REVIEW OF PARASITES FOUND IN EXTINCT ANIMALS: WHAT CAN BE REVEALED.

Parasitism is inherent to life and observed in all species. Extinct animals have been studied to understand what they looked like, where and how they lived, what they fed on, and the reasons they became extinct. Paleoparasitology helps to clarify these questions based on the study of the parasites and microorganisms that infected those animals, using as a source material coprolites, fossils in rock, tissue, bone, mummy, and amber, analyses of ancient DNA, immunodiagnosis, and microscopy.

Parasitism and host behavior in the context of a changing environment: The Holocene record of the commercially important bivalve Chamelea gallina, northern Italy.

Rapid warming and sea-level rise are predicted to be major driving forces in shaping coastal ecosystems and their services in the next century. Though forecasts of the multiple and complex effects of temperature and sea-level rise on ecological interactions suggest negative impacts on parasite diversity, the effect of long term climate change on parasite dynamics is complex and unresolved. Digenean trematodes are complex life cycle parasites that can induce characteristic traces on their bivalve hosts and hold potential to infer parasite host-dynamics through time and space. Previous work has demonstrated a consistent association between sea level rise and increasing prevalence of trematode traces, but a number of fundamental questions remain unanswered about this paleoecological proxy. Here we examine the relationships of host size, shape, and functional morphology with parasite prevalence and abundance, how parasites are distributed across hosts, and how all of these relationships vary through time, using the bivalve Chamelea gallina from a Holocene shallow marine succession in the Po coastal plain. Trematode prevalence increased and decreased in association with the transition from a wave-influenced estuarine system to a wave-dominated deltaic setting. Prevalence and abundance of trematode pits are associated with large host body size, reflecting ontogenetic accumulation of parasites, but temporal trends in median host size do not explain prevalence trends. Ongoing work will test the roles of temperature, salinity, and nutrient availability on trematode parasitism. Parasitized bivalves in one sample were shallower burrowers than their non-parasitized counterparts, suggesting that hosts of trematodes can be more susceptible to their predators, though the effect is ephemeral. Like in living parasite-host systems, trematode-induced malformations are strongly aggregated among hosts, wherein most host individuals harbor very few parasites while a few hosts have many. We interpret trace aggregation to support the assumption that traces are a reliable proxy for trematode parasitism in the fossil record.

Planktonic foraminifera eDNA signature deposited on the seafloor remains preserved after burial in marine sediments.

Environmental DNA (eDNA) metabarcoding of marine sediments has revealed large amounts of sequences assigned to planktonic taxa. How this planktonic eDNA is delivered on the seafloor and preserved in the sediment is not well understood. We address these questions by comparing metabarcoding and microfossil foraminifera assemblages in sediment cores taken off Newfoundland across a strong ecological gradient. We detected planktonic foraminifera eDNA down to 30 cm and observed that the planktonic/benthic amplicon ratio changed with depth. The relative proportion of planktonic foraminiferal amplicons remained low from the surface down to 10 cm, likely due to the presence of DNA from living benthic foraminifera. Below 10 cm, the relative proportion of planktonic foraminifera amplicons rocketed, likely reflecting the higher proportion of planktonic eDNA in the DNA burial flux. In addition, the microfossil and metabarcoding assemblages showed a congruent pattern indicating that planktonic foraminifera eDNA is deposited without substantial lateral advection and preserves regional biogeographical patterns, indicating deposition by a similar mechanism as the foraminiferal shells. Our study shows that the planktonic eDNA preserved in marine sediments has the potential to record climatic and biotic changes in the pelagic community with the same spatial and temporal resolution as microfossils.

Helminth eggs from early cretaceous faeces.

The exceptional fossil site of Las Hoyas (upper Barremian, Cuenca, Spain) yields abundant small to medium vertebrate coprolites, hindering the search for parasites. We studied the contents of 29 coprolites that were previously classified into distinct morphotypes. Several parasitic eggs were retrieved from two of these coprolites, confirming the second record of digenea trematode eggs and nematode (ascaridid) eggs from an Early Cretaceous locality. The cylindrical coprolite containing anisakid eggs was likely produced by a crocodylomorph as the parasite host, whereas the bump-headed lace coprolite indicates the role of a fish as an intermediary or definitive host of the trematodes and ascaridids. These trace and body fossils show that the Las Hoyas 126-129 Ma lacustrine ecosystem documents the early connection between basal Gonorynchiformes fish and digenetic trematodes.

Lesions in osteoderms of pampatheres (Mammalia, Xenarthra, Cingulata) possibly caused by fleas.

