Untitled

Abstract

Museum specimens of Ligula (Pseudophyllidea, Ligulidae), a fish parasite tapeworm, that have been preserved in ethanol or fixed permanently in formalin up to 24 years were used for DNA extraction and molecular characterization. DNA was amplified via PCR from samples collected from different fish hosts that lived in both salt and fresh water bodies in the Chinese Qinghai-Tibet Plateau, Russia, and England. Phylogenetic analyses based on partial nucleotide sequences of the 5'-end of nuclear 28S rRNA gene and the mitochondrial cytochrome c oxidase subunit I (COI) gene support the morphologically based taxonomy that groups the Chinese Ligula within the same species as their Europe counterpart: Lingula intestinalis. No nucleotide variation was detected in either the 28S rRNA gene or the COI gene among the seven plerocercoid samples, suggesting a considerable genetic homogeneity among Ligula from different regions. Our results show that geographic isolation, affinity of hosts, and host habitats are not reliable taxonomic criteria for Ligula classification. Our data also indicate a low genetic diversity in the Ligula prevalent in China and in Europe. Our experiments demonstrate that endogenous DNA from specimens that were subjected to permanent formalin fixation can be routinely amplified from parasitic tapeworms, suggesting that fixation time in formalin may not be a critical factor affecting DNA degradation in such museum specimens.

The use of museum specimens with high-throughput DNA sequencers

“…Unlike other aDNA approaches using materials exposed to the environment for hundreds or thousands of years, researchers utilizing museum specimens have been able to incorporate nuclear loci in their work since the mid-1990s (e.g., Roy et al, 1994; Taylor et al, 1994; Mundy et al, 1997) in the form of microsatellite allele frequency data. Improved laboratory methods led to the use of nuclear DNA (nDNA) sequences in the early 2000s (Li et al, 2000; Vallianatos et al, 2002; de la Herrán et al, 2004), and the number of studies using multiple loci has increased since then. Research using many museum samples (> 100) has also become more common (e.g., Godoy et al, 2004; Miller et al, 2006).…”

Sequence and Haplotype Analyses of Ligula intestinalis in Acanthobrama marmid (Cyprinidae) in Turkey

“…Studies on L. intestinalis species are mostly anatomical-morphologically based in Turkey [ 47 ], and the molecular studies are limited [ 48 ]. However, genetic markers such as ITS and CO1 have been frequently used in recent years to accurately determine the taxonomic positions of organisms [ 20 ]. The traditional classification of ligulid cestodes Ligula (Bloch, 1782) and Digramma (Cholodkovsky, 1914) is a controversial issue.…”

“…Several studies have been carried out on the molecular identification of L. intestinalis infection, which causes serious yield and economic losses in fish facilities. Li et al [ 20 ], certain nucleotides of 393 bp for the CO1 gene of seven L. intestinalis samples were directly sequenced from each sample. Interestingly, no nucleotide variation was detected in the sequences of the CO1 gene among the seven Ligula samples studied.…”

“…Molecular techniques are used to identify species that cannot be distinguished morphologically according to their genetic differences [ 19 ]. Nuclear 28S rDNA and mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) gene sequencing of L. intestinalis are used for molecular identification of the parasite [ 20 ]. In the case that both the existing fish species in aquatic ecosystems and the species that are grown intensively in net cages are exposed to this parasitic effect, molecular identification is important avoid economic depreciation and to take the necessary measures [ 18 ].…”