- Access to this full-text is provided by Springer Nature.
- Learn more
Download available
Content available from Scientific Reports
This content is subject to copyright. Terms and conditions apply.
1
Vol.:(0123456789)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports
Fungi anaesthesia
Andrew Adamatzky1* & Antoni Gandia2
Electrical activity of fungus Pleurotus ostreatus is characterised by slow (h) irregular waves of baseline
potential drift and fast (min) action potential likes spikes of the electrical potential. An exposure of the
myceliated substrate to a chloroform vapour lead to several fold decrease of the baseline potential
waves and increase of their duration. The chloroform vapour also causes either complete cessation of
spiking activity or substantial reduction of the spiking frequency. Removal of the chloroform vapour
from the growth containers leads to a gradual restoration of the mycelium electrical activity.
Most living cells are sensitive to anaesthetics1–3. First experiments on anaesthesia of plants have be done by
Claude Bernard in late 1800s4. Later experiments on amoeba5 shown that weak concentration of narcotics causes
the amoebae to spread out and propagate in a spread condition while narcotic concentrations led to cessation
of movements. During last century the experimental evidences mounted up including anaesthesia of yeasts1,
various aquatic invertebrates6, plants3,7, protists8, bronchial ciliated cells9. A general consensus now is that any
living substrate can be anaesthetised3. e question remains, however, how exactly species without a nervous
system would respond to exposure to anaesthetics.
In present paper we focus on fungi anaesthesia. Why fungi? Fungi are the largest, most widely distributed, and
oldest group of living organisms10. Smallest fungi are microscopic single cells. e largest (15 hectares) mycelium
belongs to Armillaria gallica (synonymous with A. bulbosa, A. inata, and A. lutea)11 and the largest fruit body
belongs to Phellinus ellipsoideus (formerly Fomitiporia ellipsoidea) which weighs half-a-ton12.
Fungi exhibit a high degree of protocognitive abilities. For example, they are capable for ecient exploration
of conned spaces13–17. Moreover, optimisation of the mycelial network18 is similar to that of the slime mould
Physarum polycephalum19 and transport networks20. erefore, we can speculate that the fungi can solve the
same range of computational problems as P. polycephalum21, including shortest path22–26, Voronoi diagram27,
Delaunay triangulation, proximity graphs and spanning tree, concave hull and, possibly, convex hull, and, with
some experimental eorts, travelling salesman problem28. e fungi’s protocognitive abilities and computational
potential make them fruitful substrates for anaesthesia because they might show us how non-neuron awareness
is changing under eects of narcotics.
We use extracellular electrical potential of mycelium as indicator of the fungi activity. Action potential-like
spikes of electrical potential have been discovered using intra-cellular recording of mycelium of Neurospora
crassa29 and further conrmed in intra-cellular recordings of action potential in hyphae of Pleurotus ostreatus
and A. gallica30 and in extra-cellular recordings of fruit bodies of and substrates colonised by mycelium of P.
ostreatus31. While the exact nature of the travelling spikes remains uncertain we can speculate, by drawing analo-
gies with oscillations of electrical potential of slime mouldPhysarum polycephalum32–35, that the spikes in fungi
are triggered by calcium waves, reversing of cytoplasmic ow, translocation of nutrients and metabolites. Stud-
ies of electrical activity of higher plants can brings us even more clues. us, the plants use the electrical spikes
for a long-distance communication aimed to coordinate the activity of their bodies36–38. e spikes of electrical
potential in plants relate to a motor activity39–42, responses to changes in temperature43, osmotic environment44,
and mechanical stimulation45,46.
e paper is structured as follows. We present the experimental setup in “Methods” section. Analysis of the
electrical activity of the intact non-anesthetised and anaesthetised fungi is given in “Results” section. “Discus-
sion” section present some critique and directions for further research.
Methods
A commercial strain of the fungus Pleurotus ostreatus (collection code 21-18, Mogu S.r.l., Italy), previously
selected for its superior tness growing on the targeted substrate, was cultured on sterilised hemp shives con-
tained in plastic (PP5) lter patch microboxes (SacO2, Belgium) that were kept in darkness at ambient room
temperature c.22
◦
C. Aer one week of incubation, a hemp brick well colonised by the fungus was manually
crumbled and spread on rectangular fragments, c.
