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The bite of the honeybee: 2-heptanone secreted from honeybee mandibles during a bite acts as a local anaesthetic in insects and mammals.

Papachristoforou A, Kagiava A, Papaefthimiou C, Termentzi A, Fokialakis N, Skaltsounis AL, Watkins M, Arnold G, Theophilidis G - PLoS ONE (2012)

Bottom Line: We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels.Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone.Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece. papachri@legs.cnrs-gif.fr

ABSTRACT
Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation-the isolated rat sciatic nerve-the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

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The evoked nCAP from the isolated rat sciatic nerve exposed to saline and 2-H. a) The amplitude of the nCAP, baseline to peak, was used as the main parameter to quantify the vitality of the sciatic nerve fibres during exposure to 2-H. The record presents the decrease in the amplitude nCAP during exposure of the sciatic nerve to 3.41 mg/mL 2-H. The first arrow indicates the beginning of the application and the second arrow indicates the time when the nerve was washed and bathed in normal saline. During exposure to 2-H, records were taken at a rate of 1 nCAP per 20s. After 2-H was replaced with normal saline, measurements were taken every 15 min. Vertical scale bar: 3 mV. Horizontal scale bar: 6 ms. b). Diagrammatic representation of the three-chamber recording bath made of Plexiglass. It consists of the recording (R), the perfusion (P) and the stimulating chambers (S), separated by two partitions. The sciatic nerve was placed along the three chambers which were filled with oxygenated saline to cover the nerve. The dimensions of each chamber were 26×26×10 mm (length. width, depth), total volume 10 mL. The cover (c) made of Plexiglass was used to close hermitically the whole recording system, while the air inside the bath was saturated with 2-H, to eliminate the evaporation of 2-H in the perfusion chamber.
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pone-0047432-g005: The evoked nCAP from the isolated rat sciatic nerve exposed to saline and 2-H. a) The amplitude of the nCAP, baseline to peak, was used as the main parameter to quantify the vitality of the sciatic nerve fibres during exposure to 2-H. The record presents the decrease in the amplitude nCAP during exposure of the sciatic nerve to 3.41 mg/mL 2-H. The first arrow indicates the beginning of the application and the second arrow indicates the time when the nerve was washed and bathed in normal saline. During exposure to 2-H, records were taken at a rate of 1 nCAP per 20s. After 2-H was replaced with normal saline, measurements were taken every 15 min. Vertical scale bar: 3 mV. Horizontal scale bar: 6 ms. b). Diagrammatic representation of the three-chamber recording bath made of Plexiglass. It consists of the recording (R), the perfusion (P) and the stimulating chambers (S), separated by two partitions. The sciatic nerve was placed along the three chambers which were filled with oxygenated saline to cover the nerve. The dimensions of each chamber were 26×26×10 mm (length. width, depth), total volume 10 mL. The cover (c) made of Plexiglass was used to close hermitically the whole recording system, while the air inside the bath was saturated with 2-H, to eliminate the evaporation of 2-H in the perfusion chamber.

Mentions: Because the in vitro experiments indicated that 2-H was 2.82 times less effective than lidocaine for hNav1.6 channels, we used an ex vivo preparation–the rat sciatic nerve fibres isolated in a three-chamber recording bath (Fig. 5 b)–to compare the local anaesthetic action of 2-H and lidocaine. The nerve compound action potential (nCAP, Fig. 5a) was used as an index of nerve fibre viability in the recording bath. The advantage of the recording chamber is that the nCAP amplitude remained constant for over 24 h for nerves incubated in saline and stimulated continuously using normal (1 Hz) supramaximal stimulation. After 24 h of continuous stimulation, the amplitude of the nCAP began to gradually decrease due to natural nerve-fibre inactivation [19]. When the nerve fibres were incubated in saline with 3.41 mg/mL 2-H, there was a drastic decrease in the amplitude of the nCAP (Fig. 5, first arrow), and the nCAP was eliminated completely within 220 to 230 s, indicating that all sciatic nerve fibres were inactivated. In this case, the chambers with the saline (the nerves and the recording electrodes) were closed hermetically, using a cover (Fig. 5 b), to prevent evaporation of 2-H. At this stage, the nerves recovered quickly once the sciatic nerve fibres were washed out and the 2-H/saline mixture was replaced with normal saline (Fig. 5 a, second arrow).


