Microbial Communities in Pre-Columbian Coprolites

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures.     

Environmental correlates of diet in the swordtail characin (Corynopoma riisei, gill)

In the sexually dimorphic swordtail characin (Corynopoma riisei, Gill), males are equipped with an opercular flag-ornament that has been suggested to function as a food-mimic since females bite at the ornament during courtship. However, virtually nothing is known about the diet in wild populations of this species. In this study, we first investigated composition of and variation in the diet of C. riisei across 18 different populations in Trinidad, using gut content analyses. We then related variation in gut content to habitat features of populations to investigate the potential link between environmental conditions and prey utilization. Our results showed that the dominating food type in the gut was various terrestrial invertebrates, both adults and larvae, but we also document substantial variation in prey types across populations. Furthermore, a canonical correlation analysis revealed a relationship between environmental characteristics and diet: populations from wider and more rapidly flowing streams with more canopy cover tended to have a diet based more on ants and mosquitoes while populations from narrow and slow flowing streams with little canopy cover tended to have a diet based more on springtails, mites and mayfly larvae. Our results add novel information on the ecology of this interesting fish and suggest the possibility of local adaptation reflecting differences in prey availability across natural populations.     

Diet strongly influences the gut microbiota of surgeonfishes

Intestinal tracts are among the most densely populated microbial ecosystems. Gut microbiota and their influence on the host have been well characterized in terrestrial vertebrates but much less so in fish. This is especially true for coral reef fishes, which are among the most abundant groups of vertebrates on earth. Surgeonfishes (family: Acanthuridae) are part of a large and diverse family of reef fish that display a wide range of feeding behaviours, which in turn has a strong impact on the reef ecology. Here, we studied the composition of the gut microbiota of nine surgeonfish and three nonsurgeonfish species from the Red Sea. High‐throughput pyrosequencing results showed that members of the phylum Firmicutes, especially of the genus Epulopiscium, were dominant in the gut microbiota of seven surgeonfishes. Even so, there were large inter‐ and intraspecies differences in the diversity of surgeonfish microbiota. Replicates of the same host species shared only a small number of operational taxonomic units (OTUs), although these accounted for most of the sequences. There was a statistically significant correlation between the phylogeny of the host and their gut microbiota, but the two were not completely congruent. Notably, the gut microbiota of three nonsurgeonfish species clustered with some surgeonfish species. The microbiota of the macro‐ and microalgavores was distinct, while the microbiota of the others (carnivores, omnivores and detritivores) seemed to be transient and dynamic. Despite some anomalies, both host phylogeny and diet were important drivers for the intestinal microbial community structure of surgeonfishes from the Red Sea.     

Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites.

On the Middle-Age site of Namur (Belgium) the analysis of coprolites revealed the presence of many well-preserved Ascaris eggs. Following rehydratation of the coprolite samples, 104 eggs were collected and extracted with an ultrasonication and phenol-chloroform based method. Three overlapping fragments of the 18S rRNA gene and one fragment of the cytochrome b gene have been reproducibly amplified, cloned and sequenced. The analysis of these sequences confirms the identification of the eggs as coming from Ascaris. Our study reveals that coprolites can be an interesting source of parasites that can be readily identified using molecular approaches. The study of ancient DNA from helminth parasites is of interest as it may answer long-standing questions in the history of infectious diseases and gives a possibility to compare these ancient sequences with those of modern populations.     

First record of Mesozoic scroll coprolites: classification,characteristics, elemental composition and probable producers

Multiple, small, cylindrical scroll coprolites having rounded and tapering ends and pertaining to a new ichnotaxon have been recovered from the Upper Triassic Tiki Formation of India. This is the first record of scroll coprolites from the Mesozoic. Based on cross‐sectional geometry, external surface textures, and internal morphology, these coprolites are subdivided into three morphotypes. The coprolites contain several kinds of undigested food material in the form of ganoid fish scales, teeth, lower jaw and skeletal remains of various osteichthyans, chondrichthyans, archosauriforms and indeterminate reptiles. These inclusions are embedded in the groundmass separated by thin mucosal layers. The groundmass contains abundant gas vesicles, and secondarily‐filled shrinkage cracks. EDS analysis shows that the overall composition of the coprolites reflects Ca, P, C and O, suggesting calcium phosphate mineralogy, though other elements such as Fe, Mn, Al, Si are present in lesser proportions. Based on their similarity with the scrolled faeces of extant euryhaline hammerhead sharks, it is deduced that these coprolites were produced by euryhaline hybodontid sharks. At least two hybodontid taxa, Lonchidion and Pristrisodus, show high prevalence in the Tiki vertebrate fauna, suggesting that these were the possible producers. As the coprolite inclusions contain remains of other aquatic animals, these carnivorous hybodonts constituted the dominant predators of the Tiki aquatic ecosystem.     

