Knockdown of Fruit Flies by Imidacloprid ... - ACS Publications

Sep 23, 2015 - National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia 20110, United States. •S Supporti...
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Knockdown of Fruit Flies by Imidacloprid Nanoaerosol Victor N. Morozov*,†,‡ and Igor L. Kanev† †

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia 142290 National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia 20110, United States

‡

S Supporting Information *

ABSTRACT: This report describes the effects of nanoaerosol particles (NAPs) from imidacloprid (IMI) on fruit flies. NAPs were produced using a newly developed generator which employs electrohydrodynamic atomization of IMI solution in ethanol. Exposure of Drosophila melanogaster to the IMI NAPs at a concentration of C = 2.7 ± 0.1 ng/cm3 caused knockdown in half of the flies in T50 = 88 ± 14 min at 22 °C and in T50 = 36 ± 2 min at 33 °C. A number of special experiments precluded IMI volatilization and contact or oral action of IMI upon exposure to the NAPs. It was shown that only the fraction of NAPs in the size range of 7−300 nm is responsible for the knockdown and that dependence of T50 on the NAPs’ fraction mass follows Haber’s rule, C × T50 = const. Comparison with the oral doses obtained when flies were fed an IMI-sucrose mixture revealed that the inhaled doses that caused knockdown were 2 orders of magnitude lower than the oral ones. This new technology may be used to quickly eliminate insects with nanoaerosols of nonvolatile insecticides in greenhouses and other closed environments.

1. INTRODUCTION The development of nanoaerosol technology opens up new ways to expose insects to insecticides. Instead of being orally applied, the insecticide can be introduced into the insect trachea or applied on the insect body in the form of nanoaerosol particles (NAPs) even if the insecticide is not volatile. Imidacloprid (IMI) presents a good example of such a nonvolatile insecticide. This insecticide belongs to the class of chemicals called the neonicotinoids, which act on the central nervous system of insects.1 Its binding to postsynaptic nicotinic acetylcholine receptors (nAChRs) induces neuronal hyperexcitation, resulting in convulsions, paralysis, and death of the insect. In conventional usage, insects consume IMI by digesting plants which were treated with IMI or through direct contact with the insecticide.2 Studies of the effects of nanoaerosolized insecticides are in their infancy: we found only one publication on this topic. Posgai et al. demonstrated penetration of aerosolized quantum dots into the trachea of Drosophila melanogaster3 and found that exposure of flies to silver NAPs increased the level of heat shock proteins 70 (Hsp70) in the flies. Thus, inhaled silver NAPs initiated oxidative stress similar to that which resulted from feeding the silver nanoparticles to the fly larvae.4 One problem in studying the biological effects of nanoaerosolized insecticides is the absence of good equipment for working with NAPs: generators, dosimeters, exposure chambers, etc. We recently developed a new type of nanoaerosol generator specifically designed to atomize biological and biologically active substances.5,6 It permits stable, long-term production of NAPs from virtually any soluble substance under mild conditions. Here we report how this technique may be applied to treat insects with nanoaerosolized nonvolatile insecticides. We demonstrate for the first time that a © 2015 American Chemical Society

nonvolatile insecticide in a nanoaerosolized form quickly kills fruit flies by penetrating into their breathing system. We show that the amount of IMI penetrating as NAPs into the insect trachea and causing knockdown in flies is less than 1/100 of that upon oral consumption and that the dependence of knockdown time on the IMI NAPs follows Haber’s law.

2. MATERIALS AND METHODS 2.1. Materials. Imidacloprid (IMI) was purchased in a local shop under the commercial name of “Biotlin” (produced by the CJSC “Avgust”, Russia). The IMI concentration in the commercial solution was found to be 18% from its optical density at 270 nm and the extinction coefficient,7 ε = 22,054 L mol−1cm−1. The substance purity and identity was verified by measuring its melting point, (Tm = 140 ± 0.5 °C) and by FTIR spectroscopy (see the Supporting Information). Sucrose, yeast extract, agar, and sodium fluorescein were obtained from Sigma-Aldrich. 2.2. Growth of Fruit Flies. A wild type of Drosophila melanogaster caught in a field near Pushchino, Moscow region (Russia) was bred in the laboratory. The stock of flies was grown on media containing 6 g of yeast extract, 22 g of semolina, 2 g of agar, 6 g of sugar, 0.15 mL of concentrated acetic acid, and one ripe minced banana added to 200 mL of distilled water. After heat treatment and cooling the semisolid mixture was placed at the bottom of a plastic jar, and its surface was dusted with dry baker’s yeast. The plastic jar was plugged with a perforated cap to allow air access. The average mass of Received: Revised: Accepted: Published: 12483