The bite of the honeybee: 2-heptanone secreted from honeybee mandibles during a bite acts as a local anaesthetic in insects and mammals.

Papachristoforou A, Kagiava A, Papaefthimiou C, Termentzi A, Fokialakis N, Skaltsounis AL, Watkins M, Arnold G, Theophilidis G - PLoS ONE (2012)

The evoked nCAP from the isolated rat sciatic nerve exposed to saline and 2-H. a) The amplitude of the nCAP, baseline to peak, was used as the main parameter to quantify the vitality of the sciatic nerve fibres during exposure to 2-H. The record presents the decrease in the amplitude nCAP during exposure of the sciatic nerve to 3.41 mg/mL 2-H. The first arrow indicates the beginning of the application and the second arrow indicates the time when the nerve was washed and bathed in normal saline. During exposure to 2-H, records were taken at a rate of 1 nCAP per 20s. After 2-H was replaced with normal saline, measurements were taken every 15 min. Vertical scale bar: 3 mV. Horizontal scale bar: 6 ms. b). Diagrammatic representation of the three-chamber recording bath made of Plexiglass. It consists of the recording (R), the perfusion (P) and the stimulating chambers (S), separated by two partitions. The sciatic nerve was placed along the three chambers which were filled with oxygenated saline to cover the nerve. The dimensions of each chamber were 26×26×10 mm (length. width, depth), total volume 10 mL. The cover (c) made of Plexiglass was used to close hermitically the whole recording system, while the air inside the bath was saturated with 2-H, to eliminate the evaporation of 2-H in the perfusion chamber.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472974&req=5

pone-0047432-g005: The evoked nCAP from the isolated rat sciatic nerve exposed to saline and 2-H. a) The amplitude of the nCAP, baseline to peak, was used as the main parameter to quantify the vitality of the sciatic nerve fibres during exposure to 2-H. The record presents the decrease in the amplitude nCAP during exposure of the sciatic nerve to 3.41 mg/mL 2-H. The first arrow indicates the beginning of the application and the second arrow indicates the time when the nerve was washed and bathed in normal saline. During exposure to 2-H, records were taken at a rate of 1 nCAP per 20s. After 2-H was replaced with normal saline, measurements were taken every 15 min. Vertical scale bar: 3 mV. Horizontal scale bar: 6 ms. b). Diagrammatic representation of the three-chamber recording bath made of Plexiglass. It consists of the recording (R), the perfusion (P) and the stimulating chambers (S), separated by two partitions. The sciatic nerve was placed along the three chambers which were filled with oxygenated saline to cover the nerve. The dimensions of each chamber were 26×26×10 mm (length. width, depth), total volume 10 mL. The cover (c) made of Plexiglass was used to close hermitically the whole recording system, while the air inside the bath was saturated with 2-H, to eliminate the evaporation of 2-H in the perfusion chamber.
Mentions: Because the in vitro experiments indicated that 2-H was 2.82 times less effective than lidocaine for hNav1.6 channels, we used an ex vivo preparation–the rat sciatic nerve fibres isolated in a three-chamber recording bath (Fig. 5 b)–to compare the local anaesthetic action of 2-H and lidocaine. The nerve compound action potential (nCAP, Fig. 5a) was used as an index of nerve fibre viability in the recording bath. The advantage of the recording chamber is that the nCAP amplitude remained constant for over 24 h for nerves incubated in saline and stimulated continuously using normal (1 Hz) supramaximal stimulation. After 24 h of continuous stimulation, the amplitude of the nCAP began to gradually decrease due to natural nerve-fibre inactivation [19]. When the nerve fibres were incubated in saline with 3.41 mg/mL 2-H, there was a drastic decrease in the amplitude of the nCAP (Fig. 5, first arrow), and the nCAP was eliminated completely within 220 to 230 s, indicating that all sciatic nerve fibres were inactivated. In this case, the chambers with the saline (the nerves and the recording electrodes) were closed hermetically, using a cover (Fig. 5 b), to prevent evaporation of 2-H. At this stage, the nerves recovered quickly once the sciatic nerve fibres were washed out and the 2-H/saline mixture was replaced with normal saline (Fig. 5 a, second arrow).

Bottom Line: We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels.Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone.Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece. papachri@legs.cnrs-gif.fr

ABSTRACT
Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation-the isolated rat sciatic nerve-the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.

Show MeSH
Related in: MedlinePlus