Imidacloprid is a widely used pesticide. It is a derivative

1. The effects of imidacloprid on chicken embryo developmentAmber VanceDr. Katrina Lustofin, Advisor Introduction Results Conclusions Imidacloprid is a widely used pesticide. It is a derivative of nicotine and is similar in structure. Imidacolprid interacts with the acetylcholine receptor, which Is widely conserved across species (Seifert and Stollberg, 2004, 18). Imidacloprid reacts with the acetylcholine receptor of insects much more readily than with the structurally distinct vertebrate receptor, making it a safer pesticide (Seifert and Stollberg, 2004, 18). The acetylcholine receptor is located at the intersection of nerve and muscle cells and propagates signal transfer (Arias et al., 2011, 5263). When nicotine interacts with this receptor, it actively competes with acetylcholine and alters the function of the receptor so that less sodium flows into the cells which results in decreased embryonic movement (Ejaz and Woong, 2006, 107) and subsequent lower birth weight (Lambers and Clark, 1996, 115). Imidacloprid reacts with this receptor in an analogous fashion because of their structural similarities. A specific concern about imidacloprid is that it may cause similar developmental defects as the known teratogen nicotine (Figure 1). For developmental studies, chicken embryos are a model organism because they are inexpensive, easy to control with dosing, sensitive to toxins, and are vertebrates (Ejaz and Woong, 2006, 108). Hypothesis: Imidacloprid will adversely affect chicken embryo development in a way similar to nicotine. Survivorship of treated embryos was not significantly different from control (p=0.298). The hypothesis that imidacloprid would adversely affect chicken embryo development was supported by this experiment. Low doses of imidacloprid had significant adverse effects on embryo size, as shown by embryonic mass and bone lengths. A lower malformation rate was observed at this low dose. However, there was no significant effect on mortality or bone width at the 2.50% concentration. The fact that imidacloprid did not decrease survivorship is important because it means imidacloprid is not lethal, and that the embryonic development is able to be observed. Imidacloprid caused developmental delays, or overall smaller embryos (Figure 3, 4, 6). The effects of imidacloprid on bone length were overall statistically significant for embryos at all treatment levels. The malformations observed in the imidacloprid treatment group were similar to those observed in the nicotine treated embryos (curled toes). However, severe malformations (stumpy legs) were only observed in the nicotine group. The doses of imidacloprid (2.50%, 3.75%, and 5.00%) are much greater than treatments used commercially (Seifert and Stollberg, 2005, 21). Also, the discrepancy between trial 1 and 2 may indicate some inconclusiveness. There was a significant difference between the average mass (p=0.030) and bone lengths (p=0.030) of the embryos between trial 1 and trial 2. Because the embryos from each trial were different, it is difficult to draw conclusions based on the summed data. The difference between trials could be due to the eggs having been laid by different hens or because of the time of year the trials were conducted (October vs. March). Structure of imidacloprid 18 20 9 20 17 Mass was significantly impacted by treatment (p=0.003). Specifically, 2.5% imidacloprid differed from the control (p=0.001) and nicotine differed from the control (p=0.007). Other treatments were not significantly different from control. Length of the tibia (p = 0.001), metatarsus (p = 0.001), and phalanx (p<0.001) were all significantly affected by treatment. Figure 4 shows a trend which was present for all bone lengths: as the concentration of imida- cloprid increased, bone length increased. This trend was observed for measurements of the metatarsus, phalanx, and tibia. Nicotine treated embryos had significantly shorter bones compared to the control (p≤0.001 for all bone lengths). Bones of embryos dosed with 2.50% imidicloprid were also significantly shorter than the control (tibia p<0.001, metatarsus p=0.005, phalanx p<0.001). Width of all bones was not affected. Figure 1: Examples of malformations of embryos treated with nicotine Normal phalanges Curled phalanges Severely malformed phalanges Literature Cited Methods Test Groups White leghorn chicken embryos were obtained from OSU. There were 12 eggs in each treatment group. Two replicas were completed. Embryos were dosed on day 5 in the air space above the yolk, and specimens were harvested on day 19. Candling Eggs were held over a light to determine the location of the embryo and air space. Movement, vasculature, bacteria presence, and eye structure were also observed. Dosing and Harvesting Eggs were injected with the control solution and concentrations of imidacloprid via a clean diabetic syringe. The hole made was covered with paraffin wax. Embryos were harvested on day 19 and weighed. Embryos were skinned, eviscerated, and stored in 95% ethanol for 3 days. Staining and Measuring Embryonic cartilage was stained with alcian blue and bone was stained with alizarin red (MacLeod, 1980). Measurements of the length and width of the right tibia, metatarsus, and phalanx were taken using Luminera software. Statistics Linear regression and ANOVA statistical tests were run on SPSS software and used for analysis of all resulting data. Arias HR, Gu RX, Feuerbach D, Guo BB, Ye Y, Wei DQ. 2011. Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor. Biochemistry 50: 5263-5278. Ejaz S, Woong LC. 2006. Diminished embryonic movements of developing embryo by direct exposure of sidestream whole smoke solutions. Archives of Toxicology 80: 107-114. Lambers DS, Clark KE. 1996. The maternal and fetal physiological effects of nicotine. Seminars in Perinatology 2(2): 115-126. MacLeod, MJ. 1980. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red. Teratology. 22:299-301. Seifert J, Stollberg J. 2005. Antagonism of a neonicotinoid insecticide imidacloprid at neuromuscular receptors. Environmental Toxicology and Pharmacology 20: 18-21. Treatment affected overall malformation rate (p=0.002). Embryos in the nicotine group were significantly malformed compared to the control (p<0.001). Embryos in the 2.50% imidacloprid group were not malformed significantly when compared to the control (p=0.107). Acknowledgements Special thanks to my advisor, Dr. Lustofin, for her support and advice. Acknowledgement to the MC Biology Department for generous funding of the project. Appreciation goes out to Dr. McShaffrey for his feedback and leadership of Capstone Class 2012. Thanks to Professor Jarrell for her help in securing supplies. Thanks to the senior Capstone Class for your encouragement. Also, thanks to the Writing Center for their help in improving each draft. Mass and length of tibia demonstrated a significant linear relationship (p=0.003) which was conserved across test groups. Intuitively, this relationship makes sense—larger embryos have larger bones.