3.1. Agrochemicals Considered Harmless to Bees
Pymetrozine (Plenum WG-50
®). This agrochemical is a systemic pesticide of the pyridine-azomethin family and is considered harmless to bees. In this experiment, a non-overlap procedure was used with a CS duration of 3 s, a US duration of 2 s, and an intertrial interval of 10 min. Forager honey bees
Apis mellifera: Hybrid var. Buckfast were used. The bees were collected in glass vials at the laboratory feeder approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then placed in a harness, fed 1.8 M sucrose, and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally and mixed with sucrose to make it palatable. Two doses were used. In one, the recommend field dose was used (5 μL of 0.3 g L
−1, 0.16 g L
−1 of pymetrozine) and in the other, 100× the recommended field dose was used (5 μL of 30 g L
−1, 0.14 g L
−1 of pymetrozine) [
14].
Experiment 1 investigated the effects of pymetrozine on simple Pavlovian conditioning where honey bees were trained to associate a CS with a US. The control group without the chemical learned well, with more than 70% of bees responding (
Table 1, item 3). The model fit the learning data well; R squared is about 0.97, and ability to learn (B4) is about 73% (
Table 2, item 3). A pretreatment with a field dose of pymetrozine decreased the learning rate (B2) three times in comparison with the control group (
Table 3, item 2); other coefficients did not differ. A pretreatment with 100× the field dose of pymetrozine practically prevented learning, having lowered the number of conditioned responses (CR) to 15% with a further decrease on trials 11 and 12, so that the fit was much worse in comparison with the control group as well as the group pretreated with a field dose (
Table 2, item 2). Ability to learn was significantly lower in comparison with the control group (
Table 3, item 3) as well as with the field dose group (
Table 3, item 1).
Table 2.
Learning curve modeling. The data from
Table 1 are analyzed below and coefficients are listed for B2, B3 and B4. The source listed is the original source of the data, and can be found in the references.
Table 2.
Learning curve modeling. The data from Table 1 are analyzed below and coefficients are listed for B2, B3 and B4. The source listed is the original source of the data, and can be found in the references.
No | Source | Experiment/chemicals | Model’s coefficients (with standard error) | R squared |
---|
B2 | B3 | B4 |
---|
1. | [14] | a pretreatment: a field dose of pymetrozine | 0.30 ± 0.06 | 0.0 ± 11.0 | 63.0 ± 4.4 | 0.90 |
2. | [14] | a pretreatment: 100× the field dose of pymetrozine | 0.35 ± 0.31 | 2.3 ± 3.6 | 14.0 ± 2.8 | 0.44 |
3. | [14] | a pretreatment: sucrose only | 0.97 ± 0.14 | 6.0 ± 3.9 | 72.7 ± 1.4 | 0.97 |
4. | [14] | a pretreatment: a field dose of pymetrozine | 0.34 ± 0.15 | 5.9 ± 9.4 | 66.1 ± 7.6 | 0.80 |
5. | [14] | a pretreatment: 100× the field dose of pymetrozine | The data are not fitted with the model. |
6. | [14] | control, sucrose only | 0.70 ± 0.09 | 1.0 ± 4.8 | 85.0 ± 2.0 | 0.98 |
7. | [9] | a US: 6.25% Bioganic | 1.22 ± 0.81 | 10.2 ± 2.3 | 1.0 ± 0.8 | 0.70 |
8. | [9] | a US: 1.56% Bioganic | 2.73 ± 10.75 | 9.9 ± 12.9 | 29.2 ± 4.1 | 0.19 |
9. | [9] | a US: sucrose only | 0.67 ± 0.12 | 0.0 ± 12 | 64.0 ± 2.2 | 0.97 |
10. | [9] | a US: 6.25% Bioganic | 0.25 ± 0.19 | 9.1 ± 2.2 | −1.2 ± 2.7 | 0.41 |
11. | [9] | a US: 1.56% Bioganic | The data are not fitted with the model. |
12. | [9] | a US: sucrose only | 1.45 ± 0.38 | 10.1 ± 5.8 | 75.0 ± 2.0 | 0.94 |
13. | [9] | a CS: citronella odor | 0.60 ± 0.08 | 0.0 ± 10.4 | 76.9 ± 2.1 | 0.96 |
14. | [9] | a CS: 6.25% Bioganic odor | 0.86 ± 0.12 | 29.8 ± 3.0 | 83.9 ± 1.2 | 0.98 |
15. | [15] | a CS+: pignut | 1.10 ± 0.16 | 9.1 ± 4.8 | 92.1 ± 1.7 | 1.00 |
16. | [15] | a CS+: sweet fennel | 2.61 ± 0.94 | 10.0 ± 4.7 | 80.9 ± 1.5 | 0.97 |
17. | [16] | a pretreatment: 10 μL dicofol | 0.83 ± 0.15 | 4.0 ± 3.8 | 56.0 ± 1.5 | 0.95 |
18. | [16] | a pretreatment: sucrose | 1.64 ± 0.50 | 20.1 ± 5.7 | 80.0 ± 1.9 | 0.99 |
19. | [16] | a pretreatment: 10 μL dicofol | 1.12 ± 0.26 | 10.1 ± 4.9 | 75.3 ± 3.3 | 0.98 |
20. | [16] | a pretreatment: sucrose | 0.44 ± 0.22 | 11.6 ± 7.8 | 90.2 ± 16.6 | 0.95 |
21. | [10] | a CS: butyric acid | 0.59 ± 0.09 | 2.0 ± 5.3 | 87.6 ± 2.4 | 0.96 |
22. | [10] | a CS: DEET | 1.99 ± 0.41 | 14.6 ± 3.2 | 74.1 ± 1.1 | 0.99 |
23. | [10] | a CS: cinnamon | 0.60 ± 0.06 | 10.1 ± 3.8 | 95.7 ± 1.7 | 0.99 |
24. | [17] | a CS: citronella | 0.79 ± 0.15 | 4.7 ± 4.4 | 58.6 ± 1.7 | 0.96 |
25. | [17] | a CS: cinnamon | 0.52 ± 0.12 | 4.1 ± 5.5 | 63.1 ± 2.8 | 0.91 |
26. | [18] | a pretreatment: fluvalinate | 0.41 ± 0.09 | 39.4 ± 1.8 | 73.5 ± 3.0 | 0.98 |
27. | [18] | a pretreatment: acetone | 0.71 ± 0.13 | 62.5 ± 1.7 | 91.8 ± 1.4 | 0.98 |
28. | [18] | a pretreatment: flucythrinate | 0.39 ± 0.30 | 24.6 ± 3.7 | 46.7 ± 6.8 | 0.84 |
29. | [18] | a pretreatment: cyfluthrin | 0.41 ± 0.12 | 2.2 ± 4.4 | 68.6 ± 7.4 | 0.84 |
30. | [18] | a pretreatment: cypermethrin | 0.45 ± 0.26 | 32.4 ± 4.8 | 66.2 ± 7.1 | 0.88 |
31. | [18] | a pretreatment: permethrin | 0.64 ± 0.43 | 40.8 ± 5.1 | 65.0 ± 4.5 | 0.81 |
32. | [18] | a pretreatment: fenvalerate | 0.42 ± 0.16 | 26.6 ± 3.9 | 70.4 ± 6.2 | 0.95 |
33. | [19] | a pretreatment: water | 0.37 ± 0.13 | −0.1 ± 3.1 | 70.0 ± 13.1 | 0.99 |
34. | [19] | a pretreatment: acetone | 0.46 ± 0.25 | 0.0 ± 6.4 | 74.1 ± 18.8 | 0.97 |
35. | [19] | a pretreatment: 0.01% coumaphos | 0.51 ± 0.50 | 0.0 ± 15.2 | 77.5 ± 34.2 | 0.89 |
36. | [19] | a pretreatment: 0.1% coumaphos | 0.45 ± 0.1 | 0.3 ± 2.8 | 82.0 ± 8.9 | 0.99 |
37. | [19] | a pretreatment: 10% coumaphos | 0.51 ± 0.43 | 0.0 ± 9.5 | 63.9 ± 23.5 | 0.93 |
38. | [19] | a pretreatment: water | 0.53 ± 0.35 | 0.3 ± 7.7 | 63.4 ± 17.6 | 0.95 |
39. | [19] | a pretreatment: acetone | 0.37 ± 0.17 | −0.6 ± 4.3 | 78.1 ± 19.0 | 0.98 |
40. | [19] | a pretreatment: 0.005% diazinon | 0.52 ± 0.36 | 0.0 ± 8.9 | 71.4 ± 21.6 | 0.94 |
41. | [19] | a pretreatment: 0.01% diazinon | 0.52 ± 0.23 | 0.3 ± 4.5 | 57.3 ± 10.8 | 0.98 |
