Method for training honeybees to respond to olfactory stimuli and enhancement of memory retention therein
View Patent ↗A specialized conditioning protocol for honeybees that is designed for use within a complex agricultural ecosystem. This method ensures that the conditioned bees will be less likely to exhibit a conditioned response to uninfected plants, a false positive response that would render such a biological sensor unreliable for agricultural decision support. Also described is a superboosting training regime that allows training without the aid of expensive equipment and protocols for training in out in the field. Also described is a memory enhancing cocktail that aids in long term memory retention of a vapor signature. This allows the bees to be used in the field for longer durations and with fewer bees trained overall.
1. A method for detecting agricultural volatile and semi-volatile organic compounds, the method comprising:
(a) training bees to exhibit a proboscis extension response when exposed to a selected volatile or semi-volatile organic compound (VOC/SVOC), the training comprising:
exposing a bee to background air;
exposing the bee to the selected VOC/SVOC; and
exposing the bee to said selected VOC/SVOC while stimulating an antennae of the bee with a sugar solution to produce a trained bee;
(b) exposing the trained bee to a sample; and
(c) observing a response of the trained bee to the exposure of the sample;
(d) wherein the presence of the selected VOC/SVOC from the sample will cause the trained bee to exhibit a proboscis extension response.
2. A method as recited in claim 1 , wherein said sugar solution comprises glucose and water.
3. A method as recited in claim 2 , wherein said sugar solution further comprises a biochemical supplement selected from the group consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine.
4. A method as recited in claim 1 , wherein said background air comprises a carrier gas containing compounds from nonspecific plants.
5. A method as recited in claim 1 :
wherein said background air comprises a carrier gas containing compounds from a healthy sample plant; and
wherein said sample comprises a potentially infected sample plant.
6. A method as recited in claim 1 , further comprising:
feeding a bee a pre-training solution of water, a sugar and a biochemical supplement selected from the group consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine.
7. A method as recited in claim 6 , wherein said biochemical supplement comprises a combination of at least two supplements selected from the group of supplements consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine.
8. A method as recited in claim 1 , further comprising:
verifying the training of the trained bee by exposing the bee to the selected VOC/SVOC.
9. A method as recited in claim 1 , further comprising:
verifying the training of the trained bee by exposing the bee to the selected VOC/SVOC; and
reinforcing the training of the bee by administering a sugar reward.
10. A method of conditioning honeybees to respond to olfactory stimulus of interest, comprising:
collecting bees for conditioning;
feeding the collected bees a solution of water, sugar and a biochemical supplement selected from the group consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine;
starving the bees;
exposing the bees to an olfactory stimulus of interest; and
applying a sugar solution to at least one antennae of a bee during exposure of the bee to the olfactory stimulus of interest;
wherein a bee associates the olfactory stimulus of interest with the application of sugar reward and exhibits a proboscis extension response when exposed to the olfactory stimulus of interest.
11. A method as recited in claim 10 , wherein said sugar comprises glucose and said sugar solution comprises glucose and water.
12. A method as recited in claim 11 , wherein said sugar solution further comprises a biochemical supplement selected from the group consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine.
13. A method as recited in claim 10 , wherein said biochemical supplement comprises a combination of at least two supplements selected from the group of supplements consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine.
14. A method as recited in claim 10 , further comprising:
repeating said exposure of the bees to an olfactory stimulus of interest and said application of a sugar solution to at least one antennae of the bee during exposure of the bee to the olfactory stimulus of interest steps more than one time.
15. A method as recited in claim 10 , further comprising:
exposing the honeybees to compounds emanating from a host-specific plant pathogen infection olfactory stimulus of interest within a carrier gas containing compounds from a uninfected host plant.
16. A method as recited in claim 10 , further comprising:
verifying the training of the trained bee by exposing the bee to the selected olfactory stimulus of interest.
17. A method as recited in claim 10 , further comprising:
verifying the training of the trained bee by exposing the bee to the selected olfactory stimulus of interest; and
reinforcing the training of the bee by administering a sugar reward.
18. A method for detecting agricultural volatile and semi-volatile organic compounds, the method comprising:
(a) training bees to exhibit a proboscis extension response when exposed to a selected volatile or semi-volatile organic compound (VOC/SVOC), the training comprising:
feeding collected bees a solution of water, glucose and a biochemical supplement selected from the group consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine;
exposing a bee to background air;
exposing the bee to the selected VOC/SVOC; and
exposing the bee to said selected VOC/SVOC while stimulating an antennae of the bee with a solution of water, glucose and a biochemical supplement selected from the group consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine to produce a trained bee;
(b) exposing the trained bee to a sample; and
(c) observing a response of the trained bee to the exposure of the sample;
(d) wherein the presence of the selected VOC/SVOC from the sample will cause the trained bee to exhibit a proboscis extension response.
19. A method as recited in claim 18 , wherein said background air comprises compounds emanating from a host-specific plant pathogen infection, within a carrier gas containing compounds from healthy nonspecific plants.
20. A method as recited in claim 18 , wherein said biochemical supplement comprises a combination of at least two supplements selected from the group of supplements consisting of: caffeine, juvenile hormone III, octopamine, protein kinase A (PKA), protein kinase C (PKC), cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), nitric oxide, and nicotine.