-diamide 7 has potent activity on three mutant GABA receptors that are resistant to GABA receptor-directed non-competitive antagonists. Additionally, mutations G336M in M3, I277F and L281C in M1 reduce the activity of fluralaner on RDL GABA receptors, while having only a small impact on the activity of fipronil. Further molecular modeling suggests that meta-diamide binds to T9’ to S15’ region in M2, close to the avermectin target site and different from the classical convulsant site [28]. Homology modeling also suggests that fluralaner might share the same binding site as meta-diamide 7 [28]. According to most recent Mode of Action Classification Scheme from the Insecticide Resistance Action Committee (IRAC) [2], GABAergic insecticides have been categorized into Groups 2A (cyclodiene and organochlorines, i.e., chlordane), 2B (phenylpyrazoles, i.e., fipronil), while glutamate-gated chloride channel activators, avermectins and milbemycins, are in Group 6. As of this writing, isoxazolines and meta-diamides have not received a category from IRAC, but these compounds will probably be assigned to a new category because of their novel actions on RDL GABA receptors. Int. J. Environ. Res. Public Health 2017, 14, 154 16 of 17

5. Conclusions In this study, the toxicity of fluralaner against Ae. aegypti, An. gambiae, and D. melanogaster was assessed in various exposure routes. Compared to fipronil, fluralaner had a more slowly developing toxicity, and generally a 7- to 100-fold lower potency in adult bioassays. These findings suggest that fipronil would be a better overall mosquitocide than fluralaner in the absence of resistance. The data also imply that the moderate potency, low contact toxicity, and slow action of fluralaner might preclude its use as a mosquitocide for vector control, despite its favorable mammalian selectivity and lack of cross resistance in rdl carrying insects. For larval assays on Ae. aegypti, the concentration leading to 50% paralysis of intact larvae was 10-fold higher than that on headless larvae, so the cuticle seems to be an important factor influencing fluralaner toxicity. Compared to fluralaner, fipronil was 10- to 13-fold less potent in larval assays, which differed from the results seen in adult mosquito bioassays. In synergism assays, DEF was found to increase fluralaner toxicity by 3.8–8 fold in feeding assays on each day of exposure, with other synergists less active. This finding suggests that carboxylamidases might be involved in the metabolism of fluralaner in mosquitoes. This study provides additional evidence for selectivity and lack of cross resistance of fluralaner. It was tested on mammalian GABAA α1β3γ2 receptors, which gave an IC50 value larger than 30 µM. Additionally, feeding and glass contact assays of fluralaner against susceptible and rdl strains of D. melanogaster showed similar activity, consistent with the equal sensitivity of larval CNS recordings towards fluralaner in both strains.

Acknowledgments: This research was funded by Deployed War Fighter Research Program under USDA Specific Cooperative Agreements 58-0208-0-068 and 58-0208-5-001 to Jeffrey Bloomquist. Author Contributions: Shiyao Jiang, Maia Tsikolia, Ulrich R. Bernier, and Jeffrey R. Bloomquist conceived and designed the experiments; Shiyao Jiang performed the experiments; Shiyao Jiang and Jeffrey R. Bloomquist analyzed the data; Miai Tsikolia and Ulrich R. Bernier contributed reagents/materials/analysis tools; Shiyao Jiang and Jeffrey R. Bloomquist wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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