42. | [19] | a pretreatment: 0.025% diazinon | 0.84 ± 0.12 | −0.1 ± 3 | 83.1 ± 3.6 | 1.00 |
43. | [19] | a pretreatment: hexane only | 0.57 ± 0.27 | 0.2 ± 6.9 | 73.4 ± 14.3 | 0.97 |
44. | [19] | a pretreatment: 0.005% diazinon | 0.61 ± 0.21 | 5.0 ± 1.9 | 31.4 ± 3.5 | 0.98 |
45. | [19] | a pretreatment: 0.07% coumaphos | 1.42 ± 0.15 | −0.01 ± 1.9 | 65.8 ± 1.4 | 1.00 |
46. | [20] | a pretreatment: sucrose | 0.21 ± 0.08 | −0.7 ± 7.2 | 79.0 ± 12.1 | 0.90 |
47. | [20] | a pretreatment: endosulfan | 1.26 ± 4.68 | −0.6 ± 5.8 | 3.7 ± 2.0 | 0.05 |
48. | [20] | a pretreatment: decis | 0.24 ± 0.09 | 0.0 ± 12.0 | 52.0 ± 7.8 | 0.89 |
49. | [20] | a pretreatment: baytroid | There is no data because learning did not occur. |
50. | [20] | a pretreatment: sevin | There is no data because learning did not occur. |
51. | [20] | a CS: hexanal | 0.19 ± 0.09 | 5.3 ± 7.0 | 78.2 ± 14 | 0.91 |
52. | [20] | a CS: endosulfan | 0.22 ± 0.05 | 1.7 ± 4.1 | 73.2 ± 6.3 | 0.96 |
53. | [20] | a CS: decis | 0.18 ± 0.09 | 10 ± 6.1 | 72.5 ± 14.6 | 0.91 |
54. | [20] | a CS: baytroid | 0.18 ± 0.05 | −0.6 ± 4.0 | 74.8 ± 9.3 | 0.96 |
55. | [20] | a CS: sevin | 0.17 ± 0.06 | 0.0 ± 12.5 | 85.8 ± 14.6 | 0.93 |
56. | [20] | a US: sucrose | 0.16 ± 0.05 | 0.0 ± 4.8 | 85.2 ± 14.0 | 0.95 |
57. | [20] | a US: endosulfan | 1.18 ± 1.61 | 2.9 ± 10.3 | 35.9 ± 6.0 | 0.28 |
58. | [20] | a US: decis | 0.18 ± 0.07 | 1.4 ± 4.6 | 64.5 ± 10.1 | 0.93 |
59. | [20] | a US: baytroid | 0.26 ± 1.03 | 10.0 ± 2.5 | 3.9 ± 13.6 | 0.45 |
60. | [20] | a US: sevin | 1.91 ± 3.11 | 5.0 ± 3.4 | 14.1 ± 2.3 | 0.70 |
61. | [11] | a pretreatment: 0 μg tebufenozide | 1.89 ± 0.45 | 12.1 ± 5.2 | 92.4 ± 1.7 | 0.96 |
62. | [11] | a pretreatment: 16 μg tebufenozide | 1.05 ± 0.08 | 3.5 ± 2.7 | 90.1 ± 1.0 | 0.99 |
63. | [11] | a pretreatment: 24 μg tebufenozide | 0.36 ± 0.12 | 11.0 ± 6.9 | 70.6 ± 5.1 | 0.88 |
64. | [11] | a pretreatment: 32 μg tebufenozide | 0.25 ± 0.12 | 9.0 ± 8.0 | 71.5 ± 10.1 | 0.84 |
65. | [11] | a pretreatment: 69.4 μg tebufenozide | 0.35 ± 0.07 | 26.0 ± 5.0 | 94.9 ± 3.8 | 0.95 |
66. | [11] | a pretreatment: 131 μg tebufenozide | 0.71 ± 0.23 | 8.6 ± 6.8 | 57.1 ± 2.8 | 0.84 |
67. | [11] | a US: 0 μg tebufenozide | 1.04 ± 0.13 | 3.0 ± 4.1 | 85.5 ± 1.5 | 0.98 |
68. | [11] | a US: 16 μg tebufenozide | 1.12 ± 0.3 | −0.7 ± 6.8 | 65.5 ± 2.4 | 0.91 |
69. | [11] | a US: 24 μg tebufenozide | 0.66 ± 0.1 | 3.0 ± 3.6 | 61.2 ± 1.6 | 0.96 |
70. | [11] | a US: 32 μg tebufenozide | 0.49 ± 0.16 | 6.1 ± 8.7 | 70.4 ± 4.6 | 0.85 |
71. | [11] | a US: 69.4 μg tebufenozide | 0.510.1 | 1.8 ± 4.8 | 61.8 ± 2.4 | 0.94 |
72. | [11] | a US: 131 μg tebufenozide | 0.64 ± 0.13 | 7.9 ± 5.3 | 71.0 ± 2.3 | 0.94 |
73. | [11] | a pretreatment: 0 μg diflubenzuron | 1.3 ± 0.19 | 0.0 ± 4.3 | 82.4 ± 1.5 | 0.97 |
74. | [11] | a pretreatment: 3.4 μg diflubenzuron | 0.72 ± 0.15 | 6.6 ± 5.3 | 65.2 ± 2.2 | 0.93 |
75. | [11] | a pretreatment: 8.5 μg diflubenzuron | 0.34 ± 0.13 | 16.8 ± 7.5 | 74.4 ± 6.1 | 0.85 |
76. | [11] | a pretreatment: 16 μg diflubenzuron | 0.67 ± 0.19 | 16.2 ± 7.4 | 78.6 ± 3.2 | 0.88 |
77. | [11] | a pretreatment: 32 μg diflubenzuron | 1.03 ± 0.4 | 12.1 ± 7.3 | 59.5 ± 2.6 | 0.81 |
78. | [11] | a pretreatment: 69.4 μg diflubenzuron | 0.31 ± 0.1 | 18.4 ± 3.8 | 55.4 ± 3.5 | 0.90 |
79. | [11] | a US: 0 μg diflubenzuron | 1.35 ± 0.43 | 12.3 ± 5.2 | 58.6 ± 1.8 | 0.89 |
80. | [11] | a US: 3.4 μg diflubenzuron | 2.06 ± 4.15 | 20.2 ± 4.6 | 29.3 ± 1.5 | 0.28 |
81. | [11] | a US: 8.5 μg diflubenzuron | 1.99 ± 2.17 | 3.7 ± 8.2 | 32.3 ± 2.7 | 0.55 |
82. | [11] | a US: 16 μg diflubenzuron | 0.16 ± 0.28 | 21.9 ± 6.3 | 43.7 ± 17.1 | 0.43 |
83. | [11] | a US: 32 μg diflubenzuron | 1.69 ± 2.7 | 7.9 ± 12.6 | 33.7 ± 4.2 | 0.30 |
84. | [11] | a US: 69.4 μg diflubenzuron | The data are not fitted with the model. |
85. | [11] | a pretreatment: 0 μg tebufenozide | 1.46 ± 0.31 | 9.4 ± 4.9 | 78.5 ± 1.7 | 0.95 |
86. | [11] | a pretreatment: 16 μg tebufenozide | 0.77 ± 0.2 | 6.8 ± 7.2 | 71.2 ± 2.9 | 0.89 |
87. | [11] | a pretreatment: 24 μg tebufenozide | 0.34 ± 0.06 | 9.7 ± 4.2 | 81.0 ± 3.3 | 0.97 |
88. | [11] | a pretreatment: 32 μg tebufenozide | 0.36 ± 0.15 | 5.7 ± 8.3 | 60.2 ± 6.2 | 0.81 |
89. | [11] | a pretreatment: 69.4 μg tebufenozide | 0.91 ± 0.18 | 10.1 ± 4.6 | 68.3 ± 1.7 | 0.94 |
90. | [11] | a pretreatment: 131 μg tebufenozide | 0.61 ± 0.2 | 4.5 ± 7.2 | 56.6 ± 3.2 | 0.85 |
91. | [11] | a US: 0 μg tebufenozide | 1.34 ± 0.2 | 3.6 ± 4.2 | 81.8 ± 1.4 | 0.97 |
92. | [11] | a US: 16 μg tebufenozide | 1.05 ± 0.47 | 10.1 ± 9.2 | 62.3 ± 3.3 | 0.77 |
93. | [11] | a US: 24 μg tebufenozide | 1.35 ± 0.89 | 6.0 ± 11.7 | 56.4 ± 4.0 | 0.65 |
94. | [11] | a US: 32 μg tebufenozide | 0.35 ± 0.08 | 10.3 ± 3.8 | 58.6 ± 2.8 | 0.94 |
95. | [11] | a US: 69.4 μg tebufenozide | 0.8 ± 0.18 | 5.0 ± 5.1 | 58.5 ± 2.0 | 0.92 |
96. | [11] | a US: 131 μg tebufenozide | 0.79 ± 0.32 | 13.3 ± 7.0 | 54.5 ± 2.7 | 0.79 |
97. | [11] | a pretreatment: 0 μg diflubenzuron | 0.26 ± 0.13 | 12.8 ± 5.7 | 52.6 ± 7.0 | 0.80 |
98. | [11] | a pretreatment: 3.4 μg diflubenzuron | 0.9 ± 0.32 | 14.5 ± 6.8 | 61.2 ± 2.6 | 0.83 |
99. | [11] | a pretreatment: 8.5 μg diflubenzuron | 0.3 ± 0.08 | 10.4 ± 5.3 | 70 ± 5.0 | 0.92 |
100. | [11] | a pretreatment: 16 μg diflubenzuron | 1.09 ± 0.32 | 3.2 ± 5.8 | 54.4 ± 2.1 | 0.89 |
101. | [11] | a pretreatment: 32 μg diflubenzuron | 0.55 ± 0.22 | 9.0 ± 8.8 | 61.0 ± 4.2 | 0.79 |
102. | [11] | a pretreatment: 69.4 μg diflubenzuron | 0.76 ± 0.24 | 14.2 ± 5.7 | 56.1 ± 2.3 | 0.85 |
103. | [11] | a US: 0 μg diflubenzuron | 0.38 ± 0.11 | 4.9 ± 5.1 | 51.9 ± 3.5 | 0.89 |
104. | [11] | a US: 3.4 μg diflubenzuron | 0.46 ± 0.13 | −0.3 ± 3.2 | 28.8 ± 1.8 | 0.89 |
105. | [11] | a US: 8.5 μg diflubenzuron | 4.35 ± 13.18 | 4.0 ± 3.8 | 27.2 ± 1.2 | 0.80 |
106. | [11] | a US: 16 μg diflubenzuron | 0.14 ± 0.16 | 21.2 ± 4.0 | 46.9 ± 15.1 | 0.69 |
107. | [11] | a US: 32 μg diflubenzuron | 0.62 ± 0.66 | 15.2 ± 6.7 | 30.3 ± 3.0 | 0.35 |
108. | [11] | a US: 69.4 μg diflubenzuron | 2.3 ± 5.41 | 3.8 ± 11.2 | 25.4 ± 3.6 | 0.27 |
Table 3.