July 3, 2015 September 20, 2015 September 23, 2015 September 23, 2015 DOI: 10.1021/acs.est.5b03219 Environ. Sci. Technol. 2015, 49, 12483−12489

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Environmental Science & Technology

Figure 1. Exposure of fruit flies to nanoaerosol. A. Schematic of simultaneous exposure of fruit flies to IMI nanoaerosol at different temperatures. B. Image of differential exposure chambers used to study effects of temperature and carbon dioxide on toxicity of IMI NAPs.

one fly varied from 0.8 to 1.3 mg in different populations used throughout this study. We will henceforth consider each fly to weigh 1 mg on average. 2.3. Nanoaerosol Generation. The basic design and performance characteristics of the nanoaerosol generator used in these experiments were described in our recent papers.5,6 Its operational principle is based on electrohydrodynamic atomization of solutions followed by gas-phase neutralization of the electrospray products with small counterions also generated by electrospraying. Briefly, a substance solution is placed in a capillary to which a high (usually positive) voltage is applied, resulting in destabilization of the solution meniscus at the capillary end and ejection of a cloud of highly charged micrometer-sized droplets. The droplet size quickly decreases while the number of droplets increases in a series of electrostatic decays caused by repulsion of charges in the drying microdroplets. These events result in the formation of a cloud of highly charged nanoclusters of dry residues. The nanoclusters are neutralized with counterions produced by electrospraying a volatile solvent or another solution, yielding a nearly neutral nanoaerosol. It has been demonstrated that biological substances retain their structure and functional activity upon such atomization and that negligible amounts of reactive oxygen species accompany the process.8 Commercial IMI solution was diluted to 0.5% or to 0.1% (19.5 mM and 3.9 mM, respectively) with 96% ethanol and placed in both capillaries in the generator. Currents of 90 nA and 40 nA were passed through the positively and negatively charged capillaries, respectively. In the experiments designed to evaluate NAP deposition in the fly trachea, the NAPs were generated from the acidic form of fluorescein (0.3% dissolved in 96% ethanol). The generator was typically set to produce aerosols at a flow rate of 2 L/min if not stated otherwise. 2.4. Nanoaerosol Characterization. Nanoaerosol size distribution was measured by a scanning mobility particle sizer (SMPS, purchased from HCN Co., Ltd., Icheon-si, Gyeonggido, South Korea) equipped with a soft X-ray neutralizer. It consumed nanoaerosol at a rate of 1 L/min and had a sheath flow rate of 10 L/min. The mass concentration of aerosol in the size range of 10−300 nm was calculated by integrating the spectra. Larger aerosol particles were characterized with a laser counter (Aerotrak 9303 counter, TSI Inc., Shoreview, MN). The total mass concentration of aerosol was measured with a piezobalance dust monitor (Kanomax, model 3521, Andover, NJ). The stability of NAP concentration in long-term exposure was monitored with a quartz microbalance dosimeter, as

described in ref 5. Considering the low volatility of IMI (vapor pressure of 1.5 × 10−9 mm Hg at 20 °C), only 10 ng will evaporate into 1 L of air to saturate it at room temperature. Thus, with a total average concentration of IMI aerosol ∼2.7 ± 0.1 μg/L, the decrease in size and mass of NAPs due to IMI evaporation may be ignored. A SmartSPM-1000 atomic force microscope (AIST-NT. Co, Moscow, Russia) was used to image NAPs on the mica surface. NAPs were deposited onto freshly cleaved mica by directing a jet of NAPs from the generator for 30 min. The mica was then scanned in tapping mode at a frequency of 250−300 kHz while keeping the environmental humidity below 50%. 2.5. Exposure System. As shown in Figure 1B, the exposure system consisted of a glass tube with ends plugged with corks through which inlet and outlet holes were drilled. The holes were covered with a fine mesh to keep the flies in the tube. To study the effects of temperature and carbon dioxide, the differential exposure system illustrated in Figure 1A and B was employed that consisted of two chambers. In evaluating the temperature effects on IMI toxicity, one chamber contained a jacket filled with water running through a thermostat. Both tubes were connected in parallel to the same nanoaerosol generator via a Y-connection. The flow through each chamber was measured by a flowmeter (EW-32900−44, Cole Parmer Instrument Co., Vernon Hills, IL) and adjusted with a valve to keep the flow rate through each chamber equal (∼1 L/min). After passing through the flow meter, nanoaerosol was collected on a HEPA filter. In evaluating the effects of carbon dioxide on the neurotoxicity of IMI NAPs, 70 cm3/min of CO2 was introduced into flow of NAPs through one tube. In order to exclude IMI vapor from the factors responsible for fly knockdown a special experiment was performed where an additional chamber with flies was attached in series with the first one to receive exhaust air. In this experiment the IMI nanoaerosol which had passed though the first chamber filled with flies was filtered out by a HEPA filter, and clean air containing IMI vapor entered the second chamber with flies. Caution: Nanoaerosolized imidacloprid should be considered hazardous to humans and should be handled accordingly. Experiments should be performed within a ventilated box in a closed system. 2.6. Estimation of IMI Consumption by Feeding. In order to estimate oral working doses flies were placed inside a plastic jar 4 cm in diameter, as shown in the Supporting Information. The open side of the jar was covered with polyester mesh made of single fibers, with open windows of 200 12484

