Paleoparasitological investigations on the Neolithic lakeside settlement of La Draga (Lake Banyoles,Spain)

Abstract

Paleoparasitological analyses were conducted on samples from the Neolithic lakeside settlement of La Draga in Spain (5320–4980 BC). Conventional microscopic analysis revealed the presence of tapeworms (genus Taenia/Echinoccocus and Diphyllobothrium), roundworms (genus Trichuris, Capillaria, and Ascaris), rumen fluke (genus Paramphistomum), and Acanthocephalan (genus Macracanthorhynchus). In addition, genetic analysis demonstrated the presence of the lancet liver fluke (Dicrocoelium dendriticum) and the human pinworm (Enterobius vermicularis) at the site. These results represent the first parasitological data from a Neolithic lakeside settlement related to the Cardial Pottery Culture. Some parasites are comparable with those retrieved from Neolithic lakeside sites studied in France, Germany, and Switzerland, which stem from and are posterior to the Linear Band Keramic Culture. However, some taxa were identified here for the first time during the Neolithic period, or represent the oldest mention of these parasites. This new paleoparasitological contribution reinforces our knowledge of intestinal parasites in Neolithic populations and provides new data on their history.

Keywords

archaeology cardium pottery culture helminths neolithic paleoparasitology Spain

Introduction

Paleoparasitology enables us to detect the presence of parasites in archaeological and paleontological samples in order to access the hygiene level, health status, and living conditions of ancient populations (Dittmar et al., 2011). While the parasite markers can be of various types (macroremains, biomolecules, or resistant/dissemination forms), the majority of the studies concern the recovery and identification of the preserved eggs produced by adult intestinal worms parasitizing the gastrointestinal tract of humans or animals (Le Bailly and Araujo, 2016).

Among the periods of profound changes in the history of humanity, the Neolithic transition is of particular interest. Indeed, the adoption of a sedentary lifestyle, the domestication process, the growing proximity between human and animal populations, and also between animals themselves raise questions regarding the emergence and the transmission of pathogens (Zammit, 2005). This postulate is supported by the fact that many parasites need animals as definitive or intermediate hosts to resume their lifecycles. However, many parasites are common to animals and humans. During the past 20 years, several analyses have been carried out on archaeological sites dated to the Neolithic period (Bouchet et al., 2003). Owing to exceptional preservation conditions, lakeside settlements have been a particular focus of this study and have supplied abundant data. Between 1995 and 2005, paleoparasitological investigations were carried out on several Neolithic lakeside settlements in France (Bouchet, 1997; Bouchet et al., 1995; Dommelier et al., 1998; Dommelier-Espejo, 2001; Dommelier-Espejo and Pétrequin, 2016), Switzerland, and Germany (Le Bailly, 2005; Le Bailly and Bouchet, 2004; Le Bailly et al., 2003). These investigations revealed the presence of several taxa of human and animal gastrointestinal parasites, including roundworms, tapeworms, and flukes.

European neolithization occurred following two migration routes: the continental Danubian route and the littoral Mediterranean route associated with the Impressa and Cardial cultures (Price, 2000; Tresset and Vigne, 2011). Up to now, paleoparasitological studies have only provided data on lakeside settlements related to the Danubian route. In this article, we report the first paleoparasitological analysis carried out on a Neolithic lakeside settlement linked to the Mediterranean route of neolithization. The purposes of this study are to determine the biodiversity of human and animal gastrointestinal parasites, evaluate the health status of the population, and reveal evidence of the organization of the site by highlighting concentrations of parasite remains using an innovative mapping method. In a broader perspective, the results of this study could provide a new starting point for the comparison of parasite biodiversity between the two neolithization routes and could help us to elucidate how parasites were diffused throughout Western and Central Europe during the Holocene.

Materials and methods

The site of La Draga

The Neolithic site of La Draga is located in Catalonia (Spain), on the edge of the periglacial lake of Banyoles, at the foothill of the Pyrenees. It was discovered in 1990 during the development of a park beside the lake. The lake extends over 2150 m long and 775 m wide, with a perimeter of 7.4 km (Figure 1). During the 1990s, landscaping around the lake helped to uncover several archaeological sites dated from 5400 to 5000 BC (Tarrús et al., 1994). This discovery was the first evidence of a Neolithic lakeside installation in the Iberian Peninsula. The nature of the deposit allowed for the exceptional preservation of the remains, comparable with the Circum-Alpine and Jurassian lakeside sites, and offered the possibility to study settlement patterns and the economic exploitation of the surrounding resources by the lakeside inhabitants (Saña, 2011; Saña et al., 2014; Tarrús, 2008; Tarrús et al., 1994). Many poles and numerous plant and animal macrofossils have been found, as well as an exceptionally conserved yew bow (Taxus baccata) and oak paddle (Quercus subg. Quercus) (Palomo et al., 2013; Piqué et al., 2015). On the basis of pottery and lithic industry finds, supported by radiocarbon dating, the site has been assigned to the late Cardial culture of the Western Mediterranean (Tarrús et al., 1994), that is, between 5430 and 5020 BC (Morales Hidalgo et al., 2010; Palomo et al., 2014). The habitation level is about 20–30 cm thick and lies directly on a lacustrine chalk layer (Tarrús et al., 1994). Part of the Neolithic village is now underwater as a result of changes in the lake level over time. The emerged occupation level contained habitation elements with several houses, including floors and ceiling levels.

Figure 1.

Location of the site of La Draga in the NE Iberia and distribution of the four excavated sectors (A, B, C, and D) from 1990 to 2014.

The site was excavated in four sectors: sector A, located on the east of the site over a surface of around 284 m², was excavated from 1991 to 1995; sector B, with a surface of 126 m², was excavated from 1997 to 2005 and is located in the west part of the site; sector C, which is the only submerged area, was studied from 1994 to 2005 and comprises a surface of about 310 m². Finally, sector D corresponds to an area of 56 m² and was excavated from 2010 to 2012. Only the latter sector is studied here (Figure 1).