In this study, the first records of lesions in osteoderms of Holmesina, a group of fossil cingulates related to armadillos, possibly caused by the action of penetrating fleas (Siphonaptera) are described. Three individuals of Holmesina cryptae (Pampatheriidae) were collected from Quaternary sediments in Lapinha Cave (Iramaia, Bahia state, Brazil). Their osteoderms were analyzed by stereomicroscope and scanning electron microscopy and alterations on their surfaces were recognized. We found 63 marks distributed in 23 of 1300 analyzed osteoderms (approximately 1.8% of the total of osteoderms), characterized by vertical cavities with well-delimited circular borders similar to those lesions made by Tungidae fleas in extant mammals. These records indicate that there was an interaction between penetrating fleas and pampatheres during the Quaternary in Brazilian Intertropical Region, and contribute to the understanding of the evolution of these ectoparasites and the relationship with their hosts.

An encrusting kleptoparasite-host interaction from the early Cambrian.

Parasite-host systems are pervasive in nature but are extremely difficult to convincingly identify in the fossil record. Here we report quantitative evidence of parasitism in the form of a unique, enduring life association between tube-dwelling organisms encrusted to densely clustered shells of a monospecific organophosphatic brachiopod assemblage from the lower Cambrian (Stage 4) of South China. Brachiopods with encrusting tubes have decreased biomass (indicating reduced fitness) compared to individuals without tubes. The encrusting tubes orient tightly in vectors matching the laminar feeding currents of the host, suggesting kleptoparasitism. With no convincing parasite-host interactions known from the Ediacaran, this widespread sessile association reveals intimate parasite-host animal systems arose in early Cambrian benthic communities and their emergence may have played a key role in driving the evolutionary and ecological innovations associated with the Cambrian radiation.

Cold climate adaptation is a plausible cause for evolution of multicellular sporulation in Dictyostelia.

Unicellular protozoa that encyst individually upon starvation evolved at least eight times into organisms that instead form multicellular fruiting bodies with spores. The Dictyostelia are the largest and most complex group of such organisms. They can be subdivided into 4 major groups, with many species in groups 1-3 having additionally retained encystment. To understand fitness differences between spores and cysts, we measured long-term survival of spores and cysts under climate-mimicking conditions, investigated spore and cyst ultrastructure, and related fitness characteristics to species ecology. We found that spores and cysts survived 22 °C equally well, but that spores survived wet and dry frost better than cysts, with group 4 spores being most resilient. Spore walls consist of three layers and those of cysts of maximally two, while spores were also more compacted than cysts, with group 4 spores being the most compacted. Group 4 species were frequently isolated from arctic and alpine zones, which was rarely the case for group 1-3 species. We inferred a fossil-calibrated phylogeny of Dictyostelia, which showed that its two major branches diverged 0.52 billion years ago, following several global glaciations. Our results suggest that Dictyostelium multicellular sporulation was a likely adaptation to a cold climate.

Parasites in micromammal fecal pellets throughout the Late Holocene ("Cueva Peligro" paleontological site, Patagonia, Argentina).

Parasite remains in micromammal fecal pellets collected from the paleontological site "Cueva Peligro" (CP 43°40'18"S, 66°24'52"W), Chubut Province, Argentina, were examined. The samples were obtained from two grids, dated between 1220 ± 7014C yr B.P. to modern dates. Fecal pellets were whole processed, rehydrated, homogenized, and examined via light microscopy. Eggs of parasites found were measured and photographed. Fecal pellets belong to one or more insectivore to omnivore unidentified micromammal species, possibly sigmodontine rodents. A high number of helminthes species was recorded (11 species), eight nematodes, two anoplocephalid cestodes and one acanthocephalan species. The sigmodontine-parasite relationship varied throughout the studied period and between studied grids. This is the first time that Gongylonema sp. and Syphacia sp. are reported from ancient times from Patagonia. The obtained results contribute to the knowledge of parasite assemblages associated to native South American sigmodontine rodents and the zoonoses present in the area throughout the lasts 1200 years.

Paleomicrobiology: Diagnosis and Evolution of Ancient Pathogens.

The last century has witnessed progress in the study of ancient infectious disease from purely medical descriptions of past ailments to dynamic interpretations of past population health that draw upon multiple perspectives. The recent adoption of high-throughput DNA sequencing has led to an expanded understanding of pathogen presence, evolution, and ecology across the globe. This genomic revolution has led to the identification of disease-causing microbes in both expected and unexpected contexts, while also providing for the genomic characterization of ancient pathogens previously believed to be unattainable by available methods. In this review we explore the development of DNA-based ancient pathogen research, the specialized methods and tools that have emerged to authenticate and explore infectious disease of the past, and the unique challenges that persist in molecular paleopathology. We offer guidelines to mitigate the impact of these challenges, which will allow for more reliable interpretations of data in this rapidly evolving field of investigation.

Two new species of ascaridoid nematodes in Brazilian Crocodylomorpha from the Upper Cretaceous.

