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BioMetalsAn International Journal on the Role ofMetal Ions in Biology, Biochemistry andMedicine ISSN 0966-0844 BiometalsDOI 10.1007/s10534-014-9805-9

Iron-based granules in body of bumblebees

Petr Jandacka, Barbora Kasparova,Yvonna Jiraskova, Katerina Dedkova,Katerina Mamulova-Kutlakova & JanaKukutschova

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Iron-based granules in body of bumblebees

Petr Jandacka • Barbora Kasparova • Yvonna Jiraskova •

Katerina Dedkova • Katerina Mamulova-Kutlakova •

Jana Kukutschova

Received: 3 June 2014 / Accepted: 20 October 2014

� Springer Science+Business Media New York 2014

Abstract The paper deals with the presence of iron-

based granules in body parts of bumblebees. Two

groups of bumblebees were collected from their

natural habitat, industrial landscape, and from a

breeding station. Detection of the magnetic particles

was performed by a vibratory magnetometer and their

morphology and elemental composition was analysed

by scanning electron microscopy with EDX micro-

analysis. By means of the EDX spectra, wild bumble-

bees were found to have many magnetic and non-

magnetic particles on their body, containing Fe, O, Al,

Si, Bi, Mg, K, and Ni, likely having origin in the

industrial pollution of the environment. In the case of

bred bumblebees the presence of iron-rich granules,

which occurred more abundantly in subsurface tissues

on the head and wings, was observed. Phase analysis

based on X-ray diffraction shows that iron-based

granules contain magnetite and wuestite and Moss-

bauer spectroscopy admits a superparamagnetic form

of these minerals. Magnetoreception, i.e. the sensory

function of these granules, is discussed within the

paper.

Keywords Magnetoreception � Bumblebee �Biomineralisation � Iron granules

Introduction

Bumblebees (Bombidae sp.) have recently been the

subject of numerous studies in part due to the

increased interest in their protection. They belong to

the insect Hymenoptera order and to the Apidae

family, so their relatives are e.g. honeybees (Apis

mellifera). The honeybee species exhibit excellent

orientation in their habitats and one of the cues for this

property could be a magnetoreception based on

presence of iron-containing particles in their body.

Since the iron-based magnetoreception was already

studied in detail on bodies of honeybees, it could be

possible to expect a very similar occurrence and

significance of iron-based particles in the bodies of

bumblebees (Hsu and Chan 2011; Hsu et al. 2007; Hsu

and Li 1994).

The presence of iron-based magnetic structures in

many living organisms including human beings was

P. Jandacka (&)

IT4Innovations Centre and Institute of Physics,

VSB - Technical University of Ostrava, 17. listopadu

15/2172, 708 33 Ostrava, Czech Republic

e-mail: petr.jandacka@vsb.cz

B. Kasparova � K. Dedkova � K. Mamulova-Kutlakova �J. Kukutschova

Nanotechnology Centre, VSB - Technical University

of Ostrava, 17. listopadu 15/2172, 708 33 Ostrava,

Czech Republic

Y. Jiraskova

Institute of Physics of Materials, Academy of Sciences

of the Czech Republic, Zizkova 22, 616 62 Brno,

Czech Republic

123

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DOI 10.1007/s10534-014-9805-9

Author's personal copy

previously described by some research studies (Safar-

ik and Safarikova 2002). Their role is still subject to

discussion, but one of their functions could be

magnetoreception, which may be connected with the

movement of magnetic crystals in tissues. Proven

magnetoreception exhibits magnetotactic bacteria via

magnetite or greigite magnetosomes arranged in

crystal chains. These bacterial cells passively rotate

in a water environment in the direction of geomagnetic

field lines (Bazylinski and Frankel 2004; Faivre and

Schuler 2008). Magnetite structures were also

detected in the pigeon upper beak, and iron-containing

otoliths and intracellular ferritin corpuscles were

found in the inner ear of birds (Fleissner et al. 2003;

Hanzlik et al. 2000; Harada 2008; Lauwers et al.

2013).

In insects, the magnetic crystals were detected via

magnetic precipitation methods and magnetic reso-

nance in body parts of ants (Acosta-Avalos et al. 1999;

Wajnberg et al. 2005). In ant’s antennae, their main

sensory organ, ferro/ferri-magnetic crystals likely

composed of magnetite/maghemite, hematite, goethite

and others minerals were found (de Oliveira et al.

