Symposium Ancient : Paleosciences April 12th, 2016

PALEOPARASITOLOGY from Ancient to Future

Míriam J. Álvarez-Martínez M.D., Ph.D.

Microbiology Department Hospital Clinic, Barcelona (Spain) ISGlobal (Barcelona Institute for Global Health) School of Medicine-University of Barcelona [email protected] ESCMID eLibrary by author ESCMID eLibrary by author ESCMIDProfessors Luiz Fernando Ferreira, KarleLibrary Jan Reinhard, Adauto Araújo by author OUTLINE

1. What is Paleoparasitology? 2. Origin of Parasites in Humans • Heirloom parasites & Souvenir parasites 3. Parasites as Markers of Prehistoric Migrations 4. Preservation of Paleoparasitological Remains a) Parasites characteristics b) Microenviroments & Recovery of Parasites – /Latrine soil /Mummified bodies 5. Techniques in Paleoparasitology: Ancient DNA (aDNA) 6. Some Paleoparasitological Studies ESCMID7. Conclusions eLibrary by author PALEOPARASITOLOGY 1 • Study of parasites in the remains of humans and other animal species recovered from archaeological or paleontological sites or any source in which they have remained preserved. • Derived from the Greek παληος (ancient) παρασιτος (next to bread) λογς (study)

• Field of knowledge splitted from Paleopathology from the finding of parasite forms in archeological ESCMIDmaterial. eLibrary by author ESCMIDSir Marc Armand RUFFER (1845-1917) eLibrary by author • Sir Marc Armand Ruffer-pioneering work in 1910- publication finding of Schistosoma haematobium eggs in the kidney tissue of Egyptian from the 20th Dynasty, dated to circa 1250 to 1100 BC

ESCMID eLibrary by author ORIGIN OF PARASITES IN HUMANS 2

First hominids in Africa were hosts for some species of parasites. Inherited from pre-hominids or acquired from enviroment

1. Phylogenetic Route Various parasites species persisted in Homo sapiens, inherited ancestrally and originating during pre-hominid times. These parasites are also found in phylogenetically proximate hosts.

2. Ecological Route New parasites acquired from the enviroment began to infect humans as the latter conqueres new spaces , ie during the exploration of new ESCMIDterritories and contact with different eLibraryanimal species and their parasites. by author Preservation of paleoparasitologicalHEIRLOOM PARASITES remains

• Some infectious agents of human disease have coevolved with species in the Homo lineage for over 400,000 years. • Most common parasites had a concomitant origin with earlier species in the human evolutionary line. • These long-term, coevolved parasites are called “Heirloom Parasites” • As humans evolved, their heirloom parasites evolved with them. • This is the case for Enterobius vermicularis (pinworm) and Pediculus humanus (head lice)

Pediculus humanus found in a partially mummified Enterobius vermicularis found in a human human body, Brazil, dated of ESCMIDcoprolite dated of 6,000 years, Chile Prof. AraujoeLibrary pictures 12,000 years by author Preservation of paleoparasitologicalSOUVENIR PARASITES remains

• Humans have acquired other parasites during their biological and social history, called “Souvenir parasites”. • Species that occur in humans commonly, arriving through - switching from other animal species (animal domestication). • This was considered the origin of two species of taeniid tapeworms (Taenia saginata and T. solium) in humans that use cattle and pigs as intermediate hosts.

