Gymnophalloides seoi eggs from the stool of a 17th century female mummy found in Hadong, Republic of Korea

It was previously reported that paleoparasitological clues for parasites infecting humans could be found in the feces of mummies of the Joseon Dynasty (1392-1910) in the Republic of Korea. Here, we report the presence of trematode eggs, including Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi (a human parasite known in Korea since 1993) in the feces of a recently excavated female mummy in Hadong, Republic of Korea. This is the first report of the discovery of a G. seoi infection in a human mummy. Since Hadong is currently not an endemic area for G. seoi, we speculate that the parasite might have occurred frequently along coastal areas of the Korean peninsula several hundred years ago and that the endemic areas contracted to, more or less, restricted regions since that time.     

Paleoparasitological report on the stool from a Medieval child mummy in Yangju, Korea

Previous studies have successfully shown evidence for parasitic infections in human remains from various archaeological sites. However, in the case of Korea, since there have been very few paleoparasitological reports published, pre-20th century parasitic infection patterns remain obscure. Therefore, in order to partly fill this gap, we are reporting on a case of paleoparasitic infection from the feces of a 15th century child mummy from Yangju, Korea. In the course of the present study, we found the eggs of Clonorchis sinensis, Ascaris lumbricoides, and Trichuris trichiura in the feces of the mummy. Trichuris trichiura eggs were found in far greater numbers than other parasite eggs; in fact, intact bipolar plugs were clearly observed and even the larvae were still visible in some eggs. The eggs of C. sinensis and A. lumbricoides were also well preserved, though not in as great a number. Since we could find a number of well-preserved larvae-containing eggs, we are encouraged that successful extraction, amplification, and sequence determination of ancient DNA from the paleoparasite eggs might be possible in future studies. With additional paleoparasitological investigation using feces from Korean mummies, we hope that a history of parasite infection in Korea will be reconstructed.     

Paleoparasitological Studies on Mummies of the Joseon Dynasty,Korea

Paleoparasitology is the application of conventional or molecular investigative techniques to archeological samples in order to reveal parasitic infection patterns among past populations. Although pioneering studies already have reported key paleoparasitological findings around the world, the same sorts of studies had not, until very recently, been conducted in sufficient numbers in Korea. Mummified remains of individuals dating to the Korean Joseon Dynasty actually have proved very meaningful to concerned researchers, owing particularly to their superb preservation status, which makes them ideal subjects for paleoparasitological studies. Over the past several years, our study series on Korean mummies has yielded very pertinent data on parasitic infection patterns prevailing among certain Joseon Dynasty populations. In this short review, we summarized the findings and achievements of our recent paleoparasitological examinations of Joseon mummies and discussed about the prospects for future research in this vein.     

Diagnosing ancient diphyllobothriasis from Chinchorro mummies

Diphyllobothrium pacificum has been reported as a human parasite from coprolites and skeletons in Peru and Chile. Our analysis of Chinchorro mummies from Chile provides the oldest evidence of D. pacificum directly associated with human mummies. These mummies date between 4,000 and 5,000 years ago. The basis for our diagnosis is presented. We find that the size of the eggs in the mummies is smaller than other discoveries of D. pacificum. We suggest that this is due to the peculiar circumstances of preservation of parasite eggs within mummies and the release of immature eggs into the intestinal tract as the tapeworms decompose after the death of the host. This information is important to consider when making diagnoses from mummies.     

Brief communication: Twentieth-century replication of an Egyptian mummy: Implications for paleopathology

Replication in a modern human cadaver of ancient Egyptian mummification focused on tools used by ancient Egyptian embalmers, the use of natron (a mixture of sodium carbonate, bicarbonate, and chloride) in the preparation of the mummy, surgical procedures in the removal of the viscera and brain, and histologic examination of the viscera. The first three areas have been reported separately (Brier and Wade [1997] ZAS 124:89–100). In this paper, we demonstrate a degree of histologic preservation comparable to that seen in Egyptian mummies, indicating the effectiveness of ancient mummification and that the histologic appearance of such mummies is little altered by the passage of millennia. Am J Phys Anthropol 107:417–420, 1998. © 1998 Wiley-Liss, Inc.     