2010; Wajnberg et al. 2004). By means of magneti-

sation of honeybees, it was observed that their bodies

exhibit magnetisation (Gould et al. 1978). The study of

composition of honeybees’ magnetic structures was

addressed by Kuterbach and Walcott (Kuterbach and

Walcott 1986), who discovered that particles isolated

from the cells of honeybees’ abdomen contain iron.

Their subsequent Mossbauer spectroscopic analysis

showed that at least 95 % of the present iron is in the

form of hydrous iron oxide, not magnetite. The

ferromagnetic material was also detected using a

magnetic resonance method (El-Jaick et al. 2001). Hsu

and Li (1994) discovered that the abdomen cells

contain trophocytes rich in clusters of superparamag-

netic particles connected with their cell membrane,

thus reaching a hypothesis of the magnetosensoric

function of superparamagnetic trophocytes (Hsu et al.

2007). Subsequently they found that the alternative

external magnetic field induces size fluctuations of

superparamagnetic clusters. These size fluctuations

lead to the increase of the Ca2? concentration in cells,

which influences neural processes around cells. Gen-

erally, magnetic crystals may not in fact play

magnetoreception role in insects, though presence of

magnetite crystals in ant’s antennae makes sense (de

Oliveira et al. 2010). Currently, the main effort of

research on magnetoreception is focused on the

evaluation of the radical-pair/cryptochrome hypothe-

sis. This hypothesis is based on photochemical reac-

tions inside the honeybees’ eyes influenced by

geomagnetic field (Valkova and Vacha 2012).

In bumblebees (Bombus sp.), the iron biominerals

were already discovered in abdominal cells (Hsu

2004; Walcott 1985). According to electron micros-

copy, the granules are approximately 1 lm in size,

often with spherical shape and associated with the

membranes, and located namely under the cuticle on

the ventral side of the abdomen. Elemental analysis

revealed that these granules contain Fe, P and Ca.

From the point of view of magnetoreception, the

orientation of bumblebees in the environment is based

mainly on their optical and olfactory abilities, but their

good orientation abilities in a total darkness with

limited olfactory information available indicate that

bumblebees use a magnetic compass that is indepen-

dent of light (Chittka et al. 1999).

The aim of this study is to detect, by means of

magnetic measurements and several experimental

techniques, whether bodies of bumblebees contain

magnetic particles or aggregates, as a potential

component of their geomagnetic sensor.

Experimental

Bumblebee samples

Bumblebee species Bombus terrestris, and its individ-

ual body parts, were used for the study. The first group

of bumblebees was wild, caught specifically in a post-

mining landscape in Ostrava, Czech Republic. The

death was caused by inhalation of ethyl acetate.

Drying proceeded under normal conditions (at room

temperature and 40–60 % humidity). The second

group were bumblebees of a special breed from the

Research Institute for Fodder Crops (VUP) in Brno-

Troubsko, Czech Republic. Bumblebees were bred

there at 25 �C and 50–60 % humidity. They were fed

by sugar solution and pollen, which is collected from

honeybees. The age of the bred bumblebees for the

purpose of the measurements was about 3 months.

The three following casts were available: queens,

workers (female) and males in a number of 10 of each.

Their death was induced by freezing.

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Magnetic model substances

Fresh equine spleen ferritin served as a model

substance (at concentration of 53 ± 3 mg/mL of

saline solution, \0.9 % of NaCl, product number:

F4503) and superparamagnetic magnetite (at concen-

tration of 5.0 ± 0.3 mg/mL of toluene, product num-

ber: 700320) were provided by Sigma Aldrich. The

size of superparamagnetic magnetite crystals, having a

size range of 5–18 nm, was measured using the laser

analyser Malvern Zetasizer NanoZS (type ZEN3600).

Measurement techniques

To confirm the presence of magnetic granules, a

combination of four experimental methods was per-

formed—vibrating sample magnetometry (VSM),

scanning electron microscopy (SEM), Mossbauer

spectroscopy and X-ray powder diffraction (XRPD).

The magnetometry serves as an input method for

detection of magnetic particles in the body parts of

bumblebees (head, thorax, abdomen and rest body-

parts—limbs and wings). The data from magnetic

measurements were processed in Matlab 7.0. Physical

quantities and their units are presented in the SI unit

system.

