Fossil insect eyes shed light on trilobite optics and the arthropod pigment screen

Article metrics

Abstract

Fossilized eyes permit inferences of the visual capacity of extinct arthropods1,2,3. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization4,5,6,7. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Structure of compound eyes of fossil and extant crane-flies.
Fig. 2: Chemistry of ommatidial lenses of fossilized crane-flies.
Fig. 3: Chemistry of corneal facet walls of a fossilized crane-fly.

Data availability

The fossil crane-flies analysed in this study are permanently accessioned into the collections of Museum Salling, Fur Museum (Fur, Denmark) and Museum Mors, Mo-clay Museum (Nykøbing Mors, Denmark). Our comparative trilobite fossil samples are housed in the research collections of the Department of Geology, Lund University. All supporting data are available from the corresponding author upon reasonable request.

Change history

  • 27 August 2019

    Owing to a technical error, this Letter was not published online on 14 August 2019, as originally stated, and was instead first published online on 15 August 2019. The Letter has been corrected online.

References

  1. 1.

    Lee, M. S. Y. et al. Modern optics in exceptionally preserved eyes of Early Cambrian arthropods from Australia. Nature 474, 631–634 (2011).

  2. 2.

    Paterson, J. R. et al. Acute vision in the giant Cambrian predator Anomalocaris and the origin of compound eyes. Nature 480, 237–240 (2011).

  3. 3.

    Anderson, R. P., McCoy, V. E., McNamara, M. E. & Briggs, D. E. G. What big eyes you have: the ecological role of giant pterygotid eurypterids. Biol. Lett. 10, 20140412 (2014).

  4. 4.

    Towe, K. M. Trilobite eyes: calcified lenses in vivo. Science 179, 1007–1009 (1973).

  5. 5.

    Gál, J., Horváth, G., Clarkson, E. N. K. & Haiman, O. Image formation by bifocal lenses in a trilobite eye? Vision Res. 40, 843–853 (2000).

  6. 6.

    Schoenemann, B., Clarkson, E. N. K. & Horváth, G. Why did the UV-A-induced photoluminescent blue-green glow in trilobite eyes and exoskeletons not cause problems for trilobites? PeerJ 3, e1492 (2015).

  7. 7.

    Schoenemann, B. & Clarkson, E. N. K. Vision in fossilised eyes. Earth Environ. Sci. Trans. R. Soc. Edinb. 106, 209–220 (2017).

  8. 8.

    Land, M. F. & Nilsson, D.-E. Animal Eyes (Oxford Univ. Press, 2002).

  9. 9.

    Schoenemann, B., Pärnaste, H. & Clarkson, E. N. K. Structure and function of a compound eye, more than half a billion years old. Proc. Natl Acad. Sci. USA 114, 13489–13494 (2017).

  10. 10.

    Nilsson, D.-E. & Kelber, A. A functional analysis of compound eye evolution. Arthropod Struct. Dev. 36, 373–385 (2007).

  11. 11.

    Vopalensky, P. & Kozmik, Z. Eye evolution: common use and independent recruitment of genetic components. Phil. Trans. R. Soc. Lond. B 364, 2819–2832 (2009).

  12. 12.

    Clements, T. et al. The eyes of Tullimonstrum reveal a vertebrate affinity. Nature 532, 500–503 (2016).

  13. 13.

    Ziegler, I. Genetic aspects of ommochrome and pterine pigments. Adv. Genet. 10, 349–403 (1961).

  14. 14.

    Struwe, G., Hallberg, E. & Elofsson, R. The physical and morphological properties of the pigment screen in the compound eye of a shrimp (Crustacea). J. Comp. Physiol. 97, 257–270 (1975).

  15. 15.

    Lindgren, J. et al. Molecular preservation of the pigment melanin in fossil melanosomes. Nat. Commun. 3, 824 (2012).

  16. 16.

    Glass, K. et al. Direct chemical evidence for eumelanin pigment from the Jurassic period. Proc. Natl Acad. Sci. USA 109, 10218–10223 (2012).

  17. 17.