Learning curve comparison. The significance level of each comparison is found below. Item numbers correspond to
Table 1 and
Table 2. Values in bold italics are considered significant,
p < 0.05.
Table 3.
Learning curve comparison. The significance level of each comparison is found below. Item numbers correspond to Table 1 and Table 2. Values in bold italics are considered significant, p < 0.05.
No | Agrochemicals compared | Significance level, p |
---|
B2 | B3 | B4 |
---|
1. | item 1, a field dose of pymetrozine—item 2, 100× the field dose of pymetrozine | 0.88 | 0.86 | 2.3 × 10−8 |
2. | item 1, a field dose of pymetrozine—item 3, sucrose only | 0.00035 | 0.66 | 0.17 |
3. | item 2, 100× the field dose of pymetrozine—item 3, sucrose only | 0.069 | 0.50 | 0.00005 |
4. | item 4, a field dose of pymetrozine—item 6, sucrose only | 0.054 | 0.67 | 0.25 |
5. | item 7, 6.25% Bioganic—item 8, 1.56% Bioganic | 0.89 | 0.98 | 0.00008 |
6. | item 7, 6.25% Bioganic—item 9, sucrose only | 0.52 | 0.42 | 6.5 × 10−10 |
7. | item 8, 1.56% Bioganic—item 9, sucrose only | 0.85 | 0.58 | 0.00004 |
8. | item 10, 6.25% Bioganic—item 12, sucrose only | 0.014 | 0.87 | 1.1 × 10−14 |
9. | item 13, citronella odor—item 14, 6.25% Bioganic odor | 0.088 | 0.014 | 0.010 |
10. | item 15, pignut—item 16, sweet fennel | 0.14 | 0.89 | 0.0001 |
11. | item 17, 10 μL dicofol—item 18, sucrose only | 0.15 | 0.030 | 1 × 10−8 |
12. | item 19, 10 μL dicofol—item 20, sucrose only | 0.093 | 0.88 | 0.41 |
13. | item 21, butyric acid—item 22, DEET | 0.0076 | 0.056 | 0.00045 |
14. | item 21, butyric acid—item 23, cinnamon | 0.93 | 0.069 | 0.014 |
15. | item 22, DEET—item 23, cinnamon | 0.0085 | 0.92 | 3.2 × 10−9 |
16. | item 24, citronella—item 25, cinnamon | 0.18 | 0.94 | 0.18 |
17. | item 26, fluvalinate—item 27, acetone | 0.094 | 0.000014 | 0.0006 |
18. | item 26, fluvalinate—item 28, flucythrinate | 0.95 | 0.007 | 0.0069 |
19. | item 26, fluvalinate—item 29, cyfluthrin | 0.99 | 0.00005 | 0.56 |
20. | item 26, fluvalinate—item 30, cypermethrin | 0.89 | 0.23 | 0.37 |
21. | item 26, fluvalinate—item 31, permethrin | 0.63 | 0.81 | 0.16 |
22. | item 26, fluvalinate—item 32, fenvalerate | 0.96 | 0.018 | 0.66 |
23. | item 27, acetone—item 28, flucythrinate | 0.36 | 0.00001 | 0.0013 |
24. | item 27, acetone—item 29, cyfluthrin | 0.13 | 1.3 × 10−6 | 0.015 |
25. | item 27, acetone—item 30, cypermethrin | 0.40 | 0.001 | 0.012 |
26. | item 27, acetone—item 31, permethrin | 0.88 | 0.008 | 0.0023 |
27. | item 27, acetone—item 32, fenvalerate | 0.20 | 0.00003 | 0.010 |
28. | item 28, flucythrinate—item 29, cyfluthrin | 0.95 | 0.0046 | 0.061 |
29. | item 28, flucythrinate—item 30, cypermethrin | 0.88 | 0.23 | 0.083 |
30. | item 28, flucythrinate—item 31, permethrin | 0.64 | 0.033 | 0.055 |
31. | item 28, flucythrinate—item 32, fenvalerate | 0.93 | 0.72 | 0.033 |
32. | item 29, cyfluthrin—item 30, cypermethrin | 0.89 | 0.0017 | 0.82 |
33. | item 29, cyfluthrin—item 31, permethrin | 0.63 | 0.00044 | 0.69 |
34. | item 29, cyfluthrin—item 32, fenvalerate | 0.96 | 0.0032 | 0.86 |
35. | item 30, cypermethrin—item 31, permethrin | 0.72 | 0.26 | 0.89 |
36. | item 30, cypermethrin—item 32, fenvalerate | 0.92 | 0.38 | 0.67 |
37. | item 31, permethrin—item 32, fenvalerate | 0.65 | 0.058 | 0.50 |
38. | item 33, water—item 34, acetone | 0.76 | 0.99 | 0.87 |
39. | item 33, water—item 35, 0.01% coumaphos | 0.76 | 0.99 | 0.85 |
40. | item 33, water—item 36, 1% coumaphos | 0.65 | 0.93 | 0.49 |
41. | item 33, water—item 37, 10% coumaphos | 0.77 | 0.99 | 0.83 |
42. | item 34, acetone—item 35, 0.01% coumaphos | 0.93 | 0.97 | 0.93 |
43. | item 34, acetone—item 36, 1% coumaphos | 0.97 | 0.97 | 0.72 |
44. | item 34, acetone—item 37, 10% coumaphos | 0.92 | 0.99 | 0.75 |
45. | item 35, 0.01% coumaphos—item 36, 1% coumaphos | 0.92 | 0.99 | 0.90 |
46. | item 35, 0.01% coumaphos—item 37, 10% coumaphos | 0.99 | 0.99 | 0.76 |
47. | item 36, 1% coumaphos—item 37, 10% coumaphos | 0.90 | 0.98 | 0.51 |
48. | item 38, water—item 39, acetone | 0.70 | 0.92 | 0.60 |
49. | item 38, water—item 40, 0.005% diazinon | 0.98 | 0.98 | 0.79 |
50. | item 38, water—item 41, 0.01% diazinon | 0.98 | 0.99 | 0.78 |
51. | item 38, water—item 42, 0.025% diazinon | 0.45 | 0.96 | 0.39 |
52. | item 39, acetone—item 40, 0.005% diazinon | 0.98 | 0.98 | 0.79 |
53. | item 39, acetone—item 41, 0.01% diazinon | 0.98 | 0.99 | 0.78 |
54. | item 39, acetone—item 42, 0.025% diazinon | 0.45 | 0.96 | 0.39 |
55. | item 40, 0.005% diazinon—item 41, 0.01% diazinon | 0.98 | 0.99 | 0.78 |
56. | item 40, 0.005% diazinon—item 42, 0.025% diazinon | 0.45 | 0.96 | 0.38 |
57. | item 41, 0.01% diazinon—item 42, 0.025% diazinon | 0.45 | 0.96 | 0.39 |
58. | item 43, hexane only—item 44, 0.05% diazinon | 0.91 | 0.54 | 0.046 |
59. | item 43, hexane only—item 45, 07% coumaphos | 0.051 | 0.98 | 0.63 |
60. | item 44, 0.05% diazinon—item 45, 0.7% coumaphos | 0.051 | 0.97 | 0.62 |
61. | item 46, sucrose—item 47, endosulfan | 0.83 | 0.99 | 0.00017 |
62. | item 46, sucrose—item 48, decis | 0.81 | 0.96 | 0.08 |
63. | item 47, endosulfan—item 48, decis | 0.81 | 0.96 | 0.077 |
64. | item 51, hexanal—item 52, endosulfan | 0.78 | 0.66 | 0.75 |
65. | item 51, hexanal—item 53, decis | 0.94 | 0.62 | 0.78 |
66. | item 51, hexanal—item 54, baytroid | 0.92 | 0.47 | 0.84 |
67. | item 51, hexanal—item 55, sevin | 0.86 | 0.72 | 0.71 |
68. | item 52, endosulfan—item 53, decis | 0.70 | 0.27 | 0.96 |
69. | item 52, endosulfan—item 54, baytroid | 0.92 | 0.47 | 0.84 |
70. | item 52, endosulfan—item 55, sevin | 0.86 | 0.72 | 0.71 |
71. | item 53, decis—item 54, baytroid | 0.99 | 0.16 | 0.90 |
72. | item 53, decis—item 55, sevin | 0.92 | 0.48 | 0.53 |
73. | item 54, baytroid—item 55, sevin | 0.90 | 0.96 | 0.53 |
74. | item 56, sucrose—item 57, endosulfan | 0.54 | 0.80 | 0.010 |
75. | item 56, sucrose—item 58, decis | 0.82 | 0.84 | 0.25 |