Mammalian fertilization as seen with the scanning electron microscope

For several years we have been looking at mammalian gametes and their interactions with the scanning electron microscope (SEM). Examining the images produced by the SEM has given us a three-dimensional view of sperm, eggs, and egg investments. We are particularly impressed with the structural variation among gametes of different mammalian species. In this short report we examine the structure of mammalian spermatozoa, eggs, zonae pellucidae, and cumuli. Our observations and those of others have led us to believe that variation in gamete structure and function may have evolved as a mechanism for reproductive isolation of mammalian species.     

Histological analysis and staining techniques modified and verified on ancient mummified tissues to study microorganism infestations.

The purpose of this work is to give a brief account of the possibility to estimate the preservation of human mummified tissues using histological analysis. This method can be useful to identify injuries and to plan qualified conservative actions on ancient human remains. Some preliminary results are presented here regarding the study on 16 ancient mummies from the Egyptian Museum of Turin. Samples of mummified tissues were taken without damaging the remains; they were hydrated again and dyed with histological techniques which were specifically modified and verified in same cases. Our research identifies some agents (biological and chemical) that appear to be responsible, among others, of mummified tissue destruction. The microscopic examination reveals features that might refer to fungal and bacterial infestation. Using special staining methods on seried sections we were able to suspect, at least in one case, the presence of biologically active forms. Microbiological assays confirm the vitality of fungi. Histological tissue analysis can then be useful to guide any conservative intervention for preservation and protection of the integrity of biological remains from museum collections.     

Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos

Faecal examinations for helminth eggs were performed on 1869 people from two riverside localities, Vientiane Municipality and Saravane Province, along the Mekong River, Laos. To obtain adult flukes, 42 people positive for small trematode eggs (Opisthorchis viverrini, heterophyid, or lecithodendriid eggs) were treated with a 20-30 mg kg(-1) single dose of praziquantel and purged. Diarrhoeic stools were then collected from 36 people (18 in each area) and searched for helminth parasites using stereomicroscopes. Faecal examinations revealed positive rates for small trematode eggs of 53.3% and 70.8% (average 65.2%) in Vientiane and Saravane Province, respectively. Infections with O. viverrini and six species of intestinal flukes were found, namely, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus caninus, Prosthodendrium molenkampi, and Phaneropsolus bonnei. The total number of flukes collected and the proportion of fluke species recovered were markedly different in the two localities; in Vientiane, 1041 O. viverrini (57.8 per person) and 615 others (34.2 per person), whereas in Saravane, 395 O. viverrini (21.9 per person) and 155207 others (8622.6 per person). Five people from Saravane harboured no O. viverrini but numerous heterophyid and/or lecithodendriid flukes. The results indicate that O. viverrini and several species of heterophyid and lecithodendriid flukes are endemic in these two riverside localities, and suggest that the intensity of infection and the relative proportion of fluke species vary by locality along the Mekong River basin.     

First insights into the metagenome of Egyptian mummies using next-generation sequencing

We applied, for the first time, next-generation sequencing (NGS) technology on Egyptian mummies. Seven NGS datasets obtained from five randomly selected Third Intermediate to Graeco-Roman Egyptian mummies (806 BC–124AD) and two unearthed pre-contact Bolivian lowland skeletons were generated and characterised. The datasets were contrasted to three recently published NGS datasets obtained from cold-climate regions, i.e. the Saqqaq, the Denisova hominid and the Alpine Iceman. Analysis was done using one million reads of each newly generated or published dataset. Blastn and megablast results were analysed using MEGAN software. Distinct NGS results were replicated by specific and sensitive polymerase chain reaction (PCR) protocols in ancient DNA dedicated laboratories. Here, we provide unambiguous identification of authentic DNA in Egyptian mummies. The NGS datasets showed variable contents of endogenous DNA harboured in tissues. Three of five mummies displayed a human DNA proportion comparable to the human read count of the Saqqaq permafrost-preserved specimen. Furthermore, a metagenomic signature unique to mummies was displayed. By applying a “bacterial fingerprint”, discrimination among mummies and other remains from warm areas outside Egypt was possible. Due to the absence of an adequate environment monitoring, a bacterial bloom was identified when analysing different biopsies from the same mummies taken after a lapse of time of 1.5 years. Plant kingdom representation in all mummy datasets was unique and could be partially associated with their use in embalming materials. Finally, NGS data showed the presence of Plasmodium falciparum and Toxoplasma gondii DNA sequences, indicating malaria and toxoplasmosis in these mummies. We demonstrate that endogenous ancient DNA can be extracted from mummies and serve as a proper template for the NGS technique, thus, opening new pathways of investigation for future genome sequencing of ancient Egyptian individuals.     