Stratigraphic indications and dates show that the site was continuously settled without interruption. Nevertheless, two phases can be singled out as they were identified all over the site, except for sector A on account of the bad preservation of the wooden structures. The older layer, identified as layer VII, is studied here and corresponds to the collapse of the architectural wooden structures and dates from 5320 to 4980 cal. BC. The more recent phase corresponds to a travertine block layer, dated from 5210 to 4800 cal. BC (Palomo et al., 2014). Layer VII presents exceptional conservation of the archaeological remains due to the significant clay fraction in the sediment. Furthermore, layer VII corresponds to an accumulation of residues of human and animal activities, as shown by the analyses of seeds, macroremains, and the faunal spectra (Antolín, 2013). All these factors were propitious to a paleoparasitological investigation of the site of La Draga.

Sampling strategies and material

During the excavation campaign in 2012, systematic sampling was adopted consisting of four samples per square meter. This strategy aimed to provide homogeneous sampling, in order to produce distribution maps of the possible recovered taxa and compare the results with other archaeological artifacts in the sector. Samples are stored and available at the Chrono-environment laboratory (Besancon, France).

Extraction method for parasite egg analysis

In total, 72 samples were selected and prepared according to the rehydration–homogenization–microsieving (RHM) method as detailed by Dufour and Le Bailly (2013). In total, 5 g of sediments was first rehydrated in a solution consisted of 50 mL 0.5% trisodic phosphate (TSP) and 50 mL 5% glycerinated water for 1 week. A few drops of 10% formalin solution were added to avoid fungi and bacteria development. During the homogenization step, samples were crushed in mortar and submitted to ultrasounds (50–60 Hz) for 1 min. Finally, samples were filtered through a column with four sieves with mesh sizes of 315, 160, 50, and 25 µm. The remains contained in the 50- and 25-µm sieves were conserved for the analysis and transferred into 4 mL polyvinyl chloride (PVC) tubes. In total, 10 slides of each fraction (i.e. 20 slides per sample) were examined under a light microscope (Leica DM-2000 LED) coupled with a digital camera (Leica ICC50-HD).

DNA analysis of ancient parasites

In total, five samples from La Draga were tested for ancient parasite DNA as described in Côté et al. (2016). In brief, after grinding 2–10 g of sediments, the DNA was purified using the PowerMax Soil DNA isolation kit (Mobio), and the extract was concentrated using QG (Qiagen) and isopropanol as a binding buffer, PE (Qiagen) as a washing buffer, and EB (Qiagen) + 0.05% Tween 20 as an elution buffer on Qiaquick columns (Qiagen). Multiplex polymerase chain reaction (PCR) was performed to target 15 species (Taenia saginata, T. solium, T. asiatica, Echinococcus granulosus, E. multilocularis, D. latum, D. nihonkaiense, D. dendriticum, Enterobius vermicularis, Trichuris trichiura, Fasciola hepatica, F. gigantica, Dicrocoelium dendriticum and D. chinensis) and 1 genus (Ascaris sp.) of gastrointestinal helminths. Amplicons were then processed to generate Ion Torrent libraries and were sequenced on an Ion Torrent Personal Genome Machine as described. Sequence analysis was performed using a homemade script, which combined bioinformatics tools (featureCounts, VarScan2, Samtools; Koboldt et al., 2012; Li et al., 2009; Liao et al., 2014).

Results

Microscopic examination of the samples

In total, 67 samples (93%) yielded positive results and allowed for the retrieval of at least one taxon. A total of seven taxa of gastrointestinal parasites were identified at least at the genus level with optical microscopy. For each taxon, the eggs were photographed and counted precisely in order to be exploited with a geographical information system (GIS), in order to better understand their spatial distribution and compare it with other analyses, such as archaeozoology (Table 1). Regarding the nature of the groundmass (with a significant proportion of clay) and the thickness (20–30 cm) of layer VII, we can consider that the recovered eggs are not contaminations.

Table 1.

Average concentrations of the different parasite egg species found at the site of La Draga, in relation to the number of analyzed samples (Nb.) per square meter.

Square (m²)	Nb.	Trichuris sp.	Macracanthorhynchus sp.	Capillaria sp. punctuated type	Capillaria sp. reticulated type	Ascaris sp.	Taenia sp.	Diphyllobothrium sp.	Paramphistomum sp.
IJ78	2	7	0	0.5	0	0	1	0.5	0
IJ79	2	19	0.5	0	0	0	0	0	0
IJ80	1	23	1	0	0	0	0	1	0
IJ81	3	13	0	0	0	0.3	0	0.7	0
JA78	1	13	0	0	0	0	0	0	0
JA79	2	13	0.5	0	1	0	0	1	0
JA80	5	5	0.4	0	0.2	0	0.2	0	0
JA81	3	16.3	0.6	0	0	0	0.3	0	0
JB78	3	4.3	0	0.3	0	0.3	0.6	0	0
JB79	1	5	0	0	0	0	2	0	0
JB80	1	4	0	0	0	0	0	0	0
JB81	2	9.5	0	1	0	0	1	0	0
JC78	3	4	0.3	0	0.3	0	0	0	0
JC80	2	9.5	1.5	0	0.5	0	1	0	0
JC81	2	23	0	0	0	0	0.5	1.5	0
JD78	2	7.5	0	0	0	0	0.5	0	0
JD79	3	28.3	0.3	0	0	0.6	1.3	0.6	0
JD80	1	0	0	0	0	0	0	0	0
JD81	2	44	1	0	0	2	0	0.5	0.5
JE78	3	48.6	1	1	0	2.3	1	0.3	0
JE79	1	34	0	0	0	8	0	0	1
JE80	1	77	0	0	1	93	6	0	0
JE81	2	56	8.5	0.5	0	12	0.5	0	0
JF78	4	29.25	0.5	0	0	2	0	0.25	1
JF79	1	72	4	0	0	4	0	0	2
JF80	3	37.3	1.6	0.3	0	5	3	0	1
JF81	3	50.3	2.3	0.3	0.3	19.3	2	0	2
JG79	1	20	0	0	0	0	0	0	1
JG80	1	37	2	0	0	3	1	0	0
JG81	2	48.5	0.5	0	1	57.5	0.5	0	1
JH80	1	18	0	0	0	2	1	0	2
JH81	4	18	0.5	0	0.5	0.5	0.5	0.5	1

