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Apis Mellifera Olfactory Discrim

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					                                                                                                                                                                                                                                                                   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.

				
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