Magnetic measurements were performed using a

vibrating magnetometer VSM EV9 Microsense (mag-

netic moment resolution 10-10 A m2, field resolution

10-4 T). Magnetic measurements were carried out at

normal as well as variable temperatures. Bumblebee

bodies were attached to the glass holder with paraffin.

This glass holder with paraffin exhibited a diamag-

netic signal of its own, therefore the holder was

measured separately and then the value of its diamag-

netic signal was subtracted from the total signal value

(holder, paraffin, and sample). The ferritin was

magnetised in liquid form in a cylindrical glass

container sealed with paraffin. Without ferritin, these

components exhibit a diamagnetic moment indepen-

dent of temperature. Superparamagnetic magnetite

was measured in dry form on diamagnetic glass of the

sample holder. Before the measurement, the holder

was soaked into the toluene-magnetite solution and

when the toluene evaporated, dry magnetite remained

on the glass surface. The temperature measurements

were performed using temperature regulation of N2

gas flow in the range of -165 to 0 �C, i.e. 108–273 K,

which is the limit for the VSM system that was used. A

very slow regime of 5 K/min was selected to ensure

the smallest difference possible between temperatures

of the sensor and samples. Uncertainties of tempera-

ture measurements are ±1 �C.

In order to determine morphology of the magnetic

structures and measure their elemental composition,

the electron microscopy SEM QUANTA 450 FEG by

FEI was used. The microscopic images were obtained

in back scattered electron (BSE) and scattered electron

(SE) regimes with subsequent EDX microanalysis of

revealed granules. For the purposes of this measure-

ment, bumblebees were coated (three times) with gold

and palladium and then they were placed on a carbon

tape and attached to a small brass target. The

microscopy was performed under magnification rang-

ing from 600 to 60009 while using a voltage of

15–20 kV.

The transmission Mossbauer spectrum was mea-

sured at room temperature using 57Co(Rh) source

moving in a constant acceleration mode. The velocity

scale was calibrated using a thin foil of a-Fe and the

isomer shift was taken with respect to its RT

Mossbauer spectrum. The measured spectrum was

evaluated within the transmission integral approach

using the program CONFIT (Zak and Jiraskova 2006).

Generally, the crystalline components can be repre-

sented by single-, double-, and/or six-line Lorentzian

subspectra determined by discrete values of hyperfine

parameters: isomer shift(s), quadrupole, and magnetic

splitting(s), corresponding to paramagnetic and/or

ferromagnetic phases.

The XRPD patterns were recorded under CoKairradiation (k = 1.789 A) using the Bruker D8

Advance diffractometer (Bruker AXS) equipped with

a fast position sensitive detector VANTEC 1. Mea-

surements were carried out in the reflection mode, and

the powder sample was placed on a rotational holder.

Phase composition was evaluated using database PDF

2 Release 2004 (International Centre for Diffraction

Data).

Results

Wild bumblebees

Magnetisation of wild bumblebees bodies exhibits

magnetic hysteresis reflecting the presence of ferro/

ferri-magnetic crystals (Fig. 1). Magnetic curves are

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influenced by the diamagnetic character of the tissue.

SEM revealed presence of various granules situated

directly on the surface of their bodies, especially on

their hairs, usually in the form of granules of

approximately the size of 1–10 lm. Examples of

EDX analysis results are presented in Fig. 2. The

presence of iron in the granules was clearly confirmed.

Detected silicates (containing mostly Si, Al, Ca, K)

probably come from dust particles and from plants

with which the wild bumblebees come into contact.

Less expected was the presence of Bi, Sb, Ni and Cr.

These elements are most likely associated with

environmental pollution (air pollution) of the area

where the wild bumblebees lived and may come e.g.

from pyrometallurgical plant or might be released by

road traffic, which can be inferred from some studies

(Kim et al. 2007; Kukutschova et al. 2011; Maher et al.

2008; Muxworthy et al. 2003; Rodriguez-Germade

et al. 2014).