Among the recovered parasites, some belonged to the Cestoda class. Embryophores of tapeworm (length = 34.88 ± 1.7 µm; width = 29.36 ± 1.4 µm) belonging to the genus Taenia sp. (Linné, 1758) or Echinococcus sp. (Rudolphi, 1801) were identified in 25 samples (34.7%; Figure 2a). Light microscopy cannot distinguish these two genera as their shape and average size are identical. In an anthropological context, these elements would be identified as Taenia sp. and would attest to the consumption of raw or undercooked beef (T. saginata) or pork (T. solium and T. asiatica; Ash and Orihel, 2007). In the present case, as the analyses were performed on samples of undetermined biological origin, these embryophores may also indicate the presence of dogs infected by tapeworms belonging to the genus Echinococcus.

Figure 2.

Photographs of the parasite eggs found in sector D of the Neolithic village of La Draga (G = x600): (a) Taenia sp., (b) Diphyllobothrium sp., (c) Capillaria sp. punctuated type, (d) Capillaria sp. reticulated type, (e) Ascaris sp., (f) Trichuris sp., (g) Paramphistomum sp., and (h) Macracanthorhynchus sp. Scale bar = 20 µm.

Operculated eggs (length = 55.9 ± 3 µm; width = 38.12 ± 3.2 µm) corresponding to the genus Diphyllobothrium sp. (Cobbold, 1858) were identified in 16 samples (22%). They all had smooth shells (Figure 2b). This tapeworm is related to the consumption of undercooked or raw fish by a fish-eating mammal (including humans), and the parasite lifecycle includes two successive intermediate hosts: first a copepod and then a freshwater fish (Gunn and Pitt, 2012). When the parasite infects humans, it can be responsible for abdominal pains, diarrhea, and anemia.

Among the Nematoda phylum, five taxa classified here under the general genus Capillaria (Zeder, 1800) have been differentiated (Figure 2c and d) in 15 samples (20.8%). The identification and taxonomy of this genus is not yet clear and changes according to the different authors. Morphologically, the eggs retrieved in La Draga can be divided into two groups: the first includes eggs with punctuated shells and the second comprises eggs with reticulated shells. Egg sizes are as diverse as the types of ornamentation. Some reticulated Capillaria sp. eggs could be a parasite of wild and domestic Bovidae, whereas the punctuated-type capillariids may be due to fish-eating habits or the presence of rodents (Bouchet, 1997; Cross, 1992; Moravec, 1981; Spratt, 2006).

Eggs of the genus Ascaris (Linné, 1758) were also identified (length = 64.68 ± 5.3 µm; width = 53.34 ± 2.9 µm; Figure 2e) in 34.7% of the samples. Currently, two species are defined: the swine species A. suum and the human species A. lumbricoides. But light microscopy cannot distinguish these two species as eggs are of similar shape and size, and this method is, therefore, not sufficient for differentiating the two species. In both cases, this parasite attests to the presence of fecal matter of human or swine origin. Because of direct oral–fecal transmission, human ascariasis reflects precarious hygiene conditions, proximity, poor management of fecal waste, and the possible use of organic waste as fertilizer for crops (Roberts and Janovy, 2009).

Trichuris sp. (Roederer, 1761) is another Nematode retrieved from samples of La Draga (Figure 2f). Whipworm eggs (length = 53 ± 2.2 µm without polar plugs; width = 27.2 ± 1 µm) were identified in 93% of the samples. Whipworm is a cosmopolitan and ubiquitous parasite with a significant number of species affecting humans and many other animals. However, the eggs of all species of Trichuris are quite similar in shape and sometimes very similar in size, such as for Trichuris trichiura, the human whipworm, and Trichuris suis, the pig whipworm. Egg size range has been established based on a survey of the parasitological literature (Acha and Szyfres, 2005; Bailenger, 1982; Beer, 1976; Brumpt, 1936; Dufour, 2015; Golvan, 1990; Mehlhorn, 2008; Soulsby, 1982; Thienpont et al., 1979). In this manner, Trichuris trichiura is defined by a width ranging between 24 and 28 µm and a length of 50–58 µm and Trichuris suis by a width of 26–31 µm and a length of 57–67 µm. Figure 3 presents the projection of the recovered eggs in these new size ranges. It shows that the majority of the eggs can be attributed to the human species Trichuris trichiura. The dynamics of whipworm infection are similar to those of the roundworm.

Figure 3.

Projection of Trichuris sp. eggs from La Draga and dimensions of T. trichiura and T. suis eggs from the literature.

One taxon from the Trematoda class was identified. Large ovoid and operculated eggs (length = 144.1 ± 9.6 µm; width = 81.93 ± 9.2 µm) were retrieved in 16 samples (22.2%). They belong to the genus Paramphistomum sp. (Fischoeder, 1901; Figure 2g). This parasite localized in the rumen exclusively affects ruminants and is often asymptomatic (Kassai, 1999).

Finally, a parasite taxon belonging to the Acanthocephalan phylum was identified. Eggs with ornamented and thick shells (length = 83 ± 4.7 µm; width = 47.21 ± 2.8 µm) were identified as Macracanthorhynchus sp. (Pallas, 1781; Figure 2h). Eggs were found in significant proportions in 22 samples (29%). This parasite affects animals, mostly pigs and wild boar, causing weight loss and enteritis, and can cause fatal peritonitis in cases of severe infections (Taylor et al., 2007).