    Pedersen, G. K. et al. Molerområdets geologi – sedimenter, fossiler, askelag og glacialtektonik. Geologisk Tidsskrift 2011, 41–135 (2011).

  18. 18.

    Freiwald, A. Insekten aus der Fur-Formation von Dänemark (Moler, ob. Paleozän/unt. Eozän?). 4. Tipulidae. Meyniana 42, 47–63 (1990).

  19. 19.

    Krzemiński, W. New fossil Tipuloidea (Diptera) from the Fur Formation of Denmark in the collection of the Natural History Museum in London. Polish J. Entomol. 70, 333–339 (2001).

  20. 20.

    Williams, D. S. Organisation of the compound eye of a tipulid fly during the day and night. Zoomorphologie 95, 85–104 (1980).

  21. 21.

    Ito, S. et al. Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples: application to chemical analysis of human hair melanins. Pigment Cell Melanoma Res. 24, 605–613 (2011).

  22. 22.

    Oakley, T. H. & Speiser, D. I. How complexity originates: the evolution of animal eyes. Annu. Rev. Ecol. Evol. Syst. 46, 237–260 (2015).

  23. 23.

    Needham, A. E. The Significance of Zoochromes (Springer, 1974).

  24. 24.

    Ren, D., Shih, C., Gao, T., Yao, Y. & Zhao, Y. Silent Stories – Insect Fossil Treasures from Dinosaur Era of the Northeastern China (Science, 2010).

  25. 25.

    Clarkson, E., Levi-Setti, R. & Horváth, G. The eyes of trilobites: the oldest preserved visual system. Arthropod Struct. Dev. 35, 247–259 (2006).

  26. 26.

    Nilsson, D.-E. in Facets of Vision (eds Stavenga, D. G. & Hardie, R. C.) 30–73 (Springer, 1989).

  27. 27.

    Speiser, D. I., Eernisse, D. J. & Johnsen, S. A chiton uses aragonite lenses to form images. Curr. Biol. 21, 665–670 (2011).

  28. 28.

    Alagboso, F. I., Reisecker, C., Hild, S. & Ziegler, A. Ultrastructure and mineral composition of the cornea cuticle in the compound eyes of a supralittoral and a marine isopod. J. Struct. Biol. 187, 158–173 (2014).

  29. 29.

    Fabritius, H.-O. et al. Functional adaptation of crustacean exoskeletal elements through structural and compositional diversity: a combined experimental and theoretical study. Bioinspir. Biomim. 11, 055006 (2016).

  30. 30.

    Ahlberg, P., Szaniawski, H., Clarkson, E. N. K. & Bengtson, S. Phosphatised olenid trilobites and associated fauna from the Upper Cambrian of Västergötland, Sweden. Acta Palaeontol. Pol. 50, 429–440 (2005).

  31. 31.

    Novellino, L., Napolitano, A. & Prota, G. Isolation and characterization of mammalian eumelanins from hair and irides. Biochim. Biophys. Acta 1475, 295–306 (2000).

  32. 32.

    Butenandt, A., Schiedt, U. & Biekert, E. Über Ommochrome, III. Mitteilung: Synthese des Xanthommatins. Justus Liebigs Ann. Chem. 588, 106–116 (1954).

  33. 33.

    d’Ischia, M. et al. Melanins and melanogenesis: methods, standards, protocols. Pigment Cell Melanoma Res. 26, 616–633 (2013).

  34. 34.

    Thiel, V. & Sjövall, P. in Principles and Practice of Analytical Techniques in Geosciences (ed. Grice, K.) 122–170 (Royal Society of Chemistry, 2015).

  35. 35.

    Lindgren, J. et al. Skin pigmentation provides evidence of convergent melanism in extinct marine reptiles. Nature 506, 484–488 (2014).

  36. 36.

    Lindgren, J. et al. Interpreting melanin-based coloration through deep time: a critical review. Proc. R. Soc. Lond. B 282, 20150614 (2015).

  37. 37.

    Lindgren, J. et al. Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur. Nature 564, 359–365 (2018).

  38. 38.