76. | item 56, sucrose—item 59, baytroid | 0.92 | 0.09 | 0.001 |
77. | item 56, sucrose—item 60, sevin | 0.59 | 0.41 | 0.002 |
78. | item 57, endosulfan—item 58, decis | 0.55 | 0.90 | 0.026 |
79. | item 57, endosulfan—item 59, baytroid | 0.64 | 0.52 | 0.052 |
80. | item 57, endosulfan—item 60, sevin | 0.84 | 0.85 | 0.006 |
81. | item 58, decis—item 59, baytroid | 0.94 | 0.13 | 0.0038 |
82. | item 58, decis—item 60, sevin | 0.60 | 0.54 | 0.0005 |
83. | item 59, baytroid—item 60, sevin | 0.64 | 0.29 | 0.51 |
84. | item 61, 0 μg tebufenozide—item 62, 16 μg tebufenozide | 0.096 | 0.17 | 0.26 |
85. | item 61, 0 μg tebufenozide—item 63, 24 μg tebufenozide | 0.008 | 0.90 | 0.002 |
86. | item 61, 0 μg tebufenozide—item 64, 32 μg tebufenozide | 6.5 × 10−8 | 0.75 | 0.069 |
87. | item 61, 0 μg tebufenozide—item 65, 69.4 μg tebufenozide | 0.008 | 0.07 | 0.56 |
88. | item 61, 0 μg tebufenozide—item 66, 131 μg tebufenozide | 0.036 | 0.69 | 2.8 × 10−9 |
89. | item 62, 16 μg tebufenozide—item 63, 24 μg tebufenozide | 0.00015 | 0.33 | 0.0017 |
90. | item 62, 16 μg tebufenozide—item 64, 32 μg tebufenozide | 0.00003 | 0.52 | 0.086 |
91. | item 62, 16 μg tebufenozide—item 65, 69.4 μg tebufenozide | 3.5 × 10−6 | 0.0009 | 0.24 |
92. | item 62, 16 μg tebufenozide—item 66, 131 μg tebufenozide | 0.19 | 0.50 | 2.3 × 10−7 |
93. | item 63, 24 μg tebufenozide—item 64, 32 μg tebufenozide | 0.52 | 0.85 | 0.94 |
94. | item 63, 24 μg tebufenozide—item 65, 69.4 μg tebufenozide | 0.94 | 0.095 | 0.0012 |
95. | item 63, 24 μg tebufenozide—item 66, 131 μg tebufenozide | 0.20 | 0.981 | 0.036 |
96. | item 64, 32 μg tebufenozide—item 65, 69.4 μg tebufenozide | 0.48 | 0.09 | 0.048 |
97. | item 64, 32 μg tebufenozide—item 66, 131 μg tebufenozide | 0.098 | 0.97 | 0.19 |
98. | item 65, 69.4 μg tebufenozide—item 66, 131 μg tebufenozide | 0.16 | 0.054 | 2.4 × 10−7 |
99. | item 67, 0 μg tebufenozide—item 68, 16 μg tebufenozide | 0.81 | 0.65 | 1.4 × 10−6 |
100. | item 67, 0 μg tebufenozide—item 69, 24 μg tebufenozide, | 0.032 | 0.99 | 1.8 × 10−9 |
101. | item 67, 0 μg tebufenozide—item 70, 32 μg tebufenozide | 0.016 | 0.75 | 0.0062 |
102. | item 67, 0 μg tebufenozide—item 71, 69.4 μg tebufenozide | 0.0046 | 0.85 | 1.3 × 10−7 |
103. | item 67, 0 μg tebufenozide—item 72, 131 μg tebufenozide | 0.043 | 0.47 | 0.00005 |
104. | item 68, 16 μg tebufenozide—item 69, 24 μg tebufenozide | 0.17 | 0.64 | 0.15 |
105. | item 68, 16 μg tebufenozide—item 70, 32 μg tebufenozide | 0.085 | 0.55 | 0.36 |
106. | item 68, 16 μg tebufenozide—item 71, 69.4 μg tebufenozide | 0.08 | 0.77 | 0.29 |
107. | item 68, 16 μg tebufenozide—item 72, 131 μg tebufenozide | 0.17 | 0.33 | 0.12 |
108. | item 69, 24 μg tebufenozide—item 70, 32 μg tebufenozide | 0.38 | 0.75 | 0.078 |
109. | item 69, 24 μg tebufenozide—item 71, 69.4 μg tebufenozide | 0.30 | 0.84 | 0.84 |
110. | item 69, 24 μg tebufenozide—item 72, 131 μg tebufenozide | 0.90 | 0.45 | 0.0025 |
111. | item 70, 32 μg tebufenozide—item 71, 69.4 μg tebufenozide | 0.92 | 0.67 | 0.12 |
112. | item 70, 32 μg tebufenozide—item 72, 131 μg tebufenozide | 0.48 | 0.86 | 0.91 |
113. | item 71, 69.4 μg tebufenozide—item 72, 131 μg tebufenozide | 0.44 | 0.40 | 0.013 |
114. | item 73, 0 μg diflubenzuron—item 74, 3.4 μg diflubenzuron | 0.028 | 0.35 | 4.5 × 10−6 |
115. | item 73, 0 μg diflubenzuron—item 75, 8.5 μg diflubenzuron | 0.0006 | 0.068 | 0.22 |
116. | item 73, 0 μg diflubenzuron—item 76, 16 μg diflubenzuron | 0.031 | 0.075 | 0.30 |
117. | item 73, 0 μg diflubenzuron—item 77, 32 μg diflubenzuron | 0.55 | 0.17 | 4.8 × 10−7 |
118. | item 73, 0 μg diflubenzuron—item 78, 69.4 μg diflubenzuron | 0.0004 | 0.0049 | 5.4 × 10−6 |
119. | item 74, 3.4 μg diflubenzuron—item 75, 8.5 μg diflubenzuron | 0.072 | 0.28 | 0.17 |
120. | item 74, 3.4 μg diflubenzuron—item 76, 16 μg diflubenzuron | 0.84 | 0.31 | 0.0028 |
121. | item 74, 3.4 μg diflubenzuron—item 77, 32 μg diflubenzuron | 0.48 | 0.55 | 0.11 |
122. | item 74, 3.4 μg diflubenzuron—item 78, 69.4 μg diflubenzuron | 0.48 | 0.55 | 0.11 |
123. | item 75, 8.5 μg diflubenzuron—item 79, 16 μg diflubenzuron | 0.17 | 0.96 | 0.55 |
124. | item 75, 8.5 μg diflubenzuron—item 77, 32 μg diflubenzuron | 0.13 | 0.66 | 0.046 |
125. | item 75, 8.5 μg diflubenzuron—item 78, 69.4 μg diflubenzuron | 0.86 | 0.85 | 0.015 |
126. | item 76, 16 μg diflubenzuron—item 77, 32 μg diflubenzuron | 0.43 | 0.70 | 0.0002 |
127. | item 76, 16 μg diflubenzuron—item 78, 69.4 μg diflubenzuron | 0.12 | 0.80 | 0.00012 |
128. | item 77, 32 μg diflubenzuron—item 78, 69.4 μg diflubenzuron | 0.11 | 0.46 | 0.36 |
129. | item 79, 0 μg diflubenzuron—item 80, 3.4 μg diflubenzuron | 0.87 | 0.27 | 2.6 × 10−10 |
130. | item 79, 0 μg diflubenzuron—item 81, 8.5 μg diflubenzuron | 0.77 | 0.39 | 2 × 10−7 |
131. | item 79, 0 μg diflubenzuron—item 82, 16 μg diflubenzuron | 0.032 | 0.26 | 0.40 |
132. | item 79, 0 μg diflubenzuron—item 83, 32 μg diflubenzuron | 0.90 | 0.75 | 0.0004 |
133. | item 80, 3.4 μg diflubenzuron—item 81, 8.5 μg diflubenzuron | 0.99 | 0.096 | 0.35 |
134. | item 80, 3.4 μg diflubenzuron—item 82, 16 μg diflubenzuron | 0.66 | 0.83 | 0.42 |
135. | item 80, 3.4 μg diflubenzuron—item 83, 32 μg diflubenzuron | 0.94 | 0.37 | 0.34 |
136. | item 81, 8.5 μg diflubenzuron—item 82, 16 μg diflubenzuron | 0.42 | 0.095 | 0.53 |
137. | item 81, 8.5 μg diflubenzuron—item 83, 32 μg diflubenzuron | 0.93 | 0.782 | 0.779 |
138. | item 82, 6 μg diflubenzuron—item 83, 32 μg diflubenzuron | 0.59 | 0.35 | 0.58 |
139. | item 85, 0 μg tebufenozide—item 86, 16 μg tebufenozide | 0.078 | 0.77 | 0.043 |
140. | item 85, 0 μg tebufenozide—item 87, 24 μg tebufenozide | 0.0053 | 0.96 | 0.52 |
141. | item 85, 0 μg tebufenozide—item 88, 32 μg tebufenozide | 0.007 | 0.71 | 0.014 |
142. | item 85, 0 μg tebufenozide—item 89, 69.4 μg tebufenozide | 0.14 | 0.92 | 0.0005 |