Genetic analyses of the samples

Only one sediment sample out of five yielded a positive result and led to DNA amplification (Côté, 2015). Ancient DNA from T. saginata and Trichuris trichiura was identified. These samples are consistent with microscopic observations and provide a specific diagnosis of the parasites. Ancient Ascaris sp. DNA was also retrieved; however, as with microscopy, it was not possible to discriminate between A. lumbricoides and A. suum. In addition to these first taxa, genetic sequences of Dicrocoelium dendriticum (the lancet liver fluke) and E. vermicularis (the human pinworm) were recovered, which complement the parasite biodiversity list. The lancet liver fluke belongs to the Trematoda class. It is a parasite of ruminants (essentially bovidae) which requires two intermediate hosts to resume its lifecycle, first a land snail and then an ant. In ruminants, the adult parasite grows in the bile ducts and can be responsible for various symptoms in case of severe infections, such as weight loss, anemia, and cirrhosis (Kaufmann, 1996; Nozais et al., 1996). Human infections are very rare because they require the ingestion of an infected ant; however, it is possible to retrieve Dicrocoelium eggs transiting in human intestines after the consumption of infected sheep liver, for example. The pinworm E. vermicularis is a nematode specific to humans, presenting a single host lifecycle. The direct fecal–oral transmission of the parasite (by eggs released onto the anal region) reveals physical hygiene problems (Mehlhorn, 2001; Roberts and Janovy, 2009). The results of the paleogenetic analysis complete the parasite biodiversity in La Draga, with two additional taxa for which no eggs were observed during the microscopic examination.

Discussion

Paleoparasitological analyses

The paleoparasitological analysis (microscopy and paleogenetic) of samples from the lakeside settlement of La Draga revealed the presence of at least nine gastrointestinal helminth taxa. The presence of these parasites is linked to the lifestyle, dietary habits, and hygiene of the population. Some of the recovered parasites, such as geohelminths (roundworm and whipworm), develop in cases of poor sanitary conditions and inadequate management of organic matter, whereas others (tapeworms) are linked to the diet of human or animals. As an example, the fish tapeworm (Diphyllobothrium) is related to ichtyophagy and can infect humans as well as other fish-eating mammals (such as canidae or mustelidae). Archaeozoological studies showed that fishing was a sporadic activity at La Draga (Buxó et al., 2014). This could appear to be in contradiction with the lakeside localization of the site; however, during the neolithization process, a change occurred in the diet, switching from fishing and hunting to a diet based on terrestrial resources and domestic animal products (Tresset and Vigne, 2011). This shift involved a decrease in the consumption of fish and correlates with the scarcity of fish tapeworm eggs in the samples. Finally, mustelidae bones as well as domestic dog remains were recovered at the site (Antolín et al., 2014; Buxó et al., 2014; Saña, 2011; Tarrús et al., 2006). The presence of Diphyllobothrium could also relate to the consumption of fish by these mammals. The tapeworm genus Taenia is related to the consumption of raw or undercooked pork or beef by humans. The paleogenetic results indicate the presence of the species T. saginata (beef tapeworm) which is linked to the consumption of beef. The presence of cattle was primarily shown by archaeozoology.

Other parasites are directly linked to the presence of livestock (cattle or pig) at or in close proximity to the site or can be related to the consumption of wild ruminants. This is the case for the flukes (Dicrocoelium sp. and Paramphistomum sp.) as well as for Macracanthorhynchus sp. and potentially Ascaris sp. The archaeozoological study of La Draga demonstrated the presence of several species of wild and domestic ruminants at the site in low proportions, such as the aurochs (Bos primigenius), deer (Cervus elaphus), roe deer (Capreolus capreolus), and ibex (Capra pyrenaica; Buxó et al., 2014; Tarrús et al., 2006). Nevertheless, archaeozoological remains are mostly made up of domestic species, in particular sheep and goat, while cattle and pig are also present but in lower proportions (Antolín et al., 2014; Saña, 2011).

Spatial analysis of the residues using the kriging method

The methodology applied for the microscopic examination of the samples, with systematic counting of the residues for a constant number of reads, allowed for the development of an original way of analyzing the distribution of eggs at the site. As it is not possible to sample the whole sector, some parts yielded no samples (Figure 4a). Therefore, the kriging method was chosen to represent the results. This statistical estimation method performs a spatial interpolation of a variable (in our case, the number of eggs). It considers the distance between the data and the estimation point, as well as the distance between the data themselves. The ordinary kriging method was applied, associated with the spherical semi-variogram model (Arnaud and Emery, 2000; Cressie, 1990), and conducted with the ArcGIS 10.1 (Esri) software. The fish tapeworm Diphyllobothrium sp. was excluded from this analysis because of its low representation in the samples as only the main taxa were considered, that is, Paramphistomum sp., Taenia sp., Ascaris sp., Macracanthorhynchus sp., and Trichuris sp. Also, Capillaria sp. residues were not included because of our incapacity to determine them precisely.

Figure 4.

Kriging maps of the parasite eggs retrieved from the site of La Draga: (a) position of the samples collected for the paleoparasitological analyses, (b) kriging map of Paramphistomum sp. eggs, (c) kriging map of Taenia sp. eggs, (d) kriging map of Ascaris sp. eggs, (e) kriging map of Macracanthorhynchus sp. eggs, and (f) kriging map of Trichuris trichiura sp. eggs.

Maps obtained from the kriging method highlight a large concentration area in the northeastern part of the excavated area for all species independently, with a more or less important spread (Figure 4). As layer VII is uniform and does not present any particular dip, the northeastern egg concentration cannot correspond to an accumulation area resulting from the site topography.