Breed bumblebees

Magnetic and chemical characterisation

In this phase of the research, it was apparent that it was

necessary to work with a control group of bumblebees

that had not been exposed to much environmental

pollution. Magnetisation of these clean bodies from

breed bumblebees exhibits paramagnetic or diamag-

netic magnetisation (Figs. 3, 4, 5, 6, 7, 8) affected by

diamagnetic tissue. The same results were obtained for

all three groups (queens, workers and males). The

paramagnetic signal is stronger in the case of head and

wings than in the case of thorax and abdomen. SEM

observations using BSE and SE modes revealed, that

in subsurface layers in hair roots on wings (in front

edge of the upper wings) and on the head, iron-based

granules can be found (Figs. 4, 6). From the EDX

spectra (Figs. 4, 6), it can be assumed that these

granules may be based on compounds of Fe, Mg, Si, P,

Ca, O, K or Cr. The EDX measurement is in

accordance with the results of magnetic measurements

except for the fact that magnetic structures and their

signal are stronger in lighter weight parts (head, wing),

because they are not shielded by the diamagnetic

signal of the tissue. In the case of males the approx-

imate weight percentage ratio of caput/thorax/abdo-

men/rest of the body (wings and legs) is 11/29/62/\1.

Workers’ ratio is 22/51/28/\1, and queens’ ratio

10/42/47/\1.

Mineralogical composition of iron-based granules

To explain an importance of detected iron-based

granules we focused on those in wings since they have

a better volume ratio granules/tissue. It seems to be

better to use surface methods that allow for aiming the

subsurface granules and analyse them directly, as

provided by Raman microscopy, which enables a point

phase analysis of a single granule approximately down

to 500 nm in size. However, this method did not

provide us any results due to unsuccessful focusing of

granules and insufficient energy of the laser that has to

screen the cuticle. Based on such a failure, volume

methods for determination of mineralogical composi-

tion of iron-based granules in the wings had to be used.

Firstly, the magnetic method was selected for the

estimation of mineral composition of the discovered

granules. The aim was to evaluate whether thermo-

magnetic curves for superparamagnetic magnetite or

ferritin (equine spleen ferritin was used as a model

substance) have a similar progress as thermomagnetic

curves from the wings of bumblebees. Constant field

of 0.02 T was chosen as it represents an approximate

linear boundary of ferritin magnetisation (Brem et al.

2006), for magnetite it is 0.2 T (Jandacka et al. 2013),

where the measured data can then be fit to Curies law

(Kittel 2004). All three measurements are influenced

by a diamagnetic moment b (A m2) independent of the

Fig. 1 Magnetisation of selected body parts of wild bumble-

bees. Diamagnetic moment of the glass holder and paraffin

presented in figure is subtracted from total moments. A fit is

represented by equation m = -4.14 9 10-7 9 B -

7.91 9 10-9, R2 = 0.999. Residual diamagnetic moment from

tissue has an impact on the ferromagnetic moment

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Fig. 2 Examples of SEM images of particles (designated by white arrows) situated on surface hairs of wild bumblebees and their EDX

spectra. Samples were coated with Au and Pd and put on carbon tape fixed on a brass sample holder

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Fig. 3 Magnetisation of breed bumblebee—head. Diamagnetic

moment (glass holder and paraffin) is subtracted from total

magnetic moments. Residual diamagnetic moment from tissue

has an impact on the paramagnetic moment

Fig. 4 SEM images of bred bumblebee—head (queen) with

subsurface iron granule (white points in photo) displayed in

BSED and SE regimes. Through the combination of these

regimes, it is possible to distinguish Fe-containing granules

located in subsurface structures of hairs. EDX spectra of such

particles are presented. Samples were coated with Au and Pd

and put on carbon tape fixed on a brass sample holder

Fig. 5 Magnetisation of bred bumblebee—wing. Diamagnetic

moment is subtracted from total magnetic moments. Residual

diamagnetic moment from tissue has an impact on the

paramagnetic moment. Note worker means female

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Fig. 6 SEM images of subsurface Fe-containing granules on a

wing (white points in photo) in bred bumblebee sample (queen)

displayed in BSE and SE regimes. EDX spectra of such granules

are presented. Samples were coated with Au and Pd and put on

carbon tape fixed on a brass sample holder

Fig. 7 Magnetisation of bred bumblebee—thorax. Diamag-

netic moment of glass holder and paraffin is subtracted from

total magnetic moments. Note worker means female

Fig. 8 Magnetisation of bred bumblebee—abdomen. Diamag-

netic moment of glass holder and paraffin is subtracted from

total magnetic moments. Note worker means female

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temperature originating from paraffin, glass and

