title / The use of super-ligands to disrupt pheromone communication systems
/ DEFRA
project code / VC0417
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
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Project title / The use of super-ligands to disrupt pheromone communication systems
DEFRA project code / VC0417
Contractor organisation and location / Central Science Laboratory
Sand Hutton
York
Total DEFRA project costs / £ 108,396
Project start date / 01/07/01 / Project end date / 31/03/03
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 3
Projecttitle / The use of super-ligands to disrupt pheromone communication systems
/ DEFRA
project code / VC0417
1. Defra has a statutory responsibility to secure safe, efficient and humane methods of controlling pests. Traditional methods of controlling mammalian pests such as poisoning and trapping are often ineffective, environmentally hazardous, socially unacceptable or uneconomic and there are increasing demands for effective, non-lethal approaches to be developed. Finding such alternatives is important for the future of effective and socially acceptable wildlife management.
2. Vertebrate semiochemicals (e.g. pheromones) play a subtle but significant role in controlling the behaviour and physiology of mammals. Rodents, because of their cryptic lifestyle, rely heavily on these signals for both communication and the co-ordination of reproductive and physiological development within the colony. In mice the pheromonal activity is mediated by complexes of biologically active small molecules (ligands) held within carrier proteins (Major Urinary Proteins; MUPs), which are released in large quantities in rodent urine.
3. This project’s predecessor, VC0411, used computational chemistry techniques (QSAR) to predict the binding strength of molecules (ligands) for the carrier, MUP, protein. This model has been used to identify molecules with greater affinity for the MUP core than its natural pheromonal cargo and hence have the predicted ability to displace those pheromones thereby disrupting this scent communication system of target species.
4. β ionone was identified as a relatively effective molecule for the displacement of pheromones from MUPs. Its ability to displace pheromones and our discovery that it readily contaminates untreated scent marks over a relatively wide area makes this a candidate material for disrupting scent mark communication although its true practical utility remains to be proven.
5. The model developed in project VC0411 requires further refinement to increase its effectiveness in predicting putative super-ligands. New and more powerful programmes are constantly under development and CSL has very recently acquired DRAGON which calculates a large number of varied molecular descriptors that can help design more potent ligands.
CSG 15 (Rev. 6/02) 3
Projecttitle / The use of super-ligands to disrupt pheromone communication systems
/ DEFRA
project code / VC0417
Scientific report (maximum 20 sides A4)
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CSG 15 (Rev. 6/02) 3
Projecttitle / The use of super-ligands to disrupt pheromone communication systems
/ DEFRA
project code / VC0417
1. Introduction
Nocturnal habits and a cryptic lifestyle have led to the evolution of olfaction as the major communication system in rodents. Mice mark territories and maintain social structure using urine in the form of scent marks (Hurst 1897). Repeated marking in one area often leads to the development of urine pillars. Mouse urine contains large quantities of protein known as MUPs (major urinary protein). MUPs are polymorphic and belong to the lipocalin family of proteins (Flower 1996); they facilitate the gradual release of volatile pheromones into the environment long after the signaller has left the area. They contain a calyx with a b-barrel structure that readily binds and transport hydrophobic ligands (Robertson et al. 1998).
Thiazole (2-sec-butyl-4,5-dihydrothiazole) and brevicomin (3,4-dihydro-exo-brevicomin) are two androgen-dependent pheromones (Robertson et al. 1993), which are found only in male mouse urine. They are known to promote inter-male aggression (Novotny et al. 1985) and to stimulate oestrus in female mice (Jemiolo et al. 1985). Disruption of the pheromone-urine complex using chemicals with a higher binding affinity (super-ligands) than the naturally occurring pheromones may provide a subtle means of manipulating problematic populations that rely heavily on urine based communication systems. A previous Defra funded project (VC0411) used QSAR (Quantitative Structure Activity Relationship) to explain the relationship between chemical characteristics of a molecule and its affinity for binding to MUPs. This study showed that increasing the negativity of the ionisation potential (HOMO) improved the binding of ligands to the pheromone-binding site.
Analysis of urine posts by GC-MS revealed quantities of menadione (vitamin K3), believed to have originated from mouse diet. Further analysis using fresh urine, showed that menadione competes with naturally occurring pheromones such as thiazole and brevicomin causing their rapid release from the MUP complex (Robertson et al. 1998). Behavioural observations showed that mice could detect subtle differences between whole urine and urine mixed with menadione (Hurst et al. 1998); their latency to approach displaced scent marks increased in response to the flux of pheromones released. Other studies (Cavaggioni et al. 1990) have shown that some naturally occurring volatiles also have the capacity to preferentially bind to MUP complexes, however their impact on pheromone release has not been quantified.