Two new ascaridoid species, Bauruascaris cretacicus n. gen. et n. sp., and Bauruascaris adamantinensis n. gen. et n. sp., are described based on the fossils of eggs preserved in 80-70 million year old phosphatized coprolites of Crocodyliformes, chronologically assigned to the Upper Cretaceous (Campanian/Maastrichtian age), collected from sedimentary rocks of the Bauru Group, Adamantina Formation in the municipality of Santo Anastácio, in the southwestern region of the state of São Paulo, Brazil, South America. This paper describes the oldest ascaridoid species ever recorded in Crocodylomorpha. Hence, this article contributes to the body of knowledge about the evolutionary history of this nematode group. It also offers a clue about the composition of the parasite fauna of these reptiles from the Late Cretaceous, which is still unknown despite numerous studies about various aspects of their biology and the pioneering paleoparasitological analysis of animal coprolites by South American researchers.

First record of Acanthocephala parasites eggs in coprolites preliminary assigned to Crocodyliformes from the Adamantina Formation (Bauru Group, Upper Cretaceous), São Paulo, Brazil.

This study presents the oldest record of Acanthocephala parasite eggs in coprolites preliminary assigned to Crocodyliformes, recovered in the region of Santo Anastácio Municipality, Southwestern São Paulo State. For this, a paleoparasitological investigation was carried out on 53 mineralized coprolites (complete or fragmented), with round shape or cylindrical shape of rounded or pointed ends, 0.2 - 3.9 cm in length x 0.1 - 2.4 cm in diameter, 3.7 grams in weight, and absence of food remains. Individual samples of the surface and internal portions of each coprolite were extracted by electric drill, dissociated with Cloridic Acid 10% solution, washed with Distilled Water, and filtered in granulometric screen Mesh / Tyler 325. After laboratory processing, the sediments retained on the granulometric screen was studied with Glycerin under optical microscopy, and the presence of four Acanthocephala eggs could be observed in sample of only one of these ichnofossils. All specimens were well preserved and showed 72.5 - 85 µm in length x 27.5 - 50 µm in width, elliptical shape, three concentric and thick shells, and embryos in their interior. This study inaugurates investigations and knowledge about Paleoparasitology in Crocodyliformes coprolites from the Bauru Group, Upper Cretaceous from the Paraná Basin.

Recovering parasites from mummies and coprolites: an epidemiological approach.

In the field of archaeological parasitology, researchers have long documented the distribution of parasites in archaeological time and space through the analysis of coprolites and human remains. This area of research defined the origin and migration of parasites through presence/absence studies. By the end of the 20th century, the field of pathoecology had emerged as researchers developed an interest in the ancient ecology of parasite transmission. Supporting studies were conducted to establish the relationships between parasites and humans, including cultural, subsistence, and ecological reconstructions. Parasite prevalence data were collected to infer the impact of parasitism on human health. In the last few decades, a paleoepidemiological approach has emerged with a focus on applying statistical techniques for quantification. The application of egg per gram (EPG) quantification methods provide data about parasites' prevalence in ancient populations and also identify the pathological potential that parasitism presented in different time periods and geographic places. Herein, we compare the methods used in several laboratories for reporting parasite prevalence and EPG quantification. We present newer quantification methods to explore patterns of parasite overdispersion among ancient people. These new methods will be able to produce more realistic measures of parasite infections among people of the past. These measures allow researchers to compare epidemiological patterns in both ancient and modern populations.

Apex Predators, Rockshelters, and Zoonoses in the Patagonian Holocene.

Carnivorous mammals are a trophic guild with an important role in the dissemination of parasite infective stages (larvae, eggs, cysts, and oocysts). In the present study, new samples of coprolites attributed to carnivorous mammals, obtained from 2 archaeological caves, were analyzed for the presence of parasites with the aim to increase the knowledge about parasites in rockshelters that could have spread to humans and other mammals. To this purpose, fragments of 3 coprolites from Cerro Casa de Piedra, cave 5 and cave 7, were examined. Coprolites were rehydrated in aqueous trisodium phosphate and processed by spontaneous sedimentation. High parasite richness was observed and new parasite species for archaeological contexts were found. The parasitological findings in Puma concolor coprolites associated with caves suggest the importance of these carnivores in the dissemination of parasites in areas with high re-use of space and steady conditions of temperature, humidity, and radiation.

Amblyomma birmitum a new species of hard tick in Burmese amber.

Amblyomma birmitum sp. nov. is formally described as a new record from 99 Ma old Burmese amber from Myanmar. This confirms the presence of the extant hard tick genus Amblyomma C.L. Koch, 1844 (Ixodida: Ixodidae) in the Late Cretaceous. This discovery is placed in its wider context and some reports of fossil hard ticks, such as a Hyalomma C.L. Koch, 1844 in Eocene Baltic amber, are misidentifications. The genus Amblyomma belongs to the clade Metastriata, a group which probably also accommodates two extinct genera, Cornupalpatum Poinar and Brown, 2003 and Compluriscutata Poinar and Buckley, 2008, also found in Burmese amber. All three fossils are thus only a little younger than published molecular divergence time estimates (ca. 124 ± 17 Ma) for the Metastriata lineage. Amblyomma has a largely Gondwanan distribution today. However, in some biogeographical scenarios, e.g. the Samafrica model, its predicted radiation time postdates the dissolution of the original Gondwana supercontinent raising questions about how its current distribution pattern was achieved.

