# Evolutionary History of Terrestrial Pathogens and Endoparasites as Revealed in Fossils and Subfossils

Authors: George Poinar
Journal: Advances in Biology
 (2014)

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## Abstract

The present work uses fossils and subfossils to decipher the origin and evolution of terrestrial pathogens and endoparasites. Fossils, as interpreted by morphology or specific features of their hosts, furnish minimum dates for the origin of infectious agents, coevolution with hosts, and geographical locations. Subfossils, those that can be C14 dated (roughly under 50,000 years) and are identified by morphology as well as molecular and immunological techniques, provide time periods when humans became infected with various diseases. The pathogen groups surveyed include viruses, bacteria, protozoa, fungi, and select multicellular endoparasites including nematodes, trematodes, cestodes, and insect parasitoids in the terrestrial environment.


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## Body

## 1. Introduction

The world is filled with pathogens that have been around for millions of years, yet we know very little about their origins and evolution. The present work uses fossils and subfossils to decipher the origin and evolution of terrestrial pathogens and endoparasites. Fossils furnish minimum dates for the origin of infectious agents, coevolution with hosts, and geographical locations. Subfossil remains, those that can be C14 dated (roughly under 50,000 years), provide time periods when humans became infected with various diseases. The detection of fossil infectious agents is based on morphology or specific features of their hosts while molecular and immunological techniques can be used as well in searching for subfossil pathogens and parasites.Arthropods in amber that were vectoring vertebrate pathogens have provided unique information on the origin and evolution of human and animal diseases [1]. Such detailed preservation is due to the instant death of the vector, its rapid desiccation, and impregnation by natural fixatives. Dates for the various amber sites mentioned in the present work are Dominican, 20–15 mya based on foraminifera and 45–30 mya based on coccoliths; Mexican, 22–26 mya; Baltic, 40–50 mya; Myanmar (Burmese), 97–110 mya; and Lebanese, 130–135 mya.The pathogen groups surveyed include viruses, bacteria, protozoa, and fungi in the terrestrial environment. Also included are multicellular endoparasites such as nematodes, trematodes, cestodes, and select insect parasitoids. Examples are included when both the pathogen/parasite and host occur together as determined by morphological, molecular, or immunological evidence. Reports of disease in the fossil record based on host signs have been summarized previously [2–4].


## 2. Pathogens

### 2.1. Viruses

Viruses are probably quite ancient and may have existed some 4 billion years ago with bacteria as hosts. The nearly 2000 species of viruses infecting plants and animals today are probably a conservative estimate of their diversity [5]. All estimates on the evolutionary history of viruses are based on molecular analyses since fossils of such small, fragile structures are almost nonexistent. Evidence of some viruses has been found in subfossil remains based on DNA analysis [4] but the only fossil evidence of infectious diseases caused by viruses associated with their hosts is insect polyhedrosis, polynida, and iridoviruses. The polyhedra of nuclear polyhedrosis viruses (Baculoviridae) and cytoplasmic polyhedrosis viruses (Reoviridae) are just large enough to be detected with the light microscope. Based on the size of the polyhedra, the particular organs in which they occur, and the type of insect hosts, it was possible to identify a cytoplasmic polyhedrosis virus infecting a biting midge (Figures 1, 2, and 3) and a nuclear polyhedrosis virus infection in a sand fly (Figures 4 and 5), both in Early Cretaceous amber from Myanmar [6]. These records provide a minimum date for the origin of the Baculoviridae and Reoviridae and suggest that some human arboviruses could have evolved first as pathogens of bloodsucking flies.Figure 1
Early Cretaceous Myanmar amber biting midge (Protoculicoidessp.; Diptera: Ceratopogonidae) that contains polyhedra of a cytoplasmic polyhedrosis virus in its midgut cells.Figure 2
Group of polyhedra (arrow) of a cytoplasmic polyhedrosis virus in the midgut cells of the Early Cretaceous biting midge in Figure1.Figure 3
Detail of polyhedra of a cytoplasmic polyhedrosis virus in the midgut cells of an Early Cretaceous biting midge in Figure1.Figure 4
An Early Cretaceous Myanmar amber sand fly (Diptera: Psychodidae) infected with a nuclear polyhidrosis virus.Figure 5
Detail of polyhedra of a nuclear polyhedrosis virus in the Early Cretaceous sand fly in Figure4.A specialized group of viruses that are carried by parasitic wasps of the families Braconidae and Ichneumonidae are the Polydnaviridae. Genomes of polydnaviruses are integrated into the wasp genome and virus replication occurs in the reproductive system of the female wasp. During oviposition, the virus is introduced along with the wasp eggs into the body of potential hosts. The virus replicates in the host and affects the host immune system by preventing it from encapsulating and destroying the wasp eggs. The transfer of these virus particles from wasp to host was first observed in 1975 with the ichneumonid,Casinaria infesta, and its plutellid moth host [7] and in 1976 with the braconidPhanerotoma flavitestacea and its pyralid moth host,Paramyelois transitella [8]. Since that time, the field has expanded and the polydnaviruses are now included in the new discipline of Paleovirology, which infers viral evolution from host genome sequence data [9, 10].Presently the family Polydnaviridae contains two genera. The genusBracovirus occurs in the calyx cells and fluid of the female wasps of the family Braconidae and the genusIchnovirus is associated with female wasps of the family Ichneumonidae. Because of the close association between the virus and members of these two families, one can assume that these viruses existed in ancient wasps of these lineages, such as the fossil braconid and ichneumonid wasps depicted in Dominican amber (Figures 6 and 7) and even in the primitive braconid wasp in 100 million year old amber from Myanmar (Figure 8).Figure 6
A female braconid wasp (Hymenoptera: Braconidae) in Dominican amber that is probably carrying the genome of aBracovirus in its reproductive system.Figure 7
A female ichneumonid wasp (Hymenoptera: Ichneumonidae) in Dominican amber that is probably carrying the genome of anIchnovirus in its reproductive system.Figure 8
A primitive braconid wasp (Hymenoptera: Braconidae) in Early Cretaceous Myanmar amber that could be carrying the genome of a polydnavirus in its reproductive system.A group of invertebrate pathogenic viruses whose presence often can be determined by color changes of the host are the iridoviruses. These double-stranded DNA viruses are icosahedral and hexagonal in cross section. The isopod iridescent virus infects terrestrial pillbugs and sowbugs and in the later stages of infection turns normal brown hosts to various shades of violet, blue, and purple [11]. In fact, an isopod thus colored can be assured of having an iridoviral infection. The bluish color is the result of paracrystalline arrays of viral particles (virions) filling the epidermal and other cells of the isopod that is eventually killed by the infection. An isopod in Cretaceous amber from Myanmar that has iridescent blue areas on its body is assumed to be infected with an ancient strain of the isopod iridescent virus (Figure 9). This is another example of identifying fossil viruses using indirect, but reliable evidence derived from our present knowledge.Figure 9
A bluish tinted terrestrial isopod in Early Cretaceous Myanmar amber that could be infected with the isopod iridescent virus.


### 2.2. Bacteria

Bacteria are ubiquitous and there are numerous reports of fossil associations between putative bacteria and various animals and plants [2, 3]. However, most of these reports refer to signs indicating a possible bacterial infection and actual bacteria are absent. One example is the dark areas on the body of the Burgess Shale onychophoran,Aysheaia pedunculata, that are thought to represent bacterial infections [12]. Other examples of indirect presence of bacteria are fossil fish with luminescent organs similar to those on their present day descendants that are operated by luminescent bacteria [3] or insects producing flatus in fossilized resin [13].Filamentous cells occurring in the marine environment some 3.6 billion years ago represent some of the earliest known bacteria [14]. When terrestrial bacterial first appeared and formed associations with animals and plants is unknown since fossil bacteria are quite difficult to detect. Their small size and delicate structure (except for spores) are not prone to fossilization. Evidence of the early evolution of some specific lineages of pathogenic bacteria has appeared in fossilized resin.The oldest known definite fossil disease-producing bacteria are associated with an entomopathogenic nematode of theHeterorhabditis clade in Early Cretaceous amber [15]. Several infective stages of the insect pathogenic nematode,Proheterorhabditis burmanicus, were preserved as they emerged from their beetle host in Myanmar amber (Figure 10). Extant members of theHeterorhabditis clade carry and release spores of the luminescent bacterium,Photorhabdus luminescens, into the body cavity of insects. The bacteria multiply and kill the insects within 24 hours. The nematodes feed on the combined bacteria host breakdown tissues and the newly formed infective stage juveniles carry these bacterial cells into a new host. Bacterial cells in the fossil beetle (Figure 11) adjacent to the nematode resembled those of extantPhotorhabdus spp. and are presumed to have been introduced into the beetle byProheterorhabditis burmanicus.Figure 10
An Early Cretaceous Myanmar amber entomopathogenic nematode,Proheterorhabditis burmanicus (arrow), adjacent to its beetle host.Figure 11
Bacterial cells ofPhotorhabdus inside the Early Cretaceous Myanmar amber beetle shown in Figure 10.Ticks (Figure12) carry a number of bacterial pathogens. One of special interest is the spirochete,Borrelia burgdorferi, the causal agent of Lyme disease. An ancient spirochete with characters ofBorrelia was found in the midgut and hemocoel of a larval hard tick belonging to the genusAmblyomma (Arachnida: Ixodidae) in Dominican amber (Figures 13 and 14) [16]. The presence of spirochetes in the hemocoel indicated that the tick was a competent vector since spirochetes do not enter the hemocoel of incompetent vectors [17]. Vertebrate hosts of extantAmblyomma ticks are reptiles, birds, and mammals and remains of all three of these groups occur in Dominican amber [18]. This discovery shows how ancient some lineages of tick-vectored pathogens can be. It is likely that genetic material of this fossil spirochete contributed to the genome of extantBorrelia lineages, including those species that infect humans. The earliest record of Lyme disease was recently reported from the Tyrolean Iceman, a 5,300-year-old mummy discovered in 1991 in the Italian Alps. During a genomic analysis of the Iceman, sequences corresponding to ~60% of the genome ofBorrelia burgdorferi indicated that the Iceman suffered from Lyme borreliosis [19].Figure 12
LarvalAmbylomma hard tick in Dominican amber.Figure 13
Spirochetes (arrows) within the chelicerial sheath and surrounding the hypostome of a larvalAmbylomma tick in Dominican amber similar to the one shown in Figure 12. Scale bar = 16 μm.Figure 14
Spirochetes in the pharynx (arrow) of a larvalAmbylomma tick in Dominican amber similar to the one shown in Figure 12. Scale bar = 13 μm.The fossil record of hard ticks extends back to the Early Cretaceous [20, 21]. This sets a time period when these arachnids could have acquired spirochetes, followed by a period of coevolution that established the vertebrate cycle and increased vector competency. The origin of Borreliosis is a mystery. It is likely thatBorrelia began as a free-living spirochete that could have made contact with tick eggs deposited in the soil. However, so little is known about interactions ofBorrelia sp. with present day ticks that this scenario is difficult to prove. Yet ifBorrelia began as a tick parasite, it could have been passed from mother to offspring by vertical or transovarial transmission (TOT). The spirochetes may have initially served as symbiotes, since TOT is normally how symbiotic microorganisms are passed in hosts. A later evolutionary step would be entering the salivary glands where they could be transferred to vertebrates.An example of this evolutionary scenario is the establishment and transfer of rickettsial organisms in arthropods [22]. Rickettsias occur in many locations inside the alimentary tract and body tissues of a wide range of animals [23, 24]. Some symbiotic species, likeWolbachia, are beneficial and assist in the digestion of blood meals, while other rickettsias are pathogenic to both arthropod vectors and their vertebrate hosts. Finding evidence of rickettsias in the fossil record is difficult but a recent study showed various stages of a rickettsial organism in the Early Cretaceous Myanmar amber hard tick,Cornupalpatum burmanicum (Figure 15). The rickettsial cells occurred in the remains of gastric ceca (Figure 16). Cocci, diplococci, and short, rod shaped cells borne singly or in short chains were dividing by binary fission. The rods ranged from 0.4 to 0.8 μm in width by 1.3–1.7 μm in length. In some areas, ovoid cells surrounded by an apparent mucoprotein, slime layer (haloes), were present. Such “halo” cells are known to occur in rickettsial populations and are considered to represent infective stages of the disease process [22, 23]. Since the cells were developing in the gastric cecal cells, the fossil could be related to any one of the many species in the families of Rickettsiaceae or Anaplasmataceae [17, 23]. Ticks are known to vector a range of rickettsial pathogens, including Q-fever, tick typhus, and Boutonneuse fever, some of which cause diseases in reptiles [24], which makes dinosaurs possible hosts [25].Figure 15
Early Cretaceous Myanmar amber larval hard tick,Cornupalpatum burmanicum that contains rickettsial organisms.Figure 16
Short rods and cocci of a rickettsial organism in a gastric ceca of the Early Cretaceous larval hard tick shown in Figure15.Insects relying solely on blood meals throughout their entire development often harbor symbiotic microorganisms [26]. While they can be considered parasites since they develop on a combination of the blood meal and digestive secretions from the insect, they may supply essential elements to their host, since blood is considered a nutritionally inadequate diet deficient in B vitamins. Nocardiform bacteria such asRhodococcus assimilate proteins and carbohydrates in the lumen of the alimentary tract of blood-sucking triatomine bugs [27]. Species of this genus occur in soil and water as well as in the lumen of insect alimentary tracts. Past evidence of this clade was discovered when cells ofPaleorhodococcus dominicanus were found in a fecal droplet adjacent to the triatome bug,Triatoma dominicana, in Dominican amber [28, 29]. The fossil species consisted of numerous coccoid elements with associated mycelial fragments [29] (Figure 18). WhileP. dominicanus could have been beneficial if it was assisting in the breakdown of blood components, it could also have been infectious since some species ofRhodococcus are pathogens, such asR. equi that infects horses.ABacillus sporangium inside of the body of the mycetophagous nematode,Oligaphelenchoides atrebora in Mexican amber (Figure 19), could indicate the presence of an infectious disease in the entire nematode population [30].Bacillus sp. are known to infect extant nematodes [31] as well as insects and humans. However, fossil evidence of such infections is rare, even though the ancient presence ofB. sphaericus,B. subtilis, andB. circulans was found in amber, sedimentary rocks, and salt deposits [32].One of the most studied bacterial diseases is the plague, which has killed countless humans throughout history. Plague is caused by a small, pleomorphic bacterium known asYersinia pestis that is transmitted by fleas. This pathogenic strain probably evolved from one of the free-living species ofYersinia that occur in soil, fresh water, foods, fish, and various animals [33]. The chances of finding fossil cells ofY. pestis are extremely slim. Some bacterial cells attached to the proboscis of a fossilRhopalopsyllus flea in Dominican amber (Figure 20) have the morphological features ofYersinia (Figure 21) although it is not possible to confirm their identity. However, the extantRhopalopsyllus cavicola is one of 125 species of fleas capable of transmittingY. pestis, in this case to cavy rodents in the American tropics [34, 35].Sophisticated molecular and immunological methods can now be used not only to identify pathogens, but to compare various strains that appeared throughout history. One of the best sources of ancient DNA for such studies is roots and crowns of teeth from the victims of various epidemics (Figure22). These were used to characterize strains ofY. pestis that were responsible for the Black Death that struck Europe in the 14th to 17th centuries [36].Past cholera epidemics also resulted in high mortality rates (Figure23). Using a preserved intestinal portion from a victim of the 1849 cholera outbreak in Philadelphia, molecular techniques were used to reconstruct the genome of the causative organism,Vibrio cholerae [37].Teeth are also a good source of decay-causing bacteria. One report characterizes filamentous actinomycetes in a tooth from an Eocene rabbit in Montana [38], thus documenting the earliest record of tooth infection in placental mammals.


