Enzymatic phosphatization of fish scales-a pathway for fish fossilization.

Phosphatized fish fossils occur in various locations worldwide. Although these fossils have been intensively studied over the past decades they remain a matter of ongoing research. The mechanism of the permineralization reaction itself remains still debated in the community. The mineralization in apatite of a whole fish requires a substantial amount of phosphate which is scarce in seawater, so the origin of the excess is unknown. Previous research has shown that alkaline phosphatase, a ubiquitous enzyme, can increase the phosphate content in vitro in a medium to the degree of saturation concerning apatite. We applied this principle to an experimental setup where fish scales were exposed to commercial bovine alkaline phosphatase. We analyzed the samples with SEM and TEM and found that apatite crystals had formed on the remaining soft tissue. A comparison of these newly formed apatite crystals with fish fossils from the Solnhofen and Santana fossil deposits showed striking similarities. Both are made up of almost identically sized and shaped nano-apatites. This suggests a common formation process: the spontaneous precipitation from an oversaturated solution. The excess activity of alkaline phosphatase could explain that effect. Therefore, our findings could provide insight into the formation of well-preserved fossils.

Time-dependent microbial shifts during crayfish decomposition in freshwater and sediment under different environmental conditions.

Fossilization processes and especially the role of bacterial activity during the preservation of organic material has not yet been well understood. Here, we report the results of controlled taphonomic experiments with crayfish in freshwater and sediment. 16S rRNA amplicon analyzes showed that the development of the bacterial community composition over time was correlated with different stages of decay and preservation. Three dominating genera, Aeromonas, Clostridium and Acetobacteroides were identified as the main drivers in the decomposition of crayfish in freshwater. Using micro-computed tomography (µ-CT), scanning electron microscopy (SEM) and confocal Raman spectroscopy (CRS), calcite clusters were detected after 3-4 days inside crayfish carcasses during their decomposition in freshwater at 24 °C. The precipitation of calcite clusters during the decomposition process was increased in the presence of the bacterial genus Proteocatella. Consequently, Proteocatella might be one of the bacterial genera responsible for fossilization.

Elucidation of the Bridging Pattern of the Lantibiotic Pseudomycoicidin.

Lantibiotics are post-translationally modified antibiotic peptides with lanthionine thioether bridges that represent potential alternatives to conventional antibiotics. The lantibiotic pseudomycoicidin is produced by Bacillus pseudomycoides DSM 12442 and is effective against many Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. While prior work demonstrated that pseudomycoicidin possesses one disulfide bridge and four thioether bridges, the ring topology has so far remained unclear. Here, we analyzed several pseudomycoicidin analogues that are affected in ring formation via MALDI-TOF-MS and tandem mass spectrometry with regard to their dehydration and fragmentation patterns, respectively. As a result, we propose a bridging pattern involving Thr8 and Cys13, Thr10 and Cys16, Ser18 and Cys21, and Ser20 and Cys26, thus, forming two double ring systems. Additionally, we localized the disulfide bridge to connect Cys3 and Cys7 and, therefore, fully elucidated the bridging pattern of pseudomycoicidin.

Adipocere formation in biofilms as a first step in soft tissue preservation.

The preservation of soft tissue in the fossil record is mostly due to the replacement of organic structures by minerals (e.g. calcite, aragonite or apatite) called pseudomorphs. In rare cases soft tissues were preserved by pyrite. We assume that adipocere, as the shaping component, might be a preliminary stage in the pyritisation of soft tissues under anaerobic conditions. Using high-performance liquid chromatography coupled to ultraviolet and mass spectrometric detection (HPLC-UV/MS) and confocal Raman spectroscopy (CRS) we were able to demonstrate the transformation of the hepatopancreas (digestive gland) of the crayfish Cambarellus diminutus [Hobbs 1945] into adipocere within only 9 days, just inside a biofilm. Microorganisms (bacteria and fungi) which were responsible for the biofilm (Sphaerotilus [Kutzig 1833] and Pluteus [Fries 1857]) and maybe the adipocere formation (Clostridium [Prazmowski 1880]) were detected by 16S rRNA gene amplicon sequencing. Furthermore, micro-computed tomography (µ-CT) analyses revealed a precipitation of calcite and further showed that in animals with biofilm formation calcite precipitates in finer grained crystals than in individuals without biofilm formation, and that the precipitates were denser and replicated the structures of the cuticles better than the coarse precipitates.

The complex role of microbial metabolic activity in fossilization.