143. | item 85, 0 μg tebufenozide—item 90, 131 μg tebufenozide | 0.033 | 0.58 | 0.00002 |
144. | item 86, 16 μg tebufenozide—item 87, 24 μg tebufenozide | 0.064 | 0.73 | 0.039 |
145. | item 86, 16 μg tebufenozide—item 88, 32 μg tebufenozide | 0.12 | 0.92 | 0.13 |
146. | item 86, 16 μg tebufenozide—item 89, 69.4 μg tebufenozide | 0.61 | 0.70 | 0.40 |
147. | item 86, 16 μg tebufenozide—item 90, 131 μg tebufenozide | 0.58 | 0.82 | 0.0033 |
148. | item 87, 24 μg tebufenozide—item 88, 32 μgtebufenozide | 0.90 | 0.68 | 0.012 |
149. | item 87, 24 μg tebufenozide—item 89, 69.4 μg tebufenozide | 0.012 | 0.95 | 0.0057 |
150. | item 87, 24 μg tebufenozide—item 90, 131 μg tebufenozide | 0.22 | 0.54 | 0.00005 |
151. | item 88, 32 μg tebufenozide—item 89, 69.4 μg tebufenozide | 0.031 | 0.65 | 0.22 |
152. | item 88, 32 μg tebufenozide—item 90, 131 μg tebufenozide | 0.33 | 0.91 | 0.61 |
153. | item 89, 69.4 μg tebufenozide—item 90, 131 μg tebufenozide | 0.28 | 0.52 | 0.0046 |
154. | item 91, 0 μg tebufenozide—item 92, 16 μg tebufenozide | 0.58 | 0.53 | 0.0002 |
155. | item 91, 0 μg tebufenozide—item 93, 24 μg tebufenozide | 0.99 | 0.85 | 0.00013 |
156. | item 91, 0 μg tebufenozide—item 94, 32 μg tebufenozide | 0.0006 | 0.25 | 8.1 × 10−6 |
157. | item 91, 0 μg tebufenozide—item 95, 69.4 μg tebufenozide | 0.060 | 0.83 | 1.8 × 10−8 |
158. | item 91, 0 μg tebufenozide—item 96, 131 μg tebufenozide | 0.16 | 0.012 | 2.0 × 10−7 |
159. | item 92, 16 μg tebufenozide—item 93, 24 μg tebufenozide | 0.77 | 0.79 | 0.27 |
160. | item 92, 16 μg tebufenozide—item 94, 32 μg tebufenozide | 0.17 | 0.98 | 0.40 |
161. | item 92, 16 μg tebufenozide—item 95, 69.4 μg tebufenozide | 0.63 | 0.64 | 0.34 |
162. | item 92, 16 μg tebufenozide—item 96, 131 μg tebufenozide | 0.65 | 0.78 | 0.084 |
163.. | item 93, 24 μg tebufenozide—item 94, 32 μg tebufenozide | 0.55 | 0.73 | 0.66 |
164. | item 93, 24 μg tebufenozide—item 95, 69.4 μg tebufenozide | 0.56 | 0.94 | 0.65 |
165. | item 93, 24 μg tebufenozide—item 96, 131 μg tebufenozide | 0.57 | 0.60 | 0.70 |
166. | item 94, 32 μg tebufenozide—item 95, 69.4 μg tebufenozide | 0.041 | 0.42 | 0.98 |
167. | item 94, 32 μg tebufenozide—item 96, 131 μg tebufenozide | 0.21 | 0.71 | 0.31 |
168. | item 95, 69.4 μg tebufenozide—item 96, 131 μg tebufenozide | 0.98 | 0.35 | 0.25 |
169. | item 97, 0 μg diflubenzuron—item 98, 3.4 μg diflubenzuron | 0.089 | 0.85 | 0.27 |
170. | item 97, 0 μg diflubenzuron—item 99, 8.5 μg diflubenzuron | 0.80 | 0.76 | 0.058 |
171. | item 97, 0 μg diflubenzuron—item 100, 16 μg diflubenzuron | 0.034 | 0.25 | 0.81 |
172. | item 97, 0 μg diflubenzuron—item 101, 32 μg diflubenzuron | 0.27 | 0.72 | 0.32 |
173. | item 97, 0 μg diflubenzuron—item 102, 69.4 μg diflubenzuron | 0.09 | 0.86 | 0.64 |
174. | item 98, 3.4 μg diflubenzuron—item 99, 8.5 μg diflubenzuron | 0.099 | 0.64 | 0.15 |
175. | item 98, 3.4 μg diflubenzuron—item 100, 16 μg diflubenzuron | 0.68 | 0.22 | 0.057 |
176. | item 98, 3.4 μg diflubenzuron—item 101, 32 μg diflubenzuron | 0.38 | 0.62 | 0.97 |
177. | item 98, 3.4 μg diflubenzuron—item 102, 69.4 μg diflubenzuron | 0.73 | 0.97 | 0.16 |
178. | item 99, 8.5 μg diflubenzuron—item 100, 16 μg diflubenzuron | 0.038 | 0.37 | 0.016 |
179. | item 99, 8.5 μg diflubenzuron—item 101, 32 μg diflubenzuron | 0.31 | 0.89 | 0.18 |
180. | item 99, 8.5 μg diflubenzuron—item 102, 69.4 μg diflubenzuron | 0.096 | 0.63 | 0.028 |
181. | item 100, 16 μg diflubenzuron—item 101, 32 μg diflubenzuron | 0.18 | 0.59 | 0.18 |
182. | item 100, 16 μg diflubenzuron—item 102, 69.4 μg diflubenzuron | 0.42 | 0.19 | 0.59 |
183. | item 101, 32 μg diflubenzuron—item 102, 69.4 μg diflubenzuron | 0.53 | 0.62 | 0.32 |
184. | item 103, 0 μg diflubenzuron—item 104, 3.4 μg diflubenzuron | 0.64 | 0.40 | 0.00002 |
185. | item 103, 0 μg diflubenzuron—item 105, 8.5 μg diflubenzuron | 0.77 | 0.89 | 0.00009 |
186. | item 103, 0 μg diflubenzuron—item 106, 16 μg diflubenzuron | 0.23 | 0.022 | 0.75 |
187. | item 103, 0 μg diflubenzuron—item 107, 32 μg diflubenzuron | 0.73 | 0.24 | 0.0002 |
188. | item 103, 0 μg diflubenzuron—item 108, 69.4 μg diflubenzuron | 0.73 | 0.93 | 0.00005 |
189. | item 104, 3.4 μg diflubenzuron—item 105, 8.5 μg diflubenzuron | 0.78 | 0.40 | 0.47 |
190. | item 104, 3.4 μg diflubenzuron—item 106, 16 μg diflubenzuron | 0.14 | 0.0005 | 0.25 |
191. | item 104, 3.4 μg diflubenzuron—item 107, 32 μg diflubenzuron | 0.82 | 0.063 | 0.67 |
192. | item 104, 3.4 μg diflubenzuron—item 108, 69.4 μg diflubenzuron | 0.74 | 0.73 | 0.42 |
193. | item 105, 8.5 μg diflubenzuron—item 106, 16 μg diflubenzuron | 0.76 | 0.006 | 0.23 |
194. | item 105, 8.5 μg diflubenzuron—item 107, 32 μg diflubenzuron | 0.78 | 0.16 | 0.36 |
195. | item 105, 8.5 μg diflubenzuron—item 108, 69.4 μg diflubenzuron | 0.89 | 0.99 | 0.64 |
196. | item 106, 16 μg diflubenzuron—item 107, 32 μg diflubenzuron | 0.50 | 0.45 | 0.31 |
197. | item 106, 16 μg diflubenzuron—item 108, 69.4 μg diflubenzuron | 0.70 | 0.18 | 0.20 |
198. | item 107, 32 μg diflubenzuron—item 108, 69.4 μg diflubenzuron | 0.76 | 0.39 | 0.31 |
The second experiment investigated the effects of pymetrozine on complex Pavlovian conditioning where honey bees were trained to discriminate between two CSs, one of which was always paired with a US. A control group learned well (
Table 1, item 6) and learning data fit well with the model (
Table 2, item 6). A field dose of pymetrozine induced spread in the raw learning data (
Table 1, item 4) and decreased the learning rate twice (
Table 2, item 4) in comparison with the control group (
Table 3, item 4). A pretreatment with 100× the field dose of pymetrozine changed the shape of the learning curve from exponential to quadratic function. In other words, the percent of conditioned responses increased during the first five trials then diminished to zero. Our model was not able to fit this learning curve (
Table 2, item 5).
