WO2011033283A1

WO2011033283A1 - Insect conditioning system

Info

Publication number: WO2011033283A1
Application number: PCT/GB2010/051494
Authority: WO
Inventors: Mathilde Briens
Original assignee: Inscentinel Limited
Priority date: 2009-09-17
Filing date: 2010-09-08
Publication date: 2011-03-24
Also published as: GB0916330D0; GB2473627A

Abstract

An insect conditioning apparatus for conditioning insects to exhibit a measurable response to a target odour, comprising a plurality of insect holders for restraining an insect of a particular type, a means for presenting a food source to the insects, a first means for exposing a first gas stream to the insects, a second means for exposing a second gas stream to the insects and a third gas stream for exposing a third gas stream to the insects.

Description

Insect Conditioning System

Field of the Invention

The invention relates to an apparatus and method of conditioning a plurality of insects, particularly forager honeybees, to respond to a target odour for subsequent use in the detection of such odours.

Background

The use of insects, particularly of bees, for detection of odours is well-known. Insects including bees, wasps, moths, aphids etc. have much greater sensitivity to odours than the best physical analytical techniques including gas chromatography.

Such insects can be trained or conditioned to respond with high accuracy and specificity to a specific odour in a detectable manner e.g. by measuring electrical signals in antennae (electroantennography) or observing physical movements, such as proboscis extension reflex.

WO 03/056292 discloses an apparatus and system of using insects to detect odours whilst restrained in an insect holder which is removably associated with a detector apparatus. The removable nature of the holder allows the insects to be raised and conditioned at one location and transferred to another location where the detector unit is located.

However, insects for use in such detection schemes must be carefully treated with particular attention paid to their training regime and handling in order that accurate odour detection can be achieved.

A well-known method of conditioning insects, e.g. forager honeybees, so that they respond to a target odour is by a Pavlovian conditioning method. This involves presenting the insect with a food reward simultaneously with the target odour causing the insect to associate the two. Once conditioned, insects will respond when exposed to the target odour, e.g. by extending their proboscis in expectation of receiving food. This physical movement can be monitored and used to measure the detection of a target odour.

However, a difficulty with such conditioning is that great care must be taken that the insects are not inadvertently conditioned to respond to other stimuli present during conditioning. For example, if a gas containing the target odour is passed over the insect during conditioning then the insect may inadvertently associate the presentation of food with the sensation of gas being blown across the insect instead of with the target odour.

When insects are being conditioned for research purposes when volumes of insects are generally low, it is relatively straightforward to take steps to remove such associated stimuli by careful manual handling of the insects. For example, to avoid the above- mentioned difficulty it is generally known that insects must receive gas passed over them without any odour before exposing them to the gas containing the odour and the food reward.

However, known methods of conditioning insects to respond to a target odour also involve a significant amount of human input which inevitably introduces variation when conditioning a plurality of insects. Additionally, involving manual handling inevitably limits the rate at which insects can be conditioned.

Furthermore, practical application of insects in an odour detection system, e.g. for use in airports, demands that a large number of insects are used in order that detection is reliable. Additionally, because any given insect is conditioned for a single target odour at a time, many insects may need to be employed to reliably detect a range of odours.

Given the shortcomings of known conditioning methods, it would be highly desirable to devise a system for automatically conditioning a plurality of insects. However, when a plurality of such insects are to be conditioned then the design of an apparatus for automating the procedure presents a particular difficulty. It has been found that regardless of how carefully such an apparatus is manufactured, stimuli associated with the exposure to the target odour, such as the noise and vibration of valve movements, can not be eliminated entirely.

Thus it seems inevitable that such an apparatus will always produce insects exhibiting more false positive and more false negative responses compared to insects conditioned by known manual handling methods.

Summary of the Invention

In a first aspect, the invention relates to an insect conditioning apparatus for conditioning insects to exhibit a measurable response to a target odour, comprising a plurality of insect holders for restraining an insect of a particular type, a means for presenting a food source to the insects, a means for detecting a measurable response from the insects, a first means for exposing a first gas stream to the insects, a second means for exposing a second gas stream to the insects and a third gas stream for exposing a third gas steam to the insects.

The inventors have found that by arranging for a third means for exposing a third gas stream allows for a solution to the problem of associated mechanical stimuli being learned by the insects. By arranging for two gas streams to be "clean" and free of target odour while a third one contains the target odour, during automated conditioning, a first clean gas stream is exposed to the insects followed by exposing a second clean gas stream. This inevitably involves associated stimuli involved in the apparatus switching from one gas stream to another. Importantly no food is presented to the insects in this phase. Following this the insects are exposed to the gas stream containing the target odour and are presented with food.

Thus, the insects are exposed to the stimuli associated with a change of gas supply without exposure to the target odour and without being rewarded with food. This therefore teaches the insects that the associated stimuli are not correlated with the presentation of food and instead that it is the target odour alone which is associated with the food.