Bacteria play an important role in the fossilization of soft tissues; their metabolic activities drive the destruction of the tissues and also strongly influence mineralization. Some environmental conditions, such as anoxia, cold temperatures, and high salinity, are considered widely to promote fossilization by modulating bacterial activity. However, bacteria are extremely diverse, and have developed metabolic adaptations to a wide range of stressful conditions. Therefore, the influence of the environment on bacterial activity, and of their metabolic activity on fossilization, is complex. A number of examples illustrate that simple, general assumptions about the role of bacteria in soft tissue fossilization cannot explain all preservational pathways: (i) experimental results show that soft tissues of cnidaria decay less in oxic than anoxic conditions, and in the fossil record are found more commonly in fossil sites deposited under oxic conditions rather than anoxic environments; (ii) siderite concretions, which often entomb soft tissue fossils, precipitate due to a complex mixture of sulfate- and iron reduction by some bacterial species, running counter to original theories that iron reduction is the primary driver of siderite concretion growth; (iii) arthropod brains, now widely accepted to be preserved in many Cambrian fossil sites, are one of the first structures to decay in taphonomic experiments, indicating that their fossilization processes are complex and influenced by bacterial activity. In order to expand our understanding of the complex process of bacterially driven soft tissue fossilization, more research needs to be done, on fossils themselves and in taphonomic experiments, to determine how the complex variation in microbial metabolic activity influences decay and mineralization.

Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits.

PREMISE: Within a broader study on leaf fossilization in freshwater environments, a long-term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis. METHODS: We extracted surface-associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits. RESULTS: Kit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition. DISCUSSION: The success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram-positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.

DNA from resin-embedded organisms: Past, present and future.

Past claims have been made for fossil DNA recovery from various organisms (bacteria, plants, insects and mammals, including humans) dating back in time from thousands to several million years BP. However, many of these recoveries, especially those described from million-year-old amber (fossil resin), have faced criticism as being the result of modern environmental contamination and for lack of reproducibility. Using modern genomic techniques, DNA can be obtained with confidence from a variety of substrates (e.g. bones, teeth, gum, museum specimens and fossil insects) of different ages, albeit always less than one million years BP, and results can also be obtained from much older materials using palaeoproteomics. Nevertheless, new attempts to determine if ancient DNA (aDNA) is present in insects preserved in 40 000-year old sub-fossilised resin, the precursor of amber, have been unsuccessful or not well documented. Resin-embedded specimens are therefore regarded as unsuitable for genetic studies. However, we demonstrate here, for the first time, that although a labile molecule, DNA is still present in platypodine beetles (Coleoptera: Curculionidae) embedded in six-year-old and two-year-old resin pieces from Hymenaea verrucosa (Angiospermae: Fabaceae) collected in Madagascar. We describe an optimised method which meets all the requirements and precautions for aDNA experiments for our purpose: to explore the DNA preservation limits in resin. Our objective is far from starting an uncontrolled search for aDNA in amber as it was in the past, but to start resolving basic aspects from the DNA preservation in resin and search from the most modern samples to the ancient ones, step by step. We conclude that it is therefore possible to study genomics from resin-embedded organisms, although the time limits remain to be determined.

Experimental taphonomy of fish - role of elevated pressure, salinity and pH.

Experiments are reported to reconstruct the taphonomic pathways of fish toward fossilisation. Acrylic glass autoclaves were designed that allow experiments to be carried out at elevated pressure up to 11 bar, corresponding to water depths of 110 m. Parameters controlled or monitored during decay reactions are pressure, salinity, proton activities (pH), electrochemical potentials (Eh), and bacterial populations. The most effective environmental parameters to delay or prevent putrefaction before a fish carcass is embedded in sediment are (1) a hydrostatic pressure in the water column high enough that a fish carcass may sink to the bottom sediment, (2) hypersaline conditions well above seawater salinity, and (3) a high pH to suppress the reproduction rate of bacteria. Anoxia, commonly assumed to be the key parameter for excellent preservation, is important in keeping the bottom sediment clear of scavengers but it does not seem to slow down or prevent putrefaction. We apply our results to the world-famous Konservat-Lagerstätten Eichstätt-Solnhofen, Green River, and Messel where fish are prominent fossils, and reconstruct from the sedimentary records the environmental conditions that may have promoted preservation. For Eichstätt-Solnhofen an essential factor may have been hypersaline conditions. Waters of the Green River lakes were at times highly alkaline and hypersaline because the lake stratigraphy includes horizons rich in sodium carbonate and halite. In the Messel lake sediments some fossiliferous horizons are rich in FeCO3 siderite, a mineral indicating highly reduced conditions and a high pH.

Detection of methicillin-resistant coagulase-negative staphylococci harboring the class A mec complex by MALDI-TOF mass spectrometry.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

Preference of Diamondback Moth Larvae for Novel and Original Host Plant after Host Range Expansion.

Utilization of a novel plant host by herbivorous insects requires coordination of numerous physiological and behavioral adaptations in both larvae and adults. The recent host range expansion of the crucifer-specialist diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), to the sugar pea crop in Kenya provides an opportunity to study this process in action. Previous studies have shown that larval ability to grow and complete development on sugar pea is genetically based, but that females of the pea-adapted strain do not prefer to oviposit on pea. Here we examine larval preference for the novel host plant. Larvae of the newly evolved pea-adapted host strain were offered the choice of the novel host plant sugar pea and the original host cabbage. These larvae significantly preferred pea, while in contrast, all larvae of a cabbage-adapted DBM strain preferred cabbage. However, pea-adapted larvae, which were reared on cabbage, also preferred cabbage. Thus both genetic differences and previous exposure affect larval host choice, while adult choice for the novel host has not yet evolved.