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Figure 2. A. Size distribution of nanoaerosol generated from different solutions of IMI. Solid line−from a solution of 0.5% imidacloprid and 0.1% Hfluorescein, dashed line−from 0.5% IMI solution, dotted line−from 0.1% IMI. All solutions were prepared with 96% ethanol. Both negatively and positively charged microcapillaries were filled with the same solutions. Each spectrum is average of 3−5 spectra (scatter is below ±10%). B. AFM image of IMI NAPs deposited on mica.

Table 1. Mass Concentrations of NAPs in the Range of 7−300 nm and the Total Mass Concentration of Aerosols Generated from Different Solutions solution sprayed 0.5% IMI 0.5% IMI+0.1% Hfluorescein 0.1% IMI

mass concentrationa of NAPs with size of 7−300 nm, ng/cm3

total mass concentration,b ng/cm3

concentration of NAPs with size of 7−300 nm, cm−3

0.8 ± 0.1 1.0 ± 0.1

2.7 ± 0.1 1.7 ± 0.1

(6.4 ± 0.1)x105 (7.7 ± 0.3) x105

0.5 ± 0.1

0.6 ± 0.1

(6.8 ± 0.4) x105

a

3 b

Mass concentration is calculated from spectra assuming that density of dry residue is 1 g/cm . Mass concentration measured with the piezobalance dust monitor.

× 200 μm2. The container was weighed on a semimicro balance (precision 10 μg, Ohaus DV215CD, Parsippany, NJ) before and after the flies were placed inside. The flies were kept in the container and deprived of food for 2 h while their weight was periodically measured. The polyester mesh-covered portion of the container was then placed onto a feeder (see its design in the Supporting Information) to allow the flies to access the food. After the flies were allowed to feed for 1 or 2 h, the container was removed from the feeder, the air inside the feeder was blown out to bring the humidity inside the container back down to room level, and measurements of the container weight were continued. 2.7. Estimation of IMI Nanoaerosol Deposited onto the Fly Body. In order to measure how much IMI is deposited onto the fly body groups of flies were exposed to IMI nanoaerosol for different time then cold-euthanized. Approximately 30 thus exposed flies were placed into a 0.6 mL microcentrifuge tube to which 0.2 mL of washing solution-1 (water containing 0.05% Tween-20) was added. The flies were washed by vortexing them for 1−2 min. The tube was then pierced at the bottom with a needle and placed inside a larger 1.5 mL tube. The washing solution was collected on a centrifuge at 2700g for 1−2 min. To remove debris, the washing probes were further centrifuged for 5 min at 4500g, and IMI concentrations in the supernatants were determined from absorption at 270 nm using an extinction coefficient7 of ε = 22 054 L mol−1cm−1. A microspectrophotometer (NanoPhotometer P330, Implen GmbH, Munich, Germany) was used to measure spectra in small 3 μL probes of the washing solution. In control measurements, a group of flies was kept for 1.5 h in the same exposure tube blown with pure air. The control flies were then cold-euthanized and washed as described above for the flies exposed to IMI NAPs.

