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Why the term ‘iron mineral-based magnetoreception’ should not be used, not even in birds

Posted by Michael_Winklhofer on 05 Mar 2010 at 18:46 GMT

We appreciate the experimental findings by Falkenberg and coworkers and are glad to see the iron-bearing nerve terminals in a number of other bird species besides homing pigeons. In the results section, the authors interpret the avian X-ray absorption spectra cautiously but then in the discussion section they become set on an interpretation that is not supported by experimental data.

1) Falkenberg et al first write that none of the reference materials matches the avian material completely and that the avian spectra “hint towards iron(III) oxides”. But later they write: “This result clearly shows that the dendrites in the avian beak mainly contain iron(III) oxide and not pure magnetite, as predicted in several papers, where magnetite was assumed to be the only magnetic mineral underlying biological magnetoreception (24,31,45,46).” (Note: ref. 24 and 46 are the same paper).

What the XANES data actually show is that most of the iron in the dendrites is ferric, i.e. iron(III). But the results do not tell yet if the compound in question is an iron(III) oxide, or rather a iron(III) hydroxide, iron(III) oxy-hydroxide, or even iron(III) phosphate because any such ferric compound would fit the near-edge part of the dendrite spectrum between 7115 and 7125 eV. The obvious mismatch between the spectra of maghemite and of dendrite at about 7130 eV (Fig. 7) does not really support the claim made in the abstract (“We also used microscopic X-ray absorption spectroscopy analyses to identify the involved iron minerals to be almost completely Fe III-oxides”).

The point here is: The presence of ferric iron does not automatically make the case for a strongly magnetic compound, and this is what matters most in the context of magnetoreception. Among the iron(III) compounds, only maghemite is strongly magnetic, while other possible iron(III) compounds are weakly magnetic at best and therefore not interesting from the point of view of magnetoreception (although they are interesting from the point of view of biochemistry and biomineralization). The decisive question for the magnetoreception mechanism is therefore: is the compound in question magnetic at all?
As in our comment to the paper by Fleissner et al. 2007 in Naturwissenschaften (see we would like to call the authors’ attention to the possibility that a lot of ferric iron may reside in the iron-storage protein ferritin, which would contribute to the XANES spectrum as well. We suggest taking into account ferritin when seeking to explain the XANES spectra quantitatively.

Given the suite of possible Fe III compounds, we suggest to use electron diffraction to identify the structure of the compound. Diffraction patterns together with the chemical information will help to identify the mineralogical nature of the compounds.

2) Falkenberg et al write: “This view [ that maghemite may account for most of the iron inside the dendrites] is shared by Tian et al. (49), who performed various physical tests, especially SQUID measurements of isolated beak skin samples and interpreted their data on the magnetic remanence as strong evidence that additionally to magnetite a second magnetic mineral – they proposed maghemite – must be contained in the dendritic system”.

It is interesting to compare this to what was actually written in Tian et al (49): "This suggests that the remanence carriers in the samples are magnetically soft minerals (probably magnetite or maghemite). ... Remanence loss around 120 K on the cooling curves of SIRM300 K and warming curves of SIRM5 K clearly indicates the presence of magnetite in the measured samples (Figures 2 and 3). A significant rapid decay of SIRM5 K in the interval of 5-20 K on both ZFC and FC warming curves (Figure 2) suggests the dominance of superparamagnetic (SPM) particles in the samples. ... The low delta ratios (less than 2) calculated from the thermal demagnetization curves of SIRM5 K in this study do not support a chain arrangement of magnetite crystals as seen in magnetotactic bacteria (Moskowitz et al. 1993; Pan et al. 2005)."

So, since magnetite and maghemite have similar magnetic properties at room temperature (for a given domain state), Tian et al were cautious enough to not assign either mineral to their room temperature remanence curves. But the subsequent low-temperature cycling of the remanence imparted at room temperature clearly showed that magnetite carries a substantial part of the remanence. On the basis of the Moskowitz delta-ratio test, Tian et al inferred that the magnetic properties are not dominated by chains of single-domain magnetite, but this does not mean that single-domain magnetite is absent. It simply means that chains, if present, do not contribute by more than 50% or so to the remanence. To conclude, we find it inappropriate to use the paper by Tian et al as strong support for maghemite.

3) Falkenberg et al write: “The XANES spectra of the dendrites suggest that magnetite is not the main constituent. Thus, care should be taken before using the well established notion of magnetite-based magnetoreception. Rather the term ‘iron mineral-based magnetoreception’ should be used – at least in birds.”