Bioganic. These experiments investigated the effects of Bioganic
® Lawn and Garden Spray Multi-Insect Killer (Bioganic Safety Brands, Roswell, GA, USA), on honey bee learning. This agrochemical is unique because it is composed almost entirely of thyme, clover, and sesame essential oils. A non-overlap procedure was used with a CS duration of 3 s, a US duration of 2 s and an intertrial interval of 10 min. Forager Africanized honey bees (
Apis mellifera L.) were used. The bees were collected in glass vials from the sill of the laboratory colonies approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then placed in a harness, fed 1.8 M sucrose and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally and mixed with sucrose to make it palatable. Two doses were used. In one condition, a 1 μL droplet of 1.56% of Bioganic was used and in the other condition a 1 μL droplet of 6.25% of Bioganic was used [
9].
In the first experiment, citronella was used as a CS and Bioganic was used as a US. The unconditioned stimulus in the control group was sucrose. In the control group, the number of conditioned responses increased to 65% (
Table 1, item 9). The learning curve for the control group fit well with the model (
Table 2, item 9). Bioganic 1.56% increased the number of conditioned responses during the initial four trials only; next, the number of responses decreased (
Table 1, item 8). The model did not fit the learning data; R squared is 0.1859 (
Table 2, item 8). Ability to learn (B4) was more than two times lower (
Table 3, item 7) in comparison with the control group. Bioganic 6.25% was not able to serve as a US. Instead, it diminished the number of conditioned responses to zero (
Table 1, item 7). The learning curve changed into a monotonically decreasing curve with ability to learn of 1.0 (
Table 2, item 7). This coefficient B4 differed significantly in comparison with the control group (
Table 3, item 6) and Bioganic 1.56% group (
Table 3, item 5).
The second experiment with Bioganic used discrimination in bees with a CS+ of citronella, a CS− of cinnamon and a variable US. The number of conditioned responses in the control group increased to 75% at the end of learning session (
Table 1, item 12). The model fit the learning data well (
Table 2, item 12). Bioganic 1.56% destroyed learning in such a way that the percent of conditioned responses increased during the initial trials then diminished during the rest of training (
Table 1, item 11). As a result, the learning data did not fit with the model (
Table 2, item 11). Bioganic 6.25% very quickly decreased the conditioned responses to zero (
Table 1, item 10). Thus, the learning curve became a monotonously decreasing curve (
Table 2, item 10). Ability to learn differed significantly between the control group and Bioganic 6.25% group (
Table 3, item 8). In the third experiment, citronella or 6.25% Bioganic odor was used as a CS+, with sucrose as the US. Bees were conditioned very well with each odor. During Trial 1 there were no conditioned responses with citronella (
Table 1, item 13), but 30% CR with 6.25% Bioganic odor (
Table 1, item 14). Both learning curves (citronella, (
Table 2, item 13) and 6.25% Bioganic, (
Table 2, item 14)) fit very well with the model. Ability to learn and readiness to learn were significantly higher with 6.25 Bioganic (
Table 3, item 9).
Comparison of pignut and sweet fennel as a CS. Pignut (
Hyptis suaveolens (L.) Poit) and sweet fennel (
Foeniculum vulgare Mill) are essential oils that have been shown to control aphids [
26]. As the use of essential oils to control insect pests increases, it is important to evaluate their effect on honey bee learning. A non-overlap procedure was used with a CS duration of 3 s, a US duration of 2 s and an intertrial interval of 10 min. Forager Africanized honey bees (
Apis mellifera L.) were used. The bees were collected in glass vials from the sill of the laboratory colonies approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then placed in a harness, fed 1.8 M sucrose, and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally by stimulating the antennae with sucrose, and with the proboscis now extended, allowed to consume the dose. In the pignut condition, bees were fed a 1 μL droplet of 3.125% pignut oil. Bees in the sweet fennel condition were fed a 1 μL droplet of 3.125% fennel oil [
15].
Bees readily associated both odors with a reward. The number of conditioned responses was higher with pignut (
Table 1, item 15), though the number of CR with sweet fennel was also high (
Table 1, item 16). Modeling revealed that ability to learn was higher with pignut (B4 = 92.1,
Table 2, item 15) in comparison with sweet fennel (B4 = 80.9,
Table 2, item 18) and this difference was significant (
Table 3, item 10).
Dicofol (Kelthane). Dicofol is an acaricide that is chemically very close to DDT. It is generally considered harmless to honey bees, but has been shown to affect other insects by changing their natural behavior [
27]. As in the previous investigations from the senior author’s laboratory, a non-overlap procedure was used with a CS duration of 3 s, US duration of 2 s and an intertrial interval of 10 min. Forager honey bees (
Apis mellifera L.) were used. The bees were collected in glass vials from the sill of the laboratory colonies approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then placed in a harness, fed 1.8 M sucrose, and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally and mixed with sucrose to make it palatable. The dose consisted of a 10 μL droplet of 0.7 g/L of dicofol [
16].
The first experiment studied the influence of dicofol on acquisition in bees with cinnamon as a CS. A control group pretreated with sucrose yielded up to 90% of conditioned responses (
Table 1, item 18). Dicofol decreased the initial Trial 1 value as well as the maximum value of CR (
Table 1, item 17). Accordingly, readiness to learn and ability to learn were higher in the control group (
Table 2, item 18) than in the dicofol group (
Table 2, item 17), with this difference being significant (
Table 3, item 11). In the second experiment, bees were pretreated with dicofol or sucrose and taught to discriminate between the odors of cinnamon oil and a perfume. Only six trials were used in this experiment. Bees learned up to 75%–80% of CR with sucrose (
Table 1, item 20) as well as with dicofol (
Table 1, item 19). Though the coefficient B4 value was higher in the sucrose group (B4 = 90.2,
Table 2, item 20) than in the dicofol group (B4 = 75.3,
Table 2, item 19), this difference was not significant (
Table 3, item 12). We hypothesize that six CS+ trials were not enough to assess the asymptotic value correctly. An insufficient number of trials led to an underestimated value of the learning rate and, subsequently, overestimation of the B4 value.
3.3. Sublethal Amounts of Agrochemicals Known to Be Harmful to Bees
Fluvalinate, flucythrinate, cyfluthrin, cypermethrin, permethrin, fenvalerate. These pesticides are pyrethroids that are lethal to honey bees in laboratory settings [
30] but were tested at sublethal dosages to determine their effects on learning. The CS duration was 6 s, the US duration was 3 s, and an overlap procedure was used in which the US was presented 3 s after the CS was presented and both terminated together. The intertrial interval was 15 min. Eight training trials were given, but the first training trial was not reported in the original paper—
i.e., data was presented only for trials 2–8 (7 trials). Unfortunately, no unpaired or discrimination control group was employed. Forager honey bees (
Apis mellifera L.) were used. Bees exiting a colony were funneled into a closed 9 cm dia glass petri dish containing insecticide-treated filter paper where they remained for 24 h before testing. After exposure, the surviving bees were chilled, harnessed, and fed. They were tested 3 h later. Bees were exposed to LC
50 dosage of fluvalinate (10.00 mg/dish), fenvalerate (1.00 mg/dish), permethrin (0.06 mg/dish), cypermethrin (0.10 mg/dish), cyfluthrin (0.10 mg/dish), flucythrinate (1.00 mg/dish) [
18].
Acquisition was tested using a CS of thyme odor in bees pretreated with fluvalinate, flucythrinate, cyfluthrin, cypermethrin, permethrin, fenvalerate, or an acetone-only control. The data consisted of seven trials. The acetone-only control provided the most conditioned responses on Trial 1, the second actual trial (62.9%,
Table 1, item 27). Other chemicals take up position in decreasing order as follows: permethrin (41.7%,
Table 1, item 31), fluvalinate (38.5%,
Table 1, item 26), cypermethrin (34.2%,
Table 1, item 30), fenvalerate (26.7%,
Table 1, item 32), flucythrinate (24.2%,
Table 1, item 28), and cyfluthrin (2.9%,
Table 1, item 29). Each learning curve fit well with the model. Ability to learn took the same position as the Trial 1 value.
Ability to learn positioned the chemicals as follows: acetone (91.8%,
Table 2, item 27), fluvalinate (73.5%,
Table 2, item 26), fenvalerate (70.4%,
Table 2, item 32), cyfluthrin (68.6%,
Table 2, item 29), cypermethrin (66.2%,
Table 2, item 30), permethrin (65.0%,
Table 2, item 31), and flucythrinate (46.7%,
Table 2, item 28). In comparison with the control chemical acetone, readiness to learn and ability to learn were lower in fluvalinate (
Table 3, item 17), flucythrinate (
Table 3, item 23), cyfluthrin (
Table 3, item 24), cypermethrin (
Table 3, item 25), permethrin (
Table 3, item 26), and fenvalerate (
Table 3, item 27). In other words, these results show that these chemicals do really harm honey bees. Cyfluthrin was the most harmful for readiness to learn, and flucythrinate was the most harmful for ability to learn.
