Characterization of organics consistent with β-chitin preserved in the Late Eocene cuttlefish Mississaepia mississippiensis

Abstract

Background: Preservation of original organic components in fossils across geological time is controversial, but the potential such molecules have for elucidating evolutionary processes and phylogenetic relationships is invaluable. Chitin is one such molecule. Ancient chitin has been recovered from both terrestrial and marine arthropods, but prior to this study had not been recovered from fossil marine mollusks.

Methodology/principal findings: Organics consistent with β-chitin are recovered in cuttlebones of Mississaepia mississippiensis from the Late Eocene (34.36 million years ago) marine clays of Hinds County, Mississippi, USA. These organics were determined and characterized through comparisons with extant taxa using Scanning Electron Microscopy/Energy Dispersive Spectrometry (SEM/EDS), Field Emission Scanning Electron Microscopy (Hyperprobe), Fourier Transmission Infrared Spectroscopy (FTIR) and Immunohistochemistry (IHC).

Conclusions/significance: Our study presents the first evidence for organics consistent with chitin from an ancient marine mollusk and discusses how these organics have been degraded over time. As mechanisms for their preservation, we propose that the inorganic/organic lamination of the cuttlebone, combined with a suboxic depositional environment with available free Fe(2+) ions, inhibited microbial or enzymatic degradation.

Figure 1. Generalized locality map.

Generalized locality map showing sites where M. mississipiensis were collected and the geographical extent of the Jackson Group in Mississippi.

Figure 2. Fragmentary preserved cuttlebone of M. mississippiensis (MGS 1951).

Fragmentary preserved cuttlebone of M. mississippiensis (MGS 1951), late Eocene, Mississippi, USA: A, B – left lateral and ventral view, respectively.

Figure 3. SEM/EDS images comparing similar structures of M. mississippiensis and Sepia.

Sheet-like structures in between carbonate spherulites in M. mississippiensis (MGS1956, Fig. 2Aa) and Sepia sp. (Fig. 2Cb). 2B, EDS analysis of M. mississipiensis (spot a) showing phosophorus in the sheet-like structures.

Figure 4. Hyperprobe comparison of HCL de-mineralized M. mississippiensis and extant Sepia sp.

A) Hyperprobe analysis of HCL de-mineralized fossil (MGS 1951) mapped for nitrogen (red). B) Hyperprobe analysis of HCL de-mineralized extant cuttlebone mapped for nitrogen (red).

Figure 5. FTIR spectra of cuttlebones of Sepia sp. and M. mississippiensis.

Spectra of Sepia sp. (top) and M. mississippiensis (bottom). Spectra indicate aragonite. By comparison, calcite has peaks at 848 and 876 cm⁻¹and a singlet at 713 cm⁻¹ .

Figure 6. FTIR spectra of HCL de-mineralized M. mississippiensis, Sepia sp. and Loligo sp.

FTIR spectra of fossil (a) M. mississippiensis and chitin from extant (b) Sepia sp. and (c) Loligo sp. A) All spectra are consistent with beta chitin. B) FTIR spectra of the hydrogen stretching region of a, b and c showing reduction in OH of M. mississippiensis. C) FTIR spectra of a, b and c emphasizing Amide I and Amide II absorbances which are distinctive for beta chitin. D) FTIR spectra of a and b; portions of the Amide III region are missing in the fossil. Overall broadening and loss of structure of the peaks is interpreted as breakdown of chitin chains into shorter fragments.

Figure 7. IHC comparison of HCL de-mineralized M. mississippiensis and Sepia sp.

A,C,E,G,I, fossil M. mississipiensis (MGS 1951); B,D,F,H,J, extant Sepia sp. A, B) Fossil/extant incubated in chitin antibody and secondary antibody, show antibody/antigen binding. C,D) Fossil/extant incubated in secondary antibody only, no antibody/antigen binding. E,F) Fossil/extant incubated in chitinase, chitin anti-body and secondary antibody. Tissues have been digested by the chitinase giving a much reduced immunological signal. G,H) Fossil/extant incubated in chitinase, chitin antibody and secondary antibody. Tissues in these samples were not completely digested by the chitinase, eptiopes were unmasked, and show enhanced antibody/antigen retrieval. I,J) Fossil/extant incubated in human testosterone antibody and secondary antibody, shows no antibody/antigen binding.

Figure 8. Comparison of two FTIR spectra of M. mississippiensis (MGS 1951).

a) spectrum lacks Amide I and Amide II peaks, b) for comparison, spectrum shows the Amide I peak at 1656 cm⁻¹ and Amide II peak at 1545 cm⁻¹.