INTRODUCTION
2
REPORT
UNDER AGREEMENT ON COOPERATION IN SCIENCE BETWEEN FEDERAL STATE
BUDGET-FUNDED INSTITUTION OF SCIENCE PAVLOV INSTITUTE OF PHYSIOLOGY
OF THE RUSSIAN ACADEMY OF SCIENCE AND AIRES HUMAN GENOME RESEARCH
FOUNDATION
Subject:
Study of High-Frequency Electromagnetic Radiation Impact and Aires Resonators Influence on
Behavior, Genetic and Epigenetic Processes in Cells of Central and Peripheral Organs (Rattus
norvegicus and Apis mellifera L.Honey Bee Models)
STAGE FIVE (December – June, 2019): Study of Aires Resonators Influence on Stress-
Responsive hsp70 Gene Expression in the Honey Bee Brain.
INTRODUCTION
Over millions of years natural electromagnetic fields (geomagnetic field, solar radiation,
atmospheric electricity) have been a constant ecological factor, influencing condition of living
organisms and ecosystems. Evolving organisms adapted to influence thereof. Today, as a result
of progress in science and technology, there is a great number of electric devices, helping a man
in various areas of life. Most electric devices around us are sources of electromagnetic radiation
of different frequency and power. High level of electromagnetic pollution of the environment
can be harmful to living organisms. Degree of EMR impact on living beings depends on field
power and strength, oscillations frequency, exposure duration and mode of generation thereof
(pulse and continuous fields) (Kudryashov et al., 2008).
Insects (butterflies, ants, cockroaches, flies) are deemed to be the best experimental
animals to study EMR impact as they are highly sensitive to magnetic and electric fields (Kumar
et al., 2011).
It has already been proved that impact of high-frequency radiation decreases queen bee’s
fertility and leads to reduction of honey and bee-bread amount in a family (Kumar et al., 2011).
Unconditional-reflex food excitability and short-time memory of a honey bee deteriorates
(Lopatina et al., 2019). Molecular and cellular mechanisms of this phenomenon are unknown
and need careful study. Apparently, change of the natural electromagnetic background is a
stressor for bees.
Heat shock proteins (HSP) are universal stress reaction sensors. Heat shock proteins are
one of the most conservative and phylogenetically ancient proteins: homology degree of
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eucaryotes and procaryotes HSP makes more than 50%, and some domains thereof are totally
identical; structural similarity of human and mice HSP is up to 95%. A certain amount of heat
shock proteins (HSP) is continuously synthesized in any nuclear cells, in numerous intracellular
structures (in the nucleus, cytoplasm, endoplasmic reticulum, chloroplasts and mitochondria) of
all multi-cellular organisms, regardless of exposure to stress factors. Heat shock proteins are
molecular chaperones, participating in protein folding (tertiary structure formation), HSP prevent
nonspecific proteins aggregation and protect them from premature proteolysis. HSP protect the
cell from impact of mutant or misfolded proteins, from death of cells caused by stress. Increase
of HSP intracellular synthesis is caused not only by heat shock, but also by any exposure to
stress: external impact (UV, heavy metals, heat shock, amino acids), pathologic impact (viral,
bacterial and parasitic infections, inflammation, malignant transformation, autoimmune
response) or even physiological impact (growth factors, cell differentiation, hormonal
stimulation, tissue growth) (Nikitin, 2008).
HSP70 heat shock protein (which belongs to the family of proteins with molecular mass
more than 70kDa) is the most studied one. HSP70 acts as a chaperone in the cell, besides it
participates in stress-related processes, such as aggregation, deaggregation, degeneration and
restoration of three-dimensional folding (Nikitin, 2008).
According to the data base NCBI GenBank (LOC408706 heat shock protein 70Cb
ortholog), gene length of a honey bee makes 8361 bps. Length of mRNA: XM_623196.5 - 4605
bps, XM_006561162 – 4497 bps. Proteins length: XP_006561225.1 - 831 a.a., XP_623199.2 -
861 a.a.