3. RESULTS 3.1. Characterization of the Imidacloprid Nanoaerosol. The spectra of IMI NAPs used throughout this study are presented in Figure 2A. They are well reproducible and characterized by a main mode diameter of 30−40 nm and a smaller peak with a diameter of ∼200 nm. As shown in Figure 2A, aerosol particles with sizes above the upper limit of the spectrometer (300 nm) are also present. The AFM image presented in Figure 2B indicates that IMI NAPs have a smooth surface and have heights in the range of diameters measured by the spectrometer. Analysis of AFM images did not show any flat deposits that would indicate that incompletely dried micro- and nanodroplets of the IMI solution are present. The total mass concentration of IMI aerosol generated from 0.5% and 0.1% solutions of pure IMI and from a mixture of 0.5% IMI and 0.1% H-fluorescein as well as mass concentrations of NAPs obtained by integrating the spectra in Figure 2A over the whole range of sizes, 7−300 nm, with a material density of 1 g/cm3 are summarized in Table 1. It is evident that the mass concentration of NAPs with sizes in the range of 7−300 nm varies between 30% and 80% of the total aerosol mass. According to our estimates based on measurements with the laser aerosol counter, the remaining mass belongs to particles with sizes between 0.3 and 0.7 μm. Particles larger than 1 μm were not registered. It was demonstrated that neither the aerosol mass concentration measured by the quartz monitor nor spectra changed during the 2.5 h experiment 3.2. Dependence of Fly Knockdown on Temperature and Carbon Dioxide. We will further characterize knockdown by T50 − the time interval during which half of flies experience hyper-activation and paralysis making them incapable of holding on to the tube wall. The comparison of knockdown curves in Figure 3A shows that T50 decreases from 88 ± 14 min at 22 °C to T50 = 36 ± 2 min at 33 °C. Preexposure of flies to IMI NAPs at 33 °C for 15 min followed by 12485

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In order to exclude a possibility that IMI vapor contributes to killing flies yet another control experiment was made in which nanoaerosol was passed through a HEPA filter before being introduced into the container with flies. Neither knockdowns nor changes in fly behavior were observed after exposure to the filtered air for 2 h at 22 °C, while in the chamber filled with the same unfiltered nanoaerosol, all of the flies were knocked down. Thus, IMI vapor does not contribute to the fly poisoning observed (see Supporting Information for more details). In an attempt to change the breathing rate, 6.5% carbon dioxide was also added to nanoaerosol in one channel. We expected that the elevated CO2 level would accelerate metabolic activity and thus breathing rate and uptake of IMI. However, as seen in Figure 3D, addition of CO2 did not affect the knockdown curve. The lack of CO2 effects may reflect the surprisingly low sensitivity of Drosophila melanogaster metabolism to both hypoxia and hypercapnia.11 3.3. Estimation of Doses Required for Knockdown of Fruit Flies by Oral Application. A plot of the relative changes in the flies’ weight over time is presented in Figure 4. It

Figure 3. Knockdown time in IMI nanoaerosol under different conditions. A. Exposure to the same nanoaerosol at 33 °C (filled circles) and at 22 °C (empty circles). Nanoaerosol was created from a solution of IMI and H-fluorescein (5:1, w/w) in EtOH. B. For the first 15 min IMI nanoaerosol was passed through the tube at 33 °C; then the temperature was lowered to 22 °C without stopping the nanoaerosol generator (filled circles). Empty circles denote knockdown in the control tube held at 22 °C. C. Exposure was for 25 min at 22 °C, in both channels; then the temperature in one channel was raised to 33 °C and pure air was passed through the tube (filled circles). The empty circles represent flies in the control tube, through which IMI nanoaerosol was passed the entire time at 22 °C. D. Effect of CO2 on knockdown time at 22 °C. Results of two independent experiments are presented. Filled circles and triangles represent knockdown in the tube with IMI nanoaerosol to which 6.5% carbon dioxide was added. Empty circles and triangles represent controls without CO2. Gray bars indicate time intervals during which temperature was changed.