diamagnetic tissue in wings. The result of the

measurement is presented in Fig. 9. It is apparent that

superparamagnetic magnetite exhibits paramagnetic

properties and its magnetic moment m is dependent on

1/T, which is in accordance with Curies law. The

thermomagnetic curve of ferritin has a more compli-

cated progress, which is likely to be ascribed to its

threefold composition: magnetite, hematite and fer-

rihydrite (Galvez et al. 2008). The measured points

were fitted with a 4th degree polynomial because of an

untypical trend of the thermomagnetic curve. The

wings of the bumblebees queen exhibited a weak

magnetic moment influenced by several failures. Due

to a poor adhesion of paraffin on a glass holder, the

sample repeatedly fell off the holder. As a result, the

measurements had to be commenced at -150 �C. The

result indicates that magnetic granules in bumblebees

bodies may not to be ferritin based. Thermomagnetic

curves of ferritin and magnetic structures of wings are

not in accordance in terms of their process. With

respect to the general threefold composition of ferritin,

this conclusion appears very uncertain—this magnetic

measurement is not sufficiently reliable.

Fig. 9 Thermomagnetisation curves at constant external field

0.02 T from A) superparamagnetic magnetite fitted by Curies

law, a = (7.03 ± 0.01) 9 10-5 A m2 K, b = (1.296 ± 0.006) 9

10-7 A m2, magnetite mass approximately 0.02 mg, B) equine

spleen ferritin, fitted by 4-th degree polynomial, ferritin mass

approximately 3.3 mg, and C) two bumblebee queen wings fitted by

Curies law, a = (1.5 ± 0.6) 9 10-7 A m2 K, b = (9.9 ± 0.3) 9

10-9 A m2, and with 4-th degree polynomial. The D) represents

manner of fixation of wings on the glass holder using paraffin.

(standard uncertainties were presented)

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57Fe Mossbauer spectrometry is highly sensitive to

the short-range interaction in iron-containing solids

and it is also being frequently used in investigation of

various iron-based oxides and/or iron-based compos-

ites down to nano-scale objects. The bumblebee

sample for the Mossbauer measurement was prepared

from approximately six main bumblebee wings in

such a way that the upper parts of the wings have filled

the measured area as much as possible (see inset in

Fig. 10). The Mossbauer measurement has resulted in

an asymmetric doublet seen in Fig. 10. It represents

iron-based species yielding either superparamagnetic

behaviour of the nanometre size particles or paramag-

netic objects—the paramagnetic behaviour is consis-

tent with the VSM measurements. The spectrum can

be analyzed either by a broadened double-line spec-

trum, the lines of which have various intensities or by

two double-line subcomponents as depicted in Fig. 10,

both characterized again by certain asymmetry. The57Fe Mossbauer hyperfine parameters for both indi-

vidual doublets are summarized in Table 1. They do

not reflect the parameters, isomer shift and quadrupole

splitting, usually presented for Fe2? and Fe3? ions in

the iron oxides. Therefore, the phase composition(s) of

the measured iron-based object(s) in the wings, similar

to the asymmetry of the Mossbauer spectrum, are

difficult to clarify conclusively from the present

measurement. The Mossbauer measurements in the

applied magnetic field and/or low temperatures could

be helpful in future investigations.

XRPD measurement was performed on 6 pairs of

dry wings in a powder form, prepared via drying in

34 �C for 2 h and via pulverising of them in agate

mortar. This method detected three mineral phases in

the wings, see Fig. 11. The first is crystalline silicon

oxide, SiO2, and the others are magnetite, Fe3O4, and

wuestite, FeO. All of them probably create the

granules.

It is apparent that only partial success was reached

in mineralogical analysis of our samples. XRPD

method revealed that iron-based granules are com-

posed of magnetite and wuestite crystals, however

Mossbauer spectroscopy did not confirm such results

clearly, since content of Fe2? and Fe3? ions, typical

for magnetite and wuestite, is not obvious. This

ambiguity may be induced by sensitivity of Mossbauer

spectroscopy to measurement environment of iron

oxides, which is tissue of wings in this case. On the

other hand, the presence of magnetite/wuestite in

superparamagnetic forms is not excluded. We have to

admit, that an uncertainty of measurement was entered

via different form of samples (XRPD = dry powder,

Mossbauer spectroscopy = fresh wings), since the

drying and milling processes on wings may cause

recrystallisation of the granular crystals. Therefore,

the mineralogical analysis is not final and needs to be

continued in future.