    Wakamatsu, K., Ito, S. & Rees, J. L. The usefulness of 4-amino-3-hydroxyphenylalanine as a specific marker of pheomelanin. Pigment Cell Res. 15, 225–232 (2002).

  39. 39.

    Carson, F. L. & Cappellano, C. H. Histotechnology: A Self Instructional Text (American Society of Clinical Pathologists, 2015).

Download references

Acknowledgements

M. Hofstedt collected specimen FUM-N-15451; C. Tell processed our extant tiger crane-fly samples; R. Hauff and G. Dyke provided comparative ink sacs from fossil squids; and C. Rasmussen prepared the histological sections, performed the Fontana–Masson staining, and assisted during the transmission electron microscopy analysis. Financial support for this project was provided by a Swedish Research Council Grant for Distinguished Young Researchers (642-2014-3773) to J.L.

Author information

J.L. and D.-E.N. conceived the project. J.L., D.-E.N. and B.P.K. wrote the paper with contributions from J.R.L. Jr and P.A., and feedback from all authors. J.L. assembled the figures with input from all authors. B.P.S., R.L.S. and H.M. prepared and curated all fossil crane-flies. J.R.L. Jr and D.-E.N. provided comparative tiger crane-fly and sandy swimming crab samples, respectively. J.L. and D.-E.N. prepared the thin sections, and C.A., J.L. and D.-E.N. undertook the polarized light microscopic investigation. C.A., P.S. and J.L. conducted the FEG-SEM and transmission electron microscopy analyses, P.L. and C.A. performed the EBSD experiments, S.I. and K.W. carried out the AHPO and HI hydrolysis analyses, P.S. and J.L. performed the ToF-SIMS experiments, M.E.E. and S.A.H. assembled the X-ray computed microtomographic data and M.E.E. made the three-dimensional reconstructions, M.J. synthesized xanthommatin and purified both the Nephrotoma screening pigments and Taeniopygia pheomelanin, and I.R.-M. and M.J. performed the maturation experiments.

Correspondence to Johan Lindgren.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Maria McNamara, Rana Sodhi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Representative samples of fossilized crane-fly compound eyes.

ad, Photographs (overview of specimen and enlargement of eyes, respectively) of specimen MHM-18a and MHM-18b (part and counterpart) immersed in Milli-Q water. Arrowheads in b and d indicate wrinkling along the perimeter of the optical surfaces. e, f, Photographs of specimen MHM-18-6a (part) immersed in Milli-Q water. g, h, Photographs of specimen FUM-N-15451 immersed in Milli-Q water (ah, n = 23 fossils). i, FEG-SEM image and X-ray computed microtomographic rendering (inset; illustration is representative of six independently sampled datasets; the colour is artificial) of the external visual surface in specimen FUM-N-13923, showing convex ommatidial lenses. j, FEG-SEM image of the internal visual surface in FUM-N-13923. Ommatidial lenses are represented by concavities. k, Enlargement of a transversely sectioned cornea in specimen MHM-18-6a (for SEM EDS element maps; see Extended Data Fig. 2). l, Magnification of the area demarcated in k showing the granular composition of the facet wall (il, n = 4 fossils). Scale bars, 2 μm (l), 30 μm (inset of i, k), 100 μm (i, j), 200 μm (f), 300 μm (b, d, h), 1 mm (e, g), 2 mm (a, c).

Extended Data Fig. 2 FEG-SEM micrograph and single-element EDS maps from a fossilized crane-fly compound eye (specimen MHM-18-6a).

SEM EDS maps differentiating increased levels of carbon, with traces of nitrogen and sulfur, in the corneal facet walls (identified by increased colour intensity) versus calcium and oxygen enrichment (with minor amounts of magnesium) in the ommatidial lenses. Data are representative of three independent analyses. Scale bar, 50 μm.