12
×
12 cm2
, of moisturised non-woven hemp pads. When
these fragments were colonised, as visualised by white and healthy mycelial growth on surface, they were used
for experiments.
OPEN
1Unconventional Computing Laboratory, UWE, Bristol, UK. 2Institute for Plant Molecular and Cell Biology,
CSIC-UPV, Valencia, Spain. *email: andrew.adamatzky@uwe.ac.uk
Content courtesy of Springer Nature, terms of use apply. Rights reserved
2
Vol:.(1234567890)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
Electrical activity of the colonised hemp pads was recorded using pairs of iridium-coated stainless steel
sub-dermal needle electrodes (Spes Medica S.r.l., Italy), with twisted cables and ADC-24 (Pico Technology,
UK) high-resolution data logger with a 24-bit A/D converter. To keep electrodes stable we have been placing
a polyurethane pad under the fabric. e electrodes were arranged in a line (Fig.1a,b). e pairs of electrodes
were pierced through the fabric and into the polyurethane pad.
e fungal substrates pierced with electrodes was placed into 20cm by 10cm by 10cm plastic boxes with
tight lids.
We recorded electrical activity at one sample per second. During the recording, the logger has been doing
as many measurements as possible (typically up to 600 per second) and saving the average value. We set the
acquisition voltage range to 156mV with an oset accuracy of 9
µ
V at 1Hz to maintain a gain error of 0.1%.
Each electrode pair was considered independent with the noise-free resolution of 17 bits and conversion time
of 60ms. Each pair of electrodes, called channels, reported a dierence of the electrical potential between the
electrodes. Distance between electrodes was 1-2cm. In each trial, we recorded eight electrode pairs, channels,
simultaneously.
To study the eect of chloroform we soaked a piece of lter paper c. 4cm by 4cm in chloroform (Sigma
Aldrich, analytical standard) and placed the piece of paper inside the plastic container with the recorded fungal
substrate.
e humidity of the fungal colonies was 70–80% (MerlinLaser Protimeter, UK). e experiments were con-
ducted in a room with ambient temperature 21
◦
C and in the darkness of protective growing tents (Fig.1c).
We have conducted ten experiments, in each experiments we recorded electrical activity of the fungi via eight
channels, i.e. 80 recordings in total.
Figure1. Experimental setup. (a, b)Exemplar locations of electrodes. (a)Top view. (b)Side view. (c)Setup in
the grow tent.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
3
Vol.:(0123456789)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
Results
Myceliated hemp pad exhibit patterns of electrical activity similar to that of spiking neural tissue. Examples of
action potential like spikes, solitary and in trains, are shown in Fig.2.
Application of the chloroform to the container with fungi substantially aected the electrical activity of the
fungi. An example of an extreme, i.e. where almost all electrical activity of mycelium seized, response is shown
in Fig.3. In this example, the introduction of the chloroform leads to the suppression of the spiking activity and
reduction of deviation in values of the electrical potential dierences recorded on the channels.
e intact, non-anesthetised, mycelium composite (control ) shows median amplitude of the irregular move-
ments of the baseline potential is 0.45mV (average 0.64mV,
σ=0.64
), median duration 29,850s (average
67,507s,
σ
=
29,850
). Aer exposure to chloroform the baseline potential movements show median amplitude
reduced to 0.16mV (average 0.18mV,
σ=0.12
) and median duration increased to 38,507s (average 38,114s,
σ
=
38,507
). For the eight channels (pairs of dierential electrodes) recorded exposure to chloroform led to
nearly three times decrease in amplitude of the dris of baseline potential and nearly 1.3 increase in duration
of the dris. Before exposure to chloroform the mycelium composite produced fast (i.e. less than 10–20min)
spikes. Median amplitude of the spikes was 0.48mV (average 0.52mV,
σ=0.2
). Median duration of spikes was
62s (average 63s,
σ=18
), median distance between the spike 214s (average 189s,
σ=90
). Aer exposure to
Figure2. Example of spiking activity recorded from a control sample; an intact, non-anesthetised, myceliated
hemp pad. Spikes are shown by arrows.