Purpose of this work is to study stress-responsive hsp70 gene expression in the honey
bee Brain upon exposure to electromagnetic radiation, emitted by WiFi router and simultaneous
exposure to WiFi router and Aires Defender Pro resonators.
STUDY MATERIALS AND METHODS
The work was performed using 10-30 day Apis mellifera carnica worker honey bees.
Bees were bred at the bee house of the Pavlov Institute of Physiology of the Russian Academy of
Science. The bees meant for the experiment were kept in the observation queen-bee cell in the
special premise at a room temperature and automatic lighting from 8 a.m. to 8 p.m. .
5 groups of bees participated in the experiment (10-16 animal units per each group):
intact group, control group (faraday’s cage, isolation from external EMR), control group (6 Aires
Defender Pro resonators in the center of each faraday’s cage face), experimental group (WiFi
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router operating in the faraday’s cage in the 24h mode + 6 Aires Defender Pro resonators
resonators).
The following algorithm was used to study impact of electromagnetic waves on hsp70
gene expression: exposure to EMR router (2 groups of bees), control without exposure (3 groups
of bees), extraction of RNA from brains of bees of all groups, RT-PCR with electrophoretic
detection (Fig. 1, 2).
Fig. 1. Design of the experiment
Fig. 2. A – Faraday’s cage, B – Faraday’s cage + Aires Defender Pro resonators.
Extraction of total RNA from the brain of the honey bee. The bees were taken from
the hive and immediately placed into the freezer for 10 minutes. Afterwards, the head capsule
Groups of bees under study
Control Experiment
Intact Faraday’s
Faraday’s
WiFi router WiFi router + resonators
WiFi router EMR, 24h
Evaluation of hsp70 gene expression
Brain extraction
RNA extraction
cDNA preparation
PCR Electrophoresis
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was opened up and the brain was extracted. The brain was homogenized with a pestle in 1.5mcl
centrifugal tube with 200mcl homogenization buffer (Evrogen). The homogenate was incubated
at a room temperature during 10 minutes. 300mcl of chloroforme was added for deproteinization,
samples were thoroughly vortexed. The samples were incubated at a room temperature during 5
minutes. Then they were centrifugated at 13,000rpm during 3 minutes. The supernatant with
nucleic acids was transferred to a clean test tube. 500mcl of isopropyl alcohol was added, the
sample was mixed. 100mcl of 3 M Sodium acetate was added, the sample was carefully mixed.
The samples were placed into the refrigerator at -20 OS for 1 hour for nucleic acids to precipitate.
Then the samples were centrifugated at 13,000rpm during 3 minutes. The supernatant was
removed. 500mcl of 96% ethyl alcohol was added, the samples were mixed. Then they were
centrifugated at 13,000rpm during 3 minutes. The supernatant was withdrawn. 500mcl of 70%
ethyl alcohol was added, the samples were mixed. Then they were centrifugated at 13,000rpm
during 3 minutes. The supernatant was withdrawn. 500mcl of 70% ethyl alcohol was added, the
samples were mixed. Then they were centrifugated at 13,000rpm during 3 minutes. The
supernatant was withdrawn. The wash-out was repeated (steps 16-18). The sediment was diluted
in TE buffer, pH 8.0 (1 mM EDTA, 10mM tris hydrochloride, pH 8.0) at a room temperature
during 10 minutes. The samples of the total RNA, prepared in such a way, were stored at -20 OS.
Reverse Transcription. The reaction of reverse transcription was performed using the
obtained samples and the reverse transcription set (Evrogen) with a random primer (Evrogen)
according to the manufacturer’s recommendations (2 hours at 38 OS). The obtained cDNA was
stored at -20OS.
Polymerase Chain Reaction. cDNA, obtained upon reverse transcription was used as a
matrix. PCR was performed according to the manufacturer’s recommendations (Evrogen), using
Veriti 96-Well Thermal Cycler (Applied Biosystems). Primers annealing temperature was 61o C.
Cycles number - 40. Primers (10pmol/mcl, Evrogen): they were selected by T.G. Zachepilo in
the GenBank by means of PrimerBLAST online package.