Figure 4. Changes in mass with flies feeding on a 5% solution of sucrose containing 2.4 mM of IMI. Gray zones (columns) denote time periods of exposure to humid air and to IMI solution. Each point is an average of 3−4 weight measurements. Arrows indicate two possible estimates of mass changes resulting from feeding.

a rapid (within ∼5 min) decrease in temperature to 22 °C resulted in rapid knockdown of flies at room temperature, as Figure 2B illustrates. High temperature by itself was not responsible for the knockdown observed: a 2 h exposure of flies to filtered air at 33 °C did not result in any knockdown or visible changes in the flies’ behavior apart from greater activity in flights and locomotion. In another experiment, flies were first subjected to IMI NAPs at 22 °C for 25 min; nanoaerosol flow was then changed to a flow of pure air and the temperature was quickly raised to 33 °C. As seen in Figure 3C, the knockdown curve in this case is very similar to those in Figure 3A at 33 °C. As Figure 3D illustrates, addition of CO2 caused no substantial changes in the knockdown curve despite the published data on the physiological effects of hypercapnia9 (suppression of metabolically demanding functions such as egglaying, Na,K-ATPase activity, and other activities that require a high energy supply). Several control experiments were performed to exclude certain routes of fly exposure to IMI. In order to exclude a possibility that flies are poisoned by consuming IMI deposited on the chamber walls a fresh group of 17 flies was placed into the tube used in the previous experiment in which IMI nanoaerosol passed through the tube for 3 h. A small amount of food was placed inside the tube. No knockdown was observed during the first 2 h, and only two flies were found dead after 15 h of exposure. The possibility that the knockdown upon exposure to IMI NAPs was caused by the IMI deposited on the glass walls10may be, thus, ruled out.

is evident that a steady decrease in weight, presumably due to water loss, is interrupted by a jump after contact with the feeder. The jump was not the result of a humidity increase: exposure to humid air (shown by a gray column in Figure 4) resulted in a temporary increase in weight, but the curve quickly returned to the previous trend, unlike the jump after feeding. Thus, the jump after feeding may be attributed to the weight of the sucrose solution consumed by the flies. As seen in Figure 4, after 1 h of feeding on sucrose solution, each fly increased its weight by 4−7% on average, indicating that 40−70 μg of solution was consumed per mg of fly. Considering the IMI concentration of 2.4 mM in the 5% sucrose solution, each fly got approximately 0.10−0.17 nmoles of IMI or 25−43 ng/mg. When flies were allowed to feed for 2 h, they acquired 7−10% of their weight, and 50% of them experienced knockdown within the next 3 h as shown in the Supporting Information. 3.4. Estimation of IMI Mass Settled on Fruit Fly Body. In order to estimate how much IMI nanoaerosol was settled on the fly bodies flies were washed after being exposed to IMI NAPs for different time and the optical density of the washing solutions were measured. It was found that IMI was washed from the body nearly completely in the first wash; the second wash of the same flies yielded only ∼1% of the first one. As seen in Figure 5, the amount of IMI washed from fly bodies increases in proportion to the exposure time. The rate of 12486

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estimated that as much as 0.33 mL of air should pass through the fly body in 1 h to supply oxygen. If all the nanoaerosol in this volume at a concentration of 2.7 ng/mL is deposited within the fly trachea, the hourly dose should reach ∼1 ng/mg. Of course, this value should be considered an upper estimate for the inhaled dose, because the ventilatory mechanism is employed by the Drosophila only occasionally at high metabolic demand.14 The estimation of the deposited dose according to the second model is based on a comparison of the diffusion coefficients of oxygen and NAPs as well as their respective concentrations. This model does not require air motion within the breathing system. The model is presented in detail in the Supporting Information and gives a rate of IMI NAP deposition of ∼1 pg/hour-mg of fly if only the concentration of the smaller, more mobile fraction (NAPs with a size of ∼30 nm) is taken into consideration. The two estimates based on different assumptions thus give values differing by 3 orders of magnitude. We believe that the truth is somewhere between, because the gas exchange rate in small insects does not rely much on ventilation, but the insects employ ventilation during flight and running.14 Considering the great difference in the expected deposition by diffusion versus ventilation, as discussed above, even short episodes of ventilation would substantially increase NAP deposition. Both estimates, despite their difference, lead to the same conclusion, namely that the knockdown dose which the fly receives through its breathing system is substantially lower than the knockdown dose upon oral consumption. 3.6. Knockdown Time As a Function of IMI Nanoaerosol Concentration. Exposure of fruit flies to IMI nanoaerosol diluted with filtered air resulted in an extension of knockdown time that is inversely proportional to the concentration of IMI NAPs with sizes of 7−300 nm, as seen in Figure 6A. Thus, the time of response to a poison as a function of concentration is described by known Haber’s rule:15 C×T50 = constant.

