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Nucleic acid extraction from complex environmental and ancient tissue material is prone to co-extract inhibitory substances that make further molecular analysis difficult or impossible. This co-extraction occurs in both solid-phase and liquid-phase/organic nucleic acid extraction protocols. Currently, the widely used method to overcome inhibition includes the addition of bovine serum albumin (BSA) to the downstream enzymatic reactions or the dilution of the nucleic acid extracts. BSA, however, seems to reduce the inhibitory effect of certain compounds only, and excessive dilution may change the original DNA composition. In this study, we introduce an innovative new method using linear polyacrylamide (LPA) to efficiently precipitate and purify nucleic acids extracted from complex environmental and ancient tissue samples in one working step. The LPA method replaces the precipitation step in classic liquid-phase/organic extraction protocols or can be easily applied as an additional post-extraction step on impure DNA extracts. As a proof of concept, we experimented with this method on different ancient human mummy samples (bones, soft tissues and gut contents) from different time periods (5000 BC-1800 AD), as well as on complex environmental samples (e.g. soil, activated sludge and animal faeces) known to contain inhibitory compounds. We demonstrated that LPA precipitates nucleic acids, even in an aqueous ethanol solution without the addition of chaotropic salts, resulting in the recovery of highly pure DNA from all tested samples that displayed inhibition with previously published extraction protocols. Compared to the current, most widely used silica-based extraction method for ancient and sedimentary DNA, our LPA method resulted in comparable DNA qualities and overall DNA compositions (human endogenous content and microbial diversity). In conclusion, our LPA method with its high purifying capacity provides an important alternative to the commonly used DNA extraction protocols in the environmental and ancient DNA (aDNA) fields.

The Vác Mummy Collection comprises 265 well documented mummified individuals from the late 16th to the early 18th century that were discovered in 1994 inside a crypt in Vác, Hungary. This collection offers a unique opportunity to study the relationship between humans and pathogens in the pre-antibiotic era, as previous studies have shown a high proportion of tuberculosis (TB) infections among the individuals. In this study, we recovered ancient DNA with shotgun sequencing from a rib bone sample of a 18th century midwife. This individual is part of the collection and shows clear skeletal changes that are associated with tuberculosis and syphilis. To provide molecular proof, we applied a metagenomic approach to screen for ancient pathogen DNA. While we were unsuccessful to recover any ancient Treponema pallidum DNA, we retrieved high coverage ancient TB DNA and identified a mixed infection with two distinct TB strains by detailed single-nucleotide polymorphism and phylogenetic analysis. Thereby, we have obtained comprehensive results demonstrating the long-time prevalence of mixed infections with the sublineages L4.1.2.1/Haarlem and L4.10/PGG3 within the local community in preindustrial Hungary and put them in context of sociohistorical factors.

Individual repeatability characterizes many behaviors. Repeatable behavior may result from repeated social interactions among familiar group members, owing to adaptive social niche specialization. In the context of aggression, in species like field crickets, social niche specialization should also occur when individuals repeatedly interact with unfamiliar individuals. This would require the outcome of social interactions to have carry-over effects on fighting ability and aggressiveness in subsequent interactions, leading to long-term among-individual differentiation. To test this hypothesis, we randomly assigned freshly emerged adult males of the southern field cricket Gryllus bimaculatus to either a solitary or social treatment. In social treatment, males interacted with a same-sex partner but experienced a new partner every 3 days. After 3 weeks of treatment, we repeatedly subjected treated males to dyadic interactions to measure aggression. During this time, we also continuously measured the 3-daily rate of carbohydrate and protein consumption. Individual differentiation was considerably higher among males reared in the social versus solitary environment for aggressiveness but not for nutrient intake. Simultaneously, social experience led to lower within-individual stability (i.e., increased within-individual variance) in carbohydrate intake. Past social experiences, thus, shaped both behavioral individuality and stability. While previous research has emphasized behavioral individuality resulting from repeated interactions among familiar individuals, our study implies that behavioral individuality, in the context of aggression, may generally result from social interactions, whether with familiar or unfamiliar individuals. Our findings thus imply that social interactions may have a stronger effect on individual differentiation than previously appreciated.

Although malaria is one of the oldest and most widely distributed diseases affecting humans, identifying and characterizing its presence in ancient human remains continue to challenge researchers. We attempted to establish a reliable approach to detecting malaria in human skeletons using multiple avenues of analysis: macroscopic observations, rapid diagnostic tests, and shotgun-capture sequencing techniques, to identify pathological changes, Plasmodium antigens, and Plasmodium DNA, respectively. Bone and tooth samples from ten individuals who displayed skeletal lesions associated with anaemia, from a site in southern Egypt (third to sixth centuries AD), were selected. Plasmodium antigens were detected in five of the ten bone samples, and traces of Plasmodium aDNA were detected in six of the twenty bone and tooth samples. There was relatively good synchronicity between the biomolecular findings, despite not being able to authenticate the results. This study highlights the complexity and limitations in the conclusive identification of the Plasmodium parasite in ancient human skeletons. Limitations regarding antigen and aDNA preservation and the importance of sample selection are at the forefront of the search for malaria in the past. We confirm that, currently, palaeopathological changes such as cribra orbitalia are not enough to be certain of the presence of malaria. While biomolecular methods are likely the best chance for conclusive identification, we were unable to obtain results which correspond to the current authentication criteria of biomolecules. This study represents an important contribution in the refinement of biomolecular techniques used; also, it raises new insight regarding the consistency of combining several approaches in the identification of malaria in past populations.

Selection may favour individuals of the same population to differ consistently in nutritional preference, for example, because optimal diets covary with morphology or personality. We provided Southern field crickets (Gryllus bimaculatus) with two synthetic food sources (carbohydrates and proteins) and quantified repeatedly how much of each macronutrient was consumed by each individual. We then quantified (i) whether individuals were repeatable in carbohydrate and protein intake rate, (ii) whether an individual’s average daily intake of carbohydrates was correlated with its average daily intake of protein, and (iii) whether short-term changes in intake of carbohydrates coincided with changes in intake of protein within individuals. Intake rates were individually repeatable for both macronutrients. However, individuals differed in their relative daily intake of carbohydrates versus proteins (i.e., ‘nutritional preference’). By contrast, total consumption varied plastically as a function of body weight within individuals. Body weight—but not personality (i.e., aggression, exploration behaviour)—positively predicted nutritional preference at the individual level as large crickets repeatedly consumed a higher carbohydrate to protein ratio compared to small ones. Our finding of level-specific associations between the consumption of distinct nutritional components demonstrates the merit of applying multivariate and multi-level viewpoints to the study of nutritional preference.