Extended Data Fig. 3 EBSD analysis of calcified crane-fly ommatidial lenses.

a, Compound eye of specimen FUM-N-13923, indicating analysed section (horizontal white line). b, Semi-transparent ommatidial lenses and darkly pigmented corneal facet walls shown in oblique section. c, FEG-SEM micrograph of the fossilized cornea. d, e, Enlargements of the boxed areas in c. Dashed coloured lines delineate individual ommatidial lenses analysed with EBSD (d, e, n = 15 separate lenses). Pole figures (stereographic projection, upper hemisphere) illustrate the {0001} (c-axis) orientation of six representative calcite crystals. Note that the c-axis is aligned with the optical axis in all lenses (yellow asterisk denotes the fossil lens depicted in Fig. 2c). Scale bars, 200 μm (b, c), 300 μm (a).

Extended Data Fig. 4 ToF-SIMS images and spectra from a compound eye of a fossilized crane-fly (specimen MHM-18-6a).

ad, Positive-ion images representing calcium carbonate (Ca2O+ m/z 96, Ca2O2+ m/z 112, Ca2O2H+ m/z 113, Ca3O2+ m/z 152 and Ca3O3+ m/z 168) (a) and a nitrogen-containing fragment (C3H8N+ m/z 58) (b), together with a two-colour overlay image of these ions (c) in which green represents the organic compound and red calcium carbonate, and the total ion image (d) with selected regions of interest (ROIs) marked in green (corneal facet walls) and red (ommatidial lenses). e, f, Positive-ion spectra generated from the ROIs indicated in d, representing corneal facet walls (e) and ommatidial lenses (f). gj, Images of negative ions representing eumelanin (C4H m/z 49, C3N m/z 50, C3NO m/z 66, C6H m/z 73, C5N m/z 74 and C8H m/z 97) (g) and oxygen (O m/z 16) (h), together with a two-colour overlay image of these ions (i) in which green represents eumelanin and red oxygen, and the total ion image (j) with selected ROIs marked in green (corneal facet walls) and red (ommatidial lenses). k, l, Negative-ion spectra generated from the ROIs indicated in j, representing corneal facet walls (k) and ommatidial lenses (l). Data are representative of six independent measurements.

Extended Data Fig. 5 PCA of ToF-SIMS images from a compound eye of a fossilized crane-fly (specimen MHM-18a).

Score image of principal component (PC)2 (inset), showing high scores for the corneal facet walls in specimen MHM-18a (Fig. 3), and a diagram comparing the positive loadings for PC2 (n = 4 analyses) with a synthetic eumelanin reference spectrum (n = 6 measurements). Note the close similarity between the distribution of the PC2 loadings and peak intensities in the eumelanin spectrum, which indicates that the chemical properties of the facet walls (bright areas in the PC2 score image) are directly comparable to this pigment. Negative PC2 loadings were obtained for O and SiOx ions, which represent the adjacent mineral matrix. The analyses included 141 negative-ion images at nominal masses between m/z 10 and m/z 150.

Extended Data Fig. 6 PCA comparing negative-ion ToF-SIMS spectra from the corneal facet walls in a fossil crane-fly with various reference materials.

a, Score plot from an analysis that includes all major eumelanin peaks in the mass range m/z 48–146 (Supplementary Table 3). b, Associated loadings from PC1 and PC2, with glyphs indicating the ion categories to which each peak is assigned. c, Bar graph comparing the added signal intensities from all peaks in the various ion categories (error bars are ±1 s.d.). In ac, xanthommatin, n = 3 measurements; xanthommatin (matured), n = 3 measurements; Sepia eumelanin, n = 3 measurements; Sepia eumelanin (matured), n = 3 measurements; dihydroxyindole (DHI) eumelanin, n = 3 measurements; DHI eumelanin (matured), n = 3 measurements; dopamine (DA) eumelanin, n = 4 measurements; ink sac of a fossil squid from the Dorset coast, n = 3 measurements; ink sac of a fossil squid from the Posidonia Shale, n = 3 measurements; fossil crane-fly facet walls, n = 7 measurements.