Table 1. Primers
Direct/reverse Sequence Product Primer Direct Apis mellifera heat shock protein 70Cb ortholog (LOC408706), XM_623196.5
92bps AAGCACAAGCAAATGAACCACCG
Reverse CCTCGCACCTCTTCCACCAT
Direct Apis mellifera ribosomal protein L32 (RpL32), NM_001011587.1
109bps TGTGCTGAAATTGCTCATGGT Reverse AGAACGTAACCTTGCACTGG
Electrophoresis in Agarose Gel. PCR-products were mixed with the loading buffer
(Evrogen, 1:1). DNA fragments were separated in 10x15cm 1.5% agarose gel (with admixture of
ethidium bromide) with TAE buffer in the horizontal electrophoresis chamber (Helicon) at 150V
during 40 minutes. Do determine the size of the amplificated fragments, DNA-markers were
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applied on the gel: 100bps (Evrogen). Electrophoresis results were detected in the transmitted
ultraviolet, using the transilluminator (Vilber Lourmat). The gel was shot, using a digital camera.
The results were saved on the computer in JPEG format.
Data Processing. Photos of electrophoregrams were analyzed in ImageJ (NCBI). First,
paths were identified in the image, then the area of stained bands was evaluated. Normalization
was performed: ratio of area of hsp70 samples bands to the area of rp49 reference gene was
Then pairwise comparison of normalized values was performed in all groups (total 10),
using non-parametric Mann-Whitney test. Statistical analysis was performed in Statistica 10.
FINDINGS
Expression of hsp70 stress-responsive gene upon exposure of the honey bee to
electromagnetic radiation was studied by the method of RT-PCR and electrophoretic detection.
As a result of performed experiments electrophoregrams were obtained (Fig. 3-5).
Expression was compared by matching bands in electrophoregrams. Thick and intensely colored
bands refer to strong expression, while thin and weakly colored ones refer to weak expression.
Hsp70 gene expression in the intact group of bees was found in 3 samples (each sample
contains the material, consisting of 2 bees brains) of 4, i.e. the animals differ by functional state
of the CNS.
In the resonator control group (6 resonators on all faces of the faraday’s cage) hsp70
stress-responsive gene expression was more uniform than in the intact bees control group. It
might be related to influence of the faraday’s cage and resonators, equalizing functional state of
the CNS.
Intact Resonators
Fig. 3. Electrophoregram of PCR-products in the intact and resonator groups. Henceforth,
figures are samples numbers, gene under study – hsp70, reference gene – rp49.
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In the faraday’s cage control group expression of hsp70 was similar to that of the
resonator group. Thus, control groups of bees were the same, apart from the intact one.
Faraday’s cage
Fig. 4. Electrophoregram of PCR-products in the faraday’s cage group
In the router group hsp70 gene was expressed positively weaker, than in the faraday’s
cage and router+resonator groups. I.e. 24h impact of EMR, emitted by the router, caused
weakening of hsp70 expression.
Router+resonators Router
Fig.5. Electrophoregram of PCR-products in the router+resonator and router groups
It is to be noted that any change of electromagnetic background, i.e. weakening of EMF
in the faraday’s cage, change of EMR parameters in the faraday’s cage due to Aires Defender
Pro resonators, EMR increase, caused by the router, result in change of hsp70 expression in
comparison with the intact group.
Results of data processing are summarized in Fig. 6.
Fig. 6. Expression of hsp70 gene in the bee’s brain (normalized values _ standard deviation). * -
differences are significant, p<0,05, Mann-Whitney test.
router router + resonators faraday’s
cage resonators intact
Average+st. dev.,
st.units
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Thus it can be concluded as follows:
1. Level of hsp70 gene expression in the honey bee’s brain is similar in control groups.
Intact bees show greater variability of this parameter.
2. After 24-h exposure to high-frequency electromagnetic radiation of a WiFi router,
expression of hsp70 gene in the honey bee’s brain weakens which can have negative impact on
functioning of the bee’s CNS.
