GENETICS: FISH
Smalley J. V., Campanella J. J. (2005): Buccal swabbing and extraction of high quality sunfish (Lepomis) DNA for use in PCR analysis. BioTechniques 38: 189-190.
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We have developed a field sampling method for obtaining high quality DNA from sunfish (Lepomis) that employs a variation on the buccal swab method and results in the collection of DNA suitable for PCR amplification and polymorphic analysis. The ease of this method—coupled with its scalability to include large sample sizes, its ambient temperature of field storage and preservation, and its simplicity of sample transport—should make it applicable to field-oriented population and conservation genetic studies involving a wide range of fish.
Le Vin A. L., Adam A., Tedder A., Arnold K. E., Mable B. K. (2011): Validation of swabs as a non‐destructive and relatively non‐invasive DNA sampling method in fish. Molecular Ecology Resources 11: 107-109.
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Non‐destructive methods of collecting DNA from small fish species can be problematic, as fin clips can potentially affect behaviour or survivorship in the wild. Swabbing body mucus may provide a less invasive method of DNA collection. However, risk of contamination from other individuals in high density groups could give erroneous genotyping results. We compared multilocus microsatellite genotypes from the same individuals when collected at low and high density and compared this with fin clips. We found no differences between these categories, with a genotyping error rate of 0.42%, validating the use of body mucus swabbing for DNA collection in fish.
Reid S. M., Kidd A., Wilson C. C. (2012): Validation of buccal swabs for noninvasive DNA sampling of small‐bodied imperiled fishes. Journal of Applied Ichthyology 28: 290-292.
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Swab samples were collected from channel darter using a 2 mm diameter non-sterile brush (Microbrush International, Grafton, WI) and from redside dace using a 6 mm diameter sterile brush (Qiagen Buccal Collection Brushes, Toronto, Ontario, Canada). Brushes were gently rubbed inside the mouths of individual fish for 5-s.
Lieber L., Berrow S., Johnston E., Hall G., Hall J., Gubili C., Sims D. W., Jones C. S., Noble L. R. (2013): Mucus: aiding elasmobranch conservation through non-invasive genetic sampling. Endangered Species Research 21: 215-222.
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Large-scale genetic sampling by non-invasive methods is of vital importance for the conservation of vulnerable or elusive species. In the marine environment, non-invasive genetic sampling can provide a powerful alternative to conventional biopsies. We designed and implemented mucus swabbing for a free-ranging elasmobranch, thereby demonstrating the utility of this method in the field. We report the first attempt at mucus collection from 30 plankton-feeding basking sharks Cetorhinus maximus from 3 spatially distinct ‘hotspots’ in Irish waters. C. maximus DNA was successfully extracted and verified using DNA barcoding of the mitochondrial DNA cytochrome c oxidase 1 gene (99% sequence similarity) and basking shark species-specific multiplex PCRs derived from the nuclear ribosomal internal transcribed spacer 2 locus. Mitochondrial control region sequencing (1086 bp) showed that Irish samples were dominated by 2 haplotypes previously found to be globally distributed. Additionally, 1 novel haplotype was defined from western County Kerry. On-going genetic tagging will eventually provide more accurate estimates of global basking shark population structuring, abundance and behavioural ecology.
Kashiwagi T., Maxwell E. A., Marshall A. D., Christensen A. B. (2015): Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success. PeerJ 3: e1188.
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Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.
Khanam T., Davie A., McAndrew B., Penman D. (2016): DNA sampling from mucus in the Nile tilapia, Oreochromis niloticus: minimally invasive sampling for aquaculture-related genetics research. Aquaculture Research 47: 4032-4037.
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In this study, the yield and quality of DNA from skin mucus, buccal mucus and fin samples of Nile tilapia, Oreochromis niloticus L., were assessed using two types of swabbing (brush and filter paper), two DNA extraction methods (salt precipitation and HotSHOT) and two different types of molecular marker (microsatellite and single nucleotide polymorphism, SNP).
Breacker C., Barber I., Norton W. H., McDearmid J. R., Tilley C. A. (2017): A low-cost method of skin swabbing for the collection of DNA samples from small laboratory fish. Zebrafish 14: 35-41.
