Shake and stew: a non‐destructive PCR‐ready DNA isolation method from a single preserved fish larva

Abstract

A rapid non-destructive alternative to isolate DNA from an individual fish larva is presented, based on the suspension of epithelial cells through vortex forces, and the release of DNA in a heated alkaline solution. DNA from >6056 fish larvae isolated using this protocol has yielded a high PCR amplification success rate (>93%), suggesting its applicability to other taxonomic groups or sources when tissue amount is the limiting factor.

DNA barcoding and morphological identification of neotropical ichthyoplankton from the Upper Paraná and São Francisco

“…With the unfolding of the Brazilian Barcode of Life Project (BrBol; http://brbol.org/pt-br/), ichthyoplankton identification by DNA barcoding may be applicable to all river basins in Brazil providing unprecedented information on fish reproduction effects due to damming, detection of species‐specific reproduction sites, recovery of hidden fish biodiversity as well as benefiting environmental and fish management conservation in the neotropics. Moreover, the developing of non‐destructive DNA extraction from larvae (Alvarado Bremer et al , ) will allow the preservation of larvae vouchers and the performance of re‐evaluation of diagnostic morphological or meristic characteristics, helping integrate morphological and molecular ichthyoplankton taxonomy. The accuracy of species identification via DNA barcoding, however, is ultimately dependent on the accuracy of morphology‐based taxonomic assessment of voucher specimens whose barcodes are available in databases such as GenBank and BOLD.…”

Genotyping Live Fish Larvae: Non-Lethal and Noninvasive DNA Isolation from 3–5 day Old Hatchlings

“…Each larva was transferred into a 1.5-mL microcentrifuge tube (Eppendorf, Hamburg, Germany) containing 200 L sterile 20 ppt Instant Ocean artificial seawater (Spectrum Brands, Blacksburg, VA), using a new 3.0-mL disposable plastic transfer pipette with the tip cut by a sterile scalpel to produce a wide-mouth pipette (20). This first bath minimizes potential cross-contamination with cells from other fish (14). Each larva was then transferred into a second pre-labeled sterile 1.5-mL microcentrifuge tube filled with 200 L sterile 20 ppt artificial seawater for incubation (see Figure 1 inset) using a new pre-cut disposable wide-mouth pipette.…”

“…Preliminary attempts to capture aqueous DNA from the 200 L of seawater that held the live larvae were conducted using a modified shake and stew DNA isolation method (14), omitting the shaking step. PCR experiments targeting two mitochondrial DNA (mtDNA) segments, namely the cytochrome c oxidase subunit I gene ( COI ) and control region I (CR-I), using the fish universal primers and conditions as described in References 21 and 22, respectively, resulted in a very low amplification success rate of 20% for COI and failed for CR-I.…”

“…First, dissecting small fin biopsies (12) from very small specimens (i.e., zebrafish embryos) is labor-intensive and requires highly technical skills and equipment. Second, minimally invasive sampling methodologies amenable to high-throughput approaches (14,15) have only been shown to be effective on fishes older than 71 days post-fertilization (dpf), as handling of the younger, more delicate specimens is often lethal. Accordingly, a non-lethal and non-invasive genotyping protocol that can be used to genotype large numbers of live early fish larvae is still needed.…”

Use of mucus as a non‐invasive sampling method for DNA barcoding of stingrays and skates (batoid elasmobranchs)

“…However, the use of this tool is still very dependent on tissue sampling ( e.g ., muscle, scale and blood), usually obtained from dead animals in fish markets and fishing vessels or from live animals, with many challenges involving the angling and handling processes, such as physical injuries and pathology due to hook removal or retention, and post‐release survivorship (Danylchuck et al ., ). On the other hand, less invasive procedures for DNA extraction has been tested in a wide variety of vertebrates (including large species of elasmobranchs) to assess information on genetic population, forensic identification, sex determination and genetic diversity (Alvarado Bremer et al ., ; Hilsdorf et al ., ; Kashiwagi et al ., ; Lieber et al ., ; Presti et al, ). Such outcomes demonstrated the feasibility of non‐destructive approaches to DNA extraction and its use in different analyses, at a low cost and with a high degree of sensitivity and confidence.…”