Note that magnetite may still be a major magnetic constituent in case the predominant ferric compounds are nonmagnetic (see point A), which would tally with the magnetic interpretation by Tian et al. (see point B). Apart from that, one has the impression that the authors convey the notion that magnetite and maghemite are altogether different minerals, but in fact they are so closely related to each other that there is a solid-solid solution with magnetite and Fd3m maghemite as end members. Since the two minerals are structurally related and have similar lattice constants, distinction by means of electron diffraction without chemical information is hardly possible (as the authors correctly point out). Also, as the authors acknowledge, maghemite has magnetic properties similar to magnetite (at physiological temperatures). As the authors are aware of all these similarities, one is left wondering why they make such a big deal of the distinction, which to us appears overstated, particularly at this stage where it is not even clear what magnetic compound is biomineralized in birds in the first place.

Along these lines, “iron-mineral based” magnetoreception is not an emerging mechanism, unlike the authors write in their introduction. Magnetite is an iron mineral after all, and the idea has been around since the discovery of biogenic magnetite by Heinz A. Lowenstam in chiton teeth.
“Iron-mineral based” does not sound very specific to a mineralogist, given the plethora of iron-bearing minerals, most of which are not strongly magnetic. Why not call it “magnetite/maghemite based magnetoreception”? This is consistent with the diffraction data available so far. The nanocrystalline particles in the spherical clusters (referred to as “bullets”) in the iron-bearing nerve terminals have a diffraction pattern consistent with randomly oriented magnetite/Fd3m maghemite crystals (Hanzlik et al 2000), while the iron-phosphorous bearing material in the shape-bags (referred to as “platelets”) co-localized with the clusters is amorphous (Fleissner et al 2003) and thus not consistent with either magnetite or maghemite. Although the authors dogmatically rule out magnetite as dominating magnetic constituent (at least in birds), they did not consider the possibility that nanocrystalline magnetite (we are dealing with grain sizes of a few nm in the case of the clusters or “bullets”) may have rapidly oxidized to maghemite during the preparation, because oxygen was not prevented from diffusing into the tissue. It may be worthwhile to subject magnetite nanoparticles from a water based ferrofluid to the same perfusion and fixation protocol as the one applied to the beak tissue. This way, a suitable control sample would be available to test for the influence of oxidation on the spectra.

Last, we have a couple of specific questions:
1) Two spots (pt 1 and pt 3 in Fig. 5) were identified as contamination – does the Titanium map show an anomaly here as well? This would help to narrow down the source of the contamination. If so, it would be interesting to do XANES iron K-edge mapping of the Titanium tools used for tissue preparation in order to see if the Titanium tools have traces of Fe contamination.
2) It is written that the absorption spectra of the dendrites may differ depending on whether they contain only part of a dendrite. How do they differ?
3) Does the section shown in Fig. 1A have the same orientation as the one shown in Fig. 1F?

Michael Winklhofer, Ludwig-Maximilians-University, Munich, Germany
Marianne Hanzlik, Technical University Munich, Germany
Joseph L. Kirschvink, California Institute of Technology, Pasadena, USA

No competing interests declared.

RE: Why the term ‘iron mineral-based magnetoreception’ should not be used, not even in birds

fleissner replied to Michael_Winklhofer on 21 Mar 2010 at 14:59 GMT

The commentators appreciate our experimental work. Thank you!
We answer to the various topics of their critique separately:

1) The main point of critique in this paragraph is that we interpret our X-ray data as evidence that a Fe(III)-oxide, presumably maghemite, is the predominant substance inside the dendrites. We also state - like we have done in previous papers - that the spatial resolution of the X-ray analysis does not yet allow for a separate investigation of the various subcellular compartments; hence the dendritic spectrum is a “mixture” of the contribution from bullets, platelets, vesicle and biological matrix around the dendrite.

Winklhofer et al. propose to consider also other Fe(III) compounds instead of the assumed maghemite. We agree that the absorption edge position only indicates the ferric nature of the dendrites (Fe(III)). The attribution of the dendrites to a Fe(III)-oxide structure includes also the analysis of the EXAFS region of the spectrum which features a strong peak compatible to a Fe-O bond distance. Iron compounds with phosphor or sulfur as main constituents can also be excluded, because neither phosphor nor sulfur is correlated to dendrite positions in XRF maps.
Of course the presence of ferric iron (oxides) does not automatically make the case for a strongly magnetic compound. The magnetic nature was postulated by us on the basis of other observations (e.g., Fleissner et al. 2003) and motivated the selection of magnetite and maghemite as reference samples in the presented paper. However also other standard compounds including ferritin and hemoglobin where measured and none of them made a better fit in the complete XANES region than maghemite.