### 2.3. Protozoa

Protozoa, with some 120,000 described species, are an ancient group with a fossil record extending back to the Archean Eon, some 2 billion years ago. Protozoa probably originated in the sea and soon afterwards formed symbiotic associations with terrestrial life forms. Eventually, they entered the lumen of the alimentary tract of invertebrates and developed on partially digested food. Some of these became permanent residents in the guts of various insects such as termites, where the protozoa served a mutualistic role of digesting cellulose. Other protozoans invaded the internal organs of their hosts and became parasites. During their evolution, some became closely associated with blood-sucking arthropods, which transported them into vertebrates where they became established as true parasites with the potential of causing disease. A few invaded the tissues of plants but the present diversity of plant parasitic protozoa is quite low.Today a number of arthropods vector parasitic protozoa and early lineages of some of these became entrapped in amber, thus providing clues to the evolution of human diseases. Two basic types of development evolved between arthropod vectors and their protozoan parasites. The first is where the protozoans only develop in the gut lumen of the vector. Examples are members of the trypanosome lineage that includeLeishmania vectored by sand flies. The second is where the protozoans penetrate the gut wall and undergo development in the body cavity of the vector. Examples are the various malarial pathogens vectored by several arthropods, especially mosquitoes.The first fossil protozoan pathogen to be discovered was one of the former types that were in the gut lumen of a phlebotomine sand fly entrapped in Early Cretaceous Myanmar amber (Figure24) [39]. In the midgut lumen of the sand fly,Palaeomyia burmitis, were various stages of the leishmanial parasite,Paleoleishmania proterus [40] (Figure 25). Aside from normal promastigotes, elongate nectomonad promastigotes (Figure 26) and short procyclic promastigotes (Figure 27) were also present. Vertebrate infective amastigotes occurred in the proboscis of the fossil sand fly (Figure 28) and a view into the thoracic lumen of the fossil sand fly revealed nucleated vertebrate blood cells. Some of these cells contained developing amastigotes (Figure 29), obviously acquired during a recent blood meal. Based on morphological characteristics of the blood cells, the vertebrate host was considered to be a reptile andPaleoleishmania proterus was thought to be an ancestor of the genusSauroleishmania that infects lizards and snakes today [41].The life cycle ofPaleoleishmania proterus was probably similar to that of leishmanial parasites vectored today by sand flies [42]. Amastigotes were acquired from the blood of a vertebrate during feeding. These developed into short ovoid procyclic forms that then formed elongate nectomonad stages in the lumen of the alimentary tract of the sand fly. Metacyclic (paramastigote) forms were transferred back into a vertebrate during blood-feeding.How, when, and where protozoan vector associations originated are difficult to determine, especially when every association could have evolved slightly differently. Also past evidence of vector associations is extremely rare since most protozoans are too small and fragile to be recognized [43]. However, in some instances, clues to possible mechanisms exist. In the case ofLeishmania and sand flies, the discovery of trypanosomes associated with sand fly larvae originating from the same Early Cretaceous Myanmar amber deposits provided a possible scenario for how these flagellates became established in sand flies. The two fossil sand fly larvae were adjacent to and probably feeding on a fruiting body of a coral mushroom,Palaeoclavaria burmitis (Figure 30) [44–46]. Associated with some expelled fecal material from both larvae were numerous flagellates. Similar flagellates surrounded the bases of the fungal fruiting bodies and were associated with debris attached to the cuticle of both sand flies. After polishing away a portion of the alimentary tract of one of the amber sand flies, numerous flagellates similar to those at the bases of the fungal fruiting bodies were also discovered in the lumen of its midgut (Figure 31).Based on these findings, the following scenario for the acquisition ofLeishmania by sand flies is presented. Free-living trypanosomes developing in the same microhabitat as the sand fly larvae and club fungi were ingested by the larvae. They may have served as food at first, but eventually the trypanosomes began to multiply inside the gut of the sand fly and were carried transtadially from the larva through the pupa and into the adult. Once established in the gut of the adult sand fly, the flagellates were eventually transferred to vertebrates during blood feeding and a vector association was established. Molecular studies showing that insects were the first hosts of trypanosomatids [47] support the above scenario on the origin of parasiticLeishmania through larval acquisition of free-living trypanosomatids.Flagellate-insect associations, some of which could evolve into vector relationships, occur today as shown by free-living trypanosomatids in the alimentary tract lumen of various insects. Species of the aquatic flagellateCrithidia can be found inside the gut lumen of mosquito larvae andHerpetomonas spp. occur in the larval habitats, as well as inside the gut lumen of fruit flies (Drosophilidae), scuttle flies (Phoridae), and muscid flies (Muscidae) [48]. Transtadial transmission ofCrithidia fasciculata was experimentally achieved in the mosquito,Culiseta incidens [49]. Not only protozoa but also bacteria are known to be transmitted transtadially from larvae into adultPhlebotomus duboscqi [50]. The trypanosomatid,Leptomonas ctenocephali, is carried through the pupal stage into the adult dog flea,Ctenocephalides canis, and when mealworm larvae were experimentally infected withLeptomonas pyrrhocoris, they carried the flagellates though the pupal and into the adult stage [51].The scenario of insect larvae acquiring free-living flagellates that are eventually carried into the adult stage may be how many insect vectors originally acquired pathogenic microorganisms. When such microbes were first transmitted into vertebrates, they would have had to deal with immune responses, which was probably achieved by entering specific tissues like the liver or blood cells. How many attempts it took before free-living trypanosomatids were established in sand flies and a vector relationship was established will never be known. However, it is obvious that a combination of confluent events resulted in successful host-vector associations.So when did sand flies began to transmitLeishmania? A minimum date of 100 million years can be established from actual fossils in Early Cretaceous Myanmar amber. However, the fossil record of sand flies extends back to 125 million year Early Cretaceous Lebanese amber [52], although some suspect that sand flies existed in the Jurassic [53] or even in the Triassic [54].As pathogens coevolve with their vectors, it is only natural that they would coevolve with their vertebrate hosts. Thus, successful establishment ofLeishmania in mammals may have been predisposed by first infecting reptiles, especially sincePaleoleishmania proterus infected reptiles in the Early Cretaceous [41]. Reptiles were probably the original hosts ofLeishmania [55, 56]. This scenario is supported by molecular data [57] and numerous reptilian trypanosome infections transmitted by sand flies today [58–61].Did several foci of sand fly-Leishmania evolve over time in different geographical areas or was this vector association a single event? Two theories supporting the latter scenario were proposed on the place of origin ofLeishmania. One proposes a Palearctic origin with reptiles as the first vertebrate hosts. The pathogen was later carried across Beringia into the Nearctic and then into the Neotropical region via the Panamanian land bridge [62]. The second theory placed the origin ofLeishmania in the Neotropical region with sloths as the first vertebrate hosts. Infected porcupines and other mammals carried the infections across the Panamanian land bridge into North America and eventually across Beringia into Eurasia [63]. The presence ofPaleoleishmania in Early Cretaceous Myanmar amber sand flies supports the former of these two theories.If leishmaniasis did evolve in the Old World, just when it arrived in the New World is a mystery. However, a Dominican amber fossil provides a minimum date of some 20–30 million years. The evidence was in aLutzomyia sand fly that was vectoringPaleoleishmania neotropicum [64] (Figure 32). In the proboscis of this Dominican amber sand fly were promastigotes (Figure 33), paramastigotes (Figure 34), and a group of amastigotes (Figure 35). Amastigotes are only formed in the vertebrate host and are normally ingested with the blood meal. The flagellates may have been multiplying in the proboscis since inLeishmania chagasi, amastigotes may divide during their transformation into promastigotes [65–67]. The promastigotes and paramastigotes (metacyclic stages) are produced inside the gut of the sand fly and indicate that there were at least two blood meals taken. Whether the origin and evolution of the New World sand flyLeishmania were the same as those of the Old World cannot be determined but they could have evolved independently of one another.Biting midges are also vectors of trypanosomes, especially those infecting birds. Some idea of the antiquity of biting midges transmitting trypanosomes was the discovery of the Early Cretaceous Myanmar amberLeptoconops nosopheris. The body of the fossil biting midge had partially cleared, revealing trypanosomes ofPaleotrypanosoma burmanicus in its midgut (Figure 36). Trypanosomes also occurred in the head, salivary glands, and salivary ducts ofL. nosopheris and some flagellates were in a salivary droplet that collected on the tip of the proboscis (Figure 37) [68]. Extant species ofLeptoconops feed on reptiles, birds, and mammals; however, based on its age and characters resembling those found on extant reptile feeders, the vertebrate host ofPaleotrypanosoma burmanicus was suspected of being a reptile [69, 70]. Molecular techniques indicated that vertebrate parasitism by trypanosomes evolved multiple times in the past [71]. It is possible that biting midges were the original ancestral vectors of theTrypanosoma lineage, sinceLeptoconops clades extend back at least 120 million years [72].Flies are not the only insect vectors of trypanosomes. Kissing bugs (Hemiptera: Reduviidae) are well known for their bloodsucking habits (Figure38) and they transmit the devastating Chagas disease (Trypanosoma cruzi) in the American tropics. Two fecal droplets containing metatrypanosomes ofTrypanosoma antiquus (Figure 39) were adjacent to a fifth instar nymph ofTriatoma dominicana in Dominican amber (Figure 17). The fossil metatrypanosomes resemble those ofT. cruzi. Analysis of mammalian hairs adjacent to the fecal droplets indicates that the vertebrate host for both the triatomine and trypanosomatid probably was a bat. Extant triatomines not only feed on bats but also are the suspected vectors of all New World bat trypanosomes [73–75]. It is possible that some lineages of trypanosomes evolved in bats and that these mammals were the original vertebrate hosts in Central and South America. Additional evidence of a long association between bats and trypanosomes is the absence of disease symptoms in these mammals [73]. Records of Chagas disease in humans date back to 4,000-year-old mummies in the Atacama Desert [76].Figure 17
A Kissing bug,Triatoma dominicana, in Dominican amber with an actinobacteria infection.Figure 18
Filaments of the actinobacteria,Paleorhodococcus dominicanus (arrows) in an anal deposit of the Dominican amber Kissing bug shown in Figure 17. Bar = 7 μm.Figure 19
ABacillus sporangium developing inside the body cavity of the mycetophagous nematode,Oligaphelenchoides atrebora, in Mexican amber.Figure 20
A flea(Rhopalopsyllus sp.) in Dominican amber.Figure 21
Bacterial cells (arrow) attached to the tip of the proboscis of the Dominican amber flea shown in Figure20.Figure 22
Human teeth from a victim of the plague discovered in a 16th century burial pit in Tuscany. Such teeth are the “treasure trove” for archaeologists wishing to obtain DNA from ancient remains (photo courtesy of Debi Poinar).Figure 23
DNA analysis showed that this individual from a burial pit in Tuscany died from cholera around 1850 (photo courtesy of Debi Poinar).Figure 24
The Early Cretaceous Myanmar amber sand fly,Palaeomyia burmitis, with stages of the trypanosomatid,Paleolishmania proterus.Figure 25
Promastigotes of the trypanosomatid,Paleolishmania proterus, developing in the midgut of the Early Cretaceous sand fly shown in Figure 24.Figure 26
Elongate nectomonad promastigotes ofPaleolishmania proterus developing in the midgut of the Early Cretaceous sand fly shown in Figure 24.Figure 27
Short procyclic promastigotes ofPaleolishmania proterus developing in the midgut of the Early Cretaceous sand fly shown in Figure 24.Figure 28
Amastigotes (arrow) ofPaleolishmania proterus acquired from a vertebrate host in the proboscis of the Early Cretaceous sand fly shown in Figure 24.Figure 29
Amastigotes (small dark bodies [arrows]) ofPaleolishmania proterus developing inside vertebrate blood cells in the thoracic lumen of the Early Cretaceous sand fly shown in Figure 24.Figure 30
One of two Early Cretaceous Myanmar amber sand fly larvae that contained flagellates inside the lumen of their alimentary tracts. Similar flagellates were associated with the adjacent club fungus.Figure 31
Flagellates inside the lumen of the alimentary tract of the Early Cretaceous sand fly larva shown in Figure30.Figure 32
Lutzomyia adiketis, the Dominican amber sand fly vector ofPaleoleishmania neotropicum.Figure 33
Four promastigotes ofPaleoleishmania neotropicum in the proboscis of the Dominican amber sand fly shown in Figure 32.Figure 34
Two paramastigotes ofPaleoleishmania neotropicum in the proboscis of the Dominican amber sand fly shown in Figure 32.Figure 35
A cluster of amastigotes (arrow) ofPaleoleishmania neotropicum in the proboscis of the Dominican amber sand fly shown in Figure 32.Figure 36
The Early Cretaceous Myanmar amber biting midge,Leptoconops nosopheris, a vector of the trypanosomatid,Paleotrypanosoma burmanicus.Figure 37
Stages ofPaleotrypanosoma burmanicus in a salivary secretion protruding from the proboscis of the Early Cretaceous biting midge shown in Figure 36.Figure 38
Kissing bugs (Hemiptera: Reduviidae), such as this extantDipetalogaster sp., transmitTrypanosoma cruzi to humans in the American tropics.Figure 39
Metatrypanosomes ofTrypanosoma antiquus in a fecal droplet of the Dominican amberTriatoma dominicana shown in Figure 17.Other types of flagellates have been discovered in triatomines. Epimastigotes of a species ofBlastocrithidia were detected in an anal droplet ofPanstrongylus hispaniolae in Dominican amber (Figures 40 and 41) [77]. The fossil flagellates are similar in size and shape to the epimastigotes ofBlastocrithidia triatomae that were described from the intestine ofTriatoma infestans [78].Blastocrithidia can be pathogenic in triatomes when populations built up in the midgut [79].Figure 40
A Kissing bug,Panstrongylus hispaniolae, in Dominican amber.Figure 41
Epimastigotes (arrows) of aBlastocrithidia sp. protozoan in the anal droplet ofPanstrongylus hispaniolae depicted in Figure 40. Bar = 77 μm.One of the most notorious protozoan diseases of humans is malaria and aside from episodic epidemics of plague and cholera, malaria continues to cause human suffering in many parts of the world today. That may be because malarial organisms are the ultimate pathogens. Few other pathogens have the ability to change their surface coat so readily as modern dayPlasmodium.A separate lineage of ancient malarial organisms is vectored by biting midges that date back 100 million years. These primitive malaria-biting midge associations still survive today in various parts of the world. The earliest evidence of malaria was obtained from a partially cleared female biting midge (Protoculicoides sp.) in Early Cretaceous Myanmar amber [80] (Figure 42). A number of developing oocysts and sporozoites of the malarial parasite,Paleohaemoproteus burmacis, were inside the abdomen of the biting midge (Figure 43). Morphological features of the vector were used to determine that the host was large and cold-blooded [81]. Likely hosts in Early Cretaceous Myanmar were lizards, crocodilians, and dinosaurs [25].Figure 42
The Early Cretaceous Myanmar amber biting midge (Protoculicoides sp.) with its malarial pathogen,Paleohaemoproteus burmacis.Figure 43
An oocyst ofPaleohaemoproteus burmacis developing in the body cavity of the Early Cretaceous biting midge depicted in Figure 42. Sporozoites are developing inside the oocyst.The development ofPaleohaemoproteus burmacis was probably very similar to malarial parasites vectored by biting midges today. During blood feeding, the female vector acquires vertebrate blood cells infected with gametocytes. After their release from the vertebrate blood cells in the stomach of the biting midge, the gametocytes unite and initiate the zygote or sporogonic stage of the life cycle. The zygote differentiates into a motile ookinete, which penetrates the epithelial cells of the insect’s midgut and forms an oocyst. Vertebrate-infective sporozoites develop within the oocysts (Figure 43) and when released, they migrate to the salivary glands. They are introduced into a vertebrate during subsequent blood meals.Paleohaemoproteus burmacis has many characteristics of the extant genusHaemoproteus that are vectored by biting midges to birds and reptiles [82–87]. Today haemoproteid malaria caused byHaemoproteus spp. is commonly transmitted byCulicoides biting midges [82, 88].DiscoveringPlasmodium malaria in the Dominican amber mosquito,Culex malariager, showed that not all lineages of human malaria evolved in the Old World [89, 90] (Figure 44) and were later introduced into the New World. Now we know that at least one lineage ofPlasmodium malaria was being vectored by mosquitoes in the Americas some 20–30 mya [90]. Inside the fossilCulex malariager were various stages (oocysts, sporozoites, ookinete, and microgametocyte) ofPlasmodium dominicana. Two oocysts were entire (Figure 45); however, two others had ruptured and groups of sporozoites were moving into the hemocoel (Figure 46). Some of the released sporozoites had managed to reach the salivary glands before the mosquito became entrapped.Figure 44
The Dominican amber mosquito,Culex malariager, a vector of the malarial pathogen,Plasmodium dominicana.Figure 45
An oocyst ofPlasmodium dominicana in the hemocoel of the Dominican amber mosquito shown in Figure 44.Figure 46
Sporozoites emerging from an oocyst ofPlasmodium dominicana in the Dominican amber mosquito shown in Figure 44.The large, pedunculated oocysts alignedPlasmodium dominicana with the extant bird parasite,Plasmodium juxtanucleare, which is transmitted byCulex spp. [83, 91]. This latter species had been assigned a basal position among avian-infectingPlasmodium species [91], suggesting that the genusPlasmodium could have evolved with birds in the Mesozoic with Cretaceous culicine mosquitoes as vectors [92].ThePlasmodium dominicana lineage could easily have been transferred from birds to mammals by culicine mosquitoes since such lateral transfers of malaria from birds to mammals occur naturally and have been demonstrated experimentally [83, 93, 94]. Once established in mammals, anopheline mosquitoes, such as the Dominican amberAnopheles dominicanus, could have transmittedPlasmodium from simians to humans [95]. Another possible pathway is that anopheline mosquitoes could have acquired infections directly from birds and passed them on to mammals, including simians. So the evolution of human malaria in the New World could have begun in birds and then shifted to mammals and finally to humans. Human malaria in the Old World may have evolved by a completely different pathway but probably also existed millions of years ago. The discovery ofPlasmodium falciparum malaria in Egyptian mummies using antigen immunoassay techniques establishes the disease in humans at a minimum date of 3200 B.C. [96, 97].Fossils provide no clues of howPlasmodium evolved from free-living ancestors; however, I believe that the original host in a vector association is the one in which the sexual stages of the parasite occur, which in the case of malaria would be the mosquito. A possible scenario is that the free-living aquatic ancestors ofPlasmodium were first acquired by mosquito larvae. The parasites were then carried through the pupa stage into the next generation of adults and this single host cycle was established. Eventually, some sporozoites entered the salivary glands and were transferred into vertebrates. Their establishment in the liver and blood cells served to protect them from host defense responses.An opposing but less likely evolutionary scenario is thatPlasmodium was originally a blood parasite of vertebrates that reproduced by merozoite division. Mosquitoes ingested vertebrate blood cells infected with merozoites and over time the released merozoites began to diversify and eventually a sexual stage was established in mosquitoes. This resulted in the establishment of a sporogonic stage with the production of vertebrate-infective sporozoites.There are numerous types of malaria in a wide range of vertebrates; however, very few have left a fossil record. A rare malarial pathogen was discovered in a Dominican amber streblid bat fly,Enischnomyia stegosoma (Figure 47) [98, 99]. Two ovoid oocysts ofVetufebrus ovatus attached to the midgut wall of the bat fly contained short, stubby sporozoites (Figure 48) and similar sporozoites in the salivary glands and ducts indicated that representatives of Neotropical Hippoboscoidea were vectoring malaria to bats in the Cenozoic.Figure 47
A Dominican amber bat fly,Enischnomyia stegosoma, vector of the malarial pathogen,Vetufebrus ovatus.Figure 48
Two oocysts with developing sporozoites ofVetufebrus ovatus in the Dominican amber bat fly shown in Figure 47.Protozoans cause a number of alimentary tract infections in all vertebrates; however, the origins of such infections are difficult to determine. They probably date back to the early evolution of vertebrates since the passage of microbes into the lumen of the alimentary tract would have occurred from eating infested food. The establishment of some of these microbes, at least for short periods, would have occurred multiple times. Reptiles were one of the early hosts of intestinal protozoan diseases [100] as shown by the discovery of mature cysts ofEntamoebites antiquus in a dinosaur coprolite from the Early Cretaceous Bernissart Iguanodon shaft in Belgium [101] (Figure 49). The fossil cysts resembled species in the widespread modern genusEntamoeba that infect amphibians, reptiles, birds, and mammals. The extantE. invadens destroys leucocytes and causes disease in reptiles when it invades the liver and causes hepatitis and nephritis. Common symptoms of gastrointestinal infections in lizards and snakes are vomiting and severe diarrhea [102–104].Entamoebites antiquus provides a minimum date when pathogenic protozoa were established in the gut of vertebrates.Figure 49
An Early Cretaceous cyst ofEntamoebites antiquus recovered from a dinosaur coprolite at the Bernissart Iguanodon shaft in Belgium.There is widespread subfossil evidence of intestinal pathogens based on samples from coprolites, latrines, and cesspits. Using an immunoassay test for determining the presence of adhesion antigens, the pathogenicEntamoeba histolytica could be detected in human samples from Brazil 6000 to 5000 BP [105]. A review of human intestinal parasites from around the world based on the microscopic examination of treated samples has been recently summarized [106].Gregarines are a very complex group of protozoans that evolved as monoxenous parasites of invertebrates [107, 108]. Their origins are enigmatic since there are no closely related free-living forms; however, since they parasitize a wide variety of marine and terrestrial invertebrates, gregarines are certainly an ancient group. Gregarines parasitize a wide range of insects today, including a number of cockroaches, which may have been one of the original host groups [109]. Discovering trophozoites and oocysts ofPrimigregarina burmanica from an Early Cretaceous Myanmar amber cockroach showed that the basic developmental stages of extant gregarines were firmly established some 100 million years ago [110, 111] (Figures 50 and 51).Figure 50
An Early Cretaceous Myanmar amber cockroach adjacent to a gametocyst (arrow) of the gregarine protozoan parasite,Primigregarina burmanica.Figure 51
Detail of the gametocyst of the gregarine parasite,Primigregarina burmanica that was released from the gut of the Early Cretaceous cockroach shown in Figure 50. Arrow shows a spore-filled sporoduct.