This study used mice as the model system and aimed to test the potential of the super-ligands identified by the QSAR in silico generated model, to preferentially bind to the MUPs, displacing the naturally bound pheromones thiazole and brevicomin and thereby disrupting the MUP-pheromone communication system.
2. Methods
· Molecular structure of ligands tested
Menadione
Vitamin K3Formula C11H8O2
Molecular weight 172.2
Yellow powder
Melting point 105 oC
No detectable odour
(Robertson et al. 1998) /
β Ionone
Formula C13H20O
Molecular weight 192.3
Light yellow liquid
Boiling point 229 0C
Violet odour
(Cavaggioni et al. 1990) /
Geosmin
Formula C12H22OMolecular weight 182.3
Liquid
Earthy odour
(Cavaggioni et al. 1990) /
Isobutyl methoxypyrazine
Formula C9H14N2O
Molecular weight 166.2
Liquid
Bell pepper odour /
Pseudoephedrine
Formula C19H15NOMolecular weight 165.2
White crystalline solid
Melting point 118 – 120 oC
No detectable odour /
Ergothioneine
Amino acidFormula C9H15N3O2S
Molecular weight 229.3
Solid
Melting point 255 – 259 oC
No detectable odour /
· Urine collection
Thirty Balb/c male mice were used for urine collection. They were singly housed in RB3 cages and maintained on a 12-hour light : dark cycle. The animals were maintained on a low vitamin K diet and water ad libitum. Urine was collected by bladder palpation, and stored in eppendorffs at –80oC until required.
· Pheromone extraction and GC-MS analysis
Each urine spot (15 µl urine and 15 µl ethanol) was washed with 3 x 90 µl water and the wash collected in an eppendorff. All eppendorffs used had been previously soaked for at least 24 hours in chloroform before use, to minimise leaching of plasticizers into the chloroform-extracted urine. Chloroform (100 µl) was added to the wash and the mixture vortexed for 10 min then left to stand for 1 hour. The samples were spun for 3 min at 1300 rpm and 70 µl of the chloroform layer removed and transferred to a glass sample vial. The sample was further diluted with 80 µl chloroform and 10 µl of the internal standard (hexachlorobenzene; 10 µg/ml) as an internal standard, to give a final sample volume of 160 µl. Vials were sealed with an airtight cap and stored at –20oC.
GC-MS analysis was used to measure the concentration of bound thiazole and brevicomin within the artificial scent marks. The instrument used was a Thermoquest GCQ – Thermoquest GC fitted with an ion trap detector, the column was run with a stream of helium at 40 cm/sec. Samples (1 µl) were injected into the column at an oven temperature of 50 oC and held for 1 min. The temperature was ramped at 10 oC/min to 160 oC followed by 25 oC/min to 280 oC and held for 3.2 min. The mass detector was run in full scan mode, with positive ion monitoring of 50 to 290 mass units and electron impact ionisation of 70 eV. As neither brevicomin nor thiazole was commercially available, the concentrations of bound pheromones were quantified by calculating their relative peak area in relation to a known internal reference standard (HCB). Mass spectra of super-ligands were compared to those obtained from analysis of authentic samples.
· Pheromone release profiles over a 24-hour period
Initial trials were conducted to determine the release profiles of thiazole and brevicomin over a 24-hour period. Artificial scent marks were made in the depressions of a porcelain dimple plates using 15 µl of urine mixed with 15 µl of ethanol and left to air dry for 0 (samples extracted after 3 min), 0.5, 1, 2, 4, 6, 8, 16, 20 and 24 hours, there were six replicates for each time point (Figures 1a & b). All samples were prepared for GC-MS analysis following the same procedure described above. The majority of pheromones were released within the first 10 hours; therefore all future analysis was conducted over a maximum of 6 hours.
Figure 1a: The 24-hour release profile for thiazole, calculated by reference to the peak area of an internal standard HCB.
Figure 1b: The 24-hour release profile for brevicomin, calculated by reference to the peak area of an internal standard HCB.
