Training Scent: Targets: Training Scent: Targets: Apis Mellifera Olfactory Discrimination in Benzene Benzene DNT Toluene Toluene DNT Control Control Nitrobenzene Benzene Toluene 2, 4, Dinitrotoluene Chemicals with Similar Molecular Structures as 2,4,Dinitrotoluene Blank Blank (DNT) Nitrobenzene 4,Nitrotoluene Nitrobenzene DNT 4,Nitrotoluene DNT Control Control Blank Blank Vernon Grant & Adam Jones University of Montana, Project TRAIN Undergraduate Research Fellow 2,Nitrotoluene DNT 2,Nitrotoluene DNT DNT Control 2, Nitrotoluene 4, Nitrotoluene 2,4,6, Trinitrotoluene 2, Nitrobutane Control Blank (TNT) Blank Toluene, or Benzene, or 2,Nitrotoluene, or Figure 1. Chemical structures of some of the training Abstract *All targets positions were randomly 4,Nitrotoluene scents tested. selected *Nitrobenzene was not tested with DNT This experiment was a behavioral study of honeybee Apis mellifera sensory Introduction discrimination. We wanted to test the specificity of the honeybee’s olfactory receptors Figure 2. Break down of each training scent and the targets that triggered when exposed to 2,4, Dinitrotoluene (DNT). It was thought that this research were tested in the field with that particular scent. This experiment was a behavioral study of the honeybee Apis may also reveal an odor mimic for DNT that can be used when conditioning honeybees mellifera’s sensory discrimination. We wanted to test the specificity of the to detect explosives such as TNT. We hypothesized that a particular region of the honeybee’s olfactory receptors that are triggered when exposed to 2,4, Discussion and Conclusions molecular structure of DNT is binding to a specific chemoreceptor on the honeybee Dinitrotoluene (DNT), a contaminant of 2, 4, 6 Trinitrotoluene (TNT). It was antennae. A randomized cafeteria trial was devised in which the honeybees were Our research has provided significant insight into the use of thought that this research might also reveal an odor mimic for DNT that can trained to chemicals with a molecular structure similar to DNT: Benzene, Toluene, honeybees as biosensors for explosives detection. Our data suggests that be used in the training of honeybees to detect explosives such as TNT. We Nitrobenzene, 2,Nitrotoluene, and 4,Nitrotoluene (Figure 2). Nonparametric statistics Apis Mellifera generalizes between certain chemicals with sufficiently similar hypothesized that a particular region of the molecular structure of DNT is were used to analyze our data, which suggested that Apis Mellifera generalizes molecular structures, when conditioned to those chemicals. However, it is binding to a specific chemoreceptor on the honeybee antennae. Similar between certain chemicals with sufficiently similar molecular structures, when not clear what region of the molecule the honeybee the receptor is specific research has identified common odor signatures within explosives for use in conditioned to those chemicals. to, nor do we know the specific receptor that is triggered. We do know developing analogous odor mimics for use in canine detection (Harper et al., chemicals that are potentially generalized by the honeybee have either a 2005). methyl, or nitrate group attached to the aromatic Benzene ring. Currently, biosensors such as dogs, rats, bees, and certain plants We were limited in this study by the ever-changing weather, and at the surpass instrumental sensors in detection of trace explosives (D.S. Moore, Results mercy of the many forest fires in the nearby Bitteroot Valley. Moving from the 2004). It has been demonstrated that bees, for example, are capable of field to the laboratory using the Proboscis Extension Response System detecting explosive odorants at concentrations below most instruments and (PERS) may help to eliminate some of the uncontrollable variables such as Each day, week, morning and afternoon had unusual highs and lows on each at levels comparable to dogs (Bromenshenk, 2003). weather and temperature. The PERS system also has the ability to expand target. A randomized cafeteria trial was devised in which the honeybees were on the variety of surrogates tested, such as 2,Nitrobutane, which has a trained to chemicals with molecular structures similar to that of DNT: Figure 5 shows the average “hits” and standard deviations for each target methyl and nitrate group but is not attached to the aromatic Benzene ring. Benzene, Toluene, Nitrobenzene, 2-Nitrotoluene, and 4-Nitrotoluene (Figure when the hive was trained to each particular scent. Future research may provide more insight into this phenomenon by 2). These chemicals were thought to be similar enough to DNT to potentially choosing DNT as the exclusive conditioned stimulus (CS). More hives need be used as an odor mimic. This research’s primary goal is to identify odor Figure 6 illustrates the results of the reverse trial when DNT became the to be replicated using DNT as the CS with the same targets: 2,Nitrotoluene, mimics for use as training aids for honeybee conditioning. conditioned stimulus. 