Hounsfield Units ranges in CT-scans of bog bodies and mummies

Mummification processes, either artificial or natural, preserve the tissues from postmortem decay, but change them from their original state. In this study we provided the first comprehensive set of Hounsfield Unit (HU) ranges specific for tissues mummified under different environmental conditions (peat bog, cold-dry and hot-dry environment). We also analyzed the impact of different museal preservation techniques on the HU ranges, as e.g. in the Tollund Man and Grauballe Man, two bog bodies from Denmark. The HU results for mummies were compared with HU results from forensic cases, cremated and inhumated ancient human skeletal remains, and fossil animal bones. Knowledge of the typical HU range for the different tissues in mummies may help to avoid misinterpretation of increased or reduced radiodensity as evidence of paleopathological conditions. Finally, we demonstrate the practical benefit of using our re-defined HU ranges by showing the improved results of 3D visualization from automatic segmentation in an Inca mummy from Mount Llullaillaco.     

Comparative morphology of minute intestinal fluke eggs that can occur in human stools in the republic of Korea

The egg morphology of minute intestinal flukes (MIF) that can occur as human infections in the Republic of Korea, i.e., Metagonimus yokogawai, M. miyatai, M. takahashii, Heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus, Stictodora fuscata, Pygidiopsis summa, and Gymnophalloides seoi, was studied in comparison with Clonorchis sinensis. The adult worms were obtained from residents of endemic areas, and their intrauterine eggs were studied and measured using light microscopy; the length, width, length-width ratio (LWR), and Faust-Meleney index (FMI). Several specimens were processed for scanning electron microscopy (SEM), and before gold-coating, the uterine portion of each fluke was etched with a sharp pin in order to expose the eggs. The MIF eggs were ovoid, pyriform, or elliptical with a size range of 21-35×12-21 μm. S. fuscata eggs revealed the highest FMI (largest in the area) and lowest LWR, whereas P. summa eggs showed the lowest FMI and medium LWR. SEM revealed that G. seoi and S. fuscata had remarkably clean shell surface lacking the muskmelon-like structure which is prominent in C. sinensis eggs. In Metagonimus spp., H. continua, H. nocens, and S. falcatus eggs, minute surface ridges were recognizable though less prominent compared with C. sinensis. On the surface of P. summa eggs, thread-like curly structures were characteristically seen. The results revealed that important differential keys for MIF eggs include the length, width, area (FMI), shape of the eggs, and the extent of the muskmelon-like structure or ridges on their shell surface and operculum.     

Mummified and skeletal southern elephant seals (Mirounga leonina) from the Victoria Land Coast,Ross Sea,Antarctica

We report on an accumulation of mummified southern elephant seals (Mirounga leonina) from Inexpressible Island on the Victoria Land Coast (VLC), western Ross Sea, Antarctica. This accumulation is unusual, as elephant seals typically breed and molt on sub‐Antarctic islands further north and do not currently occupy the VLC. Prior ancient DNA analyses revealed that these seals were part of a large, Antarctic breeding population that crashed ~1,000 yr ago. Radiocarbon dates for Inexpressible Island mummies range from 380 to 3,270 yr before present, too old to have been created by Scott's Northern Party in 1912 and varying too widely in age to represent a catastrophic death assemblage. Skeletal measurements reveal that most Inexpressible Island mummies are adult or subadult males. The presence of male elephant seals on Inexpressible Island until several hundred years ago suggests that, at a minimum, it served as a haul‐out site for the large Antarctic population and may have hosted a breeding colony. The conditions that allowed this Antarctic population to use the Ross Sea, the factors spurring its decline, and the implications for the adaptability and sensitivity of the species to environmental change all merit further study.     