We therefore stick with our original opinion: Maghemite - as the strongest magnetic compound among the Fe(III)-minerals - appears to be the best interpretation of the experimental data.

As soon as our group will have access to the new hard X-ray Micro/Nano-Probe beamline at PETRA III (DESY), probably from fall 2010 onwards, we will be able to measure XRF, XANES and also X-ray diffraction with sub-micrometer spatial resolution. This should facilitate us to image and measure the subcellular compartments separately and will hopefully enable us to attribute the specific iron oxides to the various iron-containing substructures inside the sensory dendrites in the beak.

The suggestion to use "electron diffraction" is most welcome, and we have in fact applied this method. The results on the platelets are, however, not yet satisfying, because of the preparational precautions and rapid radiation damage. Even a repetition of the diffraction analysis of the bullets showed their rapid decay during the procedure. All what we have seen so far support our view of a crystalline nature of the platelets.

Until then, our XANES-spectra must be evaluated as the best method of choice for characterizing the dendritic iron. We show in the PlosOne manuscript that in various avian species, a similar composition of maybe a little magnetite and much more maghemite occurs, like we have reported before for homing pigeons in our Fleissner et al 2007 paper. Our previous measurements (Hanzlik et al. 2001) were performed at the electron microscope under the assumption that magnetite will be the only key mineral for the magnetoreceptor transduction processes. But this method is obviously not adequate for this problem as on one hand the nano-crystalline structure is rapidly destroyed, and on the other hand little quantities of magnetite and maghemite cannot be discriminated, as the commentators acknowledge.

2) Tian et al. have applied biophysical methods to verify (and with a main focus on) magnetite inside the pigeon sensory dendrites. However, they interpret their results: “….this suggests that the remanence carriers in the samples are magnetically soft minerals (probably magnetite or maghemite)..” They do not exclude maghemite or deliver arguments to insist on magnetite as sole magnetic iron compound inside the dendrites. This in combination with our finding that a Fe(III)-compound is definitely dominating over Fe(II/III)-compounds justifies our wording “strong evidence”. If you see it in the “naked” relation to the text of the Tian et al. paper, we confess: one may understand this phrasing as an overestimating of Tian's text.

But what Tian et al. explicitly exclude is a major contribution of a chain of single domain magnetite: ”The thermal demagnetization curves ….in this study do not support a chain arrangement of magnetite crystals as seen in magnetotactic bacteria…”

3) We do not "make a big deal of the distinction" between magnetite and maghemite, we only react with our statements to the former (and obviously still ongoing) language use that non-biochemical magneto receptive processes are exclusively based on magnetite, which is by our findings highly unlikely. The suggestion “magnetite/maghemite based magnetoreception” has no advantage over the term “iron-mineral based” because the discussion here itself demonstrates nicely, that firstly this type of magnetoreceptor is a matter of “iron-minerals”, and secondly, there are still so many unanswered detailed questions about the functional meaning of the different and numerous compounds involved in biological iron metabolism. Therefore, from our point of view “iron-mineral based” is the only appropriate term considering the state of the current hard experimental evidence existing in this field of science.Thus, the term “iron-mineral based” makes most sense for this type of magnetoreceptor.

Winklhofer et al. refer to the discovery of biogenic magnetite in radula teeth. We simply do not understand the relation of this result to magneto receptive processes.

We also do not understand that Winklhofer et al. refuse to acknowledge the necessity of another magnetic compartment inside the dendrites. The bullets and the here assembled nanomagnets are too small, too sparse and - most important - they are attached to random sites around the dendritic membrane. Thus, they cannot serve as the sole basis for a direction-sensitive excitation, because, a single dendrite could then only signal the magnetic field strength without any directional information. The clear 3D-alignment of the dendritic fields, which provides strong evidence for a potential directionality of the system, was not yet known, when we wrote the earlier papers together with the Munich colleagues; this latter finding was published in our manuscript Fleissner et al 2007. Winklhofer et al. insist that these second type of magnetic compartments, which we name platelets according to their regular shape, contains “amorphous iron”. We do not know any sound experimental results, which would support this assumption.

In their assessment, Winklhofer et al. propose several experiments, which might be very helpful. We would encourage them to consider contributing their own efforts to accomplish these analyses.

No competing interests declared.