### 2.4. Fungi

Fungal pathogens are ubiquitous and parasitize a wide range of animals and plants; however, actual evidence of parasitism is rare. Most fossil examples of animal-parasitic fungi occur on arthropods in amber and in many cases, the fungi can usually be placed in extant families and genera. The oldest evidence of animal parasitism by fungi is the insect pathogen,Paleoophiocordyceps coccophagus, parasitizing a scale insect in Early Cretaceous Myanmar amber [112]. Two synnemata covered with a palisade of elongated cylindrical phialides bearing conidia at their tips had emerged from the insect (Figures 52 and 53). Evidence indicated that the hypocrealean ancestor ofP. coccophagus evolved in the Early Jurassic (193 mya) as a plant parasite that later shifted to animal hosts [112].Figure 52
Two extended synnemata of the Early Cretaceous Myanmar amber fungus,Paleoophiocordyceps coccophagus, emerging from the top of a scale insect (Albicoccidae).Figure 53
A layer of conidiogenous cells and conidia on the surface of one of the synnemata of the Early Cretaceous fungus shown in Figure52.Additional examples of fungal pathogens of insects in amber include a member of the Entomophthorales infecting a winged termite in Dominican amber [113]. The termite was covered with a white mat of mycelium that had produced budding conidia, characteristic of extant members of the genusEntomophthora (Figure 54). Another Entomophthorales infection was found on a fungus gnat in Dominican amber. The fungus formed a felt-like hymenium over the back of the fly (Figure 55), which is very similar to the growth pattern ofEntomophthora infections on extant fungus gnats [6].Figure 54
A Dominican amber termite bearing white colonies of an Entomophthorales fungus.Figure 55
An entomophthorales infection of a fungus gnat in Dominican amber. The fungus formed a felt-like hymenium (arrow) on the fly.A Dominican amberAzteca ant heavily infected with a species of the extant genusBeauveria (Figure 56) establishes a minimum date for the appearance of that clade. Conidiophores and conidia of the fungus covered portions of the ant’s body [114] (Figure 57).Figure 56
Dominican amberAzteca worker ant infected with aBeauveria fungus.Figure 57
Conidiophores and conidia of theBeauveria fungus growing on the Dominican amberAzteca ant depicted in Figure 56.The Trichomycetes comprise a group of cosmopolitan, microscopic fungi that parasitize a wide range of arthropods. A member of this order found on aCulex mosquito in Dominican amber establishes a minimum date for this group. Several types of thalli had emerged from between the junctions of the abdominal tergites on the back of the mosquito [6] (Figure 58). Some were slender, elongate forms that were producing trichospores while others were short and swollen (Figure 59).Figure 58
Group of thalli (arrow) of a trichomycete fungus emerging between the abdominal tergites of aCulex mosquito in Dominican amber.Figure 59
Slender, narrow thalli, and a short, swollen thallus of the trichomycete fungus parasitizing the Dominican amberCulex mosquito depicted in Figure 58.Some additional examples of fungal pathogens in Dominican amber are a synnemata produced by aHirsutella-like fungus on aTroctopsocopsis bark louse (Figure 60) and an undescribed fungus emerging from a winged ant (Figure 61).Figure 60
Synnematium of aHirsutella-like fungus parasitizing a bark louse in Dominican amber.Figure 61
A fungus sporulating on the abdomen of a winged ant in Dominican amber.Evidence of pathogenic fungi attacking nematodes was found among a population of the mycetophagous nematode,Oligaphelenchoides atrebora, in Mexican amber [30]. The inner tissues of infected nematodes had been replaced by fungal mycelia (Figure 62), some of which were sporulating (Figure 63) [115].Figure 62
Fungal hyphae inside the body of the nematode,Oligaphelenchoides atrebora, in Mexican amber.Figure 63
Developing spore of a fungal pathogen inside the body of the nematode,Oligaphelenchoides atrebora, in Mexican amber.Ancient fungal pathogens of vertebrates are quite rare; however, a case of coccidioidomycosis was reported in two Holocene bison dated at 8500 BP [116]. Spheres and endospores of the pathogen were found in the mandibles and adjoining bones of the bison. The subfossil stages were similar to those of extant species ofCoccidioides, a genus that infects a wide range of vertebrates, including humans. This discovery sets a locality and minimum time for the occurrence of vertebrate parasitic Ascomycetes. It is interesting that humans are considered one of the vectors of this disease, dispersing it as they migrated between North and South America [116]. Skeletal pathology in a Native American dated at 1,000 to 1,400 A.D. was attributed to coccidioidmycosis when a microscopic examination revealed both spherules and endospores ofCoccidioides immitis [117].There are numerous reports of fossil fungi on plants dating from the Palaeozoic onwards. However, few of these show the actual pathogen in association with their plant host. Many of these reports have been summarized previously [3, 118]. The fossil record of fungi begins in the Ordovician, some 460 mya [119], although molecular estimates of fungal phylogeny place the origin of terrestrial fungi (Ascomycota, Basidiomycota, and Glomales) in the Proterozoic [120].The origins of plant pathogenic fungi may have begun with simple hosts such as algae and fungi. Fungal parasitism of Early Devonian algae (400 mya) probably represents one of the first host parasitic associations with fungi [121]. The same is true for mycoparasitism where fungi parasitize other fungi. Records of such associations have been found in Devonian deposits [122] and in Early Cretaceous amber, where an agaric mushroom was parasitized by a fungus that in turn was parasitized by a second hyperparasitic fungus [123] (Figure 64). Fungi were probably the first parasites of vascular plants. An early record of pathogenic fungi associated with a vascular plant is the 400 mya DevonianPaleopyrenomycites devonicus that was developing in the cortex of aerial stems and rhizomes of the primitiveAsteroxylon (Figure 65) [124]. Rust and smut fungi are also probably very primitive although thus far, only a Tertiary record of a smut fungus infecting an Eocene angiosperm in British Columbia is known [125].Figure 64
Parasitic and hyperparasitic fungi (arrows) developing on an agaric mushroom in Early Cretaceous Myanmar amber.Figure 65
Perithecia of the Devonian ascomycetous fungus,Paleopyrenomycites devonicus, developing in the vascular plant,Asteroxylon, at the Early Devonian Rhynie chert site (Photo courtesy of T. N. Taylor).Spot fungi are an interesting heterogeneous group that evolved various methods of either penetrating surfaces of leaves and petals or entering through natural openings (stomata). Fossil records of leaf spot fungi have been reviewed and date back to the Late Carboniferous [3, 126]. Some fossil spot fungi occurred on a petal in Mexican amber and on a dicot and monocot leaf in Dominican amber [127]. Several pycnidia ofLeptothyrites dominicanus were present on the monocot leaf (Figure 66) and one pustule contained conidiophores and conidia (Figure 67).Figure 66
Five spherical, dark pycnidia of the coelomycete,Leptothyrites dominicanus, parasitizing a monocot leaf in Dominican amber.Figure 67
Conidia (arrows) inside one of the pycnidia ofLeptothyrites dominicanus shown in Figure 66.Most groups of parasitic fungi probably evolved from saprophytic species developing on dead arthropods and plants. Physiological adaptations allowing them to attack dying or weakened animals and plants could have been further modified for entering and obtaining nourishment from healthy organisms.