Paramphistomum sp. is exclusively present on the east side of the excavated area, with higher concentrations on the northeastern side, in squares JF79, JF80, JF81, and JH81 (Figure 4b). The concentration of this animal parasite in this part of the sector could be related to a stalling area. However, in these squares, the presence of an important accumulation of caprinae bones, as revealed by the archaeozoological study, seems to attest to a waste area (butchery, discarded animal feces, etc.), rather than a breeding area. This part of the excavated area is also in contact with sector B which contains more cattle bones (Antolín et al., 2014). Future studies in sector B could help to verify the nature of this accumulation of rumen fluke eggs.

Taenia sp. eggs are present in almost the whole area, but zones of higher concentration appear in squares JE80, JE81, JF80, and JF81 (Figure 4c). As the paleogenetic study identified T. saginata, a species infecting humans, this strongly suggests the presence of fecal matter of human origin mixed with the archaeological layer, with a higher concentration in the northeastern part of the excavated sector. This could be the result of a deliberate accumulation in this part of the site.

In the same part of the site, eggs of the roundworm genus Ascaris were recovered, with the highest concentrations in the same squares JE80, JE81, JF80, and JF81. However, the extent of Ascaris eggs is less important and does not cover the entire area (Figure 4d). Neither microscopic analysis nor paleogenetics were able to discriminate the roundworm species. Because of the presence of human tapeworm in the same area, the Ascaris eggs may correspond to the human species A. lumbricoides. However, eggs of Macracanthorhynchus sp., a parasite currently related to the pig, were also retrieved in this area (Figure 4e). In these circumstances, the roundworm eggs could thus be of pig origin and correspond to A. suum. Moreover, archaeozoological analysis revealed the presence of a large number of swine bones in this sector (Antolín et al., 2014). Finally, it is important to keep in mind that current swine parasites could have been more present in humans in the past. This was already presumed to be the case with the giant kidney worm (Dioctophyma renale), a common carnivorous (canidae, mustelidae) parasite retrieved from human coprolites at the Neolithic site of Arbon-Bleiche 3 (Le Bailly et al., 2003).

The spatial representation of the whipworm residues (Trichuris sp.) shows the presence of parasite eggs in a large part of the excavated area, but here again, the highest concentrations are in squares JE80, JE81, JF80, and JF81 (Figure 4f). An additional square (JE78) shows a high concentration in the southern part of the excavated area. As the egg measurements and the paleogenetic approach were consistent with the human species Trichuris trichiura, these results suggest that human fecal matter was discharged in these areas.

In light of these results, spatial representation with the kriging method tends to show the existence of a discharge area for organic matter, in which both human and animal waste was deposited. This also tends to attest to fecal waste management by the inhabitants of the site. These results are in accordance with the archaeozoological study which revealed that the inhabitants kept herds in the village and gathered butchery remains (Antolín et al., 2014). Despite this management, which usually aims to reduce the risks of contamination by parasites related to sanitation, such as whipworm, roundworm, and pinworm, the degree of infestation of the inhabitants appears high with the co-occurrence of several gastrointestinal parasites among which the whipworm, the roundworm, and the pinworm present oral–fecal transmission (Euzéby, 2002; Nozais, 1998).

Comparison with Circum-Alpine Neolithic lakeside settlements

The Neolithic site of La Draga shows important parasitic biodiversity, and comparisons can be made on some parasites observed up to now in other European lakeside sites. Microscopic analysis showed that common parasite diversity is about 53.8% between La Draga and north Alpine lakeside sites. However, some parasites can be debated. Because of the lakeside situation of the site, the fish tapeworm genus Diphyllobothrium, related to fish consumption, is of particular interest. In La Draga, the fish tapeworm was identified in 20% of the studied samples, and eggs were recovered in small proportions (Table 1). In the north Alpine lakeside settlements, the frequency of fish tapeworm in the samples varied between 6% and 9% up to 73%, and the number of eggs varied between two and several hundred eggs per sample (Le Bailly, 2005, 2011). In the sites of Arbon-Bleiche 3 (Switzerland) and Torwiesen II (Germany), dated to around 3400 and 3300 BC, the high frequencies of fish tapeworm (70% and 73%, respectively) were explained by the important role of fishing in the diet, particularly at Arbon-Bleiche 3 where the percentage of fish was estimated at around 22% of the diet (Hüster-Plogmann and Leuzinger, 1995; Le Bailly et al., 2005). This may be correlated to an economic and ecological crisis, with unfavorable climatic conditions for cultivation and animal husbandry between the 37th and the 33rd centuries BC (Magny, 2004), thus furthering fishing and hunting as the first food resources, accompanied by a transition from the Pfyn to the Horgen culture (Le Bailly et al., 2007; Schibler et al., 1997; Schibler and Chaix, 1995). In the sites of Chalain (Jura region in France), 73.7% of the samples yielded positive analyses of Diphyllobothrium eggs in station 4, dated between 3050 and 3000 BC, and some contained several hundred eggs (Dommelier-Espejo, 2001). This period corresponds not only to the introduction of the Ferrières culture through the Jura Mountains from the region of Languedoc-Roussillon (Pétrequin and Pétrequin, 1988) but also to a brief climatic depression phase (Magny, 2004). As cultural transitions were often contemporaneous with phases of unfavorable climatic conditions (Arbogast et al., 1996), it is not yet possible to discern whether variations in egg concentrations are due to climatic or anthropogenic forcing.

In the case of La Draga, climatic studies attest to favorable conditions for agriculture (Antolín et al., 2014; Revelles, 2016; Revelles et al., 2016) and breeding with an intensive mixed farming model (Antolín et al., 2014; Höbig et al., 2012; Perez-Obiol et al., 2011; Revelles et al., 2016). The differences observed in local resource management, especially in fish exploitation, can thus be explained by the different environmental conditions between the late sixth millennium BC in Catalonia to fourth to third millennia BC in the Circum-Alpine region or by the stability of the population not undergoing a cultural transition.

