3.4. Biochemical composition of the carcasses: Spectral characterization.
There are two main hypotheses that consider the biochemical components of the soft body as the key factor of preservation. The first one is focused on the presence of recalcitrant tissues such as chitin (Butler et al., 2015), while the second one emphasizes the stability of proteins tanned by aluminium ions (Wilson, Butterfield, 2014; Naimark et al., 2016a). To evaluate the importance of the two mechanisms, we assessed the sustainability of chitin and proteins in the carcasses with contrasting degrees of preservation (Figure 3 A, B) using FT-IR spectroscopy (see Materials and Methods) (Figure 3 C-H).
In all carcasses (except for the sediment-free control), the FT-IR profiles consisted of two parts: the bands corresponding to chemical bonds in organic molecules, and the bands that correspond to the mineral (inorganic) components of the sediment. We first considered the organic part (Figure 3 C-E).
The spectra of all the studied A. salina samples from the different sediments were similar. The spectral pictures of the excellently preserved montmorillonite-hosted specimens and poorly preserved clinochlore-hosted samples showed little to no difference (Figure 3 D, E). In the carcasses from different clays with different degree of preservation, the same peaks were present, and the differences affected only the intensities of the peaks. Moreover, the best and worst preserved specimens belonging to groups 1 and 4-5 from the kaolinite also had very similar FT-IR spectra (Figure 4). The spectra also did not differ much from the two controls: live nauplii and the decayed sediment-free control (Figure 3 C). The protein characteristic bands (known as Amid I and II between 1600 and 1700 cm-1 and 1510 and 1580 cm-1 respectively; Figure 3E) varied little across the carcasses from different sediments. The same is true for the chitin characteristic bands (between 3400 and 3500 cm-1 and 2950-2800 cm-1 (Negrea et al., 2015)) (Figure 3D).
The second, inorganic part of the spectra occupies intervals at wavelength <1300 cm-1. In all exhumed carcasses, these profiles repeated the spectra for the corresponding sediments (Figure 3F, G). In the SEM images, we could see small (less than 1 µm) sediment particles that stayed attached to the organic surface (Figure 2 G) even after multiple intensive rinsings. So, can the similarity of the spectra of carcasses and sediments be explained by mere adherence of inorganic particles to the carcasses? Probably not, because the 1-day control specimens, which had been kept in the kaolinite sediment for one day after death and then rinsed according to the common protocol, showed a much less pronounced signature of kaolinite (Figure 3 H). This means that mineral particles did not adhere to a nauplial carcass immediately after deposition, but it took some time for the minerals to form strong mineral-organic bonds or to become incorporated into organic tissues.
Overall, the results presented in this section do not demonstrate any simple relationship between the rate of decomposition of chitin and/or proteins and the degree of preservation of the carcasses.