· Pheromone release in the presence and absence of super-ligands
Artificial scent marks were made in the depressions of a porcelain dimple plate using 15 µl of urine mixed with 15 µl super ligand (2.9 mM in ethanol). Control samples were run alongside; here urine was mixed with ethanol only. Samples were left under ambient conditions for 0 hours (samples extracted after 3 min) 0.5, 1, 2 and 6 hours; there were six replicates for each time point before extraction and analysis by GC-MS. Comparisons were made between treatment and controls at each time point using an independent t-test including a test for unequal variances. Analysis of covariance was used to compare the most effective super-ligands; pheromone levels at 0 hours in control samples were used as a covariate to compensate for the variation observed between artificial scent marks.
· No-choice test to assess the impact of pheromone release on mouse behaviour
Twelve singly housed male Balb/c mice were used throughout the course of the experiment and were given a no-choice test over 4 days to determine their response to pheromone super-ligand treated urine. Two aliquots (30 µl) of the test substance were placed in the central two wells of a porcelain dimple plate; this was covered in fine mesh to prevent mice from coming into direct contact with the artificial scent mark. Plates were left to air dry for 30 min, before being stuck to the wall of the test arena using Velcro strips. Feed hoppers and water bottles were removed from their home cage for the duration of the trial. Combinations of treatment were:
1. Blank (2 x 30 µl of ethanol)
2. Urine (2x (15 µl urine + 15 µl ethanol))
3. Urine & ligand (2 x (15 µl of urine + 15 µl of test ligand))
4. Ligand (2 x (15 µl ethanol and 15 µl of test ligand)
The order of presentation was randomly allocated and each animal was given one plate per day. Each mouse was marked with purple dye to allow rapid identification by video surveillance software and equipment. Whilst the test arena was built within the home cage, mice were held within their nest-box. The trial commenced when the mouse was released from the nest box. Each animal was videoed for 5 minutes after first entering the test arena. Food and water were returned to the home cage once the dimple plate was removed. Video footage was analysed using Ethovision™ software and the test subjects’ latency to approach the test arena and the scent mark area, the number of visits to each zone, and the time spent in each zone quantified. Comparisons between treatments were made using a one-way ANOVA.
Figure 2: Showing the design of the arena for a no-choice test
3. Results
· Pheromone release in the presence and absence of ligands
Results for thiazole and brevicomin levels for each super-ligand are shown in tables 1 to 4. Thiazole levels were significantly lower in menadione treated urine compared with untreated in all time points after 30 minutes (0.5 hours: t (10) = 4.97, P = 0.001; 1 hour: t (10) = 4.23, P = 0.002; 2 hours: t (5.77) = 6.06, P = 0.001; 6 hours: t (5.01) = 5.70, P = 0.002). Brevicomin levels were significantly lower in menadione treated urine compared with controls after 30 minutes (0.5 hours: t (10) = 7.15, P = 0.000; 1 hour: t (5.56) = 5.95, P = 0.001; 2 hours: t (5.12) = 9.59, P = 0.000; 6 hours: t (5) = 5.43, P = 0.003). β ionone (Figure 4a & b) also significantly reduced thiazole levels in treated urine after 30 minutes (0.5 hours: t (7.19) = 4.65, P = 0.002; 1 hour: t (10) = 5.05, P = 0.000; 2 hour: t (10) = 9.32, P = 0.000; 6 hour: t = (10) = 7.01, P = 0.000). β ionone significantly reduced brevicomin levels after 30 minutes (0.5 hour: t (5.31) = 8.70, P = 0.000; 1 hour: t (5.84) = 5.11, P = 0.002; 2 hour: t (9.24) = 6.71, P = 0.000; 6 hour: t (10) = 2.59, P = 0.027). All other super-ligands tested were not consistently effective over all time points.
Comparisons were made between menadione and β ionone using analysis of covariance to determine which super-ligand was most effective at displacing naturally occurring pheromones. Menadione displaced significantly more thiazole than β ionone over time (1 hour: F (1, 10) = 11.96, P = 0.009; 2 hour: F (1, 10) = 9.33, P = 0.016; 6 hour: F (1, 10) = 26.80, P = 0.001). Menadione displaced significantly more brevicomin after 0.5 and 1 hours than β ionone (F (1, 10) = 6.95, P = 0.03, F (1, 10) = 24.54, P = 0.01) respectively, after which any differences between super-ligands was not significant. There was no significant difference over time between pheromone release from control urine run along side menadione and β ionone treated urine.