ESCMIDPaleolithic- hunters & gatherers eLibraryNeolithic- agriculture & livestock by author PARASITES MARKERS OF PREHISTORIC MIGRATIONS 3 • Host-specific parasites of humans are used to track ancient migrations

Ancient migrations to the Americas • Timing of the arrival of humans in the New World has been the focus of a long-standing and intense debate. • Accepted hypothesis that populations of prehistoric humans crossed into the Americas when climate conditions created the Bering Land Bridge at least 13, 000 years ago. • People could have reached the Americas more than 7,000 years ago using alternative routes avoiding the extreme cold of the Arctic. • From the parasitological perspective evidence shows that populations of pre-historic humans were infected by several host-specific intestinal parasites found in both North and South American archaeological sites. The life cycle of these exclusively human parasites could not have been maintained among human groups during the crossing ESCMIDfrom Siberia to Alaska via Beringia. eLibrary by author ESCMID eLibrary by author 2008

Three general migration routes of humans in prehistory hypothesized from Asia to the Americas. (a) Represents the longest-standing hypothesis of a migration route through Beringia and into North America at the time of low sea level and glacial maximum (b) Parasite probe data are consistent with a Coastal migration or ESCMID(c) Trans-Pacific migration eLibrary by author • Climate and soil temperature are limiting factors for human hookworm and whipworm, being host-specific and without intermediate hosts. • High soil moisture and moderate temperature are necessary for eggs or larvae to reach the infective stage.

• Eggs of E. vermicularis are infective as soon as they are expelled from female worms; in the external environment, they can infect humans immediately through contaminated hands, soil, water, any kind of object or as an aerosol. • Therefore, pinworm eggs are not dependent on climate conditions.

• Human hookworm and whipworm have never been reported in arctic or subarctic people. • Prehistoric infection with Hookworms, Trichuris and Strongyloides shows a North to South America distribution dated to 7000 years ago. • Because these worms are host-specific, they necessarily entered the New World through the migrations of human hosts. • Because the extracorporeal maturation of parasites requires warm conditions, migration routes through Beringia and into the center of North American continent were discounted. ESCMID• Transpacific migration or a Coastal migration, eLibrary was proposed. by author BC BC

BC ESCMID eLibraryProf Araujo© by author PRESERVATION PALEOPARASITOLOGICAL REMAINS 4 • Conditions in the preservation of parasites vary from region to region. • Crucial for preservation is the rapid interruption of decay.

• Limited to places of non moisture, high aridity, or frozen conditions. – combination of high and low temperatures, with dryness, result in rapid desiccation, hindering the activity of catalytic enzymes and destructive (ie deserts).

• Tropical climates are unsuitable for the preservation of organic matter. Combination of heavy rainfall, abundant insect life, and acid soil usually accelerate the process of ESCMIDdestruction of organic material. eLibrary by author ESCMID eLibrary by author Preservation of paleoparasitological remains PARASITES CHARACTERISTICS 4a

• Paleoparasite evidence consists – mostly eggs, rarely of larvae, of intestinal parasites – chitinous shells of ectoparasites (lice, and flies)

• Some parasites species produce protective structures that guard against decay. – Eggs of Ascaris, Capillaria, Trichuris, Oxiuros and Taenia have a good chance of recovery.

• Thin walled eggs, Enterobious or Strongyloides preserve less well.

• Problematic aspect of preservation: life cycle – Ancylostoma eggs hatch soon after deposition_ will not be found (not in latrine sediments, but in mummies and coprolites) – Strongyloides females lay eggs which hatch in intestine before ESCMIDdefecation__best condition_larvae willeLibrary be found by author Preservation of paleoparasitological remains: MICROENVIROMENTS COPROLITESPreservation of paleoparasitological DIAGNOSES remains 4b

• Comes from Greek κπρος-kopros (feces) and λιφος-litos (rock). • It refer to hardened, stony feces. • Dehydrated by rapid loss of water , or mineralized, when the organic matter has better replaced and shaped by minerals. • Named by British Rev. William Buckland, geologist , paleontologist and “dinosaur hunter” in 19th cent. – ….coprolites, petrified feces found inside and outside animals, often reveal to us not only the nature of the ingestion, displaying parts of other animals that were consumed, but even the nature of the mucosal surface of the gut and prove that the ingestion was done in those times more as less as in our times. (Wucherer, 1866) ESCMID eLibrary by author ESCMIDChame 2003. Mem InsteLibraryOswald Cruz 98 suppl 1. by author Recovery of parasites from coprolites