Extended Data Fig. 7 Experimentally treated N. suturalis corneas.

ai, Light microscopy images showing the visual appearance of the corneas (n = 1,300 sampled crane-flies) after the following sequential treatments: aqueous extraction with grinding (a), proteinase K and DTT (step 2; Supplementary Information) (b), protease and DTT (step 4) (c), chitinase (step 5) (d), proteinase K and DTT (step 6) (e), chitinase (step 7) (f), chitinase (step 9) (g), protease and DTT (step 11) (h) and heptane and HCl(aq) (i). Inset shows a FEG-SEM micrograph of the isolated corneal facet walls. All experiments were repeated twice independently with similar results. Scale bars, 30 μm (inset of i), 500 μm (a).

Extended Data Fig. 8 PCA comparing negative-ion ToF-SIMS spectra from N. suturalis screening pigments with various reference materials.

a, Score plot of an analysis that includes all major eumelanin and pheomelanin peaks in the mass range m/z 48–146 (Supplementary Table 3). b, Associated loadings from PC1 and PC2, with glyphs indicating the ion categories to which each peak is assigned. c, Bar graph comparing the added signal intensities from all peaks in the various ion categories (error bars are ±1 s.d.). In ac, Nephrotoma sub-cornea (extract), n = 3 measurements; Nephrotoma facet walls (extract), n = 3 measurements; xanthommatin, n = 3 measurements; Sepia eumelanin, n = 3 measurements; Nephrotoma facet walls, n = 3 measurements; Taeniopygia pheomelanin, n = 3 measurements; cysteinylDOPA (cysDOPA) (1:1) pheomelanin, n = 3 measurements; DHI eumelanin, n = 3 measurements; cysteinyldopamine (cysDA) (1:1) pheomelanin, n = 4 measurements; dopamine (DA) eumelanin, n = 4 measurements; DOPA eumelanin, n = 3 measurements.

Extended Data Fig. 9 Histological sections through N. suturalis compound eyes stained with Fontana–Masson.

a, Untreated section. b, Section bleached with hydrogen peroxide. c, Bleached section stained with Fontana–Masson. Note darkening of the corneal cuticle in the facet walls, which indicates the presence of melanin. Images are representative of three independent experiments. Scale bar, 50 μm (c).

Extended Data Fig. 10 FEG-SEM and polarized light micrographs of trilobite, fossil crane-fly and extant decapod ommatidial lenses.

a, FEG-SEM micrograph of a holochroal eye (specimen LO 12437t) of the trilobite Telephina bicuspis (Middle Ordovician period, Norway). b, Enlargement of fractured ommatidial lenses (a, b, n = 3 fossils). c, Single-spot SEM EDS spectrum acquired from the lens marked by a yellow asterisk in b, showing carbon, oxygen and calcium intensities that are consistent with calcite (compare with Fig. 2b). Platinum and palladium peaks derive from the sample pre-treatment (coating). Data are representative of six independent measurements. d, Plane-polarized light microscopy section through ommatidial lenses in a holochroal eye (specimen LO 12439t) of the trilobite Nileus armadillo (Middle Ordovician period, Sweden). e, Equivalent cross-polarized light micrograph. f, Plane-polarized light microscopy section through ommatidial lenses in an eye (specimen LO 12438t) of Tbicuspis. g, Equivalent cross-polarized light micrograph. h, Plane-polarized light microscopy section through a second eye (specimen LO 12437t) of Tbicuspis. i, Equivalent cross-polarized light micrograph, showing calcite crystal growth beyond the preserved lenses (arrowheads; Supplementary Information). j, Plane-polarized light microscopy section through an ommatidial lens in a schizochroal eye (specimen LO 12440t) of the trilobite Phacops latifrons (Middle Devonian period, Germany). k, Equivalent cross-polarized light micrograph. l, Plane-polarized light microscopy section through ommatidial lenses in an eye of a fossil crane-fly (specimen FUM-N-13923). m, Equivalent cross-polarized light micrograph. Data in dm are representative of three independent analyses. n, Cross-polarized light micrograph of calcite crystals in a compound eye of the extant sandy swimming crab, L. depurator (n = 3 specimens). Scale bars, 30 μm (l, n), 100 μm (d, f, h, j), 200 μm (b), 500 μm (a).

Supplementary information

Supplementary Information

This file contains supplementary text Part A-F and includes supplementary tables S1-S3.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.