Intestinal microbiota composition in fishes is influenced by host ecology and environment

The digestive tracts of vertebrates are colonized by complex assemblages of micro-organisms, collectively called the gut microbiota. Recent studies have revealed important contributions of gut microbiota to vertebrate health and disease, stimulating intense interest in understanding how gut microbial communities are assembled and how they impact host fitness (Sekirov et al. 2010). Although all vertebrates harbour a gut microbiota, current information on microbiota composition and function has been derived primarily from mammals. Comparisons of different mammalian species have revealed intriguing associations between gut microbiota composition and host diet, anatomy and phylogeny (Ley et al. 2008b). However, mammals constitute <10% of all vertebrate species, and it remains unclear whether similar associations exist in more diverse and ancient vertebrate lineages such as fish. In this issue, Sullam et al. (2012) make an important contribution toward identifying factors determining gut microbiota composition in fishes. The authors conducted a detailed meta-analysis of 25 bacterial 16S rRNA gene sequence libraries derived from the intestines of different fish species. To provide a broader context for their analysis, they compared these data sets to a large collection of 16S rRNA gene sequence data sets from diverse free-living and host-associated bacterial communities. Their results suggest that variation in gut microbiota composition in fishes is strongly correlated with species habitat salinity, trophic level and possibly taxonomy. Comparison of data sets from fish intestines and other environments revealed that fish gut microbiota compositions are often similar to those of other animals and contain relatively few free-living environmental bacteria. These results suggest that the gut microbiota composition of fishes is not a simple reflection of the micro-organisms in their local habitat but may result from host-specific selective pressures within the gut (Bevins & Salzman 2011).     

Multi‐proxy analyses of Late Cretaceous coprolites from Germany

A total of 462 coprolites from three localities exposing Upper Cretaceous deposits in the Münster Basin, northwestern Germany, have been subjected to an array of analytical techniques, with the aim of elucidating ancient trophic structures and predator–prey interactions. The phosphatic composition, frequent bone inclusions, size and morphology collectively suggest that most, if not all, coprolites were produced by carnivorous (predatory or scavenging) vertebrates. The bone inclusions further indicate that the coprolite producers preyed principally upon fish. Putative host animals include bony fish, sharks and marine reptiles – all of which have been previously recorded from the Münster Basin. The presence of borings and other traces on several coprolites implies handling by coprophagous organisms. Remains of epibionts are also common, most of which have been identified as the encrusting bivalve Atreta. Palynological analyses of both the coprolites and host rocks reveal a sparse assemblage dominated by typical Late Cretaceous dinoflagellates, and with sub‐ordinate fern spores, conifer pollen grains and angiosperm pollen grains. The dinoflagellate key taxon Exochosphaeridium cenomaniense corroborates a Cenomanian age for the Plenus Marl, from which most studied coprolites derive. The findings of this study highlight the potential of a multi‐proxy approach when it comes to unravelling the origin, composition and importance of coprolites in palaeoecosystem analyses.     

Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis

Symbiotic bacteria often help their hosts acquire nutrients from their diet, showing trends of co-evolution and independent acquisition by hosts from the same trophic levels. While these trends hint at important roles for biotic factors, the effects of the abiotic environment on symbiotic community composition remain comparably understudied. In this investigation, we examined the influence of abiotic and biotic factors on the gut bacterial communities of fish from different taxa, trophic levels and habitats. Phylogenetic and statistical analyses of 25 16S rRNA libraries revealed that salinity, trophic level and possibly host phylogeny shape the composition of fish gut bacteria. When analysed alongside bacterial communities from other environments, fish gut communities typically clustered with gut communities from mammals and insects. Similar consideration of individual phylotypes (vs. communities) revealed evolutionary ties between fish gut microbes and symbionts of animals, as many of the bacteria from the guts of herbivorous fish were closely related to those from mammals. Our results indicate that fish harbour more specialized gut communities than previously recognized. They also highlight a trend of convergent acquisition of similar bacterial communities by fish and mammals, raising the possibility that fish were the first to evolve symbioses resembling those found among extant gut fermenting mammals.     

Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection

The gut microbiotas of zebrafish and mice share six bacterial divisions, although the specific bacteria within these divisions differ. To test how factors specific to host gut habitat shape microbial community structure, we performed reciprocal transplantations of these microbiotas into germ-free zebrafish and mouse recipients. The results reveal that communities are assembled in predictable ways. The transplanted community resembles its community of origin in terms of the lineages present, but the relative abundance of the lineages changes to resemble the normal gut microbial community composition of the recipient host. Thus, differences in community structure between zebrafish and mice arise in part from distinct selective pressures imposed within the gut habitat of each host. Nonetheless, vertebrate responses to microbial colonization of the gut are ancient: Functional genomic studies disclosed shared host responses to their compositionally distinct microbial communities and distinct microbial species that elicit conserved responses.     

Contrasting Population and Diet Influences on Gut Length of an Omnivorous Tropical Fish,the Trinidadian Guppy (Poecilia reticulata)

Phenotypic plasticity is advantageous for organisms that live in variable environments. The digestive system is particularly plastic, responding to changes in diet. Gut length is the result of a trade-off between maximum nutrient absorption and minimum cost for its maintenance and it can be influenced by diet and by evolutionary history. We assessed variation in gut length of Trinidadian guppies (Poecilia reticulata) as a function of diet, season, ontogeny, and local adaptation. Populations of guppies adapted to different predation levels have evolved different life history traits and have different diets. We sampled guppies from sites with low (LP) and high predation (HP) pressure in the Aripo and Guanapo Rivers in Trinidad. We collected fish during both the dry and wet season and assessed their diet and gut length. During the dry season, guppies from HP sites fed mostly on invertebrates, while guppies in the LP sites fed mainly on detritus. During the wet season, the diet of LP and HP populations became very similar. We did not find strong evidence of an ontogenetic diet shift. Gut length was negatively correlated with the proportion of invertebrates in diet across fish from all sites, supporting the hypothesis that guppy digestive systems adapt in length to changes in diet. Population of origin also had an effect on gut length, as HP and LP fish maintained different gut lengths even in the wet season, when their diets were very similar and individuals in both types of populations fed mostly on detritus. Thus, both environment and population of origin influenced guppies gut length, but population of origin seemed to have a stronger effect. Our study also showed that, even in omnivorous fish, gut length adapted to different diets, being more evident when the magnitude of difference between animal and plant material in the diet was very large.     

Pyrosequencing faecal DNA to determine diet of little penguins: is what goes in what comes out?

DNA barcoding of faeces or stomach contents is an emerging approach for dietary analysis. We pyrosequenced mtDNA 16S markers amplified from faeces of captive little penguins (Eudyptula minor) to examine if recovered sequences reflect the proportions of species consumed. We also analysed wild little penguin faeces collected from 100 nests in southeast Australia. In the captive study, pilchards were the primary fish fed to the penguins and DNA sequences from pilchard were the most common sequences recovered. Sequences of three other fish fed in constant mass proportions (45:35:20) were all detected, but proportions of sequences (60:6:34) were considerably different than mass proportions in the diet. Correction factors based on relative mtDNA density in the fish did not improve diet estimates. Consistency between replicate samples suggests that the observed bias resulted from differences in prey digestibility. Detection of DNA from fish consumed before the penguins were brought into captivity indicates that a DNA signal in faeces can persist for at least 4 days after ingestion. In the wild-collected faeces, 24 distinct fish and 1 squid were identified; anchovy, barracouta and pilchard accounted for over 80% of these sequences. Our results highlight that DNA sequences recovered in dietary barcoding studies can provide semi-quantitative information on diet composition, but these data should be given wide confidence intervals.     