Coprolite – Rehydration in 0,5 % Na3SO4 (72h)

Addition drops of acetic formalin

Flotation Sedimentacion ESCMID20 slides in all fields for eLibrarysample by author ESCMID eLibrary by author Preservation of paleoparasitological remains: MICROENVIROMENTS MUMMIFIEDPreservation of paleoparasitological BODIES remains4b

• _ Well-preserved dead bodies. The majority found in dry places (sands of deserts or dry caves) where desiccation has taken place rapidly, doing naturally what Egyptians did by artifice. • The basic procedure is the same: water extracted rapidly from the tissues. • Low pH and anaerobic enviroment preserving organic remains.

• References to mummification processes found in ancient documents as Genesis – Chap50 …And Joseph command his servants the physicians to embalm his father, so the physicians embalmed Israel, forty days were required for it, for so many are required for embalming… So Joseph died, being hundred and ten years old; and thet embalmed him, and he was put in a coffin in Egypt.

• If rehydratation of coprolites in mummies does not occur with trisodium phosfate, ESCMID4% solution of KOH is effective. eLibrary by author BM-London

Louvre-Paris

ESCMIDCairo MeLibrary by author Ectoparasites/ other arthropods (mites, lice & ticks)

Included in Required presence of its former host

• Nits & eggs attached to hair shaft or the fur • Clothing of mummies or tools (combs)

Store in a vial with 70-100% in ethanol ESCMID eLibrary by author Examples of rare cases of parasite preservation. (A) SEM of a head louse mummy (Pediculus humanus capitis) from a Chiribaya mummy (Southern Peru; Raoult et al. 2008). (B) Close up of the louse head, showing the bloodsucking mouthparts. (C) SEM of a flea (Pulex sp.), from a ESCMIDguinea pig mummy of the Chiribaya Culture (Southern eLibrary Peru, Dittmar et al., 2003). by author ANCIENTPreservation of DNA paleoparasitological (aDNA) remains5

• aDNA pertains to specimens range from few decades old (ie museum specimens) to roughly 100,000 years. • Any DNA reported to be millions of years old is actually an artifact (Paabo, 2004) • aDNA is post-mortem preserved genetic information • Sources – Coprolites, latrine sediments, ectoparasites, soft tissues and bones. • aDNA is subject to degradation (especially nuclear DNA) and contamination • Evidence of aDNA :T. cruzi, Leishmania sp,Trichuris, Ascaris, ESCMIDEnterobius, Pulex irritans (human fleaeLibrary) and Pediculus capitis by author Two mummies from the Chiribaya civilization are displayed in El Algarrobal museum in southern Peru in 2006. ESCMIDPhotograph by MarianaeLibrary Bazo/Reuters by author aDNAPreservationSTUDY of paleoparasitological METHODS remains

• Multiplex DNA techniques- Target sequences within the total DNA sample- Sequencing entire mitochondrial genome of a mammoth • Genomics- Amplify total DNA of a sample, including contaminants • Metagenomics- Construction of a bacterial clone library from random DNA- Sequence Neanderthal DNA. • Shotgun Sequencing- Typing entire parasitological DNA in a , latrine sediments or mummy tissue. • Pyrosequencing ESCMID eLibrary by author PALEOPARASITOLOGICAL STUDIES 6

• Chagas Disease long time before Carlos Chagas • First proof of ancient Schistosomiasis • Paleopathology of Malaria ESCMID eLibrary by author Chagas Disease Long Time before Carlos Chagas

ESCMID eLibrary by author In muscle tissue of mummies spontaneously dissected from the desert Atacama was achieved DNA amplification of kinetoplast of T. cruzi.

