Genomic paleoparasitology has traced the presence of Taenia asiatica to ancient Iran (Sasanian Empire, 2nd century CE – 6th century CE)
Dating and origin of the sample
Eight coprolites among the samples belonged to the Achaemenid era (6th century BCE – 3rd century BCE) and twenty-two to the Sasanian era (2nd century CE-6th century CE). Using visible features and microscopic images, six donkeys, seven herbivores and two birds were identified as the origins of the samples. The remaining fifteen coprolites were labeled as samples of unknown origin (result from supplementary file).
Examination of a thousand microscopic slides of rehydrated paleofeces revealed seven different species of helminths belonging to Ascaris sp., strongyles, Trichuridae, Taenia sp., Parascaris sp., fasciole sp., and some unknown nematode larvae (Fig. 2). Of thirty samples, eleven (36.6%) contained helminth eggs and larvae, including strongyle eggs and larvae in five (16.6%), roundworm eggs in four (13.3% ), Taenia spp. eggs in four (13.3%), Trichuris spp. eggs in three (10%), fasciole sp. eggs in one sample (3.3%) and unrecognized nematode larvae in one sample (3.3%) (Table 1). Strongyles were detected in all coprolites except those of birds. Trichuridae was only found in the faeces of the unidentified host. Taenia spp. eggs were seen in the unknown and donkey samples and fasciole sp. eggs in the faeces of donkeys. In total, helminths were found in 66.6% donkeys, 28.5% herbivores and 26.6% coprolites of unknown origin.
Metagenomic analysis of helminths
Various phyla of microorganisms in thirty samples were documented by metagenomic analysis. Of the coprolites studied, helminth eggs were found in eight samples (Fig. 3, Supplementary File result and Supplementary File parasite reads).
NGS has identified DNA representing Parascaris equorum (Equorum Supplementary File), four species of strongyles, including Cylicocyclus insignis, Cylicocyclus nassatus, Coronocyclus coronatus, and Cylicostephanus goldi) in the paleofeces of herbivores, donkeys and unknown hosts. Additionally, NGS has identified Haemonchus contortus and Caenorhabditis elegans in herbivores and paleofeces of unknown origin, and Toxocara canis in a donkey coprolite (Table 1). In C.goldi reads, 106,308 indels and 82,299 SNPs were detected with a read depth of up to fifty-eight (Supplementary Fig. s1). The data for the CGOC_scaffold0000014 variants is shown in Fig. 4. The VS. Goldi bed, separated by the PMD tools (Supplementary Fig. s2), showed less frequency than the total aligned reads (Fig. 5). The statistics of the total (damaged and undamaged) and reads with substitutions aligned to the C. goldi genome are summarized in the total supplementary file and the damage supplementary file. For example, 65,613 reads were mapped to the 2526 nucleotides CGOC_contig0002042, of which 1044 were damaged. According to this file, the uniformity of the distribution of reads to the different genomic parts was also apparent.
four tapeworms, Fuhrmannetta malakartis, Spirometra erinaceieuropaei, Spirometra mansonoides, and T. asiatica have been identified in the paleofeces, and Fasciola hepatic was the only trematode detected.
In general, C. goldi was the most common species (13.3%), followed by C.elegans (ten%), H. contortus, C. nassatus (6.6%), P. equorum, T. asiatica, T. canis, S. erinaceieuropaei, C. coronatus, and S. mansonoids (3.3%). NGS identified four helminths, H. contortus, F. malakartis, S. erinaceieuropaei, and S. mansonoidswhile microscopy only identified relevant genera.
As shown in Fig. s3 supplemental (more details available in asiatica snp supplemental file), 14 SNPs in ninety T. asiatica lit (additional file asiatica readings) exactly matching the SNPs linked to T. asiatica genome. This was also confirmed by the .fsa file (Supplementary file asiatica contig), which showed 27 SNPs in common with J. Asian (Supplementary Fig. s4). In a separate analysis, the 90 reads and one T. asiatica contig (TASK contig0004697) have been aligned to a T. saginata scaffold (Scaffold00355), and while the alignment was less precise than the previous analysis (less alignment length), the reads shared similar SNPs with the T. asiatica genome. (Supplementary Fig. s5).
Origin of specimens
The technical protocol for detection of fecal origin uses the outer layer of coprolites, which shows the cell layer of the host intestine. However, we could not analyze them separately due to the limited number of samples. Therefore, here we report vertebrate DNA alongside microbes (unpublished data) in twenty-five paleofeces. The remaining five samples did not contain enough data.
Technically, finding vertebrate genomic reads in herbivorous animals like donkeys cannot attribute the specimen to that animal because they may have consumed various animal feces, including humans, while grazing. Based on DNA analysis followed by microscopic and botanical findings, the possible interactions and the host-parasite relationship at that time could be imagined (Fig. 6). (Origin of additional file).