Figure3. An example showing how the electrical activity of fungi changes when chloroform is introduced. e
moment of the chloroform introduction is shown by arrow.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
4
Vol:.(1234567890)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
chloroform the mycelium composite did not show any spiking activity above level of background noise, which
was for this particular recording c. 0.05mV.
In some cases the spiking activity is diminished gradually with decreased frequency and lowered amplitude,
as exemplied in Fig.4. Typically, the intact (control) spiking frequency is a spike per 70min while aer inhala-
tion of chloroform a spike per 254min in the rst 40-50hours and decreased to nearly zero aer. e median
amplitude of intact mycelium spikes is 0.51mV, average 0.74mV (
σ
=0.59). Anaesthetised mycelium shows,
spikes with median amplitude 0.11mV, average 0.2mV (
σ=0.2
). Spikes are not distributed uniformly but
gathered in trains of spikes. In the intact mycelium there is a media of 3 spikes in the train, average number of
spikes is 4.2 (
σ=4.4
). Median duration of a spike train is 84min, average 112min (
σ=32
). Media interval
between trains is 53min, average 55s (
σ=29
). Anaesthetised mycelium emits trains with median number of
2 spikes, average 2.5 spikes, average 2.5 spikes (
σ=0.84
). A median duration of such trains is 29min, average
51min (
σ=22
). e trains appear much more rarely than the trains in the intact mycelium: median interval
between trains is 227min.
In all ten but one experiment the container remained closed for over 3–4days. By that time all kinds of
electrical activity in mycelium bound substrate extinguished and the mycelium never recovered to a functional
state. In experiment illustrated in Fig.5 we removed a source of chloroform aer 16h and kept the container
open and well ventilated for an hour to remove any traces of chloroform from the air. e intact mycelium shows
median frequency of spiking as one spike per 27min, average 24min. Median amplitude of the spikes is 3.4mV,
average 3.25mV (
σ=1.45
). e anaesthetised mycelium demonstrates electrical spiking activity reduced in
amplitude: median amplitude of spikes is 0.24mV, average 0.32mV (
σ=0.2
), and low frequency of spiking:
median distance between spikes is 38min, average 40min. Electrical activity of the mycelium restores to above
noise level c.60h aer the source of the chloroform is removed from the enclosure (insert in Fig.5). Frequency
of spikes is one spike per 82min (median), average 88min. e amplitudes of recovering spikes are 0.96mV
in median (average 0.93mV,
σ=0.08
) which are three times less than of the spikes in the mycelium before the
narcosis but nearly ve times higher than of the spike of the anaesthetised mycelium.
Discussion
We demonstrated that the electrical activity of the fungus Pleurotus ostreatus is a reliable indicator of the fungi
anaesthesia. When exposed to a chloroform vapour the mycelium reduces frequency and amplitude of its spik-
ing and, in most cases, cease to produce any electrical activity exceeding the noise level (Table1a). When the
chloroform vapour is eliminated from the mycelium enclosure the mycelium electrical activity restores to a level
similar to that before anaesthesia (Table1b).
e fungal responses to chloroform are similar to that recorded by us with slime mould Physarum polycepha-
lum (unpublished results). A small concentration of anaesthetic leads to reduced frequency and amplitude of
electrical potential oscillation spikes of the slime mould, and some irregularity of the electrical potential spikes
(Fig.6a). Large amounts of anaesthetic causes the electrical activity to cease completely and never recover
(Fig.6b). Similar electrical responses to anaesthetic again highlights the fact that slime mould exhibits same-
degree anities with fungi as with protozoa, and slime mould has been classied as fungi previously47–49.
Potential, mV
1.0
0.5
0
0.5
1.0
1.5
Time, sec
011052 1053 1054 105
Figure4. Example of reduced frequency of spiking under eect of chloroform vapour. Moment when a source
of chloroform vapour was added into the container is shown by arrow.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
5
Vol.:(0123456789)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
Results presented in the paper contribute towards lling up the gaps in the taxonomy-related studies of
anaesthesia, the living creatures from Protists to fungi to plants to insects to mammals are susceptible to anaes-
thetics. Eects of chloroform on fungi might also implicitly indicate presence of potassium channels, which are
inhibited by anaesthetics50–52.