Figure 5. Accumulation of IMI on fly bodies versus time of exposure to IMI NAPs at 22 °C. Optical density at 270 nm of the solution after washing of control flies is subtracted in calculating the deposited mass of IMI. Every point is mass averaged over 30 flies.

accumulation is 16 ± 2 ng/mg- hour at 22 °C. The theoretical estimate of an IMI NAP deposition rate of ∼1.5 ng/fly hour presented in the Supporting Information is below the experimental measurements. The discrepancy may be explained by higher deposition on wings and by contribution of other factors which the simple model does not take into account. 3.5. Estimation of Doses Settled in the Fly Trachea. Theoretical estimates of the amounts of IMI which could penetrate inside the fly trachea are presented in the Supporting Information and summarized in Table 2. The estimates were Table 2. Comparison of Knockdown Doses of IMI Measured and Theoretically Estimated for Different Application Routes doses application route

estimation

oral

experimentala (knockdown)

inhalation

theoretical estimation from ventilation ratec comparison of oxygen and NAP diffusiond experimental measurement of deposition on the fly surfacee theoretical deposition on the fly surfacef

inhalation total body surface total body surface

IMI doses and consumption rates 170a ng/mg > dose >40b ng/mg ∼1 ng/hour.mg ∼1 pg/hour.mg 16 ng/hour.mg (22 °C) 1.5 ng/hour.mg

Dose of IMI measured in extracts of dead flies after they were fed sucrose-IMI mixture (ref.1). bEstimate obtained in this study. See Supporting Information. cCalculations were made assuming that NAPs are deposited from the entire air volume from which oxygen was consumed (see Supporting Information for more details). dCalculations are based on differences in diffusion coefficients and concentration of NAPs and oxygen (see Supporting Information for more details). eWashed by Tween-20 solution from fly surface. f Calculation of diffusion flow of NAPs through unstirred layer (see details in Supporting Information). a

Figure 6. Relationship between knockdown half-time (T50) and aerosol concentration calculated by integrating NAP size distribution over the range of 10−300 nm (A) or total IMI concentration measured with the dust monitor (B). ○, aerosol generated from 0.5% IMI solution and diluted with pure air. ▲, nanoaerosol generated from 0.1% IMI solution. ■, nanoaerosol generated from IMI/fluorescein mixture (0.5% and 0.1%, w/w, respectively).

obtained using two different deposition models. The first model assumes that nanoaerosol is deposited inside the trachea and air sacs from all the air volume from which oxygen was consumed to support the normal metabolic rate. Thus, it assumes that the air is actively moved through the insect breathing system, a breathing mode further referred to as ventilation. The rate of oxygen consumption, 3.52 ± 0.07 μL O2/mg-hour,12 should be multiplied by 5 to account for the oxygen content in the air and then by 19 to account for the difference in the partial pressure of oxygen in the fly trachea (19.9 kPa according to ref 13 and 21 kPa in the air). Taking both factors into account, it was

As seen from the comparison of the T50 vs C1− plots presented in Figure 6A and B, only when T50 is plotted against the reciprocal of mass concentration for NAPs with a size of 7− 300 nm do the different IMI nanoaerosols generated from pure 0.1% and 0.5% solutions as well as from 0.5% IMI solutions containing 0.1% fluorescein fit the same line, independently of the big differences in the concentration of larger particles (see Table 1). This fact unequivocally points to the smaller, more mobile fraction of NAPs as the major knockdown factor. 12487