Coumaphos. Coumaphos is an organophosphate that has been used to control mite and beetle populations that infest beehives. The CS or US durations were not clear from the paper, or whether an overlap or non-overlapnon-overlap procedure was used. The intertrial interval was 10 min, and only 5 training trials were employed. No unpaired or discrimination control groups were employed. Forager honey bees of different ages (
Apis mellifera L.) were used, as determined by wing shape. The chemical was applied to the dorsal thorax, or by intracranial injection, and left overnight. The bees were collected in glass vials from the sill of the laboratory colony located outside or from an indoor hive, chilled to reduce activity, and then harnessed and fed. Two hours after harnessing, bees received a pre-test and the bees that extended their proboscises were used for training [
19].
Acquisition was studied in bees pretreated with an application to the thorax of water, acetone, 0.01% coumaphos, 0.1% coumaphos, or 10% coumaphos, with a CS of geraniol odor. Learning consisted of only five trials. In this experiment, zero conditioned responses were observed during the first trial with water (
Table 1, item 33), acetone (
Table 1, item 34), 0.01% coumaphos (
Table 1, item 35), 0.1% coumaphos (
Table 1, item 36), and 10% coumaphos (
Table 1, item 37). The maximum number of CR for each chemical was in the range 55%–70%. Ability to learn was 70% with water (
Table 2, item 33), 74.1% with acetone (
Table 2, item 34), 77.5% with 0.01% coumaphos (
Table 2, item 35), 82.0% with 0.1% coumaphos (
Table 2, item 36), and 63.9% with 10% coumaphos (
Table 2, item 37). The learning rate and ability to learn with 10% coumaphos were corrected. Due to the insufficient number of trials, the learning rate was underestimated and, subsequently, ability to learn was overestimated. No significant differences were found among this group of chemicals (
Table 3, items 38–47).
Diazinon. Diazinon was tested in comparison with coumaphos as an organophosphate that is known to be harmful to bees [
31]. The CS and US durations are not specified, and it is not clear whether an overlap or non-overlapnon-overlap procedure was used. The intertrial interval was 10 min, and only 5 training trials were employed. No unpaired or discrimination control groups were employed. Forager honey bees of different ages (
Apis mellifera L.) were used, as determined by wing shape. The chemical was applied to the dorsal thorax, or by intracranial injection, and left overnight. The bees were collected in glass vials from the sill of the laboratory colony located outside or from an indoor hive, chilled to reduce activity, and then harnessed and fed. Two h after harnessing, bees received a pre-test and those bees that extended their proboscises were used for training [
19].
Acquisition was studied in bees pretreated with an application to the thorax of water, acetone, 0.005% diazinon, 0.01% diazinon, or 0.025% diazinon, with a CS of geraniol odor. Learning consisted of only five trials. In this experiment, zero conditioned responses were observed during the first trial with water (
Table 1, item 38), acetone (
Table 1, item 39), 0.005% diazinon (
Table 1, item 40), 0.01% diazinon (
Table 1, item 41), and 0.025% diazinon (
Table 1, item 42). The maximum number of CR was in the range 55%–78%. The learning rate and ability to learn were corrected with water (
Table 2, item 38) and 0.01% diazinon (
Table 2, item 41). No significant differences were found among this group of chemicals (see
Table 3, items 48–57).
Diazinon, coumaphos. Acquisition was studied in bees pretreated with an intracranial injection of 1 μL of 0.005% diazinon in hexane, 0.07% coumaphos in hexane, or hexane alone, with a CS of geraniol odor. Learning consisted of only five trials. The maximum number of CR was 70% with hexane alone (
Table 1, item 43), 30% with 0.005% diazinon (
Table 1, item 44), and 65% with 0.07% coumaphos (
Table 1, item 45). With hexane alone, due to an insufficient number of trials, the learning rate was underestimated and ability to learn was overestimated, so that these values were corrected (
Table 2, item 43). With 0.07% coumaphos, the learning rate was the highest (
Table 2, item 45) and differed significantly from hexane alone (
Table 3, item 59) and 0.005% diazinon (
Table 3, item 60). At the same time, ability to learn was lower with 0.005% diazinon in comparison with hexane alone (
Table 3, item 58). Thus, diazinon was more harmful for bees than coumaphos.
Endosulfan, decis, baytroid, sevin. These insecticides were chosen because of their use to control the cotton boll weevil populations in Brazil [
32]. The CS duration was 3 s, the US duration was 2 s, the intertrial interval was 10 min and a non-overlapnon-overlap procedure was used. Forager Africanized honey bees (
Apis mellifera L.) were used. The bees were collected in glass vials from the sill of the laboratory colonies approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then placed in a harness, fed sucrose, and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally and mixed with sucrose to make it palatable [
20].
In the first experiment, acquisition was studied in bees pretreated with 0.8 μL of endosulfan, 0.1 μL of decis, 0.3 μL of baytroid, 1.5 μL of sevin, or sucrose alone, with a CS of hexanal. Learning did not occur with baytroid (
Table 1, item 49) and sevin (
Table 1, item 50)—zero CR was observed during each trial. Practically no learning occurred with endosulfan—there were only four out of 12 trials with non-zero CR values (
Table 1, item 47), thus the learning data did not fit with the model (
Table 2, item 47). The maximum value of CR was 70% with sucrose (
Table 1, item 46) and 50% with decis (
Table 1, item 48). Decis impaired learning less than other chemicals from this group: there was no significant difference between its learning curve and the learning curve of sucrose (
Table 3, item 62). In the second experiment, these chemicals were used as a CS and acquisition was studied in bees with a CS of hexanal plus 0.1 μL of endosulfan, decis, baytroid, sevin, or hexanal alone. Bees readily associated each odor with a reward. The maximum CR value was 75% with hexanal (
Table 1, item 51), 65% with endosulfan (
Table 1, item 52), 70% with decis (
Table 1, item 53), 65% with baytroid (
Table 1, item 54), and 75% with sevin (
Table 1, item 55). Each learning curve fit well with the model (
Table 2, items 51–55). No significant differences were found among these chemicals (
Table 3, items 64–73).
In the third experiment, acquisition was studied in bees with a US of sucrose plus 0.1 μL endosulfan, decis, baytroid, sevin, or sucrose only. Learning was practically absent with baytroid (
Table 1, item 59), so the learning curve became descendent (
Table 2, item 59) and ability to learn was significantly lower in comparison with sucrose (
Table 3, item 76). With sevin, learning was also absent (
Table 2, item 60), so ability to learn was close to readiness to learn (
Table 2, item 60) and significantly lower in comparison with sucrose (
Table 3, item 77). With endosulfan, the number of CR initially increased, but from the ninth trial, its value progressively decreased (
Table 1, item 57), so the fit with the model was not sufficient (
Table 2, item 57) and ability to learn was significantly lower in comparison with sucrose (
Table 3, item 74). Only decis supported learning, with a maximum number of CR of 55% (
Table 1, item 58). This value was less than with sucrose (
Table 1, item 56), but no significant differences between the model’s coefficients were found in comparison with sucrose (
Table 3, item 75).
Tebufenozide (Confirm
®2F). Tebufenozide is an insect growth regular. The CS duration was 3 s, the US duration was 2 s, the intertrial interval was 10 min and a non-overlapnon-overlap procedure was used. Honey bees (
Apis mellifera L).were collected from the sill of the laboratory colony. No attempt was made to solely focus on forager bees. As a result, a mixture of forager, nest cleaning, and guard honey bees was used. The bees were collected in glass vials approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then the bees were placed in a harness, fed 1.8 M sucrose, and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally and mixed with sucrose to make it palatable [
11].
In the first experiment, acquisition was studied in bees pretreated with a 10 μL solution containing 0 μg, 16 μg, 24 μg, 32 μg, 69.4 μg, or 131 μg of tebufenozide, and a CS of cinnamon. The maximum number of CR was 100% in the control group without tebufenozide (
Table 1, item 61), 92% with 16 μg (
Table 1, item 62), 76% with 24 μg (
Table 1, item 63), 80% with 32 μg (
Table 1, item 64), 100% with 69.4 μg (
Table 1, item 65), and 64% with 131 μg (
Table 1, item 66) of tebufenozide. Each learning curve fit well with the model (
Table 2, item 61–66). With the exception of 16 μg (
Table 3, item 84), each dose of tebufenozide significantly diminished the learning rate (
Table 3, item 85–88). Ability to learn was decreased with a dose of 24 μg (
Table 3, item 85) and 131 μg (
Table 3, item 88). There were significant differences among the model’s coefficients with different doses of the chemical (
Table 3, item 89, 90, 91, 92, 94, 95, 96, and 98).