## 2.1. Viruses

Viruses are probably quite ancient and may have existed some 4 billion years ago with bacteria as hosts. The nearly 2000 species of viruses infecting plants and animals today are probably a conservative estimate of their diversity [5]. All estimates on the evolutionary history of viruses are based on molecular analyses since fossils of such small, fragile structures are almost nonexistent. Evidence of some viruses has been found in subfossil remains based on DNA analysis [4] but the only fossil evidence of infectious diseases caused by viruses associated with their hosts is insect polyhedrosis, polynida, and iridoviruses. The polyhedra of nuclear polyhedrosis viruses (Baculoviridae) and cytoplasmic polyhedrosis viruses (Reoviridae) are just large enough to be detected with the light microscope. Based on the size of the polyhedra, the particular organs in which they occur, and the type of insect hosts, it was possible to identify a cytoplasmic polyhedrosis virus infecting a biting midge (Figures 1, 2, and 3) and a nuclear polyhedrosis virus infection in a sand fly (Figures 4 and 5), both in Early Cretaceous amber from Myanmar [6]. These records provide a minimum date for the origin of the Baculoviridae and Reoviridae and suggest that some human arboviruses could have evolved first as pathogens of bloodsucking flies.Figure 1
Early Cretaceous Myanmar amber biting midge (Protoculicoidessp.; Diptera: Ceratopogonidae) that contains polyhedra of a cytoplasmic polyhedrosis virus in its midgut cells.Figure 2
Group of polyhedra (arrow) of a cytoplasmic polyhedrosis virus in the midgut cells of the Early Cretaceous biting midge in Figure1.Figure 3
Detail of polyhedra of a cytoplasmic polyhedrosis virus in the midgut cells of an Early Cretaceous biting midge in Figure1.Figure 4
An Early Cretaceous Myanmar amber sand fly (Diptera: Psychodidae) infected with a nuclear polyhidrosis virus.Figure 5
Detail of polyhedra of a nuclear polyhedrosis virus in the Early Cretaceous sand fly in Figure4.A specialized group of viruses that are carried by parasitic wasps of the families Braconidae and Ichneumonidae are the Polydnaviridae. Genomes of polydnaviruses are integrated into the wasp genome and virus replication occurs in the reproductive system of the female wasp. During oviposition, the virus is introduced along with the wasp eggs into the body of potential hosts. The virus replicates in the host and affects the host immune system by preventing it from encapsulating and destroying the wasp eggs. The transfer of these virus particles from wasp to host was first observed in 1975 with the ichneumonid,Casinaria infesta, and its plutellid moth host [7] and in 1976 with the braconidPhanerotoma flavitestacea and its pyralid moth host,Paramyelois transitella [8]. Since that time, the field has expanded and the polydnaviruses are now included in the new discipline of Paleovirology, which infers viral evolution from host genome sequence data [9, 10].Presently the family Polydnaviridae contains two genera. The genusBracovirus occurs in the calyx cells and fluid of the female wasps of the family Braconidae and the genusIchnovirus is associated with female wasps of the family Ichneumonidae. Because of the close association between the virus and members of these two families, one can assume that these viruses existed in ancient wasps of these lineages, such as the fossil braconid and ichneumonid wasps depicted in Dominican amber (Figures 6 and 7) and even in the primitive braconid wasp in 100 million year old amber from Myanmar (Figure 8).Figure 6
A female braconid wasp (Hymenoptera: Braconidae) in Dominican amber that is probably carrying the genome of aBracovirus in its reproductive system.Figure 7
A female ichneumonid wasp (Hymenoptera: Ichneumonidae) in Dominican amber that is probably carrying the genome of anIchnovirus in its reproductive system.Figure 8
A primitive braconid wasp (Hymenoptera: Braconidae) in Early Cretaceous Myanmar amber that could be carrying the genome of a polydnavirus in its reproductive system.A group of invertebrate pathogenic viruses whose presence often can be determined by color changes of the host are the iridoviruses. These double-stranded DNA viruses are icosahedral and hexagonal in cross section. The isopod iridescent virus infects terrestrial pillbugs and sowbugs and in the later stages of infection turns normal brown hosts to various shades of violet, blue, and purple [11]. In fact, an isopod thus colored can be assured of having an iridoviral infection. The bluish color is the result of paracrystalline arrays of viral particles (virions) filling the epidermal and other cells of the isopod that is eventually killed by the infection. An isopod in Cretaceous amber from Myanmar that has iridescent blue areas on its body is assumed to be infected with an ancient strain of the isopod iridescent virus (Figure 9). This is another example of identifying fossil viruses using indirect, but reliable evidence derived from our present knowledge.Figure 9
A bluish tinted terrestrial isopod in Early Cretaceous Myanmar amber that could be infected with the isopod iridescent virus.


## 2.2. Bacteria

Bacteria are ubiquitous and there are numerous reports of fossil associations between putative bacteria and various animals and plants [2, 3]. However, most of these reports refer to signs indicating a possible bacterial infection and actual bacteria are absent. One example is the dark areas on the body of the Burgess Shale onychophoran,Aysheaia pedunculata, that are thought to represent bacterial infections [12]. Other examples of indirect presence of bacteria are fossil fish with luminescent organs similar to those on their present day descendants that are operated by luminescent bacteria [3] or insects producing flatus in fossilized resin [13].Filamentous cells occurring in the marine environment some 3.6 billion years ago represent some of the earliest known bacteria [14]. When terrestrial bacterial first appeared and formed associations with animals and plants is unknown since fossil bacteria are quite difficult to detect. Their small size and delicate structure (except for spores) are not prone to fossilization. Evidence of the early evolution of some specific lineages of pathogenic bacteria has appeared in fossilized resin.The oldest known definite fossil disease-producing bacteria are associated with an entomopathogenic nematode of theHeterorhabditis clade in Early Cretaceous amber [15]. Several infective stages of the insect pathogenic nematode,Proheterorhabditis burmanicus, were preserved as they emerged from their beetle host in Myanmar amber (Figure 10). Extant members of theHeterorhabditis clade carry and release spores of the luminescent bacterium,Photorhabdus luminescens, into the body cavity of insects. The bacteria multiply and kill the insects within 24 hours. The nematodes feed on the combined bacteria host breakdown tissues and the newly formed infective stage juveniles carry these bacterial cells into a new host. Bacterial cells in the fossil beetle (Figure 11) adjacent to the nematode resembled those of extantPhotorhabdus spp. and are presumed to have been introduced into the beetle byProheterorhabditis burmanicus.Figure 10
An Early Cretaceous Myanmar amber entomopathogenic nematode,Proheterorhabditis burmanicus (arrow), adjacent to its beetle host.Figure 11
Bacterial cells ofPhotorhabdus inside the Early Cretaceous Myanmar amber beetle shown in Figure 10.Ticks (Figure12) carry a number of bacterial pathogens. One of special interest is the spirochete,Borrelia burgdorferi, the causal agent of Lyme disease. An ancient spirochete with characters ofBorrelia was found in the midgut and hemocoel of a larval hard tick belonging to the genusAmblyomma (Arachnida: Ixodidae) in Dominican amber (Figures 13 and 14) [16]. The presence of spirochetes in the hemocoel indicated that the tick was a competent vector since spirochetes do not enter the hemocoel of incompetent vectors [17]. Vertebrate hosts of extantAmblyomma ticks are reptiles, birds, and mammals and remains of all three of these groups occur in Dominican amber [18]. This discovery shows how ancient some lineages of tick-vectored pathogens can be. It is likely that genetic material of this fossil spirochete contributed to the genome of extantBorrelia lineages, including those species that infect humans. The earliest record of Lyme disease was recently reported from the Tyrolean Iceman, a 5,300-year-old mummy discovered in 1991 in the Italian Alps. During a genomic analysis of the Iceman, sequences corresponding to ~60% of the genome ofBorrelia burgdorferi indicated that the Iceman suffered from Lyme borreliosis [19].Figure 12
LarvalAmbylomma hard tick in Dominican amber.Figure 13
Spirochetes (arrows) within the chelicerial sheath and surrounding the hypostome of a larvalAmbylomma tick in Dominican amber similar to the one shown in Figure 12. Scale bar = 16 μm.Figure 14
Spirochetes in the pharynx (arrow) of a larvalAmbylomma tick in Dominican amber similar to the one shown in Figure 12. Scale bar = 13 μm.The fossil record of hard ticks extends back to the Early Cretaceous [20, 21]. This sets a time period when these arachnids could have acquired spirochetes, followed by a period of coevolution that established the vertebrate cycle and increased vector competency. The origin of Borreliosis is a mystery. It is likely thatBorrelia began as a free-living spirochete that could have made contact with tick eggs deposited in the soil. However, so little is known about interactions ofBorrelia sp. with present day ticks that this scenario is difficult to prove. Yet ifBorrelia began as a tick parasite, it could have been passed from mother to offspring by vertical or transovarial transmission (TOT). The spirochetes may have initially served as symbiotes, since TOT is normally how symbiotic microorganisms are passed in hosts. A later evolutionary step would be entering the salivary glands where they could be transferred to vertebrates.An example of this evolutionary scenario is the establishment and transfer of rickettsial organisms in arthropods [22]. Rickettsias occur in many locations inside the alimentary tract and body tissues of a wide range of animals [23, 24]. Some symbiotic species, likeWolbachia, are beneficial and assist in the digestion of blood meals, while other rickettsias are pathogenic to both arthropod vectors and their vertebrate hosts. Finding evidence of rickettsias in the fossil record is difficult but a recent study showed various stages of a rickettsial organism in the Early Cretaceous Myanmar amber hard tick,Cornupalpatum burmanicum (Figure 15). The rickettsial cells occurred in the remains of gastric ceca (Figure 16). Cocci, diplococci, and short, rod shaped cells borne singly or in short chains were dividing by binary fission. The rods ranged from 0.4 to 0.8 μm in width by 1.3–1.7 μm in length. In some areas, ovoid cells surrounded by an apparent mucoprotein, slime layer (haloes), were present. Such “halo” cells are known to occur in rickettsial populations and are considered to represent infective stages of the disease process [22, 23]. Since the cells were developing in the gastric cecal cells, the fossil could be related to any one of the many species in the families of Rickettsiaceae or Anaplasmataceae [17, 23]. Ticks are known to vector a range of rickettsial pathogens, including Q-fever, tick typhus, and Boutonneuse fever, some of which cause diseases in reptiles [24], which makes dinosaurs possible hosts [25].Figure 15
Early Cretaceous Myanmar amber larval hard tick,Cornupalpatum burmanicum that contains rickettsial organisms.Figure 16
Short rods and cocci of a rickettsial organism in a gastric ceca of the Early Cretaceous larval hard tick shown in Figure15.Insects relying solely on blood meals throughout their entire development often harbor symbiotic microorganisms [26]. While they can be considered parasites since they develop on a combination of the blood meal and digestive secretions from the insect, they may supply essential elements to their host, since blood is considered a nutritionally inadequate diet deficient in B vitamins. Nocardiform bacteria such asRhodococcus assimilate proteins and carbohydrates in the lumen of the alimentary tract of blood-sucking triatomine bugs [27]. Species of this genus occur in soil and water as well as in the lumen of insect alimentary tracts. Past evidence of this clade was discovered when cells ofPaleorhodococcus dominicanus were found in a fecal droplet adjacent to the triatome bug,Triatoma dominicana, in Dominican amber [28, 29]. The fossil species consisted of numerous coccoid elements with associated mycelial fragments [29] (Figure 18). WhileP. dominicanus could have been beneficial if it was assisting in the breakdown of blood components, it could also have been infectious since some species ofRhodococcus are pathogens, such asR. equi that infects horses.ABacillus sporangium inside of the body of the mycetophagous nematode,Oligaphelenchoides atrebora in Mexican amber (Figure 19), could indicate the presence of an infectious disease in the entire nematode population [30].Bacillus sp. are known to infect extant nematodes [31] as well as insects and humans. However, fossil evidence of such infections is rare, even though the ancient presence ofB. sphaericus,B. subtilis, andB. circulans was found in amber, sedimentary rocks, and salt deposits [32].One of the most studied bacterial diseases is the plague, which has killed countless humans throughout history. Plague is caused by a small, pleomorphic bacterium known asYersinia pestis that is transmitted by fleas. This pathogenic strain probably evolved from one of the free-living species ofYersinia that occur in soil, fresh water, foods, fish, and various animals [33]. The chances of finding fossil cells ofY. pestis are extremely slim. Some bacterial cells attached to the proboscis of a fossilRhopalopsyllus flea in Dominican amber (Figure 20) have the morphological features ofYersinia (Figure 21) although it is not possible to confirm their identity. However, the extantRhopalopsyllus cavicola is one of 125 species of fleas capable of transmittingY. pestis, in this case to cavy rodents in the American tropics [34, 35].Sophisticated molecular and immunological methods can now be used not only to identify pathogens, but to compare various strains that appeared throughout history. One of the best sources of ancient DNA for such studies is roots and crowns of teeth from the victims of various epidemics (Figure22). These were used to characterize strains ofY. pestis that were responsible for the Black Death that struck Europe in the 14th to 17th centuries [36].Past cholera epidemics also resulted in high mortality rates (Figure23). Using a preserved intestinal portion from a victim of the 1849 cholera outbreak in Philadelphia, molecular techniques were used to reconstruct the genome of the causative organism,Vibrio cholerae [37].Teeth are also a good source of decay-causing bacteria. One report characterizes filamentous actinomycetes in a tooth from an Eocene rabbit in Montana [38], thus documenting the earliest record of tooth infection in placental mammals.