Helminth Eggs Detected in Soil Samples of a Possible Toilet Structure Found at the Capital Area of Ancient Baekje Kingdom of Korea

Although research conducted in East Asia has uncovered parasite eggs from ancient toilets or cesspits, data accumulated to date needs to be supplemented by more archaeoparasitological studies. We examined a total of 21 soil samples from a toilet-like structure at the Hwajisan site, a Baekje-period royal villa, in present-day Korea. At least 4 species of helminth eggs, i.e., Trichuris trichiura, Ascaris lumbricoides, Clonorchis sinensis, and Trichuris sp. (or Trichuris vulpis) were detected in 3 sediment samples of the structure that was likely a toilet used by Baekje nobles. The eggs of T. trichiura were found in all 3 samples (no. 1, 4, and 5); and A. lumbricoides eggs were detected in 2 samples (no. 4 and 5). C. sinensis and T. vulpis-like eggs were found in no. 5 sample. From the findings of this study, we can suppose that the soil-transmitted helminths were prevalent in ancient Korean people, including the nobles of Baekje Kingdom during the 5th to 7th century.     

Detection Trend of Helminth Eggs in the Strata Soil Samples from Ancient Historic Places of Korea

For several years, we have conducted a series of studies on the patterns of ancient parasitism prevailing in the soil of rural and urban areas of past Kingdom of Korea. Actually, during our survey of paleoparasitology in archaeological sites of Korean peninsula, numerous ancient parasite eggs were discovered in the samples from the city districts of Hansung (Joseon) and Buyeo (Baikje), the palace moat at Gyeongju (Silla), shell-midden site at Bonghwang-dong (Silla to Joseon), and the reservoir found in Hwawangsansung fortress (Silla). By the paleoparasitological studies, with respect to parasitism in the high-density populations of ancient towns and cities, we have managed to catch glimpses of the patterns prevalent therein: a serious parasitic contamination of the soil in ancient urban areas, but not in rural areas of the past. Our historical research also proposed the plausible mechanism of parasite infection very serious indeed among urban populations in Korean history. Although city dwelling doubtless has accrued significant benefits for people and populations with agriculture, it can be equally supposed that living in such highly populated areas might have facilitated the spread of parasite infection.     

Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai

We compared the DNA sequences of the genus Metagonimus: M. yokogawai, M. takahashii, and M. miyatai. We obtained 28S D1 ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (mtCOI) fragments from the adult worms by PCR, that were cloned and sequenced. Phylogenetic relationships inferred from the nucleotide sequences of the 28S D1 rDNA and mtCOI gene. M. takahashii and M. yokogawai are placed in the same clade supported by DNA sequence and phylogenic tree analysis in 28S D1 rDNA and mtCOI gene region. The above findings tell us that M. takahashii is closer to M. yokogawai than to M. miyatai genetically. This phylogenetic data also support the nomination of M. miyatai as a separate species.     

Observation with SEM of rehydrated mummy teeth

SEM observation of Egyptian mummy teeth progressively rehydrated shows the perfect conservation of their tissues. This fact amongst others permits one to introduce a new parameter using SEM to estimate the age of mummies. Structural similarity between observed teeth which are of various dates must be outlined. What is the real importance of 4000 years in the world's history?     

Checklist and Scoring System for the Assessment of Soft Tissue Preservation in CT Examinations of Human Mummies

The purpose of this study was to develop a checklist for standardized assessment of soft tissue preservation in human mummies based on whole-body computed tomography examinations, and to add a scoring system to facilitate quantitative comparison of mummies. Computed tomography examinations of 23 mummies from the Capuchin Catacombs of Palermo, Sicily (17 adults, 6 children; 17 anthropogenically and 6 naturally mummified) and 7 mummies from the crypt of the Dominican Church of the Holy Spirit of Vilnius, Lithuania (5 adults, 2 children; all naturally mummified) were used to develop the checklist following previously published guidelines. The scoring system was developed by assigning equal scores for checkpoints with equivalent quality. The checklist was evaluated by intra- and inter-observer reliability. The finalized checklist was applied to compare the groups of anthropogenically and naturally mummified bodies. The finalized checklist contains 97 checkpoints and was divided into two main categories, “A. Soft Tissues of Head and Musculoskeletal System” and “B. Organs and Organ Systems”, each including various subcategories. The complete checklist had an intra-observer reliability of 98% and an inter-observer reliability of 93%. Statistical comparison revealed significantly higher values in anthropogenically compared to naturally mummified bodies for the total score and for three subcategories. In conclusion, the developed checklist allows for a standardized assessment and documentation of soft tissue preservation in whole-body computed tomography examinations of human mummies. The scoring system facilitates a quantitative comparison of the soft tissue preservation status between single mummies or mummy collections.     