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4. DISCUSSION It was demonstrated here for the first time that once a nonvolatile insecticide is turned into a highly dispersed nanoaerosol, it substantially gains in performance (as compared to contact or oral application), enabling quick knockdown of flies with low insecticide consumption. The most important question about the mechanism of action of the IMI NAPs concerns the route whereby the NAPs reach the fly nerve cells. Three kinds of data obtained in this research exclude contact action by the IMI NAPs: (i) a linear increase in the amount of IMI washable from the fly body as a function of exposure time, (ii) a special type of dependence of knockdown time on IMI NAPs concentration, and (iii) the lack of dependence of knockdown time on the mass of IMI delivered by particles larger than 300 nm (see Figure 6B). First, if a notable amount of deposited IMI had penetrated through the cuticle, no linear time dependence of the amount of washable IMI (Figure 5) would have been observed. Second, when an insect is dusted, insecticide penetration through the cuticle depends on the contact area between the deposited particles and the insect surface, on the solubility of the insecticide in the wax layer (or on a partition coefficient), and on the concentration gradient formed.16 It is reasonable to expect that the contact area between the cuticle and the deposited insecticide would increase in proportion to the number of NAPs deposited. Because this number increases linearly with time, as Figure 5 illustrates, the penetration rate also increases in proportion to the exposure time, so that the total accumulated dose increases in proportion to the square of the time (see detailed description in the Supporting Information). Hence, T50 should decrease not in proportion to C−1 but in proportion to C−1/2. However, the data in Figure 6A contradict that expectation: no linear fit is possible when T50 is plotted as a function of C−1/2. We conclude from this discrepancy that though a substantial amount of IMI is deposited on the fly body, it is not involved in poisoning the fly. Finally, diffusion through the cuticle should not depend on particle size, yet IMI NAPs with a size larger than 300 nm do not contribute to the knockdown effect, as a comparison of the concentration dependences in Figures 6A and 6B indicates. These experimental data, combined with the results of control experiments where flies were exposed to filtered nanoaerosol or placed into a chamber with walls “contaminated” with IMI NAPs, rule out IMI vapor, oral, and contact routes and support the conclusion that the breathing system is the major target for the IMI NAPs. The temperature dependence of the knockdown time is consistent with this conclusion as well. The increase in insect metabolic rate with temperature is supported by a higher gasexchange rate accompanied by a higher rate of IMI accumulation and, hence, a decrease in T50. As seen in Figure 3A, T50 at 33 °C is 40% of that at 22 °C, roughly corresponding to an approximately 3-fold increase in fly metabolic rate17 that requires a corresponding increase in the rate of oxygen and NAPs uptake with such a temperature increase. Figure 3B also supports the idea that higher oxygen consumption at higher temperature results in an increased volume of air consumed and hence in an elevated dose of IMI NAPs deposited in fly tracheae and sacs. After flies were exposed for 15 min at 33 °C, thus receiving a dose 3 times higher because of the deeper ventilation, a return to 22 °C did not prevent early knockdown. However, the data presented in Figure 3C contradict the idea of

metabolism intensity as the only factor determining the rate of IMI uptake. Flies exposed to IMI NAPs at 22 °C for 25 min and then heated to 33 °C in pure air experienced knockdown almost immediately, as if the sensitivity to the dose accumulated at the lower temperature substantially increased with temperature. Thus, both the total dose received and the threshold knockdown concentration depend on the exposure temperature. Unfortunately, we could not find any data on the temperature dependence of the binding constant for the interaction of IMI with nAChRs to check this hypothesis. In addition to these factors, changes in temperature may affect the penetration rates and biotransformation dynamics of the insecticide.18 IMI NAPs have an LC50 that is ∼1/1000 of the LC50 for the best volatile insecticides reported.19 We speculate that the extremely high neuronal toxicity of IMI and a shortcut tracheal delivery route are responsible for the very low LC50 of IMI NAPs and their quick action. This route avoids the rapid metabolic destruction of IMI in the insect gut20 and bypasses the slow penetration rate of IMI though the cuticle. Direct penetration to the trachea and breathing sacs enables almost immediate access of IMI to the nerve cells. It is worth noting that even picogram amounts of IMI dissolved in the lymph would be enough to cause havoc in the fly nervous system. The dissociation constant characterizing the interaction of IMI with the nAChRs in Drosophila melanogaster is about 3 nM.21 Dissolution of 1 pg of IMI in ∼60 nL hemolymph22 would result in an IMI concentration of 60 nM, enough to saturate all of the nAChRs. Considering the close proximity of tracheoles and breathing sacs to the nervous cells, the local IMI concentration might be substantially higher and the time to reach the receptors very brief. These features correspond well to the observed time−concentration effects of the IMI NAPs. It is apparent that the results reported here may have important practical applications. First of all, a substantial reduction in dose creates an opportunity for better pest control without heavy contamination of the environment. Though use of IMI nanoaerosol in an open field appears problematic because it is quickly removed by air motion, there is no doubt that this insecticide form may be used effectively to control insects in a closed environment: greenhouses, vegetable storage facilities, etc. As compared to the standard spraying technique, in which IMI first penetrates soil and plants, plants exposed to NAPs will remain essentially insecticide-free because the doses required to kill insects with NAPs are much lower than those the insects receive orally by eating plants. It is important to note that the effective inhaled IMI doses are less than 1/100 of the oral ones; thus, once harmful insects are killed by the insecticide nanoaerosol and the air is cleaned, pollinators can be allowed indoors because of the extremely low amount of IMI deposited on plants and because the deposited insecticide poorly penetrates through the cuticle. Of course, care should be taken to protect humans from breathing the nanoaerosol, because its toxicity is expected to considerably exceed that of IMI taken orally. In fact, an estimation of the LC50 of nanoaersolized IMI NAPs for mammals is planned for subsequent studies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03219. 12488