3. Isolated 24-h exposure to Aires Defender Pro resonators has no impact on expression
of hsp70 gene in the honey bee’s brain.
4. Upon simultaneous 24-h exposure to Aires Defender Pro resonators and WiFi router,
expression of hsp70 gene in the honey bee’s brain increases to the control level.
DISCUSSION
Heat shock proteins play an important part in the life of a cell and the organism as a
whole. HSP participate in control of proteins quality, in protection of cells from aggregation of
misfolded proteins or in forwarding of misfolded proteins to proteasomes for proteolysis thereof.
Virtually all cell proteins at least temporarily interact with HSP70. Need in chaperones upon
exposure to stress factors increases significantly. Interaction between the protein target and
HSP70 result in stabilization of the former, then the protein folds correctly, or chaperons of a
different type are recruited, which is followed by further restoration of the intact conformation
(Nikitin, 2008). Thus decrease of transcriptional activity of hsp70 gene causes deficiency of
HSP70 protein and increase of misfolded proteins and aggregates thereof. Accumulation of
misfolded proteins and aggregates thereof in the neural tissue may result in derangement of
learning processes and memory formation.
Despite the fact that development of stress reactions is generally related to increase of
HSP70 level, there are also evidences that such conditions as hyperthermia, ageing or disease
may decrease reaction of heat shock proteins in the brain (Pardue et al., 2007). This demonstrates
that increase of heat shock proteins synthesis can be necessary in some cell reactions, however,
not in all of them (Agustiño et al., 2012). The literature also describes decrease of HSP70 in case
of cerebral ischaemia (Yang et al., 2005). These data show that increased expression of HSP70
is not critical upon early adaptation. However, regulation at later stages, including increase of
heat shock proteins number, suggests that stress proteins are of importance in facilitation of long-
term tolerance.
Decrease in number of heat shock proteins is, apparently, indicative of existence of non-
thermal physical stimuli, acting through unidentified mechanisms via low-intensity electric fields
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without direct connection between power and effect size. Since the animals were exposed to non-
ionizing radiation in their entirety, their organism could react to the stress in multiple ways
(Agustiño et al., 2012).
A number of works demonstrate negative impact of EMR on honey bees (Harst et al.,
2006). Under impact of EMR, their locomotor activity decreased, their ability to orient in space
was impaired, it took them more time to get to the hive, their ability to return to their family
degraded abruptly, they built less and became more aggressive. It is shown that bees perceive
EMR as danger signal (Favre, 2011). When exposed to EMR, egg-laying capacity of the queen
bee decreased, drone brood was observed (Halabi et al., 2013), amount of honey and bee-bread
in hives decreased abruptly (Kumar et al., 2011). Behavior deviations of a honey bee as a result
of exposure to different-frequency EMR are also shown in works of Russian researchers
(Yeskov, Bragin, 1986; Yeskov, Toboev, 2008; Lopatina et al., 2019).
Similar ecologically significant changes in behavior and reproduction of other insects
(locust, flies, ants) under EMR impact are demonstrated in the work of Cucurachi et al. (2013). It
is shown that upon exposure to high-power EMR oxidative stress and change of genes
expression are observed in Drosophila (Manta et al., 2017). CNS of insects is quite sensitive to
EMR: it is shown that it causes decrease of ability to form conditioned food reflex to olfactic and
visual stimuli in Myrmica sabuleti ants (Cammaerts et al., 2011).
Thus distortions of cognitive activity of honey bees upon long-term exposure to high-
frequency electromagnetic radiation may be related to accumulation of misfolded neuronal
proteins, caused by decrease of transcriptional activity of hsp70 gene and deficiency of HSP70
protein.
* * * * *
Results of the performed experiments expressly testify hsp70 expression normalization
under influence of Aires Defender Pro resonators. The data, obtained during the series of
experiments is partly in compliance with previously obtained data on food excitability and short-
term memory in bees upon simultaneous exposure to WiFi router and resonators. It is possible
that hsp70 normalizing action of resonators has a deferred effect on bees’ behavior.
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