Insecticides have a variety of commercial applications including urban pest control, agricultural use to increase crop yields, and prevention of proliferation of insect-borne diseases. Many pesticides in current use are synthetic molecules such as organochlorine and organophosphate compounds. Some synthetic insecticides suffer drawbacks including high production costs, concern over environmental sustainability, harmful effects on human health, targeting non-intended insect species, and the evolution of resistance among insect populations. Thus, there is a large worldwide need and demand for environmentally safe and effective insecticides. Here we show that Erythritol, a non-nutritive sugar alcohol, was toxic to the fruit fly Drosophila melanogaster. Ingested erythritol decreased fruit fly longevity in a dose-dependent manner, and erythritol was ingested by flies that had free access to control (sucrose) foods in choice and CAFE studies. Erythritol was US FDA approved in 2001 and is used as a food additive in the United States. Our results demonstrate, for the first time, that erythritol may be used as a novel, environmentally sustainable and human safe approach for insect pest control.

Copyright: © 2014 Baudier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

During an examination of the effects of commonly used non-nutritive sweeteners on the longevity of Drosophila melanogaster, we discovered that erythritol, the main component of the sweetener Truvia, was toxic when ingested by fruit flies as compared to similar concentrations of nutritive sugar controls (sucrose, corn syrup) and other non-nutritive sweeteners. We describe here the effects of erythritol and Truvia on the longevity and motor function of the fruit fly, Drosophila melanogaster. We show that when flies consumed erythritol their longevity decreased in a positive concentration-dependent manner. We also use choice tests and capillary feeding (CAFE) assays to show that flies consumed erythritol when given free access to control (sucrose) food sources and suffered decreased longevity. Consumption of erythritol is safe to humans, even when consumed at high levels [4] , [5] . Thus, we suggest erythritol has potential for use as a novel, human-safe insecticide.

Insects have significant worldwide deleterious impact on human health, agriculture, and economic growth [1] . Cost of application of insecticides for the prevention of insect damage has been estimated at $10 Billion annually in the US alone [2] . Further, widespread use of toxic insecticides continues to pose a significant threat to human health, as highlighted by recent deaths in Bihar India [3] . Thus, there is a strong need for cost-effective and human-safe insecticides to control insect pest populations.

Materials and Methods

Drosophila culturing and sample sizes for solid food studies All animals were cultured at 25°C, kept at 50–60% humidity, and were raised under a standard 12∶12 light dark cycle. For each experimental treatment n = 30 flies were tested in groups of 10 flies per tube, and three tubes per treatment. In each treatment one tube contained males, one females, and one tube contained five flies of each sex. Tubes were kept on their side to minimize subjects becoming mired in the food. Foods were replaced twice a week. The total number of fruit flies used for these experiments was 690, with 300 used for two initial trials testing mortality among store-brand sweeteners, 120 used for repeating this with sweeteners with blue dye (0.05%) and pure erythritol, 120 for choice trials and 150 for concentration trials. Standard Drosophila food for larval culturing consisted of water, cornmeal, yeast, molasses, and agar, as previously described [6]. A similar food (without molasses) also served as the base to which treatments were added. The addition of cornmeal and yeast assured the flies still received sufficient carbohydrates and protein in addition to any effects of the treatment additives. We combined Drosophila food with an equal weight/volume (0.0952 g/ml) of one non-nutritive sweetener (Truvia, Equal, Splenda, Sweet'N Low, or PureVia) or a control nutritive sweetener (controls: sucrose or corn syrup). We initially raised wild type (Canton S) larvae on the standard food and transferred 0–24 hour old adult flies to foods containing one non-nutritive sweetener or a control treatment and observed them for 65 days. Longevity assays and climbing behavioral assays were performed as previously described [6]. The number of dead flies were scored daily. Climbing behavior was assayed every second day. For climbing assays, a modified version of Le Bourg and Lints was used [7]. Groups of 10 or fewer flies were transferred to a clean, empty vial and given 18 seconds to climb 5 centimeters. The number of flies that successfully reach the 5 centimeter line were recorded. We compared the longevity of flies raised on food containing an equal weight/volume (0.0952 g/ml) of each of these sweeteners to control foods. Experimenters were blinded to treatments when assessing mortality and climbing ability. The exception was corn syrup, as it is not a white solid and can therefore be texturally discerned. This procedure was repeated with foods containing brilliant blue FCF (Fisher 50-727-25) in 0.05% weight/volume concentration [8], as well as erythritol, sucrose, Truvia, or PureVia as treatments. Flies were then examined daily for externally visible blue guts for 14 days. The number of dead flies and blue flies were scored daily.

Concentration Trials We pepared standard fly foods as previously described, then added treatments of 2 M, 1 M, 0.5 M and 0.1 M concentrations of erythritol and 0.5 M sucrose control. We placed 0 to 24 hour-old Drosophila on these foods and recorded mortality daily for 35 days as above.

Choice Experiments We prepared foods containing 2 M erythritol, 1 M erythritol and 1 M sucrose for paired presentations in open choice tests. In each treatment one food type contained 0.05% brilliant blue FCF (Fisher 50-727-25). The blue dye allowed visual confirmation of feeding on the dyed food in the pair. We presented the flies with access to two food choices by using a modified cotton stopper with approximately a 1.5 centimeter diameter hole to connect each pair of food tubes. We set up three choice trial groups: the first was between blue 1 M erythritol and non-blue 1 M erythritol foods (blue guts would confirm the blue dye did not completely inhibit feeding and confirm erythritol was being consumed), the second was between blue 1 M erythritol and non-blue 1 M sucrose foods (blue guts would confirm confirm erythritol was being consumed in the presence of sucrose), and the third was a choice between blue 1 M sucrose and non-blue 1 M sucrose, as a negative control (blue guts would confirm the blue dye did not inhibit feeding). The final choice treatment was between blue 2 M erythritol food and non-blue 1 M sucrose food (this treatment provides a comparison with the 1 M erytritol/1 M erythritol treatment as a test of dilution of toxicity by alternative food sources; blue guts would confirm confirm erythritol was being consumed in the presence of sucrose). We recorded number of flies with visible blue gut contents and mortality daily for 30 days.

CAFE experiments CAFE experiments were performed as described in [9]. Briefly, flies were held in vials with a cheesecloth bottom over water to promote high humidity; the vials were plugged with cotton and liquid food was presented using microcapillaries. Ten Canton S flies aged 0–12 hours old were used per vial. We used one vial of males and another of females for earch treatment. The experiments were conducted in a room that was kept at 25°C and 50–60% humidity. We made solutions of 5% w/v sucrose and erythritol in water, with 0.05% brilliant blue FCF dye added (Fisher 50-727-25). Each treatment solution was loaded in a 5 µl calibrated glass microcapillary tube (VWR International 53432-706) and inserted through the cotton plug on the vial such that the bottom of each capillary was accessible to the flies. We simultaneously held one control capillary of each solution in a similar CAFE apparatus without flies to quantify liquid loss to evaporation. Fly consumption was documented by capturing digital images (JPEG format, 4000X3000 pixel resolution) of the setup at one-hour intervals for six hours. We estimated the amount of liquid consumed from each pipet over time after subtraction of liquid lost from the corresponding evaporation control pipet over the equivalent amount of time. Estimates of liquid volume were taken using the ruler tool in Image J version 1.47v (NIH- http://imagej.nih.gov/ij/). We measured the distances (in pixels) from the bottom capillary tip to the 5 µl reference line, and from the bottom capillary tip to the meniscus of the dyed liquid in the capillary, in each photograph. We converted the liquid length measurement to volume relative to the length measurement for the 5 ul reference line.