## 2.3. Protozoa

Protozoa, with some 120,000 described species, are an ancient group with a fossil record extending back to the Archean Eon, some 2 billion years ago. Protozoa probably originated in the sea and soon afterwards formed symbiotic associations with terrestrial life forms. Eventually, they entered the lumen of the alimentary tract of invertebrates and developed on partially digested food. Some of these became permanent residents in the guts of various insects such as termites, where the protozoa served a mutualistic role of digesting cellulose. Other protozoans invaded the internal organs of their hosts and became parasites. During their evolution, some became closely associated with blood-sucking arthropods, which transported them into vertebrates where they became established as true parasites with the potential of causing disease. A few invaded the tissues of plants but the present diversity of plant parasitic protozoa is quite low.Today a number of arthropods vector parasitic protozoa and early lineages of some of these became entrapped in amber, thus providing clues to the evolution of human diseases. Two basic types of development evolved between arthropod vectors and their protozoan parasites. The first is where the protozoans only develop in the gut lumen of the vector. Examples are members of the trypanosome lineage that includeLeishmania vectored by sand flies. The second is where the protozoans penetrate the gut wall and undergo development in the body cavity of the vector. Examples are the various malarial pathogens vectored by several arthropods, especially mosquitoes.The first fossil protozoan pathogen to be discovered was one of the former types that were in the gut lumen of a phlebotomine sand fly entrapped in Early Cretaceous Myanmar amber (Figure24) [39]. In the midgut lumen of the sand fly,Palaeomyia burmitis, were various stages of the leishmanial parasite,Paleoleishmania proterus [40] (Figure 25). Aside from normal promastigotes, elongate nectomonad promastigotes (Figure 26) and short procyclic promastigotes (Figure 27) were also present. Vertebrate infective amastigotes occurred in the proboscis of the fossil sand fly (Figure 28) and a view into the thoracic lumen of the fossil sand fly revealed nucleated vertebrate blood cells. Some of these cells contained developing amastigotes (Figure 29), obviously acquired during a recent blood meal. Based on morphological characteristics of the blood cells, the vertebrate host was considered to be a reptile andPaleoleishmania proterus was thought to be an ancestor of the genusSauroleishmania that infects lizards and snakes today [41].The life cycle ofPaleoleishmania proterus was probably similar to that of leishmanial parasites vectored today by sand flies [42]. Amastigotes were acquired from the blood of a vertebrate during feeding. These developed into short ovoid procyclic forms that then formed elongate nectomonad stages in the lumen of the alimentary tract of the sand fly. Metacyclic (paramastigote) forms were transferred back into a vertebrate during blood-feeding.How, when, and where protozoan vector associations originated are difficult to determine, especially when every association could have evolved slightly differently. Also past evidence of vector associations is extremely rare since most protozoans are too small and fragile to be recognized [43]. However, in some instances, clues to possible mechanisms exist. In the case ofLeishmania and sand flies, the discovery of trypanosomes associated with sand fly larvae originating from the same Early Cretaceous Myanmar amber deposits provided a possible scenario for how these flagellates became established in sand flies. The two fossil sand fly larvae were adjacent to and probably feeding on a fruiting body of a coral mushroom,Palaeoclavaria burmitis (Figure 30) [44–46]. Associated with some expelled fecal material from both larvae were numerous flagellates. Similar flagellates surrounded the bases of the fungal fruiting bodies and were associated with debris attached to the cuticle of both sand flies. After polishing away a portion of the alimentary tract of one of the amber sand flies, numerous flagellates similar to those at the bases of the fungal fruiting bodies were also discovered in the lumen of its midgut (Figure 31).Based on these findings, the following scenario for the acquisition ofLeishmania by sand flies is presented. Free-living trypanosomes developing in the same microhabitat as the sand fly larvae and club fungi were ingested by the larvae. They may have served as food at first, but eventually the trypanosomes began to multiply inside the gut of the sand fly and were carried transtadially from the larva through the pupa and into the adult. Once established in the gut of the adult sand fly, the flagellates were eventually transferred to vertebrates during blood feeding and a vector association was established. Molecular studies showing that insects were the first hosts of trypanosomatids [47] support the above scenario on the origin of parasiticLeishmania through larval acquisition of free-living trypanosomatids.Flagellate-insect associations, some of which could evolve into vector relationships, occur today as shown by free-living trypanosomatids in the alimentary tract lumen of various insects. Species of the aquatic flagellateCrithidia can be found inside the gut lumen of mosquito larvae andHerpetomonas spp. occur in the larval habitats, as well as inside the gut lumen of fruit flies (Drosophilidae), scuttle flies (Phoridae), and muscid flies (Muscidae) [48]. Transtadial transmission ofCrithidia fasciculata was experimentally achieved in the mosquito,Culiseta incidens [49]. Not only protozoa but also bacteria are known to be transmitted transtadially from larvae into adultPhlebotomus duboscqi [50]. The trypanosomatid,Leptomonas ctenocephali, is carried through the pupal stage into the adult dog flea,Ctenocephalides canis, and when mealworm larvae were experimentally infected withLeptomonas pyrrhocoris, they carried the flagellates though the pupal and into the adult stage [51].The scenario of insect larvae acquiring free-living flagellates that are eventually carried into the adult stage may be how many insect vectors originally acquired pathogenic microorganisms. When such microbes were first transmitted into vertebrates, they would have had to deal with immune responses, which was probably achieved by entering specific tissues like the liver or blood cells. How many attempts it took before free-living trypanosomatids were established in sand flies and a vector relationship was established will never be known. However, it is obvious that a combination of confluent events resulted in successful host-vector associations.So when did sand flies began to transmitLeishmania? A minimum date of 100 million years can be established from actual fossils in Early Cretaceous Myanmar amber. However, the fossil record of sand flies extends back to 125 million year Early Cretaceous Lebanese amber [52], although some suspect that sand flies existed in the Jurassic [53] or even in the Triassic [54].As pathogens coevolve with their vectors, it is only natural that they would coevolve with their vertebrate hosts. Thus, successful establishment ofLeishmania in mammals may have been predisposed by first infecting reptiles, especially sincePaleoleishmania proterus infected reptiles in the Early Cretaceous [41]. Reptiles were probably the original hosts ofLeishmania [55, 56]. This scenario is supported by molecular data [57] and numerous reptilian trypanosome infections transmitted by sand flies today [58–61].Did several foci of sand fly-Leishmania evolve over time in different geographical areas or was this vector association a single event? Two theories supporting the latter scenario were proposed on the place of origin ofLeishmania. One proposes a Palearctic origin with reptiles as the first vertebrate hosts. The pathogen was later carried across Beringia into the Nearctic and then into the Neotropical region via the Panamanian land bridge [62]. The second theory placed the origin ofLeishmania in the Neotropical region with sloths as the first vertebrate hosts. Infected porcupines and other mammals carried the infections across the Panamanian land bridge into North America and eventually across Beringia into Eurasia [63]. The presence ofPaleoleishmania in Early Cretaceous Myanmar amber sand flies supports the former of these two theories.If leishmaniasis did evolve in the Old World, just when it arrived in the New World is a mystery. However, a Dominican amber fossil provides a minimum date of some 20–30 million years. The evidence was in aLutzomyia sand fly that was vectoringPaleoleishmania neotropicum [64] (Figure 32). In the proboscis of this Dominican amber sand fly were promastigotes (Figure 33), paramastigotes (Figure 34), and a group of amastigotes (Figure 35). Amastigotes are only formed in the vertebrate host and are normally ingested with the blood meal. The flagellates may have been multiplying in the proboscis since inLeishmania chagasi, amastigotes may divide during their transformation into promastigotes [65–67]. The promastigotes and paramastigotes (metacyclic stages) are produced inside the gut of the sand fly and indicate that there were at least two blood meals taken. Whether the origin and evolution of the New World sand flyLeishmania were the same as those of the Old World cannot be determined but they could have evolved independently of one another.Biting midges are also vectors of trypanosomes, especially those infecting birds. Some idea of the antiquity of biting midges transmitting trypanosomes was the discovery of the Early Cretaceous Myanmar amberLeptoconops nosopheris. The body of the fossil biting midge had partially cleared, revealing trypanosomes ofPaleotrypanosoma burmanicus in its midgut (Figure 36). Trypanosomes also occurred in the head, salivary glands, and salivary ducts ofL. nosopheris and some flagellates were in a salivary droplet that collected on the tip of the proboscis (Figure 37) [68]. Extant species ofLeptoconops feed on reptiles, birds, and mammals; however, based on its age and characters resembling those found on extant reptile feeders, the vertebrate host ofPaleotrypanosoma burmanicus was suspected of being a reptile [69, 70]. Molecular techniques indicated that vertebrate parasitism by trypanosomes evolved multiple times in the past [71]. It is possible that biting midges were the original ancestral vectors of theTrypanosoma lineage, sinceLeptoconops clades extend back at least 120 million years [72].Flies are not the only insect vectors of trypanosomes. Kissing bugs (Hemiptera: Reduviidae) are well known for their bloodsucking habits (Figure38) and they transmit the devastating Chagas disease (Trypanosoma cruzi) in the American tropics. Two fecal droplets containing metatrypanosomes ofTrypanosoma antiquus (Figure 39) were adjacent to a fifth instar nymph ofTriatoma dominicana in Dominican amber (Figure 17). The fossil metatrypanosomes resemble those ofT. cruzi. Analysis of mammalian hairs adjacent to the fecal droplets indicates that the vertebrate host for both the triatomine and trypanosomatid probably was a bat. Extant triatomines not only feed on bats but also are the suspected vectors of all New World bat trypanosomes [73–75]. It is possible that some lineages of trypanosomes evolved in bats and that these mammals were the original vertebrate hosts in Central and South America. Additional evidence of a long association between bats and trypanosomes is the absence of disease symptoms in these mammals [73]. Records of Chagas disease in humans date back to 4,000-year-old mummies in the Atacama Desert [76].Figure 17
A Kissing bug,Triatoma dominicana, in Dominican amber with an actinobacteria infection.Figure 18
Filaments of the actinobacteria,Paleorhodococcus dominicanus (arrows) in an anal deposit of the Dominican amber Kissing bug shown in Figure 17. Bar = 7 μm.Figure 19
ABacillus sporangium developing inside the body cavity of the mycetophagous nematode,Oligaphelenchoides atrebora, in Mexican amber.Figure 20
A flea(Rhopalopsyllus sp.) in Dominican amber.Figure 21
Bacterial cells (arrow) attached to the tip of the proboscis of the Dominican amber flea shown in Figure20.Figure 22
Human teeth from a victim of the plague discovered in a 16th century burial pit in Tuscany. Such teeth are the “treasure trove” for archaeologists wishing to obtain DNA from ancient remains (photo courtesy of Debi Poinar).Figure 23
DNA analysis showed that this individual from a burial pit in Tuscany died from cholera around 1850 (photo courtesy of Debi Poinar).Figure 24
The Early Cretaceous Myanmar amber sand fly,Palaeomyia burmitis, with stages of the trypanosomatid,Paleolishmania proterus.Figure 25
Promastigotes of the trypanosomatid,Paleolishmania proterus, developing in the midgut of the Early Cretaceous sand fly shown in Figure 24.Figure 26
Elongate nectomonad promastigotes ofPaleolishmania proterus developing in the midgut of the Early Cretaceous sand fly shown in Figure 24.Figure 27
Short procyclic promastigotes ofPaleolishmania proterus developing in the midgut of the Early Cretaceous sand fly shown in Figure 24.Figure 28
Amastigotes (arrow) ofPaleolishmania proterus acquired from a vertebrate host in the proboscis of the Early Cretaceous sand fly shown in Figure 24.Figure 29
Amastigotes (small dark bodies [arrows]) ofPaleolishmania proterus developing inside vertebrate blood cells in the thoracic lumen of the Early Cretaceous sand fly shown in Figure 24.Figure 30
One of two Early Cretaceous Myanmar amber sand fly larvae that contained flagellates inside the lumen of their alimentary tracts. Similar flagellates were associated with the adjacent club fungus.Figure 31
Flagellates inside the lumen of the alimentary tract of the Early Cretaceous sand fly larva shown in Figure30.Figure 32
Lutzomyia adiketis, the Dominican amber sand fly vector ofPaleoleishmania neotropicum.Figure 33
Four promastigotes ofPaleoleishmania neotropicum in the proboscis of the Dominican amber sand fly shown in Figure 32.Figure 34
Two paramastigotes ofPaleoleishmania neotropicum in the proboscis of the Dominican amber sand fly shown in Figure 32.Figure 35
A cluster of amastigotes (arrow) ofPaleoleishmania neotropicum in the proboscis of the Dominican amber sand fly shown in Figure 32.Figure 36
The Early Cretaceous Myanmar amber biting midge,Leptoconops nosopheris, a vector of the trypanosomatid,Paleotrypanosoma burmanicus.Figure 37
Stages ofPaleotrypanosoma burmanicus in a salivary secretion protruding from the proboscis of the Early Cretaceous biting midge shown in Figure 36.Figure 38
Kissing bugs (Hemiptera: Reduviidae), such as this extantDipetalogaster sp., transmitTrypanosoma cruzi to humans in the American tropics.Figure 39
Metatrypanosomes ofTrypanosoma antiquus in a fecal droplet of the Dominican amberTriatoma dominicana shown in Figure 17.Other types of flagellates have been discovered in triatomines. Epimastigotes of a species ofBlastocrithidia were detected in an anal droplet ofPanstrongylus hispaniolae in Dominican amber (Figures 40 and 41) [77]. The fossil flagellates are similar in size and shape to the epimastigotes ofBlastocrithidia triatomae that were described from the intestine ofTriatoma infestans [78].Blastocrithidia can be pathogenic in triatomes when populations built up in the midgut [79].Figure 40
A Kissing bug,Panstrongylus hispaniolae, in Dominican amber.Figure 41
Epimastigotes (arrows) of aBlastocrithidia sp. protozoan in the anal droplet ofPanstrongylus hispaniolae depicted in Figure 40. Bar = 77 μm.One of the most notorious protozoan diseases of humans is malaria and aside from episodic epidemics of plague and cholera, malaria continues to cause human suffering in many parts of the world today. That may be because malarial organisms are the ultimate pathogens. Few other pathogens have the ability to change their surface coat so readily as modern dayPlasmodium.A separate lineage of ancient malarial organisms is vectored by biting midges that date back 100 million years. These primitive malaria-biting midge associations still survive today in various parts of the world. The earliest evidence of malaria was obtained from a partially cleared female biting midge (Protoculicoides sp.) in Early Cretaceous Myanmar amber [80] (Figure 42). A number of developing oocysts and sporozoites of the malarial parasite,Paleohaemoproteus burmacis, were inside the abdomen of the biting midge (Figure 43). Morphological features of the vector were used to determine that the host was large and cold-blooded [81]. Likely hosts in Early Cretaceous Myanmar were lizards, crocodilians, and dinosaurs [25].Figure 42
The Early Cretaceous Myanmar amber biting midge (Protoculicoides sp.) with its malarial pathogen,Paleohaemoproteus burmacis.Figure 43
An oocyst ofPaleohaemoproteus burmacis developing in the body cavity of the Early Cretaceous biting midge depicted in Figure 42. Sporozoites are developing inside the oocyst.The development ofPaleohaemoproteus burmacis was probably very similar to malarial parasites vectored by biting midges today. During blood feeding, the female vector acquires vertebrate blood cells infected with gametocytes. After their release from the vertebrate blood cells in the stomach of the biting midge, the gametocytes unite and initiate the zygote or sporogonic stage of the life cycle. The zygote differentiates into a motile ookinete, which penetrates the epithelial cells of the insect’s midgut and forms an oocyst. Vertebrate-infective sporozoites develop within the oocysts (Figure 43) and when released, they migrate to the salivary glands. They are introduced into a vertebrate during subsequent blood meals.Paleohaemoproteus burmacis has many characteristics of the extant genusHaemoproteus that are vectored by biting midges to birds and reptiles [82–87]. Today haemoproteid malaria caused byHaemoproteus spp. is commonly transmitted byCulicoides biting midges [82, 88].DiscoveringPlasmodium malaria in the Dominican amber mosquito,Culex malariager, showed that not all lineages of human malaria evolved in the Old World [89, 90] (Figure 44) and were later introduced into the New World. Now we know that at least one lineage ofPlasmodium malaria was being vectored by mosquitoes in the Americas some 20–30 mya [90]. Inside the fossilCulex malariager were various stages (oocysts, sporozoites, ookinete, and microgametocyte) ofPlasmodium dominicana. Two oocysts were entire (Figure 45); however, two others had ruptured and groups of sporozoites were moving into the hemocoel (Figure 46). Some of the released sporozoites had managed to reach the salivary glands before the mosquito became entrapped.Figure 44
The Dominican amber mosquito,Culex malariager, a vector of the malarial pathogen,Plasmodium dominicana.Figure 45
An oocyst ofPlasmodium dominicana in the hemocoel of the Dominican amber mosquito shown in Figure 44.Figure 46
Sporozoites emerging from an oocyst ofPlasmodium dominicana in the Dominican amber mosquito shown in Figure 44.The large, pedunculated oocysts alignedPlasmodium dominicana with the extant bird parasite,Plasmodium juxtanucleare, which is transmitted byCulex spp. [83, 91]. This latter species had been assigned a basal position among avian-infectingPlasmodium species [91], suggesting that the genusPlasmodium could have evolved with birds in the Mesozoic with Cretaceous culicine mosquitoes as vectors [92].ThePlasmodium dominicana lineage could easily have been transferred from birds to mammals by culicine mosquitoes since such lateral transfers of malaria from birds to mammals occur naturally and have been demonstrated experimentally [83, 93, 94]. Once established in mammals, anopheline mosquitoes, such as the Dominican amberAnopheles dominicanus, could have transmittedPlasmodium from simians to humans [95]. Another possible pathway is that anopheline mosquitoes could have acquired infections directly from birds and passed them on to mammals, including simians. So the evolution of human malaria in the New World could have begun in birds and then shifted to mammals and finally to humans. Human malaria in the Old World may have evolved by a completely different pathway but probably also existed millions of years ago. The discovery ofPlasmodium falciparum malaria in Egyptian mummies using antigen immunoassay techniques establishes the disease in humans at a minimum date of 3200 B.C. [96, 97].Fossils provide no clues of howPlasmodium evolved from free-living ancestors; however, I believe that the original host in a vector association is the one in which the sexual stages of the parasite occur, which in the case of malaria would be the mosquito. A possible scenario is that the free-living aquatic ancestors ofPlasmodium were first acquired by mosquito larvae. The parasites were then carried through the pupa stage into the next generation of adults and this single host cycle was established. Eventually, some sporozoites entered the salivary glands and were transferred into vertebrates. Their establishment in the liver and blood cells served to protect them from host defense responses.An opposing but less likely evolutionary scenario is thatPlasmodium was originally a blood parasite of vertebrates that reproduced by merozoite division. Mosquitoes ingested vertebrate blood cells infected with merozoites and over time the released merozoites began to diversify and eventually a sexual stage was established in mosquitoes. This resulted in the establishment of a sporogonic stage with the production of vertebrate-infective sporozoites.There are numerous types of malaria in a wide range of vertebrates; however, very few have left a fossil record. A rare malarial pathogen was discovered in a Dominican amber streblid bat fly,Enischnomyia stegosoma (Figure 47) [98, 99]. Two ovoid oocysts ofVetufebrus ovatus attached to the midgut wall of the bat fly contained short, stubby sporozoites (Figure 48) and similar sporozoites in the salivary glands and ducts indicated that representatives of Neotropical Hippoboscoidea were vectoring malaria to bats in the Cenozoic.Figure 47
A Dominican amber bat fly,Enischnomyia stegosoma, vector of the malarial pathogen,Vetufebrus ovatus.Figure 48
Two oocysts with developing sporozoites ofVetufebrus ovatus in the Dominican amber bat fly shown in Figure 47.Protozoans cause a number of alimentary tract infections in all vertebrates; however, the origins of such infections are difficult to determine. They probably date back to the early evolution of vertebrates since the passage of microbes into the lumen of the alimentary tract would have occurred from eating infested food. The establishment of some of these microbes, at least for short periods, would have occurred multiple times. Reptiles were one of the early hosts of intestinal protozoan diseases [100] as shown by the discovery of mature cysts ofEntamoebites antiquus in a dinosaur coprolite from the Early Cretaceous Bernissart Iguanodon shaft in Belgium [101] (Figure 49). The fossil cysts resembled species in the widespread modern genusEntamoeba that infect amphibians, reptiles, birds, and mammals. The extantE. invadens destroys leucocytes and causes disease in reptiles when it invades the liver and causes hepatitis and nephritis. Common symptoms of gastrointestinal infections in lizards and snakes are vomiting and severe diarrhea [102–104].Entamoebites antiquus provides a minimum date when pathogenic protozoa were established in the gut of vertebrates.Figure 49
An Early Cretaceous cyst ofEntamoebites antiquus recovered from a dinosaur coprolite at the Bernissart Iguanodon shaft in Belgium.There is widespread subfossil evidence of intestinal pathogens based on samples from coprolites, latrines, and cesspits. Using an immunoassay test for determining the presence of adhesion antigens, the pathogenicEntamoeba histolytica could be detected in human samples from Brazil 6000 to 5000 BP [105]. A review of human intestinal parasites from around the world based on the microscopic examination of treated samples has been recently summarized [106].Gregarines are a very complex group of protozoans that evolved as monoxenous parasites of invertebrates [107, 108]. Their origins are enigmatic since there are no closely related free-living forms; however, since they parasitize a wide variety of marine and terrestrial invertebrates, gregarines are certainly an ancient group. Gregarines parasitize a wide range of insects today, including a number of cockroaches, which may have been one of the original host groups [109]. Discovering trophozoites and oocysts ofPrimigregarina burmanica from an Early Cretaceous Myanmar amber cockroach showed that the basic developmental stages of extant gregarines were firmly established some 100 million years ago [110, 111] (Figures 50 and 51).Figure 50
An Early Cretaceous Myanmar amber cockroach adjacent to a gametocyst (arrow) of the gregarine protozoan parasite,Primigregarina burmanica.Figure 51
Detail of the gametocyst of the gregarine parasite,Primigregarina burmanica that was released from the gut of the Early Cretaceous cockroach shown in Figure 50. Arrow shows a spore-filled sporoduct.