Thus, in a second aspect, the invention relates to a method of conditioning a plurality of insects of a particular type to exhibit a measurable response when exposed to a target odour, the method including the steps of exposing the insects to a first gas stream which does not contain the target odour, followed by exposing the insects to a second gas stream which does not contain the target odour, followed by simultaneously exposing the insects to a third gas stream containing the target odour and a food source.

The invention is applicable to a wide range of different insects, with the insect holders being designed and dimensioned to hold an insect of a particular type. The invention finds particular application to forager honeybees (Apis mellifera).

The invention enables a plurality of insects to be conditioned simultaneously, for example at least three, preferably at least six.

The invention may involve greater than three gas streams, however three streams are all that is necessary and so this is preferred.

The apparatus is typically encased in a gas tight housing which permits the gas streams to enter and leave but otherwise remaining air tight. This assists in preventing any surrounding odours from interfering with the conditioning process.

A wide range of types of measurable response can be employed, however proboscis extension reflex has been found to be a convenient and reproducible response.

The insects are held in respective insect holders so that they are restrained and held in place. Typically their head is exposed so that its proboscis extension can be detected and the food source can be delivered conveniently. Suitable insect holders are described in WO 03/055301. The insects in their holders may be arranged in a variety of ways, for example in a circle or in a linear array. The conditioning apparatus, in a preferred embodiment comprises a holding unit, as described in WO 03/056292 which holding unit comprises a plurality of insect holders. Such a holding unit can then be removed following conditioning and placed in a detector unit.

The gas streams are arranged so that they pass over the head of the insects so that each insect is exposed to the same gas simultaneously.

Typically the gas streams are provided by a system of pipes and valves. As it is required that the flow rate of gas remains unchanged throughout the conditioning process, the arrangement must be such as to achieve this. However, a small amount of random variation in the total flow rate could be beneficial in hiding the inevitable variation cased when switching between gas streams.

Thus, preferably the three gas streams each flow continuously with only one at a time being diverted onto the insects. Typically the other two will continue to flow but will be directed to a dump instead of passing over the insects. In another arrangement, the three gas streams each flow continuously with two at a time being diverted onto the insects whilst the third continues to flow and directed to a dump.

Typical flow rates of gas from each stream are sufficient to provide an air flow rate over the insects of from 0.5 to 5.0 ms^-1, preferably from 1.0 to 3.0 ms^-1.

The food source is typically a sucrose solution, although other similar foods can be envisaged. The food source may be delivered in a number of different ways such as passing wells of food in front of the insects along a conveyor belt manner, exposing wells of food by uncovering a closure or by moving a food well or individual spoons to the location of the insects head so the insect can feed and be stimulated to do so by the contact of the sucrose with its mouthparts and antennae.

In a preferred embodiment, the insects are exposed to more than two clean gas streams before being exposed to the target gas stream. The clean gas streams exposure stage may involve cycling two clean gas streams a number of times before then exposing the target gas stream and rewarding with food. This may be achieved by switching between two clean gas streams and thus maintaining a constant flow of clean gas. Alternatively one clean gas stream may flow continuously whilst a second clean gas stream is directed to the insects periodically, exposing the insects to a varying clean gas flow rate.

During conditioning, a gas stream, whether clean or containing the target odour or both, is typically exposed to the insects for from 1 to 30 seconds, preferably from 3 to 15 seconds. Each gas stream may be exposed to the insects for the same duration, cycling at regular intervals, or the intervals may be different or randomised, as desired. As discussed above, in some embodiments one clean gas stream may flow continuously without altering its flow rate.

The measurable response, e.g. proboscis extension reflex, can be measured in a variety of ways. For example Doppler radar, image analysis, or by including an electromagnetic radiation transmitter and receiver such that a physical movement breaks the radiation beam indicating a positive response, as described in GB 0817868.3.

The conditioning regime is preferably controlled by a programmable microcontroller. The measured responses are typically collected in electronic form and stored for analysis.

The invention will now be illustrated with reference to the following figures, in which:-

Figure 1 is a system layout of an apparatus according to the present invention.

Figure 2 is a photograph of a holding unit for use in conditioning apparatus according to the invention.

Turning to the figures, Figure 1 shows a system layout 10 of a conditioning apparatus according to the invention. The apparatus 10 includes a forager honey bee holding unit 12 to which is connected a gas inlet stream 14 and from which is connected a gas outlet stream 16. The gas outlet stream 16 passes via a flow meter 18 to a fan 20 and then to dump 22.

Feeding the gas inlet stream 14 and three gas streams 24, 26, 28 each having a respective valve 30, 32, 34 before merging with gas inlet stream 14. The valves 30, 32, 34 are arranged such that any of streams 24, 26, 28 can be directed to inlet stream 14 or be directed via fan 36 to dump 38. As is shown in Figure 1, valve 34 is directing stream 28 to inlet stream 14 whilst valves 30, 32 are directing streams 24, 26 to dump 38.

Gas streams 26 and 28 are clean, in that they contain no target odour, and stream 24 contains the target odour.

Gas streams 24 and 26 have the same flow rate.

A feeding mechanism 40 is provided to deliver the food when the insects are exposed to target odour stream 24.