With regards to directions of future research, as far as we are aware, the present paper is the rst in the eld,
and therefore it rather initiates the research than brings any closure or conclusions. We know that anaesthetics
block electrical activity of fungi (as well as slime moulds) however we do not know exact biophysical mechanisms
of these actions. e study of biophysics and molecular biology of fungi anaesthesia could be a scope for future
research. Another direction of studies could be the analysis of the decision making abilities of fungi under the
inuence of anaesthetics. An experiment could be constructed when fungal hyphae are searching for an optimal
path in a labyrinth when subjected to increasing doses of chloroform vapour. ere may be an opportunity to
make a mapping from concentrations of anaesthetic to geometry of the mycelium search path.
Figure5. Example of electrical activity of mycelium colonised hemp pad before, during and aer stimulation
with chloroform vapour. Arrow labelled ‘ON’ shows moment when a source of chloroform vapour was added
into the enclosure, ‘OFF’ when the source of chloroform was removed. e spiking activity of the mycelium
recovering from anaesthesia is zoomed in.
Table 1. Anaesthesia induced changes in electrical activity of fungi. (a)Containers with chloroform remain
sealed. (b)Lid from the container is removed aer 16h.
a b
Intact Anaesthetised Intact Anaesthetised Recovered
Baseline potential 0.64mV 0.18mV Spike amplitude 3.25mV 0.32 mV 0.93mV
Duration of oscillations
29 ·103
28 ·103
Spike frequency 24min 40min 88min
Spike amplitude 0.4mV 0
Spike duration 62s n/a
Spike frequency 214s n/a
Content courtesy of Springer Nature, terms of use apply. Rights reserved
6
Vol:.(1234567890)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
Received: 19 June 2021; Accepted: 13 December 2021
References
1. Sonner, J. M. A hypothesis on the origin and evolution of the response to inhaled anesthetics. Anesth . Analg. 107, 849 (2008).
2. Eckenho, R. G. Why can all of biology be anesthetized?. Anesth. Analg. 107, 859–861 (2008).
3. Grémiaux, A., Yokawa, K., Mancuso, S. & Baluška, F. Plant anesthesia supports similarities between animals and plants: Claude
Bernard’s forgotten studies. Plant Signal. Behav. 9, e27886 (2014).
4. Bernard, C. etal. Lectures on the Phenomena of Life Common to Animals And Plants. Translation by Hebbel E. Ho, Roger Guil-
lemin [and] Lucienne Guillemin (1974).
5. Hiller, S. Action of narcotics on the ameba by means of microinjection and immersion. Proc. Soc. Exp. Biol. Med. 24, 427–428
(1927).
6. Oliver, A., Deamer, D. & Akeson, M. Sensitivity to anesthesia by pregnanolone appears late in evolution. Ann. N. Y. Acad. Sci. 625,
561–565 (1991).
Figure6. Response of slime mould Physarum polycephalum to triuoroethane (Sigma Aldrich, UK). Electrical
potential dierence between two sites of 10mm long protoplasmic tube was measured using aluminium
electrodes, amplied and digitised with ADC-20 (Pico Technology, UK). (a)5
µ
L of triuoroethane applied to a
5mm
×
5mm piece of lter paper placed in the Petri dish with slime mould. (b)25
µ
L applied. Arrows indicate
moments when the piece of paper soaked in triuoroethane was placed in a Petri dish with the slime mould.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
7
Vol.:(0123456789)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
7. Milne, A. & Beamish, T. Inhalational and local anesthetics reduce tactile and thermal responses in Mimosa pudica. Can. J. Anesth.
46, 287–289 (1999).
8. Nunn, J., Sturrock, J. E., Wills, E., Richmond, J. E. & McPherson, C. e eect of inhalational anaesthetics on the swimming velocity
of Tetrahymena pyriformis. J. Cell Sci. 15, 537–554 (1974).
9. Verra, F. et al. Eects of local anaesthetics (lidocaine) on the structure and function of ciliated respiratory epithelial cells. Biol. Cell
69, 99–105 (1990).
