Electronic Supplementary Materials:
Light-dependent Magnetic Compass Orientation in Amphibians and Insects — Candidate Receptors
and Candidate Molecular Mechanisms
John B. Phillips1, Paulo E. Jorge1,2 and Rachel Muheim3
1 Department of Biological Sciences, Virginia Polytechnic Institute and State University, Derring Hall, Blacksburg, VA
24061, USA
2 Unidade de Investigação em Eco-Etologia, Instituto Superior de Psicologia Aplicada, R. Jardim do Tabaco, n 34, 1149049 Lisboa, Portugal
3 Department of Animal Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden
S1.1 Evidence for Magnetic Sensitivity in Retinula 1-6 Cells in the Compound Eye of the blowfly Calliphora
vicina.
Intracellular recording techniques were used to sample the effects of different alignments of a 100 μT
magnetic field on the response of R1-6 cells to light in the compound eye of the blowfly, Calliphora vicina
(Phillips 1987).
Methods
Glass microelectrodes were manufactured from borosilicate or aluminasilicate glass using a Sutter
Instruments P-80/PC microelectrode puller, filled with 3M KCl or 4M K-acetate, and used to record
intracellularly from retinula 1-6 (R1-6) cells in the compound eye of the blowfly. Data shown in Table S1 and
Figure S1 are from recordings obtained with high resistance (> 600 megaΩ) electrodes that produced stable
responses when first introduced into the preparation. The signal from the recording electrode was processed
initially by a pre-amplifier housed in an electrically shielded box adjacent to the preparation. The signal was
then routed on to a custom low-pass filter and amplifier. The recording apparatus was enclosed in a
grounded aluminum box located inside the stimulus coils (see below). The stimulus coils and recordings
systems were constructed largely of non-magnetic materials.
Magnetic stimuli were produced by a concentric five-coil system, consisting of two cube surface coils aligned
to cancel the vertical and horizontal components of the ambient field, and a 3-axis octagonal coil system that
2
could produce a 100 uT field in any alignment in 3-dimensions. Stimulus control was carried out using
custom software and hardware designed and constructed by Jon Willis. Light stimuli were produced with a
100 W tungsten/halogen light source, monochromator (Oriel), counter-rotating neutral density wedges and
shutter (Vincent Associates). Light stimuli were transmitted to the preparation by a liquid-core fiber optic
(Oriel). The output end of the fiber optic was mounted on an optical perimeter used to vary the alignment of
the fiber optic in both the horizontal and vertical planes. A lens focused the output of the fiber optic onto the
surface of the compound eye.
Blowflies, Calliphora vicina, were raised on beef liver in a laboratory colony. For the experiments, all of the
appendages were removed from the fly and the resulting openings sealed with wax. A small opening was
made at the top of the left eye, which was covered with mineral oil to prevent drying. The preparation was
positioned in the center of the five-coil system with the left eye at the center of the optical perimeter. A
reference electrode (low resistance micropipette) was inserted through a small opening in the top of the
thorax. The recording electrode was advanced down into the compound eye using a Kopf hydraulic
microdrive. Oscillation of the recording circuit (“buzzing”) was used to facilitate cell penetration. Only high
resistance electrodes presumed to have very fine tips (as indicated by strong responses and occasional
recordings from the smaller R7 and R8) provided evidence of magnetic field sensitivity (see below). Once a
cell was impaled, the alignment of the fiber optic was varied in three dimensions while presenting a
sequence of identical flashes of light. The optical axis of the cell was determined as the alignment of the fiber
optic that produced the highest amplitude of response.
The type of photoreceptor was rapidly determined using flashes of light of at least 3 different wavelengths
adjusted to the same quantal flux; the accuracy of this method for identifying cell type was confirmed in
some cells by obtaining responses to equal quantal flashes of light of wavelengths every 30 nm from 340580 nm. In recordings carried out over more than one year, only a dozen or so cells were identified as R7 or
R8 cells (complete spectra were obtained from at least one cell of each of the four subtypes, i.e., R7p, R7y,
R8p, and R8y). The small number and short duration of R7 and R8 recordings prevented systematic
sampling for magnetic field sensitivity.
To determine if the R1-6 cells are sensitive to magnetic field alignment, the peak amplitude of response to
20 ms flashes of 370 nm light (given at a rate of 1 Hz) was measured in each of 32 horizontal alignments of
100 µT magnetic field, uniformly spaced through 360° (referred to as one horizontal “sweep” of the magnetic
field). Light stimuli were adjusted to produce amplitudes of response ~60% of the maximum. In a majority of
sweeps, responses to five flashes of light were averaged in each magnetic field condition. Prior to the
beginning of each sweep, a continuous series of flashes identical to those used to sample the magnetic field
response was presented at a rate of 1 Hz until the cell’s response stabilized. During the magnetic sweep,
after every second magnetic field alignment, a control response was obtained to identical flashes of light with
the magnetic field cancelled (< 5% of the stimulus intensity) to control for changes in the overall sensitivity of
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the preparation. In both the experimental and control conditions, the first flash was presented 800 ms after
setting a new magnetic field condition. The average response obtained in each alignment of the magnetic
field was then normalized by dividing this value by either the lowest or highest of the values obtained in the
two adjacent control responses with the magnetic field cancelled (see below).