Coprolites as a source of information on the genome and diet of the cave hyena

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet.     

SL1 RNA gene recovery from Enterobius vermicularis ancient DNA in pre-Columbian human coprolites

Enterobius vermicularis, pinworm, is one of the most common helminths worldwide, infecting nearly a billion people at all socio-economic levels. In prehistoric populations the paleoparasitological findings show a pinworm homogeneous distribution among hunter-gatherers in North America, intensified with the advent of agriculture. This same increase also occurred in the transition from nomad hunter-gatherers to sedentary farmers in South America, although E. vermicularis infection encompasses only the ancient Andean peoples, with no record among the pre-Colombian populations in the South American lowlands. However, the outline of pinworm paleoepidemiology has been supported by microscopic finding of eggs recovered from coprolites. Since molecular techniques are precise and sensitive in detecting pathogen ancient DNA (aDNA), and also could provide insights into the parasite evolutionary history, in this work we have performed a molecular paleoparasitological study of E. vermicularis. aDNA was recovered and pinworm 5S rRNA spacer sequences were determined from pre-Columbian coprolites (4110 BC-AD 900) from four different North and South American archaeological sites. The sequence analysis confirmed E. vermicularis identity and revealed a similarity among ancient and modern sequences. Moreover, polymorphisms were identified at the relative positions 160, 173 and 180, in independent coprolite samples from Tulán, San Pedro de Atacama, Chile (1080-950 BC). We also verified the presence of peculiarities (Splicing leader (SL1) RNA sequence, spliced donor site, the Sm antigen biding site, and RNA secondary structure) which characterise the SL1 RNA gene. The analysis shows that the SL1 RNA gene of contemporary pinworms was present in pre-Columbian E. vermicularis by 6110 years ago. We were successful in detecting E. vermicularis aDNA even in coprolites without direct microscopic evidence of the eggs, improving the diagnosis of helminth infections in the past and further pinworm paleoepidemiological studies.     

Scanning electron microscope study of ancient parasite eggs recovered from Korean mummies of the Joseon Dynasty

We have previously shown that parasite eggs have been identified in the coprolites of Korean mummies. These eggs have shed light on parasitic infection patterns in Korean populations living several hundred years ago. We conducted a scanning electron microscopy (SEM) study on ancient Trichuris trichiura, Ascaris lumbricoides, Metagonimus yokogawai, Paragonimus westermani, and Gymnophalloides seoi eggs recovered from Korean mummies of the Joseon Dynasty. We anticipated that the taphonomic conditions of mummification would alter the eggs of certain species but not of others. Our SEM data show that each species of ancient egg exhibited different degrees of preservation. Thus, some of them, for example, M. yokogawai, exhibited a better preservation status than others, suggesting that they should be the first candidates considered when choosing subjects for future paleoparasitological studies.     

Interdemic variation in the foraging ecology of the African cyprinid, Barbus neumayeri

In the African cyprinid, Barbus neumayeri, populations from hypoxic waters have larger gills than populations from well-oxygenated streams. Differences in trophic morphology and feeding performance between these populations suggest a reduction in feeding efficiency in large-gilled fish that may reflect spatial constraints of the gills. However, this variation may also reflect interdemic variation in diets. In this study, we describe patterns of variation in diet, gut morphology, and prey availability for populations of B. neumayeri from swamp (low-oxygen) and stream (high-oxygen) sites in Kibale National Park, Uganda. Our results indicate that B. neumayeri are omnivorous, feeding primarily on benthic prey items; however, diets differed among swamp and stream sites for certain prey types. The observed dietary differences do not provide direct support for predictions based on variation in trophic musculature; hard-bodied prey were more common in low-oxygen sites. Prey availability also differed among sites; in particular, insect abundance and richness was generally lower in the swamp sites. Gut length was longer in one of the four populations, but did not conform to expectations based on diet differences. Condition and growth rates did not differ between populations from hypoxic and well-oxygenated sites, despite observed differences in prey availability and diet, suggesting that B. neumayeri may be distributed in a way that equalizes fitness among populations in different habitats.     