4,Nitrotoluene, and Nitrobenzene. Due to the small population size (7 hives), replicating additional hives may justify our work with refutable Materials and Methods 2,Nitrotoluene and 4,Nitrotoluene targets appear to have the greatest evidence. success when the hive is trained to DNT. •The hives were conditioned to associate each chemical scent with a reward. Rep or t •Using GPS, target positions were measured 100 feet from the hive and 50 feet BeeCount Target Sc ent Training Sc ent Mean N Std. Deviation from one another (Figure 3). 100ft Hive Toluene Toluene DNT 35.00 6.00 13 1 70.750 . 10 •Electronic feeders (Figure 4) were positioned approximately 10 meters behind Benz ene Total Benz ene 32.93 59.15 14 13 68.414 90.356 100ft DNT each target position. 50ft Total 17.00 56.14 1 14 87.539 . 100ft •The honeybees were trained by applying a training scent to a felt ring that was 100ft DNT Toluene 5.08 13 6.861 Mean Rank of Bee Count by Date Benz ene 17.46 13 17.558 8 DNT 31.75 4 60.191 placed around the perimeter of each electronic feeder (Figure 4). 50ft Nitrobenz ene 4.91 11 7.648 •Fifteen milliliters of Benzene, Toluene, Nitrobenzene, or 2,Nitrotoluene were 2-Nitrotoluene 19.00 9 49.930 4-Nitrotoluene 7.33 3 4.726 50ft Total 12.58 53 27.498 applied to each ring during training. Nitrobenz ene Nitrobenz ene Total 27.09 11 29.643 6 27.09 11 29.643 •The DNT training rings were created using 20g of DNT, mixed with 15mL Acetone, 2-Nitrotoluene DNT 2-Nitrotoluene 44.00 56.00 2 9 49.497 91.010 and then applied to a felt ring. 4-Nitrotoluene Total DNT 53.82 6.00 11 2 83.035 2.828 •Ten grams of 4,Nitrotoluene was also mixed with 15mL Acetone, before KEY 4-Nitrotoluene Total 156.33 96.20 3 5 205.855 167.243 4 application to a felt ring. Control Ring Toluene Benz ene 5.08 41.77 13 13 8.341 53.165 Randomly selected target positions •Each field target was a strip of cotton cording (approximately 6 inches). They DNT Nitrobenz ene 5.80 3.82 5 11 9.094 4.238 were prepared the same way as the training scents (felt ring). Electronic feeder positions 2-Nitrotoluene 4-Nitrotoluene 2.67 1.33 9 3 2.062 1.528 2 •Careful handling and storage of the chemicals was necessary in order to prevent No Ring Total Toluene 13.11 1.08 54 13 30.540 1.498 cross contamination, as contamination is a critical issue in odor signature research Figure 3. The field test design. Each feeder Benz ene DNT 17.62 4.00 13 5 26.516 8.396 (Furton et. al, 2001). was moved each morning in order to Nitrobenz ene 2-Nitrotoluene .09 2.00 11 9 .302 4.272 0 encourage hive foraging. 4-Nitrotoluene Total .00 5.22 3 54 .000 14.785 Control Ring No Ring DNT 2-Nitrotoluene 4-Nitrotoluene Toluene Benzene Total Toluene 11.56 52 37.353 Target Scent (DNT Training) Procedure Benz ene DNT 34.00 14.95 52 20 56.025 30.796 Nitrobenz ene 8.98 44 18.366 Error Bars: +/- 1 SD 2-Nitrotoluene 19.92 36 54.427 4-Nitrotoluene 41.25 12 111.954 Each morning all the feeders (3 in all) in the field were moved Total 19.79 216 49.672 approximately 10 meters to the left or right. At approximately 10:00 A.M., the Figure 5. This chart breaks down each training Figure 6. This graph shows honeybee hits on each electronic feeders were turned off and the training scent for that day was target when the hive was trained to DNT. Bee count reapplied. The honeybees were allowed to drain each feeder of it’s sugar solution. scent, and the mean number of hits on each target tested with that scent. and date are nested together and ranked each target hit. The target positions were then randomly selected and set in place. The array of targets consisted of the conditioned scent (target), DNT (positive control), a blank spot (bare ground), and a control (a strip of cotton Literature Cited Bromenshenk, J. J., Henderson, C. B., and Smith, G. C. (2003). Biological Systems, Acknowledgments cording without a scent) as illustrated in Figure 2. paper II, pp. 273-283. Alternatives for landmine detection. Santa Monica, I would like to thank Dr. Colin Henderson, Dr. Jerry Bromenshenk, Dr. Penny Kukuk, Pat After everything was set in place, we started the cameras and vacated the California: Rank Corp. Furton, K., Myers, L. (2001). The scientific foundation and efficacy of the use of canines Hurley, Project TRAIN, & Bridges to the Baccalaureate. I would also like to thank the Bee test site. The cameras collected data for 5 minutes. After the 5 minute testing as chemical detectors for explosives. Talanta, 54, 487-500. Alert Technology Team, Scott Debnam, Tyler Running Crane, and the unsung Co-author period, cameras and targets were gathered and the targets were placed in the Harper, R.J., Almirall, J.R., and Furtokn, K.G. (2005). Identification of dominant odor freezer and the field was cleared of all targets. Once everything was cleared, the Figure 4. The electronic feeder and chemicals emanating from explosives for use in developing optimal training Adam Jones. Last but certainly not least, I would like to thank my family who support me aid combinations and mimics for canine detection. Forensic Chem. Anal., 67(2), feeders were turned back on to maintain conditioning. Video data was observed felt ring used for training. 313-327. in all my academic endeavors. Moore, D.S. (2004). Instrumentation for trace detection of high explosives. Rev. Sci. and honeybees were counted at each target position. Instrum. 75(8), 2499-2512.