Mitochondrial and nuclear gene sequences have been widely used in species identification, population genetics and phylogeny of tapeworms (Caira and Jensen, 2017). They could provide important basis for the identification and classification of new tapeworm species, as well as for the accurate diagnosis of tapeworm disease, molecular epidemiological investigation and the formulation of preventive measures (Le et al., 2002). Owing to maternal inheritance, a lack of recombination and a fast rate of evolution, mitochondrial genome become a useful marker for population studies, species identification and phylogenetics (Li et al., 2019).
Chinese softshell turtle Pelodiscus sinensis, with high nutrition and medicinal value, is an excellent tonic and favored by consumers in China (Jia et al., 2005). In order to meet the market demand, the artificial breeding of Chinese softshell turtle develops rapidly. It has become an important commercial farmed aquatic species, and the annual output exceeds 300,000 tons in China since 2012 (data from the China Fishery Statistical Yearbook 2012). During a clinical sampling process of Chinese softshell turtle, we discovered and collected a tapeworm from the intestinal tract in the one (Fig. 1).
Tapeworms (Cestoda) are one of the major groups in the flatworms (Platyhelminthes: Neodermata). Tapeworms, with a cosmopolitan distribution, form a diverse group with approximately 5000 species parasitizing humans, domestic and other animals (Caira and Jensen, 2017). Tapeworms are currently classified in 19 orders, and there are about 1000 species parasitize elasmobranchs, almost 500 in teleosts as adults and four species from turtles (Caira and Jensen, 2017; Scholz and Kuchta, 2017). The adults of tapeworms parasitize in the digestive tract of vertebrates (mainly in the intestine), whereas larvae (metacestodes) in different organs and body cavity of invertebrates and vertebrates. The larvae of tapeworms are more harmful than adults, because larvae might migrate in internal organs of their intermediate hosts (Dick et al., 2006). Comparing to some pathogens, cestode-infected mortality is relatively minor in aquaculture (Scholz et al., 2021), however some tapeworms have a serious pathological effect on their hosts, resulting in slow growth, decreased resistance, squished guts of the fish including those in aquaculture (Scholz, 1999; Shinn et al., 2015). Invasive species of fish cestode would be a threat to cultured and economically important fish (Scott and Grizzle, 1979; Hoole et al., 2001; Kuchta et al., 2018). Bothriocephalus gowkongensis, as an indigenous fish disease, was firstly found in southern China, but now it has spread all over China and been found in United States, some countries of West Asia and Europe (Scott and Grizzle, 1979; Andrews et al., 1981; Shokrolahi et al., 2016). Due to the increased popularity of consumption of sashimi and the rapid development of aquaculture and global trade, fish-borne tapeworms appear more common (Scholz and Kuchta, 2016; Scholz et al., 2019). Raw meat food culture may result in parasitic disease in some areas and tapeworm disease has become a new or re-emerging infectious disease which endangers human health.
In China, the parasitism study of tapeworm was mainly in fish, and there were few parasitism reports in testudinate. It is necessary to identify species of tapeworm and advance warning about parasitic infection. The identification of tapeworm species is generally based on the traditional taxonomic methods and molecular taxonomic methods. Traditional morphological methods cannot identify tapeworms that are morphologically similar but genetically different, leading to some taxonomic controversies. Molecular classification, is simple, rapid and informative, which can be used as a powerful tool for identification and research the samples whose part is incomplete or missing.
In this study, the tapeworms which parasitize in humans, pets and fish were collected and then their mitochondrial genomes were sequenced respectively. According to the nucleotide and amino acid similarity of mitochondrial genomes, Spirometra erinaceieuropaei, Dipylidium caninum and Digramma interrupta were identified. We analyzed interspecific differences in the mitochondrial genome among four tapeworms by comparing codon usage, Ka/ Ks, and p-distance. Using protein-coding genes from the complete mitochondrial genome, the phylogenies of 66 tapeworms have been reconstructed (Raissi et al., 2021). In order to identify tapeworm quickly and efficiently, universal primers were designed according to highly homologous regions of mitochondrial genes in four tapeworms. Specific PCR primers for identification were designed according to the specific regions in the mitochondrial genomes of Pelodiscus sinensis. These primers enable easy, rapid, and accurate species identification at the molecular level. The methods established in this study can detect tapeworm from Chinese softshell turtle rapidly and accurately, providing technical support for epidemiological investigation, early clinical diagnosis, exports and imports quarantine of live aquatic animal and prevention of tapeworm.