Ancient parasites from endemic deer from "CUEVA PARQUE DIANA" archeological site, Patagonia, Argentina.

The narrow Andean-Patagonian temperate rainforest strip in the west of southern South America is inhabited by two endemic species of cervids, the southern pudu (Pudu puda) and the huemul (Hippocamelus bisulcus), both cataloged as near threatened and threatened species, respectively. One of the possible causes of their declined number is the susceptibility to livestock diseases. Significant zooarchaeological records of both deer have been found throughout the Holocene from Patagonia. The present contribution reports the first paleoparasitological results obtained from coprolites of endemic deer from the archeological site "Cueva Parque Diana," Neuquén Province, Argentina, and discusses the possible diseases found in ancient times. Thirty-four coprolites were fully processed, rehydrated, homogenized, sieved, subjected to spontaneous sedimentation, and examined by light microscopy. Thirty samples contained parasite remains. The presence of diverse parasitic diseases such as trematodioses, metastrongylosis, trichuriosis, strongylida gastroenteritis, dioctophymosis, and coccidiosis which could cause diseases in deer previous to the arrival of European livestock and the presence of zoonotic diseases in the hunters-gatherers and fishermen are discussed.

First paleoparasitological record of digenean eggs from a native deer from Patagonia Argentina (Cueva Parque Diana archaeological site).

Eggs representative of a digenean species were found in coprolites belonged to an endemic deer from Patagonia. Samples were collected from the archaeological site named "Cueva Parque Diana". This site is a cave located at the Lanín National Park, Neuquén Province, Argentina. The coprolites were dated from 2370±70 to 580±60 years B.P. The eggs were ellipsoidal, operculated, yellowish and thin-shelled. Measurements (n=65) ranged from 120.0 to 142.5 (133.2±6.53) µm long and 62.5 to 87.5 (72.6±6.15) µm wide. Eggs were well-preserved and were identified as belonged to Class Trematoda, Subclass Digenea, similar to those of Fasciola hepatica or with another species not identified at present from Patagonia. This is the first report of digenean eggs from ancient deer worldwide. The present study confirms the presence of representatives of digenean species in endemic deer from Patagonia in ancient times and the presence of a trematode disease prior to the arrival of European cattle.

Earliest Evidence of Toxocara sp. in a 1.2-Million-Yr-Old Extinct Hyena (Pachycrocuta brevirostris) Coprolite from Northwest Pakistan.

The study of fossil parasites can provide insight into the antiquity of host-parasite relationships and the origins and evolution of these paleoparasites. Here, a coprolite (fossilized feces) from the 1.2-million-yr-old paleontological site of Haro River Quarry in northwestern Pakistan was analyzed for paleoparasites. Micromorphological thin sectioning and Fourier-transform infrared spectrometry (FTIR) analysis confirms the coprolite belonged to a bone-eating carnivore, likely the extinct giant short-faced hyena (Pachycrocuta brevirostris). Parasitological analysis shows the coprolite to be positive for Toxocara sp. To our knowledge, this is the earliest evidence for Toxocara sp. found.

Paleoparasitological analysis of the extinct Myotragus balearicus Bate 1909 (Artiodactyla, Caprinae) from Mallorca (Balearic Islands, Western Mediterranean).

Myotragus balearicus (Artiodactyla, Caprinae) is an extinct caprine endemic of the Eastern Balearic Islands or Gymnesics (i.e., Mallorca, Menorca and surrounding islets, Western Mediterranean Sea). In spite of its small size, c. 50cm height at the shoulder, it was the largest mammal inhabiting these islands until the human arrival, and it had peculiar short legs and frontal vision. It disappeared between 2830 and 2210calBCE. The coprolites here studied were recovered from Cova Estreta, in Pollença, Mallorca. The samples were subjected to microscopic examination and enzyme-linked immunosorbent assays (ELISA) for E. histolytica/E. dispar, Giardia intestinalis and Cryptosporidium parvum. This study provides new paleoparasitological data from an extinct animal species of the Holocene period. The microscopy revealed one sample containing uninucleated-cyst of Entamoeba sp., whereas ELISA detected nine positive samples for Cryptosporidium sp. The finding of these protozoans can help in the discussion of its extinction cause and demonstrates the antiquity and the evolutionary history of host-parasite relationships between protozoa and caprines since the Messinian.