Muscle biopsy technique for electrophoresis analysis of fish from the genus Brycon

Abstract

Protein and mitochondrial DNA have been used as molecular markers to assess variability in stock identification studies of fishes. Protein and mtDNA used for electrophoretic analysis are extracted from tissues, which often leads to death of the individuals. In this study, we present a skeletal muscle biopsy procedure to extract mitochondrial DNA that does not require specimen sacrifice. Eighty pirapitinga-do-sul (Brycon opalinus) were biopsied by the present technique, with no mortalities recorded. Total DNA was extracted from muscle and digested by restriction enzymes ApaI and HpaI. The mtDNA fragment patterns were hybridized with 32P-labeled pirapitinga-do-sul mtDNA probes. The described technique is simple and may be useful in protocols requiring tissue extraction for DNA and protein analyses without loss of the individual investigated.<br>Variabilidade apresentada por proteínas e pelo DNA mitocondrial tem sido utilizada em estudos populacionais de peixes. A obtenção de tecido para análise eletroforética de proteína e DNA mitocondrial muitas vezes requer a morte dos indivíduos. No presente trabalho, nós apresentamos uma técnica modificada para biopsia de tecido muscular sem a morte dos animais. Oitenta Pirapitingas-do-sul (Brycon opalinus) foram biopsiadas pela presente técnica, não tendo sido observado mortalidade. O DNA total foi extraído do tecido muscular e digerido com as enzymas de restrição ApaI e HpaI. Os fragmentos de DNA mitocondrial foram hibridizados com sondas de DNA mitocondrial de pirapitinga-do-sul marcadas com P32. A técnica descrita pode ser útil em estudos genéticos em peixes

Identification and characterization of microsatellites loci in Brycon opalinus (Cuvier, 1819) (Characiforme, Characidae, Bryconiae)

“…Tissues were obtained from adipose fin biopsy stored in 95% ethanol at room temperature until use (Hilsdorf et al . 1999).…”

“…Tissues were obtained from adipose fin biopsy stored in 95% ethanol at room temperature until use (Hilsdorf et al 1999). Total DNA was extracted by the protocol described by Taggart et al (1992).…”

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales

“…However, non‐destructively DNA isolation is desirable, especially when large populations or threatened species have to be analyzed. Although DNA can be successfully obtained from muscle (H ilsdorf et al 1999) or blood samples of fish (C ummings and T horgaard 1994; E stoup et al 1996; M artinez et al 1998) without the sacrifice of the animals, adult individuals of some species are often too small to be effectively sampled by muscle biopsy or drawing blood.…”

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, 2014). 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, 2014;Hilsdorf et al, 1999;Kashiwagi et al, 2015;Lieber et al, 2013;Presti et al, 2013). 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.…”