Variation in the normalized responses in most recordings was quite small, typically in the range of +1-2%
(but see Fig. 3 in the main text). For analysis, we compared the distribution of magnetic field alignments
associated with amplitudes of response that were as low or lower than the lowest of the two adjacent control
responses (“NormLow”) with that of the magnetic field alignments associated with amplitudes that were as
higher or higher than the highest of the two adjacent control responses (“NormHigh”). Due to variation in the
stability of responses over time, data are only presented from the first 360° “sweep” of the magnetic field
sampled from each recording (Table S1 and Fig. S1). In the data shown in Table S1, the mean axis of
magnetic field alignments associated with either the NormLow or the NormHigh responses was calculated by
vector addition from the distribution of doubled angles. The non-parametric Watson U2 test was used to
determine if there was a significant difference in the unimodal or bimodal distribution of magnetic field
alignments associated with NormLow and HormHigh responses (test carried out on undoubled and doubled
angles; respectively; Batschelet 1981)
Results and Discussion
Although variation in the amplitude of response was quite small, recordings obtained with high resistance
electrodes showed a weak, but consistent, effect of the magnetic field (Table S1; and see Fig. S1). In
recordings obtained with low resistance electrodes (< 600 megaΩ), normal spectral sensitivities were
observed, but there were no consistent effects of the magnetic field on the response to light (data not
shown).
Table S1. Possible magnetic field effects on intracellular responses of blowfly R1-6 cells. NormLow and NormHigh--see
Methods. Hor – horizontal alignment of the optical axis of the cell relative to the anterior end of fly, Vert – vertical
alignment of the optical axis relative to horizontal, Axis—mean axis calculated from magnetic field alignments relative to
anterior end of fly, r – mean vector length, n – sample size. Significance by the Watson U2 test (Batschelet 1981) for
bimodal (Bimod U2) and unimodal (Unimod U2) distributions, (*) - p < 0.10, * - p < 0.05, *** - p < 0.001. 1X—response to
only one flash of light sampled in each alignment of the magnetic field. K-Ac –recording and reference electrodes filled
with 4M K acetate. † Only responses lower than either of the adjacent control responses included in the NormLow
distribution for this cell.
4
Optical Axis
NormLow
NormHigh
Statistical Comparisons
Bimod U2
Unimod U2
0.37(12)
0.180(*)
0.095(ns)
101-281°
0.37(12)
0.191*
0.084(ns)
0.67(10)
178-358°
0.26(8)
0.254*
0.061(ns)
32-212°
0.41(14)
133-313°
0.45(6)
0.163(*)
0.116(ns)
†
17°
24-204°
0.32(15)
117-297°
0.55(11)
0.250*
0.082(ns)
K-Ac, †
360°
20°
25-205°
0.34(13)
114-294°
0.30(11)
0.158(*)
0.133(ns)
7
336°
22°
96-276°
0.22(16)
5-185°
0.43(11)
0.170(*)
0.143(ns)
8
338°
27°
146-326°
0.54(7)
65-245°
0.51(14)
0.252*
0.116(ns)
9
350°
28°
150-330°
0.56(9)
77-257°
0.26(14)
0.224*
0.075(ns)
10
332°
30°
13-193°
0.48(14)
101-281°
0.22(13)
0.233*
0.091(ns)
11
335°
42°
139-319°
0.30(14)
22-202°
0.61(8)
0.183(*)
0.166(*)
12
315°
50°
164-344°
0.54(14)
78-258°
0.53(14)
0.404***
0.298**
13
346°
55°
157-337°
0.40(12)
69-249°
0.43(11)
0.225*
0.118(ns)
Cell#
Hor.
Vert.
Axis
r (n)
Axis
r(n)
1
330°
0°
86-266°
0.38(16)
17-197°
2
---
0°
17-197°
0.54(10)
3
340°
2°
90-270°
4
355°
16°
5
345°
6
Add. Info
1x
K-Ac
Two characteristics of the responses shown in Table S1 suggest that these responses, although extremely
weak, do represent an effect of magnetic field alignment on the photoreceptor’s response to light. First,
variation in R1-6 cell response was dependent on the alignment of the axis (“Bimod U2”), not polarity
(“Unimod U2”), of the magnetic field, as predicted by the RPM (Ritz et al. 2000; Rogers & Hore 2009) and
shown to be characteristic of the light-dependent magnetic compass responses of birds, amphibians, and
some insects (Wiltschko & Wiltschko 1972; Phillips 1986, Vácha et al. 1988a, Dommer et al. unpubl.).
Secondly, a reanalysis of the data carried out several years after completion of the experiments revealed
that the distributions of the mean magnetic field axes associated with low amplitude (Fig. S1a) and high
amplitude (Fig. S1b) responses exhibit regular hexagonal patterns. Although the explanation for these
patterns is by no means clear, the presence of a regular hexagonal arrangement in R1-6 morphology (Fig.