Intestinal helminths as a biomolecular complex in archaeological research

Enteric helminths are common parasites in many parts of the world and in the past were much more widespread both geographically and socially. Many enteric helminths are relatively long-lived in the human host, often benign or of low pathogenicity while producing large numbers of environmentally resistant eggs voided in the faeces or found associated with individual remains (skeletons and mummies). The combination of helminth characters offers opportunities to the field of historical pathogen research that are quite different to that of some of the more intensively studied high impact pathogens. Historically, a wealth of studies has employed microscopic techniques to diagnose infection using the morphology of the helminth eggs. More recently, various ancient DNA (aDNA) approaches have been applied in the archaeoparasitological context and these are revolutionizing the field, allowing much more specific diagnosis as well as interrogating the epidemiology of helminths. These advances have enhanced the potential for the field to provide unique information on past populations including using diseases to consider many aspects of life (e.g. sanitation, hygiene, diet, culinary practices and other aspects of society). Here, we consider the impact of helminth archaeoparasitology and more specifically the impact and potential for application of aDNA technologies as a part of the archaeologists'' toolkit.This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.     

Adhesiveness and surface patterns of eggs in neotropical freshwater teleosts

Eggs of 23 Characiformes and eight Siluriformes, belonging to nine families with diverse reproductive behaviour, were ultrastructurally analysed. The migratory species exhibited non-adhesive eggs, whereas, most of the sedentary species presented some degree of egg adhesiveness. Among the Characiformes, non-adhesive eggs showed zona radiata with pore-canals or a fibrillar net at the surface; weakly adhesive eggs presented only zona radiata with pore-canals while adhesive eggs exhibited zona radiata with apparatus like globules, filaments, villi or honeycomb-like pores depending on the systematic group. The 'jelly' coat is strongly related to the Siluriformes eggs apparently without relationship with adhesiveness. A micropylar disc was present in adhesive eggs of a few species of both Characiformes and Siluriformes. Some patterns were characteristic of the animal pole, others of the vegetal pole, and others were common to both poles. The radial ridges converging to the micropyle in Astyanax bimaculatus lacustris appear to be related to fertilization. In general, egg surface structures in the Characiformes varied according to the genus, whereas all Siluriformes showed a similar egg surface pattern, regardless of the group analysed. Multivariate analysis allowed the identification of eight clusters among the Characiformes and three among the Siluriformes showing relationships between reproductive style and egg characteristics. It is suggested that egg surface and adhesiveness may be related to reproductive patterns and to phylogenesis.     

Effects of mummy consumption on fitness and oviposition site selection on Harmonia axyridis

Intraguild predation (IGP) has been commonly reported between predators and parasitoids used as biological control agents as predators consuming parasitoids within their hosts. However, the effect of parasitoid–mummy consumption on the fitness of the predator and subsequent oviposition site selection have not been well studied. In our study, we conducted two laboratory experiments to examine the influence of Aphidius gifuensis Ashmead (Hymenoptera: Braconidae) mummies as prey on fitness and subsequently oviposition site selection of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Results indicate that when H. axyridis was reared on A. gifuensis mummies only, its larval development was prolonged, and body weight of the 4th instar larvae and newly emerged adults, and fecundity decreased. Moreover, H. axyridis did not exhibit oviposition preference on plants infested with unparasitized aphids or aphids parasitized for shorter than 9 days. However, compared with plants with mummies (parasitized ≥9 days), H. axyridis laid more eggs on plants with unparasitized aphids. In contrast, H. axyridis previously fed with A. gifuensis mummies did not show a significant oviposition preference between plants with unparasitized aphids and those with mummies (parasitized ≥9 days). Overall, our results suggest that mummy consumption reduced the fitness of H. axyridis. Although H. axyridis avoided laying eggs on plants with A. gifuensis mummies, prior feeding experience on A. gifuensis mummies could alter the oviposition site preference. Thus, in biological control practice, prior feeding experience of H. axyridis should be carefully considered for reduction of IGP and increase of fitness of H. axyridis on A. gifuensis.