La Draga differs from other perialpine Neolithic sites by the presence of the roundworm genus Ascaris, thereby making it the oldest mention of this parasite in a lacustrine context. It has been mentioned in more recent Central European sites, such as Clairvaux 8 (3900–3700 BC), Chalain 3 and 19 (3200–2980 BC; Dommelier-Espejo and Pétrequin, 2016) or Arbon-Bleiche 3 (Akeret et al., 1999). The earliest presence of the roundworm in La Draga may be linked to its proximity to the African continent, as an African origin is proposed for Ascaris sp. (Mitchell, 2013). Nevertheless, Ascaris sp. eggs have been identified in samples from the Pleistocene cave of Arcy-sur-Cure (Bouchet et al., 1996). This demonstrates that the diffusion paths, as well as the period of introduction of roundworm across the European continent, are still unclear and need to be elucidated with further paleoparasitological studies.

Finally, the recovery of the thorny-headed worm Macracanthorhynchus sp. represents the oldest occurrence of this parasite in archaeology. It was previously retrieved in Western Europe from the Roman site of Beauvais (France), dated between the AD 1st and the 3rd centuries (Dufour, 2015), from the Chehrabad salt mine in Iran, dated to the Sassanian Era (AD 4th/5th centuries; Mowlavi et al., 2015), and in America, from the Antelope Cave in Arizona dated to around AD 900 (Fugassa et al., 2011).

Conclusion

The paleoparasitological study conducted on the Neolithic lakeside site of La Draga reveals the presence of nine different taxa of intestinal worms. It provides the first parasitological data from a Neolithic archaeological site associated with the Cardial Pottery Culture. Some of the recovered taxa, such as Ascaris sp. or Trichuris trichiura, indicate hygiene-related problems, whereas other parasites are related to the diet and the presence of livestock at the site. The proportions of recovered parasites are consistent with the archaeozoological data and confirm a diet partially consisted of livestock products with a low fishing contribution.

The methodology employed in this study, including the systematic counting of the parasite residues and an original representation of the results using a GIS, demonstrates the existence of a large area of accumulation of parasite eggs in sector D. It tends to indicate the management of organic matter by the inhabitants of the site and the depositing of waste in the same area. This study enhances our knowledge of the way of life of the inhabitants of this site, but other sites attributed to the Cardial culture should also be analyzed in order to provide further evidence of parasite markers during this period of the Neolithic.

Footnotes

Funding

The fieldwork was funded by HAR2012-38838-C02-01 (CSIC, Spain) and HAR2012-38838-C02-02 (UAB, Spain) and archaeozoological analyses funded by HAR2014-60081-R (UAB, Spain). CM, paleoparasitology PhD, is funded by the Bourgogne Franche-Comte Region. Paleogenetic analysis performed by NMLC during her PhD was developed in the Jacques Monod Institute in Paris.

References

Acha

Szyfres

(2005) Zoonoses et maladies transmissibles communes à l’homme et aux animaux. Paris: OMS.

Akeret

Haas

Leuzinger

, et al. (1999) Plant macrofossils and pollen in goat/sheep faeces from the Neolithic lake-shore settlement Arbon Bleiche 3, Switzerland. The Holocene 9(2): 175–182.

Antolín

(2013) Of cereals, poppy, acorns and hazelnuts. Plant economy among early farmers (5500–2300 cal BC) in the NE of the Iberian Peninsula. An archaeobotanical approach. PhD Thesis, Universitat Autònoma de Barcelona.

Antolín

Buxó

Jacomet

, et al. (2014) An integrated perspective on farming in the early Neolithic lakeshore site of La Draga (Banyoles, Spain). Environmental Archaeology 19(3): 241–255.

Arbogast

Magny

Petrequin

(1996) Climat, cultures céréalières et densité de population au néolithique: Le cas des lacs du Jura français de 3500 à 2500 av. J.-C. Archäologisches Korrespondenzblatt 26(2): 121–144.

Arnaud

Emery

(2000) Estimation et interpolation spatiale: Methodes deterministes et methodes geostatistiques. Paris: Hermes.

Ash

Orihel

(2007) Ash & Orihel’s Atlas of Human Parasitology. Chicago, IL: ASCP Press.

Bailenger

(1982) Parasitological and Functional Coprology. 4th edition. Bordeaux: J. Bailenger.

Beer

RJS

(1976) The relationship between Trichuris trichiura (Linnaeus 1758) of man and Trichuris suis (Schrank 1788) of pig. Research in Veterinary Science 20(1): 47–54.

10.

Bouchet

(1997) Intestinal capillariasis in neolithic inhabitants of Chalain (Jura, France). The Lancet 349(9047): 256.

11.

Bouchet

Baffier

Girard

, et al. (1996) Paléoparasitologie en contexte pléistocène: Premières observations à la Grande Grotte d’Arcy-sur-Cure (Yonne), France. Comptes rendus de l’Académie des sciences. Série 3, Sciences de la vie 319(2): 147–151.

12.

Bouchet

Harter

Le Bailly

(2003) The state of the art of paleoparasitological research in the Old World. Memorias do Instituto Oswaldo Cruz 98: 95–101.

13.

Bouchet

Pétrequin

Paicheler

, et al. (1995) Première approche paléoparasitologique du site Néolithique de Chalain (Jura, France). Bulletin de la Société de Pathologie Exotique 88: 265–268.

14.

Brumpt

(1936) Précis de Parasitologie II Cinquiéme Edition. Paris: Masson et Cie Editeurs.

15.

Buxó

Piqué

Saña

(2014) La Draga: Environmental archaeology. In: Smith

(ed.) Encyclopedia of Global Archaeology. New York: Springer, pp. 4345–4352.

16.

Côté

(2015) Apports de la paléogénétique à l’étude des helminthes gastro-intestinaux anciens. PhD Thesis, Université Bourgogne Franche-Comté.

17.

Côté

NML

Daligault

Pruvost

, et al. (2016) A new high-throughput approach to genotype ancient human gastrointestinal parasites. PLoS ONE 11(1): e0146230.

18.