10. Carlile, M. J., Watkinson, S. C. & Gooday, G. W. e Fungi (Gulf Professional Publishing, 2001).
11. Smith, M. L., Bruhn, J. N. & Anderson, J. B. e fungus Ar millaria bulbosa is among the largest and oldest living organisms. Nature
356, 428 (1992).
12. Dai, Y.-C. & Cui, B.-K. Fomitiporia ellipsoidea has the largest fruiting body among the fungi. Fungal Biol. 115, 813–814 (2011).
13. Hanson, K. L. et al. Fungi use ecient algorithms for the exploration of microuidic networks. Small 2, 1212–1220 (2006).
14. Held, M., Edwards, C. & Nicolau, D.V. Examining the behaviour of fungal cells in microconned mazelike structures. In Imaging,
Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI, vol. 6859, 68590U (International Society for Optics and Photon-
ics, 2008).
15. Held, M., Edwards, C. & Nicolau, D. V. Fungal intelligence; or on the behaviour of microorganisms in conned micro-environ-
ments. J. Phys. Conf. Ser. 178, 012005 (2009).
16. Held, M., Lee, A. P., Edwards, C. & Nicolau, D. V. Microuidics structures for probing the dynamic behaviour of lamentous fungi.
Microelectron. Eng. 87, 786–789 (2010).
17. Held, M., Edwards, C. & Nicolau, D. V. Probing the growth dynamics of Neurospora crassa with microuidic structures. Fungal
Biol. 115, 493–505 (2011).
18. Boddy, L., Hynes, J., Bebber, D. P. & Fricker, M. D. Saprotrophic cord systems: dispersal mechanisms in space and time. Mycosci-
ence 50, 9–19 (2009).
19. Adamatzky, A. Developing proximity graphs by Physarum polycephalum: Does the plasmodium follow the toussaint hierarchy?.
Parallel Process. Lett. 19, 105–127 (2009).
20. Adamatzky, A. (ed.) Bioevaluation of World Transport Networks (World Scientic, 2012).
21. Adamatzky, A. (ed.) Advances in Physarum Machines: Sensing and Computing with Slime Mould (Springer, 2016).
22. Nakagaki, T., Yamada, H. & Tóth, Á. Intelligence: Maze-solving by an amoeboid organism. Nature 407, 470 (2000).
23. Nakagaki, T. Smart behavior of true slime mold in a labyrinth. Res. Microbiol. 152, 767–770 (2001).
24. Nakagaki, T., Yamada, H. & Toth, A. Path nding by tube morphogenesis in an amoeboid organism. Biophys. Chem. 92, 47–52
(2001).
25. Nakagaki, T. et al. Minimum-risk path nding by an adaptive amoebal network. Phys. Rev. Lett. 99, 068104 (2007).
26. Tero, A. et al. Rules for biologically inspired adaptive network design. Science 327, 439–442 (2010).
27. Shirakawa, T., Adamatzky, A., Gunji, Y.-P. & Miyake, Y. On simultaneous construction of Voronoi diagram and delaunay triangula-
tion by Physarum polycephalum. Int. J. Bifurc. Chaos 19, 3109–3117 (2009).
28. Jones, J. & Adamatzky, A. Computation of the travelling salesman problem by a shrinking blob. Nat. Comput. 13, 1–16 (2014).
29. Slayman, C. L., Long, W. S. & Gradmann, D. “Action potentials’’ in Neurospora crassa, a mycelial fungus. Biochim. Biophys. Acta
Biomembr. 426, 732–744 (1976).
30. Olsson, S. & Hansson, B. Action potential-like activity found in fungal mycelia is sensitive to stimulation. Naturwissenschaen 82,
30–31 (1995).
31. Adamatzky, A. On spiking behaviour of oyster fungi Pleurotus djamor. Sci. Rep. 8, 1–7 (2018).
32. Iwamura, T. Correlations between protoplasmic streaming and bioelectric potential of a slime mold, Physarum polycephalum.
Shokubutsugaku Zasshi 62, 126–131 (1949).