## 2.4. Fungi

Fungal pathogens are ubiquitous and parasitize a wide range of animals and plants; however, actual evidence of parasitism is rare. Most fossil examples of animal-parasitic fungi occur on arthropods in amber and in many cases, the fungi can usually be placed in extant families and genera. The oldest evidence of animal parasitism by fungi is the insect pathogen,Paleoophiocordyceps coccophagus, parasitizing a scale insect in Early Cretaceous Myanmar amber [112]. Two synnemata covered with a palisade of elongated cylindrical phialides bearing conidia at their tips had emerged from the insect (Figures 52 and 53). Evidence indicated that the hypocrealean ancestor ofP. coccophagus evolved in the Early Jurassic (193 mya) as a plant parasite that later shifted to animal hosts [112].Figure 52
Two extended synnemata of the Early Cretaceous Myanmar amber fungus,Paleoophiocordyceps coccophagus, emerging from the top of a scale insect (Albicoccidae).Figure 53
A layer of conidiogenous cells and conidia on the surface of one of the synnemata of the Early Cretaceous fungus shown in Figure52.Additional examples of fungal pathogens of insects in amber include a member of the Entomophthorales infecting a winged termite in Dominican amber [113]. The termite was covered with a white mat of mycelium that had produced budding conidia, characteristic of extant members of the genusEntomophthora (Figure 54). Another Entomophthorales infection was found on a fungus gnat in Dominican amber. The fungus formed a felt-like hymenium over the back of the fly (Figure 55), which is very similar to the growth pattern ofEntomophthora infections on extant fungus gnats [6].Figure 54
A Dominican amber termite bearing white colonies of an Entomophthorales fungus.Figure 55
An entomophthorales infection of a fungus gnat in Dominican amber. The fungus formed a felt-like hymenium (arrow) on the fly.A Dominican amberAzteca ant heavily infected with a species of the extant genusBeauveria (Figure 56) establishes a minimum date for the appearance of that clade. Conidiophores and conidia of the fungus covered portions of the ant’s body [114] (Figure 57).Figure 56
Dominican amberAzteca worker ant infected with aBeauveria fungus.Figure 57
Conidiophores and conidia of theBeauveria fungus growing on the Dominican amberAzteca ant depicted in Figure 56.The Trichomycetes comprise a group of cosmopolitan, microscopic fungi that parasitize a wide range of arthropods. A member of this order found on aCulex mosquito in Dominican amber establishes a minimum date for this group. Several types of thalli had emerged from between the junctions of the abdominal tergites on the back of the mosquito [6] (Figure 58). Some were slender, elongate forms that were producing trichospores while others were short and swollen (Figure 59).Figure 58
Group of thalli (arrow) of a trichomycete fungus emerging between the abdominal tergites of aCulex mosquito in Dominican amber.Figure 59
Slender, narrow thalli, and a short, swollen thallus of the trichomycete fungus parasitizing the Dominican amberCulex mosquito depicted in Figure 58.Some additional examples of fungal pathogens in Dominican amber are a synnemata produced by aHirsutella-like fungus on aTroctopsocopsis bark louse (Figure 60) and an undescribed fungus emerging from a winged ant (Figure 61).Figure 60
Synnematium of aHirsutella-like fungus parasitizing a bark louse in Dominican amber.Figure 61
A fungus sporulating on the abdomen of a winged ant in Dominican amber.Evidence of pathogenic fungi attacking nematodes was found among a population of the mycetophagous nematode,Oligaphelenchoides atrebora, in Mexican amber [30]. The inner tissues of infected nematodes had been replaced by fungal mycelia (Figure 62), some of which were sporulating (Figure 63) [115].Figure 62
Fungal hyphae inside the body of the nematode,Oligaphelenchoides atrebora, in Mexican amber.Figure 63
Developing spore of a fungal pathogen inside the body of the nematode,Oligaphelenchoides atrebora, in Mexican amber.Ancient fungal pathogens of vertebrates are quite rare; however, a case of coccidioidomycosis was reported in two Holocene bison dated at 8500 BP [116]. Spheres and endospores of the pathogen were found in the mandibles and adjoining bones of the bison. The subfossil stages were similar to those of extant species ofCoccidioides, a genus that infects a wide range of vertebrates, including humans. This discovery sets a locality and minimum time for the occurrence of vertebrate parasitic Ascomycetes. It is interesting that humans are considered one of the vectors of this disease, dispersing it as they migrated between North and South America [116]. Skeletal pathology in a Native American dated at 1,000 to 1,400 A.D. was attributed to coccidioidmycosis when a microscopic examination revealed both spherules and endospores ofCoccidioides immitis [117].There are numerous reports of fossil fungi on plants dating from the Palaeozoic onwards. However, few of these show the actual pathogen in association with their plant host. Many of these reports have been summarized previously [3, 118]. The fossil record of fungi begins in the Ordovician, some 460 mya [119], although molecular estimates of fungal phylogeny place the origin of terrestrial fungi (Ascomycota, Basidiomycota, and Glomales) in the Proterozoic [120].The origins of plant pathogenic fungi may have begun with simple hosts such as algae and fungi. Fungal parasitism of Early Devonian algae (400 mya) probably represents one of the first host parasitic associations with fungi [121]. The same is true for mycoparasitism where fungi parasitize other fungi. Records of such associations have been found in Devonian deposits [122] and in Early Cretaceous amber, where an agaric mushroom was parasitized by a fungus that in turn was parasitized by a second hyperparasitic fungus [123] (Figure 64). Fungi were probably the first parasites of vascular plants. An early record of pathogenic fungi associated with a vascular plant is the 400 mya DevonianPaleopyrenomycites devonicus that was developing in the cortex of aerial stems and rhizomes of the primitiveAsteroxylon (Figure 65) [124]. Rust and smut fungi are also probably very primitive although thus far, only a Tertiary record of a smut fungus infecting an Eocene angiosperm in British Columbia is known [125].Figure 64
Parasitic and hyperparasitic fungi (arrows) developing on an agaric mushroom in Early Cretaceous Myanmar amber.Figure 65
Perithecia of the Devonian ascomycetous fungus,Paleopyrenomycites devonicus, developing in the vascular plant,Asteroxylon, at the Early Devonian Rhynie chert site (Photo courtesy of T. N. Taylor).Spot fungi are an interesting heterogeneous group that evolved various methods of either penetrating surfaces of leaves and petals or entering through natural openings (stomata). Fossil records of leaf spot fungi have been reviewed and date back to the Late Carboniferous [3, 126]. Some fossil spot fungi occurred on a petal in Mexican amber and on a dicot and monocot leaf in Dominican amber [127]. Several pycnidia ofLeptothyrites dominicanus were present on the monocot leaf (Figure 66) and one pustule contained conidiophores and conidia (Figure 67).Figure 66
Five spherical, dark pycnidia of the coelomycete,Leptothyrites dominicanus, parasitizing a monocot leaf in Dominican amber.Figure 67
Conidia (arrows) inside one of the pycnidia ofLeptothyrites dominicanus shown in Figure 66.Most groups of parasitic fungi probably evolved from saprophytic species developing on dead arthropods and plants. Physiological adaptations allowing them to attack dying or weakened animals and plants could have been further modified for entering and obtaining nourishment from healthy organisms.


## 3. Multicellular Endoparasites

### 3.1. Nematomorpha

Hairworms are an ancient group of arthropod parasites that live inside invertebrates and usually kill the latter when their development is completed. A specimen in Early Cretaceous Myanmar amber provides a minimum date of 100 my for their existence [128]. Evidence that they parasitized cockroaches in the Tertiary is shown by the hairworm,Paleochordodes protus, emerging from a blattid in Dominican amber [129] (Figure 68). Since this group of parasites is not closely related to any existing free-living or parasite group, it is difficult to determine their origins and evolutionary development. Hairworms appear to be an ancient group that arose independently and successfully maintained themselves to the present, in spite of ongoing environmental disruptions that are destroying their host’s habitats.Figure 68
The hairworm,Paleochordodes protus (arrow), emerging from a cockroach in Dominican amber.