Cressie

(1990) The origins of kriging. Mathematical Geology 22(3): 239–252.

19.

Cross

(1992) Intestinal capillariasis. Clinical Microbiology Reviews 5(2): 120–129.

20.

Dittmar

Araújo

Reinhard

(2011) Chapter 10: The study of parasites through time: Archaeoparasitology and paleoparasitology. In: Grauer

(ed.) A Companion to Paleopathology. New York: John Wiley & Sons, pp. 170–190.

21.

Dommelier

Bentrad

Paicheler

, et al. (1998) Parasitoses liées à l’alimentation chez les populations néolithiques du lac de Chalain (Jura, France). Anthropozoologica 27: 41–49.

22.

Dommelier-Espejo

(2001) Contribution à l’étude paléoparasitologique des sites néolithiques en environnement lacustre dans les domaines jurassien et péri-alpin. PhD Thesis, Université de Reims Champagne-Ardenne.

23.

Dommelier-Espejo

Pétrequin

(2016) Analyses paléoparasitologiques à Clairvaux et à Chalain. In: Pétrequin

Pétrequin

(eds) Clairvaux et le ‘Néolithique Moyen Bourguignon’. Les Cahiers de La MSHE Ledoux. Besançon: Presses Universitaires de Franche-Comté, pp. 1175–1192.

24.

Dufour

(2015) Synthèse de données et nouvelle contribution à l’étude des parasites de l’époque romaine, et apports méthodologiques de l’extraction des marqueurs au traitement des résultats. PhD Thesis, Université Bourgogne Franche-Comté.

25.

Dufour

Le Bailly

(2013) Testing new parasite egg extraction methods in paleoparasitology and an attempt at quantification. International Journal of Paleopathology 3(3): 199–203.

26.

Euzéby

(2002) Risques parasitaires liés aux déjections d’origine humaine et animale manipulées ou épandues: Le péril fécal et le problème de l’eau. Tampa, FL: Institut Romark pour la recherche médicale.

27.

Fugassa

Reinhard

Johnson

, et al. (2011) Parasitism of prehistoric humans and companion animals from Antelope Cave, Mojave County, Northwest Arizona. Journal of Parasitology 97(5): 862–867.

28.

Golvan

(1990) Atlas de Parasitologie. Paris: Le Léopard d’or.

29.

Gunn

Pitt

(2012) Helminth parasites. In: Gunn

Pitt

(eds) Parasitology: An Integrated Approach. Chichester: John Wiley & Sons, pp. 86–136.

30.

Höbig

Weber

Kehl

, et al. (2012) Lake Banyoles (northeastern Spain): A last glacial to Holocene multi-proxy study with regard to environmental variability and human occupation. Quaternary International 274: 205–218.

31.

Hüster-Plogmann

Leuzinger

(1995) Fischerei und Fischreste in der jungsteinzeitlichen Seeufersiedlung in Arbon (TG). Archäologie der Schweiz 18(3): 109–117.

32.

Kassai

(1999) Veterinary Helminthology. Oxford: Butterworth-Heinemann.

33.

Kaufmann

(1996) Parasites of sheep and goats In: Kaufmann

(ed.) Parasitic Infections of Domestic Animals. Basel: Birkhäuser, pp. 145–201.

34.

Koboldt

Zhang

Larson

, et al. (2012) VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Research 22(3): 568–576.

35.

Le Bailly

(2005) Evolution de la relation hôte/parasite dans le système lacustre nord alpins au Néolithique (3900-2900 BC), et nouvelles données dans la détection des paléoantigènes de Protozoa. PhD Thesis, Université de Reims Champagne-Ardenne, France.

36.

Le Bailly

(2011) Les parasites dans les lacs nord alpins au Néolithique (3900-2900 BC), et nouvelles données dans la détection des paléoantigènes de Protozoa. Sarrebruck: Editions Universitaires Européennes.

37.

Le Bailly

Araujo

(2016) Past intestinal parasites. Microbiology Spectrum 4(4): 143–154.

38.

Le Bailly

Bouchet

(2004) Etude parasitologique des coprolithes humains du site néolithique Arbon-Bleiche 3. In: Jacomet

Leuzinger

Schibler

(eds) Die Jungsteinzeitliche Seeufersiedlung Arbon-Bleiche 3: Umwelt Und Wirtschaft. Fraunfeld: Archäologie im Thurgau, Band 12, pp. 372–377.

39.

Le Bailly

Leuzinger

Bouchet

(2003) Dioctophymidae eggs in coprolites from Neolithic site of Arbon–Bleiche 3 (Switzerland). Journal of Parasitology 89(5): 1073–1076.

40.

Le Bailly

Leuzinger

Schlichtherle

, et al. (2005) Diphyllobothrium: Neolithic parasite? Journal of Parasitology 91(4): 957–959.

41.

Le Bailly

Leuzinger

Schlichtherle

, et al. (2007) ‘Crise économique’ au Néolithique à la transition Pfÿn-Horgen (3400 BC): Contribution de la paléoparasitologie. Anthropozoologica 42(2): 175–185.

42.

Handsaker

Wysoker

, et al. (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16): 2078–2079.

43.

Liao

Smyth

Shi

(2014) FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30(7): 923–930.

44.

Magny

(2004) Holocene climate variability as reflected by mid-European lake-level fluctuations and its probable impact on prehistoric human settlements. Quaternary International 113(1): 65–79.

45.

Mehlhorn

(2001) Encyclopedic Reference of Parasitology: Diseases, Treatment, Therapy. New York; Heidelberg: Springer Science + Business Media

46.

Mehlhorn

(2008) Encyclopedia of Parasitology: Biology, Structure, Function, Diseases, Treatment, Therapy. 3rf Edition. New York; Heidelberg: Springer.

47.

Mitchell

(2013) The origins of human parasites: Exploring the evidence for endoparasitism throughout human evolution. International Journal of Paleopathology 3(3): 191–198.