33. Kamiya, N. & Abe, S. Bioelectric phenomena in the myxomycete plasmodium and their relation to protoplasmic ow. J. Colloid
Sci. 5, 149–163 (1950).
34. Kishimoto, U. Rhythmicity in the protoplasmic streaming of a slime mold, Physarum polycephalum. I. A statistical analysis of the
electric potential rhythm. J. Gen. Physiol. 41, 1205–1222 (1958).
35. Meyer, R. & Stockem, W. Studies on microplasmodia of Physarum polycephalum V: Electrical activity of dierent types of micro-
plasmodia and macroplasmodia. Cell Biol. Int. Rep. 3, 321–330 (1979).
36. Trebacz, K., Dziubinska, H. & Krol, E. Electrical signals in long-distance communication in plants. In Communication in plants,
277–290 (Springer, 2006).
37. Fromm, J. & Lautner, S. Electrical signals and their physiological signicance in plants. Plant Cell Environ. 30, 249–257 (2007).
38. Zimmermann, M.R. & Mithöfer, A. Electrical long-distance signaling in plants. In Long-Distance Systemic Signaling and Com-
munication in Plants, 291–308 (Springer, 2013).
39. Simons, P. e role of electricity in plant movements. New Phytol. 87, 11–37 (1981).
40. Fromm, J. Control of phloem unloading by action potentials in mimosa. Physiol. Plant. 83, 529–533 (1991).
41. Sibaoka, T. Rapid plant movements triggered by action potentials. Bot. Mag. = Shokubutsu-gaku-zasshi 104, 73–95 (1991).
42. Volkov, A. G. et al. Mimosa pudica: Electrical and mechanical stimulation of plant movements. Plant Cell Environ. 33, 163–173
(2010).
43. Minorsky, P. Temperature sensing by plants: A review and hypothesis. Plant Cell Environ. 12, 119–135 (1989).
44. Volkov, A. G. Green plants: electrochemical interfaces. J. Electroanal. Chem. 483, 150–156 (2000).
45. Roblin, G. Analysis of the variation potential induced by wounding in plants. Plant Cell Physiol. 26, 455–461 (1985).
46. Pickard, B. G. Action potentials in higher plants. Bot. Rev. 39, 172–201 (1973).
47. Whittaker, R. H. On the broad classication of organisms. Q. Rev. Biol. 34, 210–226 (1959).
48. Talbot, P. Fungi with a plasmodial thallus division myxomycota: Slime-moulds. In Principles of Fungal Taxonomy, 99–108 (Springer,
1971).
49. Bruns, T. D., White, T. J. & Taylor, J. W. Fungal molecular systematics. Annu. Rev. Ecol. Syst. 22, 525–564 (1991).
50. Jan, L. Y. & Jan, Y. N. How might the diversity of potassium channels be generated?. Trends Neurosci. 13, 415–419 (1990).
51. Kindler, C. H., Yost, S. C. & Gray, A. T. Local anesthetic inhibition of baseline potassium channels with two pore domains in
tandem. J. Am. Soc. Anesthesiol. 90, 1092–1102 (1999).
52. Steinberg, E., Waord, K., Brickley, S., Franks, N. & Wisden, W. e role of k 2p channels in anaesthesia and sleep. Pügers Archiv-
Eur. J. Physiol. 467, 907–916 (2015).
Acknowledgements
is project has received funding from the European Union’s Horizon 2020 research and innovation pro-
gramme FET OPEN “Challenging current thinking” under grant agreement No 858132. e authors would like
to acknowledge the collaboration of Mogu S.r.l. providing the living materials used in the experiments. At the
time of experiments AG was aliated with Mogu S.r.l., Inarzo, Italy. All the experiments have been conducted
in the Unconventional Computing Lab, UWE, Bristol.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
8
Vol:.(1234567890)
Scientic Reports | (2022) 12:340 | https://doi.org/10.1038/s41598-021-04172-0
www.nature.com/scientificreports/
Author contributions
A.A. and A.G. conducted and analysed experiments. A.A. and A.G. wrote the paper.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to A.A.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access is article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
© e Author(s) 2022
Content courtesy of Springer Nature, terms of use apply. Rights reserved
1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com