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Fin clipping of live fish under anesthesia is widely used to collect samples for DNA extraction. An alternative, potentially less invasive, approach involves obtaining samples by swabbing the skin of nonanesthetized fish. However, this method has yet to be widely adopted for use in laboratory studies in the biological and biomedical sciences. Here, we compare DNA samples from zebrafish Danio rerio and three-spined sticklebacks Gasterosteus aculeatus collected via fin clipping and skin swabbing techniques, and test a range of DNA extraction methods, including commercially available kits and a lower-cost, in-house method. We verify the method for polymerase chain reaction analysis, and examine the potential risk of cross contamination between individual fish that are netted together. We show that swabbing, which may not require the use of anesthesia or analgesics, offers a reliable alternative to fin clipping. Further work is now required to determine the relative effects of fin clipping and swabbing on the stress responses and subsequent health of fish, and hence the potential of swabbing as a refinement to existing DNA sampling procedures.
Colussi S., Campia V., Righetti M., Scanzio T., Riina M. V., Burioli E. A.., Foglini C., Ingravalle F., Prearo M., Acutis P. L. (2017): Buccal swab: A tissue sampling method for refinement of experimental procedures involving rainbow trout. Journal of Applied Ichthyology 33: 515-519.
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Buccal swabbing is a minimally invasive method to obtain DNA and biological material from humans and animals, including fish. Reports on buccal swabbing in fish are few and only for a limited number of species. Rainbow trout (Oncorhynchus mykiss) is an important animal model and because the yield of DNA may vary among and within different species in individuals of different sizes, it was selected as useful to optimize the buccal DNA collection in this species. Different storage methods were evaluated, aimed at DNA preservation by limiting DNA degradation and bacterial growth, using commonly available and inexpensive reagents. DNA quality was also tested by amplification of a single‐copy nuclear gene and a mitochondrial gene. The results suggest that ethanol is the best storage choice for buccal swab sampling in fish genetic studies, as well as suitable for small‐bodied rainbow trout.
Taslima K., Taggart J. B., Wehner S., McAndrew B. J., Penman D. J. (2017): Suitability of DNA sampled from Nile tilapia skin mucus swabs as a template for ddRAD-based studies. Conservation Genetics Resources 9: 39-42.
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For conservation, ethical and welfare reasons, minimally invasive DNA sampling is becoming increasingly important in animal genetics studies. Skin mucus swabs have been used as a source for fish DNA in PCR-based studies, but their suitability for high-throughput sequencing has not been examined. DNA was extracted from skin mucus, muscle and fin samples of two Nile tilapia and used in double-digest restriction-site associated DNA (ddRAD) sequencing. Approximately 16,000 and 9000 RAD loci were retrieved from de novo and reference-based analyses respectively. The numbers of RAD loci retrieved from three tissue sources were similar, with >83 % being shared among all three samples. Minor bacterial contamination was detected in a single muscle sample (0.07 % of the total RAD loci). The data indicates that DNA derived from skin mucus can be reliably used for ddRADseq and is likely to be applicable in other similar genomic analyses.
Berger C. A., Preisfeld A. (2018): DNA isolation of mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) as an alternative method to conventional fin‐clipping. Journal of Applied Ichthyology 34: 1126-1130.
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The first objective of the actual project was to investigate if mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) is suitable for DNA extraction. In the second step, it was analyzed if the avoidance of environmentally hazardous chemicals could lead to sufficiently pure DNA isolation. Finally, it was examined if samples taken from different fish body parts yielded different grades of purity of the extracted DNA. Mucus was collected from 552 individuals of S. trutta and T. thymallus from 2012 to 2015. DNA was extracted with two different kits (E.Z.N.A.® Insect DNA Kit and my‐Budget DNA Mini Kit) and two different swabs (my‐Budget roughened laminated sterile cotton swabs and common autoclaved cotton swabs). The results showed that the my‐Budget DNA Mini Kit in combination with the my‐Budget roughened laminated sterile cotton swabs was the most suitable method. It is a non‐destructive and swift technique that results in the concentration of DNA being high and pure.
Li Y., Evans N. T., Renshaw M. A., Jerde C. L., Olds B. P., Shogren A. J., Deiner K., Lodge D. M., Lamberti G. A., Pfrender M. E. (2018): Estimating fish alpha-and beta-diversity along a small stream with environmental DNA metabarcoding. Metabarcoding and Metagenomics 2: 24262.
