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Effects of SWNT feeding on Drosophila growth, development, and longevity R. Bruce R. Michelle and Mallory E. (1) Department of Biochemistry and Cell Biology, Rice University (2) Department of Chemistry and Center for Nanoscale Science and Technology, Rice University (3) Trinity University ABSTRACT Previous research aiming at addressing biomedical applications of SWNT examined the viability of adult Drosophila after feeding SWNT during the larval stage. Although viability was unaffected, these experiments suggested that larval SWNT feeding produced an increase in longevity and a slight developmental delay. Current research has repeated these previous experiments with additional controls, as well as investigating the effects of both larval and adult SWNT feeding and adult only SWNT feeding on longevity. In order to rule out increased longevity due to the effects of caloric restriction, an additional graphite control was added to better mimic the palatability of the experimental SWNT food. Results indicate that SWNT feeding may produce a slight delay in the development of Drosophila although SWNT feeding had no effect on larval growth and adult weight at eclosion. The longevity experiments are still ongoing. Kathleen M. 1, Beckingham 2, Weisman 1, Reith 3. Harden EXPERIMENTAL PROTOCOL General protocol for all experiments given below. note: the SWNT concentrations used here were as high as could be achieved in aqueous solution and represent massive exposure 1) collection of larvae: standard egg lay plates used (see a.), eggs collected, counted, and transferred to larval food which consisted of a stiff paste of bakers’ yeast suspended in SWNT solution or control 2) transfer of larvae: larvae were transferred to food, 1 larvae per 5 mg of food, food + larvae were on 15 mL agar pads in bottles (see b.), larvae allowed to grow and pupate there 3) adult collection: as emerged as adults, collected twice daily, sorted by sex and weighed in batches of 10, virgin adults set up in batches of 10 in standard vials (see c.) with 7 mL of agar, sucrose, and 50 mg yeast +/- SWNT, transferred twice a week to new vials and scored daily for survival QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 2. Does SWNT feeding affect developmental rate of Drosophila? Figures 2 a-d below depict data from four experiments concerning the rate at which the flies emerged from their pupal cases (eclosion rate). The x-axis plots hours since the first eclosion and the y-axis plots the percentage of the total flies that have emerged at a given time period. A developmental delay is observed when a space can be seen between the red line (control-fed) and the black line (SWNT-fed), displaying a period of time in which more control flies emerged than SWNT flies. A slight developmental delay can be seen in a and c. 120 120 a. egg lay plate a. 100 100 b. % total flies emerged % total flies emerged 80 80 60 60 INTRODUCTION Single-walled carbon nanotubes (SWNT) are a newly discovered form of carbon that exhibit unique properties including high strength and fluorescence in the near-infrared (near-IR). Included in the myriad of ongoing SWNT studies, many applications for SWNT in biomedical science are being pursued, including targeting of biological molecules and imaging in biological systems. Materials science applications involve the use of SWNT in nanoscale electronic devices and high performance components. However, these growing applications bring with them increased exposure of the biosphere to SWNT and it is thus important to determine SWNT effects in biological systems. These experiments aim to address the biological effects of SWNT using the model organism, Drosophila. The experimental approach was to feed food containing high levels of SWNT and to address the effects of feeding on various aspects of viability. Additionally, we asked whether SWNT fed to animals entered the body through gut. We also explored the possibility of using near-IR to detect SWNT in the whole organism. This endeavor was successful and we were able to detect groups of and individual SWNT in the larval and adult fly tissue. 40 40 b. larval bottle c. adult vial 20 20 0 0 120 8 24 32 48 56 72 80 96 104 120 128 144 0 0 8 24 32 48 56 72 80 96 104 120 128 144 Hours 120 Hours RESULTS % total flies emerged c. 100 100 80 d. % total flies emerged 80 This general design allowed us to compare the following parameters for control and SWNT fed flies: 1) viability and growth 2) developmental rate 4) Longevity *note: the results below are from combinations of the experiments and one preliminary experiment that tested survival to adulthood 60 60 40 40 20 20 0 0 16 24 64 72 88 96 112 120 136 0 36 52 60 100 108 124 132 148 156 172 Hours Hours As can be seen from the figures above, SWNT feeding has little or no effect on rate of development. 1. Does SWNT feeding affect viability or growth of Drosophila? A. B. DROSOPHILA LIFE CYCLE Important Notes on Drosophila Life Cycle. • • • embryo develops in egg case - no net growth larvae hatches in 24 hours from egg case and commences feeding larval stages - feeding stage, animal grows 200-fold 3. Does SWNT feeding affect longevity of Drosophila? Quic kTime™ and a TIFF (Uncompres sed) decompress or are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ an d a TIFF (Uncompressed) decompressor are need ed to see this p icture. • pupal stage - animal is inert, nonfeeding and undergoes reorganization of tissue to give the adult body • adult stage, no growth but feeding and reproductive activity • *adult weight at emergence directly reflects larval growth Figure 1A. depicts a comparison of survival rates to the pupal and adult stages for Drosophila fed exclusively on SWNT yeast paste (9 ppm) or on control yeast paste throughout the entire larval period. Figure 1B. shows the average weight of newly eclosed flies fed for the entire larval period exclusively on SWNT yeast paste (12 or 24 ppm) or on control yeast paste. Flies were weighed in batches of 10 males or 10 females. Figure 3 shows the adult survival times for adult males fed control food or food containing SWNT or graphite. Accidental deaths (censored deaths) have been removed from the data. Note that SWNT feeding causes a slight decrease in longevity, comparable to that seen with graphite. Longevity data analysis is completed on experiment 4 above, indicating mild effects caused by SWNT exposure although no worse than effects caused by graphite. EXPERIMENTAL DESIGN Four feeding experiments set up to test the following: Experiment 1: designed to test the effects of SWNT feeding in larval stage only • 24 ppm SWNT solution used for preparation of larval food only, adult control food used for adult stages Experiment 2: designed to test the effects of SWNT feeding in larval and adult stages •12 ppm SWNT solution used for preparation of larval and adult food, half control and half SWNT food used for adult stages Experiment 3: designed to test the effects of SWNT feeding in adult stage only •12 ppm SWNT solution used for preparation of adult food only, control food used for larval stages Experiment 4: designed to test the effects of SWNT feeding in larval stage only with additional graphite control •12 ppm SWNT solution and 12 ppm graphite solution used for preparation of larval food only, adult control food used for adult stages CONCLUSIONS 1. Experiments indicate that even with massive SWNT exposure in food, most parameters show only minor or no effect on organism. 2. Viability remains unchanged with SWNT exposure. 3. Development remains unchanged with SWNT exposure. As shown in the figures above, feeding SWNT throughout the larval stage has no effect on growth of Drosophila. Additionally, survival to the pupal stage and to the adult stage are unaffected by SWNT feeding. ACKNOWLEDGEMENTS This research has been supported by the the Alliance for NanoHealth (NASA NNJ05HE75A). The authors are also grateful to Scott Pletcher for his help in the analysis of longevity data and Valerie Moore for her guidance and support. REFERENCES Tsyboulski, D. A., Bachilo, S. M. & Weisman, R. B. Versatile visualization of individual singlewalled carbon nanotubes with near-infrared fluorescence microscopy. Nano Lett. 5, 975-979 (2005).
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