DOI: 10.1021/acs.est.5b03219 Environ. Sci. Technol. 2015, 49, 12483−12489

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Environmental Science & Technology

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S1. Estimation of nanoaerosol deposition in fly trachea and sacs. Ventilation model. S2. Estimation of deposition rate of nanoaerosol inside fly trachea. Diffusion model. S3. Estimation of the IMI deposited on the fly body surface. S4. Rate of penetration of insecticide NAPs through the insect cuticle. S5. Method of feeding fruit flies on sucrose solution. S6. Estimation of oral doses of IMI from the fly metabolic rate. S7. Effect of IMI vapor on fruit fly survival. S8. FTIR spectrum of IMI (PDF)

(11) Van Voorhies, W. A. Metabolic function in Drosophila melanogaster in response to hypoxia and pure oxygen. J. Exp. Biol. 2009, 212, 3132−3141. (12) Hulbert, A. J.; Clancy, D. J.; Mair, W.; Braeckman, B. P.; Gems, D.; Partridge, L. Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster. Exp. Gerontol. 2004, 39, 1137−1143. (13) Lehmann, F. O. Matching spiracle opening to metabolic need during flight in Drosophila. Science 2001, 294, 1926−1929. (14) Lehmann, F. O.; Schutzner, P. The respiratory basis of locomotion in Drosophila. J. Insect Physiol. 2010, 56, 543−550. (15) Tennekes, H.; Gretton, V. A.; Stedeford, T. Hazard and risk assessment of chemical carcinogenicity within a regulatory context. In Cancer Risk Assessment: Chemical Carcinogenesis, Hazard Evaluation, and Risk Quantification; Hsu, C. H., Stedeford, T., Eds.; John Wiley and Sons: Hoboken, NJ, 2010; pp 37−65. (16) Webb, J. E.; Green, A. A. On penetration of insecticides through the insect cuticle. J. Exp. Biol. 1945, 22, 8−20. (17) Orr, P. R. Critical thermal increments for oxygen consumption of an insect. J. Gen. Physiol. 1925, 7, 731−734. (18) Khan, H. A. A.; Akram, W. The effect of temperature on the toxicity of insecticides against Musca domestica L.: Implications for the effective management of diarrhea. PloS One, April 20149(Issue 4) e95636. (19) Scharf, M. E.; Nguyen, S. N.; Song, C. Evaluation of volatile low molecular weight insecticides using Drosophila melanogaster as a model. Pest Manage. Sci. 2006, 62, 655−663. (20) Cresswell, J. E.; Robert, F. X. L.; Florancea, H.; Smirnoff, N. Clearance of ingested neonicotinoid pesticide (imidacloprid) in honeybees (Apis mellifera) and bumblebees (Bombus terrestris). Pest Manage. Sci. 2014, 70, 332−337. (21) Huang, Y.; Williamson, M. S.; Devonshire, A. L.; Windass, J. D.; Lansdell, S. J.; Millar, N. S. Molecular characterization and imidacloprid selectivity of nicotinic acetylcholine receptor subunits from the peach−potato aphid Myzus persicae. J. Neurochem. 1999, 73, 380−389. (22) MacMillan, H. A.; Hughson, B. N. A high-throughput method of hemolymph extraction from adult Drosophila without anesthesia. J. Insect Physiol. 2014, 63, 27−31.

AUTHOR INFORMATION

Corresponding Author

*Phone: 7-496-773-0623; fax: 7-496-733-0553; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate fruitful discussions with members of the Laboratory of Nanostructures and Nanotechnology: Andrei Mikheev, Yuri Shlyapnikov, and Elena Slyapnikova. We thank Dr. Alexey Zabelin from IBBP RAS for obtaining IR spectrum of IMI. The substantial contribution of Dr. Tamara Morozova to the manuscript and the assistance of Jennifer Guernsey in manuscript editing are gratefully acknowledged. We acknowledge funding from the Russian Foundation for Basic Research (Grant No. 15-29-01180).

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REFERENCES

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DOI: 10.1021/acs.est.5b03219 Environ. Sci. Technol. 2015, 49, 12483−12489