Blood puncture as a nondestructive sampling tool to obtain DNA in frogs: comparison of protocols and survival analysis

Abstract

In molecular biology studies of Anura, nondestructive methods to obtain genetic material are needed as alternatives to toe clipping. This work evaluates a nondestructive method for sampling DNA from blood puncture, comparing the performance of three different extraction protocols (Qiagen Kit, Salting-out and Chelex). We collected 134 individuals of Eleutherodactylus johnstonei, extracting blood via puncture of the medial vein using commercial-grade glucometer lancets. We extracted 100-1880 ng DNA, finding no differences between the extraction protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S mitochondrial gene. Amplification was successful for the three extraction protocols, although Chelex showed better performance, making it the most recommendable protocol for extraction of DNA from blood. The resulting sequences corresponded to those registered in the GenBank for this species. Additionally, we found no significant differences in survival or weight change between the individuals that were manipulated and a control group (mean survival 66.7% treated, 62.9% untreated). Data reveal that blood samples obtained by puncture are a convenient alternative to other tissues (phalange, buccal swab, liver) that have traditionally been used as DNA sources for anurans. The technique is applicable to small and large species, covering most anuran diversity, provides enough DNA for many genetic applications and produces no noticeable effect on the survival or performance, given that it does not affect the motor parts or the dexterity of the animals.

Inside the Redbox: Applications of haematology in wildlife monitoring and ecosystem health assessment

“…Weber et al, 2010), FTA® cards (e.g. Mendoza et al, 2012) or PAX gene blood tubes until analysis (e.g. Thach et al, 2003).…”

Skin swabbing of amphibian larvae yields sufficient DNA for efficient sequencing and reliable microsatellite genotyping

“…Recently, several alternatives have been proposed for adults, such as transferring epithelial cells from the dorsal side of salamanders to FTA cards (Davis et al, 2002; but this method requires a cloning step), blood puncture (Mendoza et al, 2012), buccal swabs (Broquet et al, 2007a;Gallardo et al, 2012;Pidancier et al, 2003) or skin swabs (Prunier et al, 2012). The last one, established only for adults, is particularly advantageous, as it allows minimizing the handling time, stress and potential injuries induced on the animal compared to buccal swabbing (Pichlmüller, unpublished data), especially for small individuals.…”

Effects of Tail Clipping on Larval Performance and Tail Regeneration Rates in the Near Eastern Fire Salamander, Salamandra infraimmaculata

“…Researchers may also clip amphibian body parts for mark-recapture studies [5,6], stable isotope analysis [7,8],or for obtaining tissue sample for genetic studies. Invasive and non-invasive techniques for sampling amphibian DNA include: tail clipping [9], toe clipping [10], blood puncture [11], skin swabbing [12], and buccal swabbing [13]. The applicability and efficiency of these methods vary among species but most are not suitable for sampling DNA from early development larval stages due to their small size and handling sensitivity.…”