48.

Morales Hidalgo

Fontanals Torroja

Oms Arias

, et al. (2010) La chronologie du Néolithique ancien cardial du nord-est de la péninsule Ibérique. Datations, problématique et méthodologie. L’Anthropologie 114(4): 427–444.

49.

Moravec

(1981) Proposal of a new systematic arrangement of nematodes of the family Capillariidae. Folia Parasitologica 29(2): 119–132.

50.

Mowlavi

Makki

Heidari

, et al. (2015) Macracanthorhynchus hirudinaceus eggs in Canine Coprolite from the Sasanian Era in Iran (4th/5th Century CE). Iranian Journal of Parasitology 10(2): 245.

51.

Nozais

(1998) Maladies parasitaires et péril fécal: Les maladies dues aux helminthes. Bulletin de la Société de Pathologie Exotique 91: 416–422.

52.

Nozais

Datry

Danis

(1996) Traité de parasitologie médicale. Paris: Pradel.

53.

Palomo

Piqué

Terradas

, et al. (2014) Prehistoric occupation of Banyoles lakeshore: Results of recent excavations at La Draga Site, Girona, Spain. Journal of Wetland Archaeology 14(1): 58–73.

54.

Palomo

Piqué

Terradas-Batlle

, et al. (2013) Woodworking technology in the Early Neolithic site of La Draga (Banyoles, Spain). In: XXXIIIe rencontres internationales d’archéologie et d’histoire d’Antibes, Antibes 20–22 October 2013. France: Association pour la promotion et la diffusion des connaissances archéologiques, pp. 383–396.

55.

Perez-Obiol

Jalut

Julia

, et al. (2011) Mid-Holocene vegetation and climatic history of the Iberian Peninsula. The Holocene 21(1): 75–93.

56.

Pétrequin

(1988) Le Néolithique des lacs: Préhistoire des lacs de Chalain et de Clairvaux (4000-2000 av. J.-C.). Paris: Éditions Errance.

57.

Piqué

Palomo

Terradas

, et al. (2015) Characterizing prehistoric archery: Technical and functional analyses of the Neolithic bows from La Draga (NE Iberian Peninsula). Journal of Archaeological Science 5: 166–173.

58.

Price

(2000) Europe’s First Farmers. Cambridge: Cambridge University Press.

59.

Revelles

(2016) Archaeoecology of Neolithisation. Human-environment interactions in the NE Iberian Peninsula during the Early Neolithic. Journal of Archaeological Science: Reports. Epub ahead of print 13 February 2016. DOI: http://dx.doi.org/10.1016/j.jasrep.2016.02.004.

60.

Revelles

Burjachs

Van Geel

(2016) Pollen and non-pollen palynomorphs from the Early Neolithic settlement of La Draga (Girona, Spain). Review of Palaeobotany and Palynology 225: 1–20.

61.

Roberts

Janovy

(2009) Foundations of Parasitology. 8th Edition. Columbus, OH: McGraw-Hill Higher Education.

62.

Saña

(2011) La gestió dels recursos animals. In: Bosch

Chinchilla

Tarrús

(eds) El Poblat Lacustre Del Neolític Antic de La Draga. Excavacions 2000–2005. Girona: Monografies Del CASC, pp. 177–212.

63.

Saña

Bogdanovic

Navarrete

(2014) Taphonomic evaluation of the degree of historical representation of the archaeological bone samples in anaerobic versus aerobic environments: The Neolithic site of La Draga (Banyoles, Spain). Quaternary International 330: 72–87.

64.

Schibler

Chaix

(1995) L’évolution économique sur la base de données archéozoologiques. In: Stökli WE, Niffeler

Gross-Klee

(eds) Die Schweiz Vom Paläolithikum Zum Mitteralter (SPM 2: Neolithikum). Basel: SGU Verlag, pp. 97–120.

65.

Schibler

Jacomet

Hüster-Plogmann

, et al. (1997) Economic crash in the 37th and 36th centuries cal. BC in Neolithic lake shore sites in Switzerland: Postpalaeolithic Europe I. Anthropozoologica 25–26: 553–570.

66.

Soulsby

EJL

(1982) Helminths, Arthropods and Protozoa of Domesticated Animals. 7th Edition. London: Baillière Tindall.

67.

Spratt

(2006) Description of Capillariid Nematodes (Trichinelloidea:Capillariidae) Parasitic in Australian Marsupials and Rodents. Auckland, New Zealand: Magnolia Press.

68.

Tarrús

(2008) La Draga (Banyoles, Catalonia), an Early Neolithic Lakeside Village in Mediterranean Europe. Catalan Historical Review 1: 17–33.

69.

Tarrús

Chinchilla

Bosch

(1994) La Draga (Banyoles): Un site lacustre du Néolithique ancien cardial en Catalogne. Bulletin de la Société Préhistorique Française 91(6): 449–456.

70.

Tarrús

Saña

Chinchilla

, et al. (2006) La Draga (Banyoles, Catalogne): Traction animale à la fin du VIè millénaire? In: Pétrequin

Arbogast

Pétrequin

, et al (eds) Premiers Chariots, Premiers Araires. La Diffusion de La Traction Animale, En Europe Pendant Les IVè et IIIè Millénaires Avant Notre ère. Paris: CNRS Editions, pp. 25–30.

71.

Taylor

Coop

Wall

(2007) Veterinary Parasitology. Oxford; Ames, IA: Blackwell Publishing.

72.

Thienpont

Rochette

Vanparijs

OFJ

(1979) Diagnosing Helminthiasis through Coprological Examination. Beerse: Janssen Research Foundation.

73.

Tresset

Vigne

(2011) Last hunter-gatherers and first farmers of Europe. Comptes Rendus Biologies 334(3): 182–189.

74.

Zammit

(2005) Les conséquences écologiques de la néolithisation dans l’histoire humaine. Bulletin de la Société Préhistorique Française 102(2): 371–379.