Fig. 10 Mossbauer spectrum of the bumblebees wings com-

posite into a sample (inset) 10 mm in diameter

Fig. 11 XRPD spectrum of bumblebee wings fragmentised to

powder form after drying

Table 1 57Mossbauer hyperfine parameters; isomer shift, d,

quadrupole splitting, D, asymmetry, D, and areas of sub-

spectra, A

d (mm/s) D (mm/s) D (-) A (%)

Doublet 1 (grey) 0.113 (07) 0.605 (23) 1.52 77.5

Doublet 2 (pattern) 0.090 (30) 0.215 (68) 1.44 22.5

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Discussion: function of the iron-based granules

To the best of our knowledge, the presence of

magnetic iron-based granules in bumblebee wings

and head was not yet observed. The results achieved

within this study are different to similar studies on

honeybees, mentioned in introduction section and it is

not consistent with the study of Walcott (1985) on

bumblebees, who revealed iron-rich granules contain-

ing P and Ca in the abdomen cuticle. We did not detect

such particles in this bodypart by means of magnetic

and other techniques.

Very recently, similar discovery was done by

Lauwers et al. (2013). They detected iron-rich gran-

ules in the inner avian ears, in cuticular plates of

receptor hair cells. This finding resembles our discov-

ery of iron-based granules in the roots of surface hairs

of bumblebees. The authors had a difficulty determin-

ing the mineral composition of the iron-rich granules,

but by means of electron diffraction, they concluded

that granules are probably composed of ferritin

molecules. The similarity between their and our

results shows that iron may be significant element

for normal evolution of surface hairs, thus the iron-

based granules in bumblebees may not be connected

with magnetosensation.

In spite of the fact that presence of strongly

magnetic magnetite in bumblebee wings was not

definitely evidenced, one can consider a sense of

magnetic granules in oscillating wings, in front edge

of the upper wings, close to head. These granules are

magnetised in a geomagnetic field and perform a

periodic motion with a frequency of approximately

220 Hz when a bumblebee flies. This motion gener-

ates an electromagnetic induction and this effect might

provide them magnetic compass information via a

head sensor (see Fig. 12). This hypothesis could be

evaluated by the behavioural experiment, when the

granules would be somehow removed from the wings.

Conclusions

In the surface tissue of the studied bumblebees, in the

roots of surface hairs, on the wings and on the head,

iron-based granules exhibiting paramagnetic magnet-

isation are located. The presence of these granules was

detected with the help of magnetometry and scanning

electron microscopy. XRPD analysis shows that

granules contain iron minerals magnetite and wuestite,

likely in superparamagnetic form according to Moss-

bauer spectroscopy. In this phase, the exact function of

these structures is very difficult to be determined, but it

can be stated that there are three options available:

directional magnetoreception (magnetic compass),

metabolic reservoir of iron for a proper function of

surface hairs and metabolic waste deposition. The

considered magnetoreception functioning of granules

located in the wings could be based on electromag-

netic induction generated by periodic motion of wings.

In further analyses, the mineralogical measure-

ments have to be done in samples from tens/hundred of

freshly killed bumblebee bodies where iron-based

granules will be present in sufficient amount, for better

reliability of analyses. For future, we do not recom-

mend to prepare the samples via drying and separation

processes, since in this case the validity of results is

threatened.

Acknowledgments This paper was elaborated in the framework

of the projects ‘‘IT4 Innovations Centre of Excellence’’, Reg. No.

CZ.1.05/1.1.00/02.0070, and Nanotechnology—the basis for

Fig. 12 Presentation of

hypothetic

magnetoreception principle

based on electromagnetic

induction activated during

bumblebee flight

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international cooperation project, Reg. No. CZ.1.07/2.3.00/20.0074

financed by the Structural Funds of EU and from the state budget of

the Czech Republic, the project Detection and characterization

ultrafine particles in living systems and their influence on

environment provided within Students Grant competition, Reg.

no. SP 2014/76, and finally Grant P108/11/1350.

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