Human residents on the Southwest coast of South America suffered American Trypanosomiasis 9,000 years ago. Spontaneously mummified body the Atacama Desert (Chile) ESCMIDGuhl et al., Lancet, 1997 eLibrary by author Visceromegalies found in Atacama’s mummies were due to chronic forms of American Trypanosomiasis

Human settlements and the construction of houses, where vector was nested, facilitated maintaining balance between the biology of Trypanosoma its vector, the environment and ESCMIDhuman eLibrary behavior. by author Geographical (A) and chronological (B) information on the cultures in South America used for the paleoepidemiological study of Trypanosoma cruzi Discrete Typing Units. ESCMID eLibrary by author Phylogenetic and network reconstruction of ancient T. cruzi cytochrome b gene fragments.

(A)Phylogenetic reconstruction discriminating the samples and controls of T. cruzi DTU (B) Haplotype network reconstruction clustering the seven TU including ancient haplotypes ESCMID(C) Table showing the percentages of DTU in theeLibrary tissues analysed. by author ESCMID PHOTOGRAPHeLibrary COURTESY DENNIS VAN GERVEN by author FirstPreservationproof of of ancient paleoparasitologicalSchistosomiasis remains

• Since the discovery of parasite eggs on mummies in the 1920s, scientists have suspected that Nubians might have been infected by schistosomiasis. Nubia, former African kingdom, A.D. 250 to 1400, in now Northern Sudan.

• Assumed that disease in Nubians by S. haematobium, that causes similar symptoms that S. mansoni, but doesn't require irrigation channels to thrive.

• The snail that transmits S. haematobium thrives better in water that's moving and well oxygenated and that is not very polluted, whereas the S. mansoni snail does very well in water that’s been standing around and has more dirt in it.

• Analysis of the mummified skin revealed traces of proteins belonging to S. mansoni—first proof that the ancient Nubians, or any ancient civilization, ESCMIDwere afflicted by schistosomiasis. eLibrary by author • Paleopathology of Malaria

Current state of the methods used to suggest the presence of malaria in human remains recovered from the archaeological record. 1. Gross examination of human remains 2. aDNA ESCMID3. Immunological methodology eLibrary by author Paleopathology of Malaria 1. Gross examination of human remains • Malaria itself is not to result in any direct response in the skeletal system

• Skeletal response related to inherited hemolytic anemias caused by the malaria.

Porotic hyperostosis and cribra orbitalia, most common bony responses found in prehistoric populations .

• Porotic hyperostosis in cranial bones: the outer table of the cranial vault thins or is completely destroyed. • Cribra orbitalia : pitting in the orbital roofs.

ESCMID eLibrary by author 2. aDNA Paleopathology of Malaria • aDNA analysis can provide promising results. Challenges associated : problems preservation,degradation, contamination, and organic inhibitors (ie tannins, humic and fulvic acids) • No studies reported hemozoin isolation with human remains in an archaeological context. The insoluble nature of hemozoin dissolves at a pH of 10–11 and the unique signature it produces when analyzed with laser desorption mass spectrometry – potential method to detect malaria in human more reliable method than aDNA. 3. Immunological Techniques Created for diagnosing malaria in living individuals produced some positive results when applied to human remains from archaeological sites. • ParaSight TM-F test is P. falciparum histidine-rich protein 2 (PfHRP II). ESCMIDContaminants and false positives –eLibraryquestions the reliability...... by author Preservation of paleoparasitologicalCONCLUSIONS remains

• Paleoparasitology interdisciplinary science • Origin of Human Parasites • from African ancestors (heirloom parasites) • from acquisition during evolution (souvenir parasites) • Parasites as probes of human prehistoric migrations • Intestinal parasites tracking human behaviour • Paleolithic__Neolithic • Coprolites, mummies and letrines as aDNA source • Existence of parasites centuries before described • Travel with return ticket ESCMID• from ancient to future and fromeLibraryfuture to ancient by author Unde Venis et Quo Vadis?

Paleoparasitus sit amet...

ESCMID eLibrary by author ACKNOWLEDGEMENTS

Profs. L. F. Ferreira, K. J. Reinhard, A. Araújo

THANK YOU FOR YOUR ATTENTION

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