Addiction of Insects for Biosensoring

Periodic Reporting for period 1 - ActIng (Addiction of Insects for Biosensoring)

Période du rapport: 2015-08-10 au 2017-08-09

The olfactory systems of animals have long been considered useful for detecting volatile compounds in the environment. This Marie Sklodowska Curie project is based on a totally new approach exploiting the associative learning and in particular to the Pavlov’s Classical Conditioning to implement next generation biosensors.
The innovative idea of the project ActIng consists in the modification of the motivation priorities of the insects by means of the administration of drugs of abuse through the creation of an Artificial Unconditioned Stimulus consisting in the withdrawal symptoms of an addicted condition that will be associated with an originally aversive or neutral volatile organic compound (VOC) as a Conditioned Stimulus (e.g. the odours of blood or explosives or other marking volatiles) to obtain a reliable and highly motivated biosensor. As a result, the insect biosensor should focus on the target VOC that has been associated with the intake of the drug. The German cockroach, Blatella germanica was chosen as model insect.

The first step was the identification of one or more psychoactive compounds and on the definition of withdrawal symptoms.The drugs selected in the project were:
• Benzene
• Chloral Hydrate
• Nicotine
• Tobacco smoke particulate matter extract (TSP)
• Heroin
• Methadone
The studies were undertaken using only males and was carried out through a common structure comprising three phases:
1. Training. Repeated administration of food integrated with the psychoactive compounds;
2. Stabilization phase consisting in starvation of the insects or in restoring of the control diet or in the maintenance of the treatment;
3. Choice test. Verification of the establishment of addiction by choice test.

The administration of drug laced food was carried out using the CaFe (Capillary Feeding) assay adapted to cockroach feeding (figures 1 A and B).
A. Chloral Hydrate
CHY did not elicit any positive appetitive response. Control food was preferred to CHY in all conditions.
B. Benzene
Control food was preferred to Benzene BNZ in all conditions. Benzene was administered as vapour and the absorption is achieved by the respiratory system. Treated insects, assayed in a two-way olfactometer, tend to actively avoid the BNZ even if specifically trained.
C. Nicotine (NIC) and tobacco smoke particulate matter extract (TSP)
The experiments on tobacco related compounds showed that the nicotine alone is not addictive. On the contrary, the data show that there is a marked preference for the TSP laced food and that there are cues that TSP induce addiction. However, no marked withdrawal symptoms were observed under the behavioural point of view (Figures 2, 3, 4).
These findings opened the way to further questions.
1 Nicotine, which is expected to be the most important addictive tobacco component, is efficiently metabolized by the cockroaches.
2 To what extent the olfaction is involved in the preference expressed for the TSP
3 Other compounds besides nicotine contribute to the development of preference/addiction for TSP.
D. Haemolimph analysis
Nicotine was injected in the haemocoel of the cockroaches (figure 5) and haemolymph was analyzed by GC. The concentration of NIC in the haemolymph decreases rapidly (figure 6). This result suggests a significant detoxification process is in place to eliminate the nicotine.
E. Olfactometer assays
The second question arising from the preference experiments was if the preference for TSP was driven by olfaction or by addiction and to what extent. An olfactometry experiment was necessary to clarify whether treatments trigger the onset of addiction for the TSP. This set of experiments was achieved using a two-way olfactometer (figure 7). Data indicated that insects do not show any significant preference for the TSP or for Control food odour regardless the training (Figure 8) so cockroaches should eat the TSP food in order to express a preference leading to think that an addictive process could have been primed.
F. TSP fractions
The third question was if other compounds in the TSP extract besides nicotine are involved in the preference for the TSP. The TSP ethanolic extract was fractionated (figure 9) and two fractions (A and B) were obtained. Nicotine is present in the fraction B.
Results showed in figure 10 (A, B, C, and D) confirmed that fraction B, and fractions A+B laced food were preferred over the control food proving that other compounds contained in the tobacco smoke are essential for the onset of nicotine addiction.
G. Heroin and Methadone (preliminary results)
Treatments with Heroin (HER) and methadone (MET) showed that both heroin and methadone were preferred over the control food at the lower concentrations (figures 11 A and B) during all the experimental period (figure 12 and 13). A significant increase in the aggressivity was observed among the individuals after the administration of both HER and MET.
It was observed an increasing uptake of HER laced food at the highest concentration, a typical sign of the onset of an addicted status.
After a 24 hours starvation period, the insects treated with the highest concentration of both MET and HER were dead or moribund. The moribund insects have injected with HER or MET solutions. Results showed that most of the treated insects recovered completely after injections. An example of recovery after injection is shown in Video 1.
Summarizing the results obtained, it can be concluded that:
1. Chloral Hydrate and Benzene did not elicit any positive appetitive response;
2. Nicotine is not an addictive compound for male cockroaches;
3. The cockroach’s detoxification system rapidly metabolizes nicotine;
4. The tobacco smoke particulate matter extract triggers interest in male cockroaches;
5. The TSP induces addiction-like effects detectable from the alteration of the feeding habits;
6. The olfaction is not involved in the preference for the TSP.
7. Cockroaches must eat the TSP to show the preference;
8. The effects induced by TSP are likely due to non-polar compounds that could act alone or in synergy with nicotine;
9. Heroin and to a lesser extent methadone are addictive for male cockroaches.

Defining and understanding the reaction of insects to different environmental stimuli associated with addiction-inducing chemicals could open new technological insights in the use of insects for beneficial purposes and will generate new approaches for interpretation of insect brain physiology.
Due to the highly speculative nature of this project, the objectives were not completely accomplished in the period covered by the Marie Sklodowska Curie fellowship. Withdrawal symptoms were identified for heroin and, to a lesser extent, for the methadone and the TSP extract. Apparently, nicotine does not induce any appreciable effect on cockroaches.
The phase of association was not achieved because of the difficulty in finding a clearly recognizable state of drug addiction that eventually was found for HER. Another critical issue was the difficulty to obtain the required licence to work with heroin and methadone, that required more than a half of the total period covered by the fellowship.
Nevertheless, the work done opened new insights on the addiction processes among insects with regard to nicotine, tobacco smoke, heroin, and methadone and allow to progress to the phase of association.

4 - Preference rate of TSP trained males between Control and TSP laced food in the choice phase

12 - Consumption of food in the five different MET treatment groups.

8 -Ternary diagrams. Integration of the time spent by insects in the three arms of the olfactometer

3 - Preference rate of NIC trained males between Control and NIC laced food in the choice phase

13 - Consumption of food in the five different HER treatment groups.

10 - Preference rate of males trained with Control, fraction A, fraction B and fractions A+B

2 - Preference rate between Control and NIC/TSP in the choice phase regarding control trained males

5 - Injection of NIC in the haemocoel of a male cockroach.

9 - HPLC profile of the TSP extract. Fractions A and B

11 - Daily individual consumption of food of methadone (A) and heroin (B) laced food

7 - Structure of the two-way olfactometer used in the assay.

1 - A - Round plastic cage used for the choice phase of the experiments B – CaFe assay.

6 - GC analysis of the haemolymph of male cockroaches.

Periodic Reporting for period 1 - ActIng (Addiction of Insects for Biosensoring)

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