S1c) and in the projections on to second order cells (Hardie 1986) is consistent with the effect of the
magnetic field originating in this class of photoreceptor cells.
5
Figure S1. Mean axes calculated from magnetic field alignments associated with (a) low amplitude (NormLow) and (b)
high amplitude (NormHigh) responses to 20 ms flashes of 370 nm light; see methods. (c) Schematic diagram of cross
section through one ommatidia showing the hexagonal array of microvillar alignments (parallel lines) in the rhabdomeres
(Rh) that make up the photopigment-containing portion of the receptors cells (RC); figure from Hardie & Kirschfeld
(1983). Double-headed arrows in (a) and (b) are the mean axes calculated for cells in Table S1 plotted as deviations
from each cell’s optical axis (black triangle at the top of each diagram).
S1.2 A variable, magnetically-sensitive screening pigment in fly photoreceptors? An alternative design for a
cryptochrome-based magnetoreceptor in flies is one in which the blue-absorbing carotenoid screening
pigment in R7y (Kirschfeld et al. 1978; McIntyre & Kirschfeld 1981; Kirschfeld et al. 1983) is replaced by
DmCRY1 in a subpopulation of these cells. The absorption spectrum of the fully oxidized form of DmCRY1 is
remarkably similar to that of the carotenoid screening pigment (compare Fig. 4 and 5a). Furthermore,
dichroic absorption of blue light (400-500 nm) by R7y indicates that screening pigment molecules (at least of
the carotenoid) are non-randomly aligned (Vogt & Kirschfeld 1983, 1984). If DmCry1 serves as the screening
pigment in a subpopulation of R7y cells (perhaps in ommatidia located along the lateral or ventral margin of
the compound eye), an effect of the magnetic field on the photo-equilibrium between the full reduced and
flavo-semiquinone forms of the flavin chromophore (Fig. 5a) would change the spectral properties of
incoming light and, thus, could alter the responses of both the R7y and underlying R8y cells. Referencing the
responses of these hypothetical cryptochrome-screened photoreceptors to those of the remaining
subpopulation of carotenoid-screened (non-magnetically sensitive) R7y and R8y cells, and/or to the UVsensitive R7p and blue-sensitive R8p cells (Hardie 1986), would make it possible to distinguish the magnetic
field’s effect from the variation in response caused by changes in background light levels.
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S1.3 Could cryptochrome function as an antenna pigment in fly photoreceptors? Behavioral responses of
flies to both magnetic and spectral light stimuli (Phillips & Sayeed 1993; Troje 1993; Fukushi 1994; Gegear
et al. 2008) are consistent with the involvement of a cryptochrome-based photosystem (Fig. 5a), while
preliminary neurophysiological responses have provided evidence for an antagonistic effect of the magnetic
field on photo-excitation pathways resulting from the absorption of light by the light-harvesting antenna
pigment and photopigment in fly photoreceptors (Fig. 3, Fig. S1; Table S1). An intriguing question is whether
these two sets of observations have a common explanation, i.e. whether DmCRY1 (or another flavin or
flavoprotein; McIntyre & Kirschfeld 1981) functions as an antenna pigment (e.g.) in a subpopulation of R7y or
R8y cells. Because the fully oxidized flavin chromophore has a peak of fluorescent emission at ~530 nm
(Saxena et al. 2005), which coincides with the λmax of the R8y photopigment (Kirschfeld et al. 1978, 1983,
Hardie 1986, McIntyre & Kirschfeld 1981), efficient transfer of energy could occur. If, however, prior to
transfer of energy to the photopigment, the flavin chromophore is reduced to the radical form, which has
relatively little fluorescent emission, there could be a decrease in the efficiency of energy transfer.
Depending on a variety of factors, including the relative time constants of these competing processes, an
antagonistic effect of the magnetic field on the photoreduction of the fully oxidized to the radical form (FADox
→ FAD•ˉ) and photooxidation of the radical form back to the full oxidized form (FAD•ˉ → FADox; Section 4)
could have corresponding effects on the efficiency of energy transfer, and thus on the response of the
photoreceptors to light. Selective screening (McIntyre & Kirschfeld 1981), and differencing of the responses
of magnetically sensitive and non-magnetic sensitive photoreceptors, could accentuate the magnetic field’s
effect.
We should emphasize that, to date, there is no evidence to suggest that the similarities between antenna
pigment and screening pigment absorption spectra in fly photoreceptors (Fig. 2 and 4), and those of type 1
insect cryptochromes (Fig. 5a), are anything but coincidence. Nevertheless, experiments to determine if
energy transfer between the antenna pigment and photopigment in R7y or R8y is sensitive to the magnetic
field alignment (as preliminary evidence from R1-6 suggests; Fig. 3, Fig. S1) and, if so, whether the
magnetic field’s effect is disrupted by exposure to low-level radio frequency fields (Ritz et al. 2004, 2009;
Vácha et al. 2009), would be relatively straight forward to carry out. These and other related experiments
should be the first steps in determining whether photoreceptors like R7y and R8y are specialized for
detection of the earth’s magnetic field.
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