A microcontroller 42 is provided to control the timings and activation of valves 30, 32, 34 and feeding mechanism 40. It is also responsible for controlling the LED brightness and to record the measured photodiode signal to indicate whether or not a proboscis extension has occurred or not.

In use the fans 36 and 20 are activated and microcontroller 42 positions valve 34 to direct clean stream 28 to inlet stream 14 to achieve the arrangement shown in Figure 1. Valves 30 and 32 are positioned to direct streams 24 and 26 to dump 38. The clean air passes over the bees in the bee holding unit and exits the holding unit to dump 22.

After a pre-set period of time, microcontroller 42 simultaneously switches valves 34 and 32 to simultaneously direct clean gas stream 28 to dump 38 and to direct clean gas stream 26 to inlet stream 14. The microcontroller may continue to simultaneously switch valves 34 and 32 to switch between the clean gas streams as many times as is desired.

The microcontroller then switches valve 30 and valve 34 simultaneously to direct target odour stream 24 to inlet stream 14. The microcontroller also simultaneously activates feeding mechanism 40 to deliver the food source to the bees. Any proboscis extension, as measured by variation in the received photodiode signal is recorded.

The process may then begin again and repeat as many times as desired until the bees are sufficiently trained.

In an alternative mode of operation fans 36 and 20 are activated and microcontroller 42 positions valve 34 to direct clean stream 28 to inlet stream 14. As before, valves 30 and 32 are initially positioned to direct streams 24 and 26 to dump 38. The clean air passes over the bees in the bee holding unit and exits the holding unit to dump 22.

After a preset period of time, microcontroller switches valve 32 to simultaneously direct clean gas streams 26 and 28 to inlet stream 14, thus increasing the flow rate of clean air passed over the bees.

The microcontroller may continue to switch valves 32 or 34 to direct one or both of clean streams to inlet 14.

The microcontroller then activates valve 30 whilst clean gas stream 28 is directed to inlet stream 14. Gas stream 24 containing the target odour then mixes with clean gas stream 28 and the mixed stream flows to inlet gas stream 14.

Other ways of switching the flow rates may be envisaged which achieve the same result as those described herein.

Figure 2 shows a holding unit 50 for use in a conditioning apparatus according to the invention. The holding unit 50 comprises a plurality of insect holders 52 each of which contains a forager honey bee 54. The bees 54 are held in position so that they cannot move the location of their bodies but with their head exposed.

The holding unit 50 comprises an inlet gas port 56 and an outlet gas port 58. Transmitters 62 are provided mounted on a printed circuit board 64. Receivers 66 are provided on the insect holders.

In use, bees are captured and placed into a respective insect holder 52. The insect holders are then snap-fit into place on the holding unit 50. The holding unit is then fitted in place within a conditioning unit housing (not shown). During conditioning gas enters gas inlet port 56, passes over the bees 54 and exits through gas outlet port. Proboscis extension is monitored by variations in the signal received from receiver 66.

Following conditioning the holding unit 50 is removed from the conditioning unit and the trained insects are placed in a detector unit for subsequent detection of odours.

Claims

1. An insect conditioning apparatus for conditioning insects to exhibit a measurable response to a target odour, comprising a plurality of insect holders for restraining an insect of a particular type, a means for presenting a food source to the insects, a first means for exposing a first gas stream to the insects, a second means for exposing a second gas stream to the insects and a third gas stream for exposing a third gas stream to the insects.

2. A method of conditioning a plurality of insects of a particular type to exhibit a measurable response when exposed to a target odour, the method comprising employing a conditioning apparatus according to claim 1 and including the steps of exposing the insects to a first gas stream which does not contain the target odour, followed by exposing the insects to a second gas stream which does not contain the target odour, followed by simultaneously exposing the insects to a third gas stream containing the target odour and a food source.

3. An apparatus according to claim 1, or a method according to claim 2, wherein the apparatus is encased in a gas tight housing which permits the gas streams to enter and leave but otherwise remaining air tight.

4. An apparatus or method according to any one of the preceding claims wherein the insects are forager honeybees (Apis mellifera).

5. An apparatus or method according to any one of the preceding claims, wherein at least three, preferably at least six, insects are conditioned simultaneously.

6. An apparatus or method according to any one of the preceding claims wherein the measurable response is proboscis extension reflex.

7. An apparatus or method according to any one of the preceding claims wherein the apparatus comprises a holding unit, which holding unit comprises a plurality of insect holders.

8. An apparatus or method according to any one of the preceding claims, wherein the gas streams are arranged so that they pass over the insects so that each insect is exposed to the same gas simultaneously.

9. An apparatus or method according to any one of the preceding claims wherein the three gas streams are arranged to each flow continuously with only one at a time being diverted onto the insects.

10. An apparatus or method according to any one of claims 1 to 8, wherein the three gas streams are arranged to each flow continuously with two at a time being diverted onto the insects.

11. An apparatus or method according to any one of the preceding claims, wherein the insects are arranged to be exposed to more than two clean gas streams before being exposed to the target gas stream.