Dataset Information

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus):A Preliminary Protocol.

ABSTRACT: This study describes a successful protocol for establishing cell lines from the threatened Triturus cristatus in terms of collection, preparing, establishing, cryopreserving, thawing and quality checking. Different parameters such as media, media change, fresh vs. cryopreserved tissue and seeding density were tested to optimize culture conditions for this species. With fresh tissue, no considerable differences in the use of two different media were found, but with cryopreserved tissue, a combination of ITS (insulin/transferrin/selenite) and 2-mercaptoethanol had a positive effect on growth. Real-time measurements on the cell lines were used, for the first time in amphibian cells, to investigate the effect of different treatments such as media change with or without washing. Media change had a positive impact on the cells, whereas the effect was negative when combined with washing. It is concluded that establishment of cell lines is possible from the great crested newt, especially when using fresh tissue, but much more challenging if the tissue has been cryopreserved. Real-time measurement during cell culture is a useful tool to visualize the sensitivity of amphibian cells during different culture treatments.

SUBMITTER: Strand J

PROVIDER: S-EPMC7912844 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (<i>Triturus cristatus</i>):A Preliminary Protocol.

Strand Julie J Callesen Henrik H Pertoldi Cino C Purup Stig S

Animals : an open access journal from MDPI 20210201 2

This study describes a successful protocol for establishing cell lines from the threatened <i>Triturus cristatus</i> in terms of collection, preparing, establishing, cryopreserving, thawing and quality checking. Different parameters such as media, media change, fresh vs. cryopreserved tissue and seeding density were tested to optimize culture conditions for this species. With fresh tissue, no considerable differences in the use of two different media were found, but with cryopreserved tissue, a ...[more]

PMID: 33535698

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Project description:Environmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species-specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and "metabarcoding" have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high-throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

Project description:BACKGROUND:The rapid radiation of crested newts (Triturus cristatus superspecies) comprises four morphotypes: 1) the T. karelinii group, 2) T. carnifex - T. macedonicus, 3) T. cristatus and 4) T. dobrogicus. These vary in body build and the number of rib-bearing pre-sacral vertebrae (NRBV). The phylogenetic relationships of the morphotypes have not yet been settled, despite several previous attempts, employing a variety of molecular markers. We here resolve the crested newt phylogeny by using complete mitochondrial genome sequences. RESULTS:Bayesian inference based on the mitogenomic data yields a fully bifurcating, significantly supported tree, though Maximum Likelihood inference yields low support values. The internal branches connecting the morphotypes are short relative to the terminal branches. Seen from the root of Triturus (NRBV = 13), a basal dichotomy separates the T. karelinii group (NRBV = 13) from the remaining crested newts. The next split divides the latter assortment into T. carnifex - T. macedonicus (NRBV = 14) versus T. cristatus (NRBV = 15) and T. dobrogicus (NRBV = 16 or 17). CONCLUSIONS:We argue that the Bayesian full mitochondrial DNA phylogeny is superior to previous attempts aiming to recover the crested newt species tree. Furthermore, our new phylogeny involves a maximally parsimonious interpretation of NRBV evolution. Calibrating the phylogeny allows us to evaluate potential drivers for crested newt cladogenesis. The split between the T. karelinii group and the three other morphotypes, at ca. 10.4 Ma, is associated with the separation of the Balkan and Anatolian landmasses (12-9 Ma). No currently known vicariant events can be ascribed to the other two splits, first at ca. 9.3 Ma, separating T. carnifex - T. macedonicus, and second at ca. 8.8 Ma, splitting T. cristatus and T. dobrogicus. The crested newt morphotypes differ in the duration of their annual aquatic period. We speculate on the role that this ecological differentiation could have played during speciation.

Dataset Information

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus):A Preliminary Protocol.

Publications

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (<i>Triturus cristatus</i>):A Preliminary Protocol.

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