### 3.2. Nematodes

Nematodes are another primitive group that has no close affinity to any extant invertebrate group and the lineage from which they evolved is probably extinct. Thus attempting to trace their origins is highly speculative, even with molecular techniques. It is possible that nematodes originated in the sea and first parasitized marine invertebrates and vertebrates in the Cambrian. The most complete fossil record of any particular nematode group is of the Mermithidae, a group well represented in amber and easily detected as they are leaving or have just emerged from their arthropod hosts.Mermithid nematodes superficially resemble hairworms in size and development. They parasitize a wide range of terrestrial invertebrates and probably evolved from free-living dorylaimids or mononchs. A recent study reported free-living dorylaimids that had entered the body cavity of some terrestrial insects, thus showing a possible first step in an evolutionary scenario indicating that mermithids evolved from free-living Dorylaimida [130].Many mermithids parasitize small flying insects, which is why the most complete record of their evolutionary history is in amber. Evidence in Early Cretaceous Lebanese amber suggests that chironomid midges or their ancestors were the original insect hosts of mermithids (Figure69) [131]. However based on the earliest fossil record of all their host groups, an estimated date for the origin of mermithids would be in the Permian [15]. Evidence that mermithids had diversified by the mid-Tertiary is provided by a wide range of parasitized hosts, including ants (Figure 70), hemipterans (Figure 71), trichopterans (Figure 72), and arachnids in Baltic and Dominican amber [15, 132].Figure 69
Parasitism of an Early Cretaceous Lebanese amber chironomid midge by the mermithid nematode,Cretacimermis libani. The nematode is coiled in the abdomen of its host.Figure 70
Parasitism of a Baltic amber ant by the mermithid nematode,Heydenius formicarius.Figure 71
The mermithid nematode,Heydenius brownii, emerging from a planthopper in Baltic amber.Figure 72
Two mermithid nematodes,Heydenius trichorosus, adjacent to their caddis fly host (Triaenodes balticus) in Baltic amber.Several clades of the nematode group Tylenchomorpha also parasitize insects and among these the acugutturids, iotonchids, allantonematids, and sphaerulariids have a fossil record. Those with the greatest selection of hosts are the Sphaerularidae and Allantonematidae. In contrast to mermithids that only mature but do not multiply in their hosts, the small size of these tylenchomorphs allows them to undergo one or even two generations inside their host. Often the juveniles can be observed within the body cavity or emerging from various parts of insects in amber. Fossil records of sphaerulariids and allantonematids include species parasitizing beetles, ants, and flies in Tertiary amber [15]. The earliest fossil record of sphaerulariids isTripius balticus that was parasitizing a sciarid fly in Baltic amber. However, based on the earliest fossil record of their host group, an estimated date for the origin of sphaerulariids would be in the Jurassic [15].The Dominican amberPalaeoparasitylenchus dominicanus establishes a location and minimum time when drosophilid flies were victimized by allantonematids. However, based on the earliest fossil record of their host group, an estimated date for the origin of allantonematids would be in the Permian [15]. Using the same criterion, an estimated date for the origin of the iotonchids would be in the Cretaceous [15]. However, the oldest fossil record of an iotonchid isPaleoiotonchium dominicanum in a parasitized fungus gnat in Dominican amber. The iotonchids are especially interesting since they provide evidence that insect parasitic tylenchomorphs evolved from mycetophagous clades. While the life cycle of the fossilPaleoiotonchium dominicanum remains unknown, it is probably similar to that of its recent descendant,Iotonchium californicum, that was studied in California [133]. This latter species has two alternating life cycles, a fungal feeding cycle with the nematodes developing on mushrooms and an insect-parasitic cycle where development only occurs inside the body of a fungus gnat that feeds on the mushroom.Iotonchium is a nematode that has the ability to utilize both fungi and insects as hosts. It appears to be at a stage in its evolution that is ready to sever the ties with the more primitive fungal portion of its life cycle.A representative of the acugutturid moth parasites,Setonema protera, also occurs in Dominican amber; however, based on the earliest fossil record of its host group, an estimated date for the origin of this group would be in the Carboniferous [15].Representatives of the nematode suborder Rhabditina are mainly microbotrophic free-living nematodes; however, some lineages developed amazing specialized patterns of parasitism. One of these lineages is a member of the family Diplogasteridae that evolved the ability to parasitize fig wasps. Diplogasterids are common inhabitants of soil and decaying organic matter and their associations with arthropods are mainly phoretic in nature. Yet some of these nematodes became parasites of fig wasps. Fig wasps are very tiny hymenopterans that develop in fig synconia within the flower clusters. Their entire life cycle takes place in the fig with female flight patterns related to entering and exiting the fig flowers as they pollinate the new flowers and deposit their eggs. TheParasitodiplogaster lineage of diplogastrids is carried by the adult fig wasps to the fig flowers and then parasitizes the developing fig wasp larvae. Originally, the nematodes were probably strictly phoretic and developed on pollen and bacteria in the fig flowers, using the wasps only for transport. At some point, juvenile nematodes emerging from eggs deposited in the fig flowers entered the fig wasp larvae and adults. That this association has continued for 20–30 million years is evident from a parasitized fig wasp surrounded by released stages of the diplogastrid,Syconema dominicana, in Dominican amber [15]. However, based on the earliest fossil record of their host group, an estimated date for the origin of this nematode lineage would be in the Eocene [15].Another unique mode of parasitism originated independently in two genera of rhabditine nematodes,Steinernema andHeterorhabditis. These two nematode genera evolved from separate lineages of free-living, bacterial-feeding, soil nematodes but developed a very similar pattern of insect parasitism. Each lineage became associated with its own specific insect-pathogenic bacteria that were originally acquired from the immediate environment. These bacteria are retained in the gut of the infective stage juveniles and released when the latter penetrate into a living insect. The released bacteria kill the insect within 24 hours and the nematodes feed on the tissues of the corpse that have been modified by the specific bacteria. These so-called entomopathogenic nematodes are sold commercially as biological control agents. The discovery of infective juveniles of one of these clades in Early Cretaceous Myanmar amber shows that entomopathogenic nematodes were present some 100 mya (Figure 10) [15]. The fossil infective stageProheterorhabditis burmanicus was adjacent to a beetle that contained bacteria in its tissues similar to those carried by extant heterorhabditids (Figure 11) [134]. Based on the earliest fossil record of their host group, an estimated date for the origin of both the heterorhabditids and steinernematids would be in the Permian [15].Helminthiasis refers to diseases of humans and animals caused by parasitic worms such as nematodes, trematodes (flukes), and cestodes (tapeworms). Past remains of vertebrate parasitic nematodes are mainly found in coprolites and corpses. Two common animal parasites are ascarids or stomach worms and oxyurids or pinworms. The oxyurids evolved from microbial soil inhabitants and it was not that large a step to shift from feeding on microbes in the environment to those in the gut lumen of invertebrates and vertebrates. The earliest record of an oxyurid parasitizing invertebrates isPaleothelastoma tipulae from an Early Cretaceous crane fly in Myanmar amber [15]. Based on the earliest fossil record of their host group, an estimated date for the origin of the invertebrate parasitic oxyurids would be in the Silurian [15].Oxyurids found in subfossil deposits show the wide range of vertebrate hosts of these parasites.Syphacia was found in the remains of a 10,000–12,000-year-old ground squirrel in Eastern Russia [135], eggs ofParapharyngodon sceleratus were recovered from 9,000-year-old coprolites of an iguanid in Brazil [136], and juveniles ofAgamofilaria oxyura were recovered from a 10,000 ± 180-year-old Shasta Ground Sloth bolus [137]. Based on the earliest fossil record of their host group, an estimated date for the origin of the vertebrate parasitic oxyurids would be in the Carboniferous [15].It is difficult to determine when humans were first infected by the oxyurid pinworm,Enterobius vermicularis. In spite of the 10,000-year-old record from North America and molecular traces ofE. vermicularis in 27 pre-Columbian coprolites ranging from 1,100 to 6,110 years in North and South America [138, 139], the first human infections were probably in the Old World, even though such records from archaeological sites are rare [106]. The oldest Old World records are from a 2100-year-old Chinese mummy [140] and surprisingly, the only report of pinworms in Egyptian mummies was from a site in Roman-occupied Egypt that dates from 30 to 395 AD [141]. However, the discovery ofE. vermicularis in a chimpanzee [142] provides an avenue for early human infection in the Old World and peoples migrating across the Bering Strait could have brought this nematode to the New World [143].Ascarids or stomach worms are probably the most common nematode parasites of vertebrates. The complicated life cycle of many ascarids, including the giant roundworm that infects humans, makes determining their early evolution difficult. Why it is necessary for the immature worms to traverse the wall of the intestine, migrate through the tissues of the host, and then return to small intestine to continue their development is perplexing. The oldest fossil evidence of this group is Early Cretaceous eggs ofAscarites spp. from a dinosaur coprolite in Belgium [101]. However, based on the earliest fossil record of their host group, an estimated date for the origin of ascarids would be in the Permian [15]. Ascarids radiated in numerous lines of vertebrates and invertebrates and have been recovered from more archaeological sites than any other human parasite. A selection of Pleistocene and Holocene sites containing the human ascarid,Ascaris lumbricoides, extending back 30,000 years [144] is presented in Table 1 and shows the geographical range of this parasite over various time periods. It has been suggested that humans acquiredA. lumbricoides from pigs, since they harbour the closely related species,A. suum. However,A. lumbricoides has been recovered from Old World chimpanzees, gorillas, and bonobos as well as from New World howler monkeys [145–147] and the original infections in humans were probably acquired from apes and simians.Table 1
Records of the human stomach worm,Ascaris lumbricoides, from global archaeological sites.
Country
Site
Date
Reference
France
Clairvaux, Jura
3600 BC
[149]
Chalain, Jura
2700–2440 BC
[149]
Bobigny
2nd century AD
[175]
Paris
14th-15th century AD
[176]
Marly-le-Roi, Yveline
17th-18th century AD
[177]
Germany
Drobintz girl
600 BC
[178]
Bremerhaven
100 BC–500 AD
[179]
Lübeck and other cities
15th century AD
[180]
England
Somerset
4100–2600 BP
[181]
Winchester
Roman period
[182]
Lindow Man
2nd century AD
[183]
York
2nd-3rd century AD
[184]
York
9th-10th century AD
[185]
Winchester
1000 years
[186]
Winchester
1100 AD
[187]
Southampton
13th-14th century AD
[188]
York
14th–16th century AD
[189]
Worcester
15th century
[190]
N. America
Kentucky Salts Cave
1125–290 BC
[191]
Tennessee Big Bone Cave
2177
±
145 BP
[192]
Arizona Elden Pueblo
1070–1250 AD
[193]
Adak Island, Alaska
840
±
40 BP
[194]
Williamsburg, Virginia
1720 AD
[195]
Africa
Kruger Cave, Rustenburg
10,000–7,000 BP
[196]
Middle East
Egypt PUM mummy
200 BC
[197]
Acre, Israel
13 century AD
[198]
S. America
Huarmey Valley, Peru
2277
BC
±
181
[199]
Gentio Cave, Brazil
3490
±
120 BP
[106]
Brazil sites
8800–7000 BP
[200]
New Zealand
Wellington
150–200 years
[201]Hookworms are another clade of nematode intestinal parasites recovered from archaeological finds and reports of these parasites have been recently summarized [106]. The two species attacking humans,Ancylostoma duodenale andNecator americanus, are usually lumped together since the stages normally recovered (eggs and first stage juveniles) are so similar that it is impossible to distinguish them by morphological methods. New World records date back to 7230 BP [148], while Old World records are slightly younger (3600 BP) [149]. Hookworms probably evolved from free-living microbotrophic soil nematodes. Based on the earliest fossil record of their host group, an estimated date for the origin of hookworms would be in the Palaeocene [15]. Again, there is a good possibility that humans originally acquired infections from apes and simians sinceN. americanus has been found in chimpanzees [150] andA. duodenale infects Bonoboas [151] and Howler monkeys [146].Another intestinal parasite that has been traced in archaeological remains is the human whipworm,Trichuris trichiura. Most findings are from the Old World, with the oldest record of 10,000–7,000 years BP coming from Africa [152], although eggs of this genus from rodent coprolites date back to 30,000 [153]. Reports of these have been recently summarized [106]. Based on the earliest fossil record of their host group, an estimated date for the origin of whipworms would be in the Carboniferous [15]. Whipworms infect a range of vertebrates, including dogs, cats, mice, pigs, and sheep. The presence of the human whipworm in Old World simians and primates [145, 147, 154–157] provides an avenue for human infections.While parasites in the lumen of the alimentary track of vertebrates can be discovered in mummies and coprolites, the remains of insect vectored nematodes are usually not available in archaeological deposits unless molecular and immunological methods are used. Filarial nematodes adjacent to their insect vectors in Dominican amber establish the presence of filariids in the Cenozoic along with their blackfly and mosquito insect vectors [15]. Based on the earliest fossil record of their host group, an estimated date for the origin of filariids would be in the Carboniferous [15].Fossil evidence of phytoparasitic nematodes is quite rare. The earliest record of plant parasitism by nematodes is the 400 mya Devonian parasite,Palaeonema phyticum, that was developing in the stomatal chambers of the Early Devonian (396 mya) land plant,Aglaophyton major. This species was an epistrate feeder that mechanically disrupted cortical cells and ingested the contents [158].Palaeonema probably evolved from free-living microbotrophic clades that entered the natural opening ofAglaophyton to escape predators and explore a new food source. Lineages with this type of behavior are rare today since most plant parasites have evolved a stylet that is used to penetrate plant tissue and withdraw cell sap. Such stylet bearing nematodes are quite rare as fossils of higher plants and thus far are only represented by a population ofOligaphelenchoides maximis inside plant tissue in Dominican amber [15]. Their early lineages were probably free-living fungal feeders.


### 3.3. Trematodes

Trematodes or flukes can also be found in ancient human remains and reports of liver flukes,Fasciola hepatica, and schistosomes,Schistosoma spp., in archaeological sites have been recently summarized [106]. It is interesting thatF. hepatica has only been recovered in Old World coprolites, in spite of its global occurrence today in cattle and sheep. Subfossils of both genera date back some 5,000 years [159, 160]. It has been suggested that humans acquired schistosomes from ruminant and rodent definitive hosts sometime in the Miocene [161]. This is quite possible since the oldest fossil evidence of this group is an egg of the trematode,Digenites proterus, recovered from a dinosaur coprolite in the Early Cretaceous Bernissart Iguanodon shaft in Belgium [101].


### 3.4. Cestodes

Cestodes or tapeworms are another parasite group that occurs in coprolites. The oldest fossil records of this group are 270 million-year-old tapeworm eggs in a shark coprolite [162], which suggests that terrestrial tapeworms might have originated in a marine habitat. Archaeological finds usually reveal the three tapeworm species,Taeina saginata,T. asiatica, andT. solium that infect humans through cattle and pigs. There is evidence that these and probably other tapeworms were originally acquired when humans devoured wild bovids and swine before these animals were domesticated [163]. Early records ofTaenia eggs in human mummies date back to 3200 BP [164].


## 3.1. Nematomorpha

Hairworms are an ancient group of arthropod parasites that live inside invertebrates and usually kill the latter when their development is completed. A specimen in Early Cretaceous Myanmar amber provides a minimum date of 100 my for their existence [128]. Evidence that they parasitized cockroaches in the Tertiary is shown by the hairworm,Paleochordodes protus, emerging from a blattid in Dominican amber [129] (Figure 68). Since this group of parasites is not closely related to any existing free-living or parasite group, it is difficult to determine their origins and evolutionary development. Hairworms appear to be an ancient group that arose independently and successfully maintained themselves to the present, in spite of ongoing environmental disruptions that are destroying their host’s habitats.Figure 68
The hairworm,Paleochordodes protus (arrow), emerging from a cockroach in Dominican amber.