In the second experiment, acquisition was studied in bees with a US of 1 μL of sucrose plus 0 μg, 16 μg, 24 μg, 32 μg, 69.4 μg, or 131 μg of tebufenozide, and a CS of cinnamon. The maximum number of CR was 92% in the control group without tebufenozide (
Table 1, item 67), 80% with 16 μg (
Table 1, item 68), 64% with 24 μg (
Table 1, item 69), 88% with 32 μg (
Table 1, item 70), 68% with 69.4 μg (table 1, item 71), and 76% with 131 69.4 μg of tebufenozide (
Table 1, item 72). Each learning curve fit well with the model (
Table 2, item 67–72). In each dose, tebufenozide significantly decreased ability to learn and, except for 16 μg, lowered the learning rate (
Table 3, item 99–103). Ability to learn was significantly lower with 24 μg (
Table 3, item 110) and 69.4 μg (
Table 3, item 113) in comparison with 131 μg of tebufenozide.
In the third experiment, discrimination was studied in bees pretreated with a 10 μL solution containing 0 μg, 16 μg, 24 μg, 32 μg, 69.4 μg, or 131μg of tebufenozide, and a CS+ of cinnamon. The maximum number of CR was 88% in the control group without tebufenozide (
Table 1, item 85), 77% with 16 µg (
Table 1, item 86), 85% with 24 µg (
Table 1, item 87), 65% with 32 µg (
Table 1, item 88), 73% with 69.4 μg (
Table 1, item 89), and 69% with 131 µg of tebufenozide (
Table 1, item 90). Each learning curve fit well with the model (
Table 2, item 85–90). The chemical significantly decreased ability to learn in the doses of 16 µg (
Table 3, item 139), 32 µg (
Table 3, item 141), 69.4 µg (
Table 3, item 142), and 131 µg (
Table 3, item 143) in comparison with the control group. The learning rate was significantly lower with 24 µg (
Table 3, item 140), 32 µg (
Table 3, item 141), and 131 µg (
Table 3, item 143). Additionally, there were significant differences in ability to learn and the learning rate among different doses of the chemical (
Table 3, item 144, 147, 148, 149, 150, 151, and 153).
In the fourth experiment, discrimination was studied in bees with a US of 1 μL of sucrose plus 0 μg, 16 μg, 24 μg, 32 μg, 69.4 μg, or 131 μg of tebufenozide, and a CS+ of cinnamon. The maximum number of CR was 88% in the control group without tebufenozide (
Table 1, item 91), 77% with 16 µg (
Table 1, item 92), 77% with 24 µg (
Table 1, item 93), 60% with 32 µg (
Table 1, item 94), 64% with 69.4 µg (
Table 1, item 95), and 64% with 131 µg (
Table 1, item 96). That said, with each dose, the number of CR reached its maximum value in the midst of the learning session then decreased. Each learning curve was fitted with the model (
Table 2, item 91–96), though with the doses of 16 µg (
Table 2, item 92), 24 μg (
Table 2, item 93), 131 µg of tebufenozide (
Table 1, item 96) a spread of CR values was found. The chemical significantly decreased ability to learn in each dose (
Table 3, item 154–158) in comparison with the control group. The learning rate was significantly lower with 32 µg (
Table 2, item 97). A significant difference in the learning rate was found between the doses of 32 µg and 69.4 µg (
Table 3, item 166). No other differences in the model’s coefficients among doses were found.
Diflubenzuron (Dimilin
®). Diflubenzuron is an insect growth regular that disrupts molting but has been shown to have no effect on honey bees [
33]. The CS duration was 3 s, the US duration was 2 s, the intertrial interval was 10 min and a non-overlapnon-overlap procedure was used. Honey bees (
Apis mellifera L.) were collected from the sill of the laboratory colony. No attempt was made to solely focus on forager bees. As a result, a mixture of forager, nest cleaning, and guard honey bees was used. The bees were collected in glass vials approximately 24 h prior to use. The vials were placed in an ice water bath to reduce activity and then the bees were placed in a harness, fed 1.8 M sucrose, and set aside. The following day, each bee received a pre-test in which the antennae was stimulated with sucrose. If a vigorous proboscis response was observed, the bee was used 10 min or more later. The 10 min delay was used to reduce the excitation induced by sucrose stimulation. The agrochemical was administered orally and mixed with sucrose to make it palatable [
11].
In the first experiment, acquisition was studied in bees pretreated with a 10 μL solution containing 0 μg, 3.4 μg, 8.5 μg, 16 μg, 32 μg, or 69.4 μg of diflubenzuron, and a CS of cinnamon. The maximum number of CR was 92% in the control group without diflubenzuron (
Table 1, item 76), 72% with 3.4 μg (
Table 1, item 74), 80% with 8.5 μg (
Table 1, item 75), 84% with 16 μg (
Table 1, item 76), 72% with 32 μg (table 1, item 77), and 72% with 69.4 μg of diflubenzuron (
Table 1, item 78). Each learning curve fit well with the model (
Table 2, item 73–78). Each dose of the chemical significantly lowered the learning rate (
Table 3, item 114, 115, 116, 118) with the exception of 32 μg (
Table 3, item 117). Ability to learn was significantly decreased with 3.4 μg (
Table 3, item 114), 32 μg (
Table 3, item 117), and 69.4 μg (
Table 3, item 118). There were also significant differences in ability to learn among different doses of the chemical (
Table 3, item 120, 124, 125, 126, and 127).
In the second experiment, acquisition was in bees with a US of 1 μL of sucrose plus 0 μg, 3.4 μg, 8.5 μg, 16 μg, 32 μg, or 69.4 μg of diflubenzuron, and a CS of cinnamon. The maximum number of CR was 68% in the control group without diflubenzuron (
Table 1, item 79), 36% with 3.4 μg (
Table 1, item 80), 36% with 8.5 μg (
Table 1, item 81), 44% with 16 μg (
Table 1, item 82), 56% with 32 μg (
Table 1, item 83), and 56% with 69.4 μg of the chemical (
Table 1, item 84). The learning curve fit very well only in the control group (
Table 2, item 79). Diflubenzuron increased the number of CR during some initial trials and decreased it during the second part of the session. Low R squared values were found for 3.4 μg (
Table 2, item 80), 32 μg (
Table 2, item 83), and especially 69.4 μg, for which the data did not fit with the model (
Table 2, item 84). Ability to learn was significantly decreased with 3.5 μg (
Table 3, item 129), 8.5 μg (
Table 3, item 130), and 32 μg (
Table 3, item 132). The learning rate was significantly lower with 16 μg in comparison with the control group (
Table 3, item 131). There were no significant differences between the model’s coefficients among different doses (
Table 3, item 133–138).
In the third experiment, discrimination was studied in bees pretreated with a 10 μL solution containing 0 μg, 3.4 μg, 8.5 μg, 16 μg, 32 μg, or 69.4 μg of diflubenzuron, and a CS+ of cinnamon. The maximum number of CR was 58% in the control group without diflubenzuron (
Table 1, item 97), 69% with 3.4 μg (
Table 1, item 98), 69% with 8.5 μg (
Table 1, item 99), 62% with 16 μg (
Table 1, item 100), 77% with 32 μg (
Table 1, item 101), and 65% with 69.4 μg of the chemical (
Table 1, item 102). Each learning curve fit well (
Table 2, item 97–102). Ability to learn did not differ significantly between the control group and 3.4 μg of diflubenzuron (
Table 3, item 169), 16 μg (
Table 3, item 171), 32 μg (
Table 3, item 172), and 69.4 μg (
Table 3, item 173). The dose of 8.5 μg increased ability to learn on the trend level (
Table 3, item 170,
p = 0.058). With the dose of 16 μg, the learning rate was higher than in the control group (
Table 3, item 171). A significant difference between ability to learn was found with some doses (
Table 3, item 178, 180). The learning rate differed significantly between 8.5 μg and 16 μg of the chemical (
Table 3, item 178).
In the fourth experiment, discrimination was studied in bees with a US of 1 μL of sucrose plus 0 μg, 3.4 μg, 8.5 μg, 16 μg, 32 μg, or 69.4 μg of diflubenzuron, and a CS+ of cinnamon. The maximum number of CR was 58% in the control group without diflubenzuron (
Table 1, item 103), 35% with 3.4 μg (
Table 1, item 104), 34% with 8.5 μg (
Table 1, item 105), 42% with 16 μg (
Table 1, item 106), 42% with 32 μg (
Table 1, item 107), and 42% with 69.4 μg of the chemical (
Table 1, item 108). With the dose of 69.4 μg of diflubenzuron, the number of CR increased during the initial trials, but decreased toward the end of the learning session (table 1, item 108). The learning curves fit well in the control group (
Table 2, item 103), with 3.4 μg (table 2, item 104), with 8.5 μg (
Table 2, item 105), and with 16 μg (
Table 2, item 106). With the dose of 32 μg, the learning data were spread around the model curve (
Table 2, item 107). With the dose of 69.4 μg, the learning curve looked like a sine wave that worsened fitting (
Table 2, item 108). Ability to learn was decreased significantly with the doses of 3.4 μg (
Table 3, item 184), 8.5 μg (
Table 3, item 185), 32 μg (
Table 3, item 187), and 69.4 μg (
Table 3, item 188). The dose of 16 μg, instead, significantly decreased the learning rate (
Table 3, item 186). Readiness to learn was higher with 16 μg in comparison with the control group (
Table 3, item 186). Additionally, ability to learn was significantly higher with 16 μg in comparison with 3.4 μg (
Table 3, item 190) and with 8.5 μg (
Table 3, item 193).