High-resolution coproecology: using coprolites to reconstruct the habits and habitats of New Zealand's extinct upland moa (Megalapteryx didinus)

Knowledge about the diet and ecology of extinct herbivores has important implications for understanding the evolution of plant defence structures, establishing the influences of herbivory on past plant community structure and composition, and identifying pollination and seed dispersal syndromes. The flightless ratite moa (Aves: Dinornithiformes) were New Zealand's largest herbivores prior to their extinction soon after initial human settlement. Here we contribute to the knowledge of moa diet and ecology by reporting the results of a multidisciplinary study of 35 coprolites from a subalpine cave (Euphrates Cave) on the South Island of New Zealand. Ancient DNA analysis and radiocarbon dating revealed the coprolites were deposited by the extinct upland moa (Megalapteryx didinus), and span from at least 6,368±31 until 694±30 (14)C years BP; the approximate time of their extinction. Using pollen, plant macrofossil, and ancient DNA analyses, we identified at least 67 plant taxa from the coprolites, including the first evidence that moa fed on the nectar-rich flowers of New Zealand flax (Phormium) and tree fuchsia (Fuchsia excorticata). The plant assemblage from the coprolites reflects a highly-generalist feeding ecology for upland moa, including browsing and grazing across the full range of locally available habitats (spanning southern beech (Nothofagus) forest to tussock (Chionochloa) grassland). Intact seeds in the coprolites indicate that upland moa may have been important dispersal agents for several plant taxa. Plant taxa with putative anti-browse adaptations were also identified in the coprolites. Clusters of coprolites (based on pollen assemblages, moa haplotypes, and radiocarbon dates), probably reflect specimens deposited at the same time by individual birds, and reveal the necessity of suitably large sample sizes in coprolite studies to overcome potential biases in diet interpretation.     

Comparative gut content analysis of invasive mosquitofish from Italy and Spain

Eastern mosquitofish (Gambusia holbrooki) are among the most widely introduced freshwater species globally. To gain a better understanding of feeding patterns in non‐native populations, and which local factors may influence them at the population level, we carried out gut content analysis on 163 specimens from nine invasive populations in Italy and Spain. Based on previous studies, we predicted that (a) mosquitofish are omnivores with a preference for detritus and cladocerans; (b) they display size‐ and population‐specific differences in gut morphologies and diet, with larger fish feeding more intensively over a wider range of prey items; and (c) some of the variation would be associated with differences in local environmental and climatic factors. Our results confirmed our first prediction, because mosquitofish fed on a variety of diet items, among which detritus and Cladocera dominated. However, not a single diet item was shared among all populations. Congruent with our second prediction, we further identified size‐ and population‐specific differences in the occurrence of some diet items and gut morphologies. However, observed patterns in dietary habits did not seem to be driven by the environmental and climatic variables we had quantified. The fairly variable diet likely aids invasion success and helps explain the ubiquity of invasive mosquitofish across Italy and Spain, as mosquitofish seem to be able to rely on whatever a local habitat provides. We further propose that size‐specific differences likely capture the substantial sexual size dimorphism (males are smaller than females), while population‐specific differences are likely the result of differences in local prey abundance. The lack of an influence of temperature on dietary habits suggests that mosquitofish feeding ecology may be less impacted by rising temperatures than other freshwater fish species. If true, then this suggests climate change‐induced effects may further exacerbate the competitive superiority of mosquitofish over native species in the future.     

河南灵井许昌人遗址鬣狗粪化石的初步研究

河南灵井许昌人遗址是我国近年来发掘的最为重要的古人类遗址之一。该遗址中出土了大量的石器、骨器、动物化石、粪便化石等遗存以及人类头盖骨化石等。我们对该遗址出土的鬣狗粪化石进行了类型学及其包含的微体化石等方面研究,从分析统计结果来看,鬣狗粪化石中包含了丰富的古信息,如古寄生虫卵、动物毛发、孢粉、植硅体、真菌等。本文主要针对鬣狗粪化石中古寄生虫卵及动物毛发进行分析,探讨了作为寄主鬣狗罹患的寄生虫病,以及鬣狗的食物来源等情况,为深入理解更新世晚期人类适应环境与气候提供新的证据。