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Environmental DNA (eDNA) metabarcoding has been increasingly applied to biodiversity surveys in stream ecosystems. In stream networks, the accuracy of eDNA-based biodiversity assessment depends on whether the upstream eDNA influx affects downstream detection. Biodiversity assessment in low-discharge streams should be less influenced by eDNA transport than in high-discharge streams. We estimated α- and β-diversity of the fish community from eDNA samples collected in a small Michigan (USA) stream from its headwaters to its confluence with a larger river. We found that α-diversity increased from upstream to downstream and, as predicted, we found a significant positive correlation between β-diversity and physical distance (stream length) between locations indicating species turnover along the longitudinal stream gradient. Sample replicates and different genetic markers showed similar species composition, supporting the consistency of the eDNA metabarcoding approach to estimate α- and β-diversity of fishes in low-discharge streams.
Domingues R. R., Garrone‐Neto D., Hilsdorf A. W., Gadig O. B. (2019): Use of mucus as a non‐invasive sampling method for DNA barcoding of stingrays and skates (batoid elasmobranchs). Journal of Fish Biology 94: 512-516.
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In this study we tested the use of mucus from five species of Neotropical marine batoid elasmobranchs to extract genomic DNA for barcoding and phylogenetic analysis. The DNA from all individuals sampled was successfully amplified and sequenced for molecular barcode, allowing 99–100% accuracy to the species level. This method proved to provide reliable and good‐quality DNA for barcoding and phylogenetic analysis of Neotropical elasmobranchs, through rapid handling and with low disturbance to animals.
Baerwald M. R., Goodbla A. M., Nagarajan R. P., Gootenberg J. S., Abudayyeh O. O., Zhang F., Schreier A. D. (2020): Rapid and accurate species identification for ecological studies and monitoring using CRISPR‐based SHERLOCK. Molecular Ecology Resources 20: 961-970.
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One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of “proof of principle” experiments to characterize SHERLOCK’s ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK’s ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.
Tilley C. A., Gutierrez H. C., Sebire M., Obasaju O., Reichmann F., Katsiadaki I., Barber I., Norton W. H. (2020): Skin swabbing is a refined technique to collect DNA from model fish species. Scientific Reports 10: 18212.
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Model fish species such as sticklebacks and zebrafish are frequently used in studies that require DNA to be collected from live animals. This is typically achieved by fin clipping, a procedure that is simple and reliable to perform but that can harm fish. An alternative procedure to sample DNA involves swabbing the skin to collect mucus and epithelial cells. Although swabbing appears to be less invasive than fin clipping, it still requires fish to be netted, held in air and handled—procedures that can cause stress. In this study we combine behavioural and physiological analyses to investigate changes in gene expression, behaviour and welfare after fin clipping and swabbing. Swabbing led to a smaller change in cortisol release and behaviour on the first day of analysis compared to fin clipping. It also led to less variability in data suggesting that fewer animals need to be measured after using this technique. However, swabbing triggered some longer term changes in zebrafish behaviour suggesting a delayed response to sample collection. Skin swabbing does not require the use of anaesthetics and triggers fewer changes in behaviour and physiology than fin clipping. It is therefore a more refined technique for DNA collection with the potential to improve fish health and welfare.
Tilley C. A., Barber I., Norton W. (2021): Skin swabbing protocol to collect DNA samples from small-bodied fish species. F1000Research 10: 1064.
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Fish species are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals to permit identification of their genotype. The current standard procedure to sample DNA is fin clipping, which involves anaesthetising individuals and removing a portion of the caudal fin. While fin clipping reliably generates good quality DNA samples for downstream applications, there is evidence that it can alter health and welfare, leading to infection and impacting on the fish’s behaviour. This in turn can result in greater variation in the data collected. In a recent study we adapted a skin swabbing protocol to collect DNA from small-bodied fish, including sticklebacks and zebrafish, without the use of anaesthetics or sharp instruments. A rayon-tipped swab was used to collect mucus from the flank of the fish, which was then used for DNA extraction. We subsequently demonstrated that compared to fin clipping, skin swabbing triggered fewer changes in stress axis activation and behaviour. We also found that data collected from fish that had been swabbed were less variable than data from fish that had been fin clipped, potentially allowing smaller sample sizes in experimental groups after using this technique, and thereby reducing animal use. Here we provide a detailed protocol explaining how to collect DNA samples from small laboratory fish using skin swabs.