## 3.2. Nematodes

Nematodes are another primitive group that has no close affinity to any extant invertebrate group and the lineage from which they evolved is probably extinct. Thus attempting to trace their origins is highly speculative, even with molecular techniques. It is possible that nematodes originated in the sea and first parasitized marine invertebrates and vertebrates in the Cambrian. The most complete fossil record of any particular nematode group is of the Mermithidae, a group well represented in amber and easily detected as they are leaving or have just emerged from their arthropod hosts.Mermithid nematodes superficially resemble hairworms in size and development. They parasitize a wide range of terrestrial invertebrates and probably evolved from free-living dorylaimids or mononchs. A recent study reported free-living dorylaimids that had entered the body cavity of some terrestrial insects, thus showing a possible first step in an evolutionary scenario indicating that mermithids evolved from free-living Dorylaimida [130].Many mermithids parasitize small flying insects, which is why the most complete record of their evolutionary history is in amber. Evidence in Early Cretaceous Lebanese amber suggests that chironomid midges or their ancestors were the original insect hosts of mermithids (Figure69) [131]. However based on the earliest fossil record of all their host groups, an estimated date for the origin of mermithids would be in the Permian [15]. Evidence that mermithids had diversified by the mid-Tertiary is provided by a wide range of parasitized hosts, including ants (Figure 70), hemipterans (Figure 71), trichopterans (Figure 72), and arachnids in Baltic and Dominican amber [15, 132].Figure 69
Parasitism of an Early Cretaceous Lebanese amber chironomid midge by the mermithid nematode,Cretacimermis libani. The nematode is coiled in the abdomen of its host.Figure 70
Parasitism of a Baltic amber ant by the mermithid nematode,Heydenius formicarius.Figure 71
The mermithid nematode,Heydenius brownii, emerging from a planthopper in Baltic amber.Figure 72
Two mermithid nematodes,Heydenius trichorosus, adjacent to their caddis fly host (Triaenodes balticus) in Baltic amber.Several clades of the nematode group Tylenchomorpha also parasitize insects and among these the acugutturids, iotonchids, allantonematids, and sphaerulariids have a fossil record. Those with the greatest selection of hosts are the Sphaerularidae and Allantonematidae. In contrast to mermithids that only mature but do not multiply in their hosts, the small size of these tylenchomorphs allows them to undergo one or even two generations inside their host. Often the juveniles can be observed within the body cavity or emerging from various parts of insects in amber. Fossil records of sphaerulariids and allantonematids include species parasitizing beetles, ants, and flies in Tertiary amber [15]. The earliest fossil record of sphaerulariids isTripius balticus that was parasitizing a sciarid fly in Baltic amber. However, based on the earliest fossil record of their host group, an estimated date for the origin of sphaerulariids would be in the Jurassic [15].The Dominican amberPalaeoparasitylenchus dominicanus establishes a location and minimum time when drosophilid flies were victimized by allantonematids. However, based on the earliest fossil record of their host group, an estimated date for the origin of allantonematids would be in the Permian [15]. Using the same criterion, an estimated date for the origin of the iotonchids would be in the Cretaceous [15]. However, the oldest fossil record of an iotonchid isPaleoiotonchium dominicanum in a parasitized fungus gnat in Dominican amber. The iotonchids are especially interesting since they provide evidence that insect parasitic tylenchomorphs evolved from mycetophagous clades. While the life cycle of the fossilPaleoiotonchium dominicanum remains unknown, it is probably similar to that of its recent descendant,Iotonchium californicum, that was studied in California [133]. This latter species has two alternating life cycles, a fungal feeding cycle with the nematodes developing on mushrooms and an insect-parasitic cycle where development only occurs inside the body of a fungus gnat that feeds on the mushroom.Iotonchium is a nematode that has the ability to utilize both fungi and insects as hosts. It appears to be at a stage in its evolution that is ready to sever the ties with the more primitive fungal portion of its life cycle.A representative of the acugutturid moth parasites,Setonema protera, also occurs in Dominican amber; however, based on the earliest fossil record of its host group, an estimated date for the origin of this group would be in the Carboniferous [15].Representatives of the nematode suborder Rhabditina are mainly microbotrophic free-living nematodes; however, some lineages developed amazing specialized patterns of parasitism. One of these lineages is a member of the family Diplogasteridae that evolved the ability to parasitize fig wasps. Diplogasterids are common inhabitants of soil and decaying organic matter and their associations with arthropods are mainly phoretic in nature. Yet some of these nematodes became parasites of fig wasps. Fig wasps are very tiny hymenopterans that develop in fig synconia within the flower clusters. Their entire life cycle takes place in the fig with female flight patterns related to entering and exiting the fig flowers as they pollinate the new flowers and deposit their eggs. TheParasitodiplogaster lineage of diplogastrids is carried by the adult fig wasps to the fig flowers and then parasitizes the developing fig wasp larvae. Originally, the nematodes were probably strictly phoretic and developed on pollen and bacteria in the fig flowers, using the wasps only for transport. At some point, juvenile nematodes emerging from eggs deposited in the fig flowers entered the fig wasp larvae and adults. That this association has continued for 20–30 million years is evident from a parasitized fig wasp surrounded by released stages of the diplogastrid,Syconema dominicana, in Dominican amber [15]. However, based on the earliest fossil record of their host group, an estimated date for the origin of this nematode lineage would be in the Eocene [15].Another unique mode of parasitism originated independently in two genera of rhabditine nematodes,Steinernema andHeterorhabditis. These two nematode genera evolved from separate lineages of free-living, bacterial-feeding, soil nematodes but developed a very similar pattern of insect parasitism. Each lineage became associated with its own specific insect-pathogenic bacteria that were originally acquired from the immediate environment. These bacteria are retained in the gut of the infective stage juveniles and released when the latter penetrate into a living insect. The released bacteria kill the insect within 24 hours and the nematodes feed on the tissues of the corpse that have been modified by the specific bacteria. These so-called entomopathogenic nematodes are sold commercially as biological control agents. The discovery of infective juveniles of one of these clades in Early Cretaceous Myanmar amber shows that entomopathogenic nematodes were present some 100 mya (Figure 10) [15]. The fossil infective stageProheterorhabditis burmanicus was adjacent to a beetle that contained bacteria in its tissues similar to those carried by extant heterorhabditids (Figure 11) [134]. Based on the earliest fossil record of their host group, an estimated date for the origin of both the heterorhabditids and steinernematids would be in the Permian [15].Helminthiasis refers to diseases of humans and animals caused by parasitic worms such as nematodes, trematodes (flukes), and cestodes (tapeworms). Past remains of vertebrate parasitic nematodes are mainly found in coprolites and corpses. Two common animal parasites are ascarids or stomach worms and oxyurids or pinworms. The oxyurids evolved from microbial soil inhabitants and it was not that large a step to shift from feeding on microbes in the environment to those in the gut lumen of invertebrates and vertebrates. The earliest record of an oxyurid parasitizing invertebrates isPaleothelastoma tipulae from an Early Cretaceous crane fly in Myanmar amber [15]. Based on the earliest fossil record of their host group, an estimated date for the origin of the invertebrate parasitic oxyurids would be in the Silurian [15].Oxyurids found in subfossil deposits show the wide range of vertebrate hosts of these parasites.Syphacia was found in the remains of a 10,000–12,000-year-old ground squirrel in Eastern Russia [135], eggs ofParapharyngodon sceleratus were recovered from 9,000-year-old coprolites of an iguanid in Brazil [136], and juveniles ofAgamofilaria oxyura were recovered from a 10,000 ± 180-year-old Shasta Ground Sloth bolus [137]. Based on the earliest fossil record of their host group, an estimated date for the origin of the vertebrate parasitic oxyurids would be in the Carboniferous [15].It is difficult to determine when humans were first infected by the oxyurid pinworm,Enterobius vermicularis. In spite of the 10,000-year-old record from North America and molecular traces ofE. vermicularis in 27 pre-Columbian coprolites ranging from 1,100 to 6,110 years in North and South America [138, 139], the first human infections were probably in the Old World, even though such records from archaeological sites are rare [106]. The oldest Old World records are from a 2100-year-old Chinese mummy [140] and surprisingly, the only report of pinworms in Egyptian mummies was from a site in Roman-occupied Egypt that dates from 30 to 395 AD [141]. However, the discovery ofE. vermicularis in a chimpanzee [142] provides an avenue for early human infection in the Old World and peoples migrating across the Bering Strait could have brought this nematode to the New World [143].Ascarids or stomach worms are probably the most common nematode parasites of vertebrates. The complicated life cycle of many ascarids, including the giant roundworm that infects humans, makes determining their early evolution difficult. Why it is necessary for the immature worms to traverse the wall of the intestine, migrate through the tissues of the host, and then return to small intestine to continue their development is perplexing. The oldest fossil evidence of this group is Early Cretaceous eggs ofAscarites spp. from a dinosaur coprolite in Belgium [101]. However, based on the earliest fossil record of their host group, an estimated date for the origin of ascarids would be in the Permian [15]. Ascarids radiated in numerous lines of vertebrates and invertebrates and have been recovered from more archaeological sites than any other human parasite. A selection of Pleistocene and Holocene sites containing the human ascarid,Ascaris lumbricoides, extending back 30,000 years [144] is presented in Table 1 and shows the geographical range of this parasite over various time periods. It has been suggested that humans acquiredA. lumbricoides from pigs, since they harbour the closely related species,A. suum. However,A. lumbricoides has been recovered from Old World chimpanzees, gorillas, and bonobos as well as from New World howler monkeys [145–147] and the original infections in humans were probably acquired from apes and simians.Table 1
Records of the human stomach worm,Ascaris lumbricoides, from global archaeological sites.
Country
Site
Date
Reference
France
Clairvaux, Jura
3600 BC
[149]
Chalain, Jura
2700–2440 BC
[149]
Bobigny
2nd century AD
[175]
Paris
14th-15th century AD
[176]
Marly-le-Roi, Yveline
17th-18th century AD
[177]
Germany
Drobintz girl
600 BC
[178]
Bremerhaven
100 BC–500 AD
[179]
Lübeck and other cities
15th century AD
[180]
England
Somerset
4100–2600 BP
[181]
Winchester
Roman period
[182]
Lindow Man
2nd century AD
[183]
York
2nd-3rd century AD
[184]
York
9th-10th century AD
[185]
Winchester
1000 years
[186]
Winchester
1100 AD
[187]
Southampton
13th-14th century AD
[188]
York
14th–16th century AD
[189]
Worcester
15th century
[190]
N. America
Kentucky Salts Cave
1125–290 BC
[191]
Tennessee Big Bone Cave
2177
±
145 BP
[192]
Arizona Elden Pueblo
1070–1250 AD
[193]
Adak Island, Alaska
840
±
40 BP
[194]
Williamsburg, Virginia
1720 AD
[195]
Africa
Kruger Cave, Rustenburg
10,000–7,000 BP
[196]
Middle East
Egypt PUM mummy
200 BC
[197]
Acre, Israel
13 century AD
[198]
S. America
Huarmey Valley, Peru
2277
BC
±
181
[199]
Gentio Cave, Brazil
3490
±
120 BP
[106]
Brazil sites
8800–7000 BP
[200]
New Zealand
Wellington
150–200 years
[201]Hookworms are another clade of nematode intestinal parasites recovered from archaeological finds and reports of these parasites have been recently summarized [106]. The two species attacking humans,Ancylostoma duodenale andNecator americanus, are usually lumped together since the stages normally recovered (eggs and first stage juveniles) are so similar that it is impossible to distinguish them by morphological methods. New World records date back to 7230 BP [148], while Old World records are slightly younger (3600 BP) [149]. Hookworms probably evolved from free-living microbotrophic soil nematodes. Based on the earliest fossil record of their host group, an estimated date for the origin of hookworms would be in the Palaeocene [15]. Again, there is a good possibility that humans originally acquired infections from apes and simians sinceN. americanus has been found in chimpanzees [150] andA. duodenale infects Bonoboas [151] and Howler monkeys [146].Another intestinal parasite that has been traced in archaeological remains is the human whipworm,Trichuris trichiura. Most findings are from the Old World, with the oldest record of 10,000–7,000 years BP coming from Africa [152], although eggs of this genus from rodent coprolites date back to 30,000 [153]. Reports of these have been recently summarized [106]. Based on the earliest fossil record of their host group, an estimated date for the origin of whipworms would be in the Carboniferous [15]. Whipworms infect a range of vertebrates, including dogs, cats, mice, pigs, and sheep. The presence of the human whipworm in Old World simians and primates [145, 147, 154–157] provides an avenue for human infections.While parasites in the lumen of the alimentary track of vertebrates can be discovered in mummies and coprolites, the remains of insect vectored nematodes are usually not available in archaeological deposits unless molecular and immunological methods are used. Filarial nematodes adjacent to their insect vectors in Dominican amber establish the presence of filariids in the Cenozoic along with their blackfly and mosquito insect vectors [15]. Based on the earliest fossil record of their host group, an estimated date for the origin of filariids would be in the Carboniferous [15].Fossil evidence of phytoparasitic nematodes is quite rare. The earliest record of plant parasitism by nematodes is the 400 mya Devonian parasite,Palaeonema phyticum, that was developing in the stomatal chambers of the Early Devonian (396 mya) land plant,Aglaophyton major. This species was an epistrate feeder that mechanically disrupted cortical cells and ingested the contents [158].Palaeonema probably evolved from free-living microbotrophic clades that entered the natural opening ofAglaophyton to escape predators and explore a new food source. Lineages with this type of behavior are rare today since most plant parasites have evolved a stylet that is used to penetrate plant tissue and withdraw cell sap. Such stylet bearing nematodes are quite rare as fossils of higher plants and thus far are only represented by a population ofOligaphelenchoides maximis inside plant tissue in Dominican amber [15]. Their early lineages were probably free-living fungal feeders.


## 3.3. Trematodes

Trematodes or flukes can also be found in ancient human remains and reports of liver flukes,Fasciola hepatica, and schistosomes,Schistosoma spp., in archaeological sites have been recently summarized [106]. It is interesting thatF. hepatica has only been recovered in Old World coprolites, in spite of its global occurrence today in cattle and sheep. Subfossils of both genera date back some 5,000 years [159, 160]. It has been suggested that humans acquired schistosomes from ruminant and rodent definitive hosts sometime in the Miocene [161]. This is quite possible since the oldest fossil evidence of this group is an egg of the trematode,Digenites proterus, recovered from a dinosaur coprolite in the Early Cretaceous Bernissart Iguanodon shaft in Belgium [101].


## 3.4. Cestodes

Cestodes or tapeworms are another parasite group that occurs in coprolites. The oldest fossil records of this group are 270 million-year-old tapeworm eggs in a shark coprolite [162], which suggests that terrestrial tapeworms might have originated in a marine habitat. Archaeological finds usually reveal the three tapeworm species,Taeina saginata,T. asiatica, andT. solium that infect humans through cattle and pigs. There is evidence that these and probably other tapeworms were originally acquired when humans devoured wild bovids and swine before these animals were domesticated [163]. Early records ofTaenia eggs in human mummies date back to 3200 BP [164].


## 4. Parasitic Arthropods

While there are many examples of parasitic wasps in the fossil record, there are very few examples of actual parasitism. Some samples occur in amber and several of these show evidence of dryinid wasp parasitism of planthoppers [3]. Evidence of an early stage ichneumonid wasp larva that was dislodged from its feeding position on the abdomen of a spider appeared in Dominican amber [165]. Such larvae first feed on spider hemolymph from the outside and then eventually consume the organs of the spider. Other evidence of spider parasitism by wasps in Dominican amber is an ichneumonid wasp larva that devoured the eggs of a spider and then pupated adjacent to the adult spider [166].WorkerFormica ants are parasitized by small braconid wasps that deposit their eggs directly into the body cavity of the host. The parasitic larvae develop in the ant’s hemocoel and when mature, emerge between the abdominal segments and form a cocoon for pupation in the soil [167]. Evidence that the ancestors of extantElasmosoma wasps have been around for some 40 million years came from a braconid wasp larva emerging from a worker ant in Baltic amber [168].Strepsiptera, a unique order of insects, have another method of parasitizing insects. They possess active first instar larvae called triungulins that search the environment for planthopper hosts. After finding and entering the body of a planthopper, the active triungulin molts into a sedimentary larva that develops to an adult inside the host. The winged adult males emerge and search for females that remain in their planthopper hosts with part of their body protruding for mating purposes. After fertilization the developing eggs hatch into a new generation of triungulins that swarm out of the female’s brood pouch prepared to search for another victim. The oldest records of actual parasitism by this group are puparia inside an Eocene ant [169] and a female inside a Baltic amber ant [170]. A group of triungulins leaving their delphacid planthopper host was captured in Dominican amber when the infected planthopper landed in some resin [171]. An empty puparium in a halictid bee shows another host group for Tertiary strepsiptera [171].A larval hymenopteran emerging from an adult trichopteran was found in Baltic amber [172]. This association is presently unknown, since today only the larval and pupal stages of trichopterans are parasitized by hymenopterans. Some tachinid fly parasitoids deposit eggs directly on their insect hosts and this behavior was noted in Dominican amber where the leaf beetle,Stenaspidiotus microptilus, had a tachinid egg attached to its pronotum [173].Some interesting arthropod parasites were recently described from dog mummies in Egypt [174]. Numerous specimens of the dog tick,Rhipicephalus sanguineus, and a specimen of the louse fly,Hippobosca longipennis, were found still attached to the remains of dogs in tombs at the El Deir site in Egypt. While only dating to Roman times, they do show vector associations and suggest possible disease transmission scenarios.


## 5. Discussion and Conclusions

Tracing the history of diseases is similar to climbing the evolutionary scale, with free-living organisms at the base and a series of mutated parasitic stages appearing along the ascent until ultimately, human pathogens appear at the peak. The origins and coevolution of infectious agents are reflected in their present life histories and associations with their hosts. Whether the infection is latent or virulent, includes single or multiple hosts, is permanently associated with the host or has a resistant free-living stage, has both an intermediate as well as a definitive host, and is vector spread or a contagion are reflected in the evolutionary history of the infectious agent, which often extends back millions of years.The evolutionary history of host-pathogen associations may also determine how long the relationships will continue under future fluctuating environmental and climatic conditions. It is often the least specialized associations that survive such changes. An example is the polydnaviruses that occur only in certain groups of parasitic wasps and probably date back to the early lineages of these wasps. While very successful now, their host specificity makes polydnaviruses especially vulnerable to extinction if conditions change. If a particular wasp lineage becomes extinct because its host is eradicated, the virus also disappears. In contrast, the iridoviruses infecting the sow bug have a wide host range, crossing phyla, and even infecting vertebrates. The origin of iridoviruses probably extends back to early invertebrate lineages and these viruses have a much better chance of survival since if one host group disappears, the infection continues in other hosts. The polyhedral viruses fall between polydnaviruses and iridoviruses in being restricted to more than one genus of insects but rarely crossing families and never crossing phyla. These viral groups survived the long period of coevolution with their hosts although others certainly became extinct along the way. Unfortunately very little is known about the evolution of pathogenic viruses in reservoir hosts and how long it took before the infection became latent or if it ever was virulent. Undetectable amounts of viruses certainly exist as latent infections in all vertebrates (reservoir hosts) and some of these eventually enter humans as highly virulent pathogens.With pathogenic bacteria, a similar situation occurs between the infectious agent and the host. Several species of the genusBacillus coevolved with insects and became overt pathogens. At least one,B. anthracis, became a vertebrate pathogen that also infects humans. The apparent absence of reservoir hosts and the persistence of the pathogen in the environment in the form of resting spores separate this group from most pathogenic forms such as plague bacteria and Lyme disease spirochetes. These traits suggest thatBacillus is in an early evolutionary stage with its hosts.Fossil evidence of protozoan infections indicates that lineages ofLeishmania evolved in their insect vectors long before they infected vertebrates. The pathway of infection was initiated through sand fly larvae feeding on flagellates in the environment. The presence of gametogenesis and syngamy ofPlasmodium in a mosquito in Dominican amber indicates the vector as the original host. Another character used to determine the original host in arthropod-vector associations is the amount of damage done to both arthropod and vertebrate hosts. The host showing the least amount of damage is considered the primary host.Parasitic helminthes obviously evolved from free-living ancestors but their origins are a mystery since no early progenitors have been found. An estimate of the date for their origins can be made from the earliest fossil record of their host group. As with the above pathogen groups, some lineages are associated with specific hosts and others with multiple hosts. An example of the former is nematodes of the genusParasitodiplogaster that coevolved with fig wasps and are at risk of extinction if their host lineage perishes. In contrast, vertebrate parasitic nematodes of the genusAscaris coevolved with a wide range of hosts and their existence is much more secure.Based on our present knowledge and evidence from the past, it is obvious that both contagion and vector-borne pathogens are quite ancient. There is no definite subfossil evidence that any of these pathogens originated in humans. Humans acquired them from reservoir hosts (zoonosis) that had asymptomatic infections after a precarious balance was established with the pathogen [174]. Such a balance is absent when the pathogens are introduced into humans and then they become overt and highly virulent.Unfortunately, we know very little about the development of human pathogens in reservoir hosts and arthropod vectors or why some vectors are more efficient carriers than others. All of these traits are connected with the coevolution of pathogens with their hosts that occurred millions of years ago.The host-parasite associations around us today represent various stages of coevolution. Some may be at the end of their evolutionary line while others may be in the middle or just beginning. Most of these associations will be unsuccessful for one reason or another while others will survive and fall into one or more of the following recognized stages of symbiotic relationships: inquilinism (two organisms living in the same habitat), commensalism (one of the two organisms benefits), mutualism (both organisms benefit), or parasitism (one organism takes nourishment from the other, which suffers). Determining the intricacies of these evolutionary stages presents a challenge to future generations of scientists.



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*Source: 181353-2014-06-12.xml*