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Journal of Fish Biology (2011) 79, 801–805

doi:10.1111/j.1095-8649.2011.03049.x, available online at wileyonlinelibrary.com

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A quick, least-invasive, inexpensive and reliable methodfor sampling Gadus morhua postlarvae for genetic analysis

L. Mirimin*, D. O’Keeffe, A. Ruggiero, M. Bolton-Warberg, S. Vartiaand R. FitzGerald

Carna Research Station, Ryan Institute, National University of Ireland, Galway,Republic of Ireland

(Received 4 November 2010, Accepted 26 May 2011)

The present study describes the successful design and testing of a quick, least-invasive, reliableand inexpensive sampling procedure for Atlantic cod Gadus morhua. This protocol can be easilyapplied to postlarval fish following a simple three-step procedure, without availing of commercialDNA extraction kits, while ensuring survival of sampled individuals. © 2011 The Authors

Journal of Fish Biology © 2011 The Fisheries Society of the British Isles

Key words: Atlantic cod; DNA extraction; sampling technique.

The use and application of genetic data have been increasingly important aspectsof the study of both wild and cultivated species. In finfishes, collection of samplesfor genetic analysis can be carried out using various tissue types, including muscle(Chapela et al., 2007), fin (Zilberman et al., 2006), gill (Martial et al., 1994), blood(Martínez et al., 1998), scales (Yue & Orban, 2001) and whole eggs (Aranishi, 2006).DNA is isolated using a range of available extraction methods (Blin & Stafford, 1976;Walsh et al., 1991; Miller et al., 1998; Tel-Zur et al., 1999), and target DNA frag-ments are subsequently amplified for DNA analysis via PCR. Such a process can becarried out successfully with a very small amount of tissue, and hence sampling tech-niques tend to be more or less invasive depending on the type of tissue and the size offish being sampled. When collecting tissue from larvae or very young and small fish,however, sampling for DNA analyses is often invasive or lethal, leading to loss ofindividuals. This is a limiting factor in experimental studies of captive stocks, wheredifferent families or strains may need to be monitored at an early life stage, and alsoin studies of wild populations, especially when dealing with species of conservationconcern. A number of ad hoc methods have been developed for least-invasive fin-fish sample collection and subsequent DNA extraction: body mucus and epithelialcells can be collected using Whatman FTA Card Technology (Lucentini et al., 2006)or using buccal swabs (Campanella & Smalley, 2006) with genomic DNA being

*Author to whom correspondence should be addressed. Tel.: 353 95 32201; email: [email protected]

801© 2011 The AuthorsJournal of Fish Biology © 2011 The Fisheries Society of the British Isles

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subsequently isolated in combination with commercially available extraction kits (LeVin et al., 2010). Nonetheless, costs of sampling material (e.g. FTA cards or swabs)and DNA extraction kits, as well as time of execution, can be prohibitive when deal-ing with large numbers of samples. The present study describes the designing andtesting of a least-invasive and inexpensive sampling procedure for a marine species,Atlantic cod Gadus morhua L. 1758, which can be easily applied following a simplethree-step procedure, without requiring expensive sampling tools or commercial DNAextraction kits. Such a method was developed in order to allow sampling and survivalof postlarval fish, for successful collection of tissue for reliable DNA analyses.

The sampling procedure consists of three main steps: (1) swabbing a fish withsterile paper, (2) enzymatic digestion of collected cells and (3) DNA extraction bychelating-resin protocol. First, small pieces of blotting paper (0·5 cm2) were cut andplaced in 1·5 ml tubes and sterilized by autoclaving at 121◦ C for 15 min, prior touse. Epithelial cells and mucus were collected by gently swabbing twice a sterilepiece of blotting paper over the head, abdomen or tail (head-to-tail direction). Thisprocess lasted a few seconds per fish and did not require anaesthesia, which tend tobe unsuitable to fish of small size (e.g. <1 g). Samples were stored at −20◦ C forup to 2 months from the day of sampling. DNA was extracted from each samplevia proteinase K digestion (2 h at 56◦ C, total volume of 200 μl, with 0·1 mg pro-teinase K) and Chelex (Bio-Rad; www.bio-rad.com) extraction procedure (10 min at99◦ C, in 10% Chelex resin) (modified from Walsh et al., 1991). Additionally, theDNA-containing aqueous solution was separated from Chelex beads by centrifugingat 8000 g for 2 min and then by transferring the supernatant into a new clean tubeor plate.

In order to evaluate effectiveness and reliability of the method, the experimentwas divided into two parts: (1) testing efficiency and reliability of the samplingmethod on postlarval fish in the presence of positive controls (i.e. fin clips from thesame fish) and (2) assessing performance of resulting molecular data in parentageassignment analyses of fish of known pedigree, while monitoring the response offish to the sampling procedure. All experimental fish were kept at an approximatetank density of one fish l−1. For part (1), a total of 15 individuals were sampledat 113 days post hatch (dph). Fish ranged between 0·33 and 1·61 g in total bodymass and between 4·3 and 5·2 cm in total body length (LT) (Table I). Fin clips(0·2 cm × 0·2 cm) were also obtained from the same fish to be used as positivecontrols. In part (2), a total of 48 individuals (c. 100 dph, averaging 1·17 ± 0·47 gin total body mass) of known pedigree (i.e. originating from two half-sibling fam-ilies) were sampled and subsequently monitored for adverse effects every 4 to 6 hup to 72 h post-sampling, by visual inspection and comparison to unsampled fish,which were kept in a separate tank with the same fish density. In order to quantifyand compare amounts of extracted DNA from swabs and fin clips, concentration ofDNA was estimated by UV absorbance, using a NanoDrop 3300 Fluorospectrometer(www.nanodrop.com). To test for successful and reliable DNA extraction, multi-locusgenotypes were obtained for three microsatellite loci: Gmo19 (Miller et al., 2000),Gmo132 (Brooker et al., 1994) and PGmo76 (Skirnisdottir et al., 2008). Each ampli-fication reaction was carried out in a total volume of 10 μl, containing c. 40–80 ngof DNA, 1× Green GoTaq buffer (Promega; www.promega.com), 3 mM MgCl2,1 μmol of each primer, 250 μM of each dNTP and 0·5 U of GoTaq DNA Poly-merase (Promega). PCR thermal conditions included an initial denaturation step of

© 2011 The AuthorsJournal of Fish Biology © 2011 The Fisheries Society of the British Isles, Journal of Fish Biology 2011, 79, 801–805

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Table I. Total lengths (LT), masses (M) and relative amount of extracted DNA from post-larval Gadus morhua (113 days post hatch) used in experiment part 1

Sample (areaof swabbing) LT (cm) M (g)

DNA from swab(ng μl−1)

DNA from fin(ng μl−1)

Cod 1 (head) 4·7 0·91 95·55 34·05Cod 2 (head) 5·1 1·61 109·92 59·50Cod 3 (head) 4·8 1·18 80·37 53·32Cod 4 (head) 5·2 1·47 43·56 45·19Cod 5 (head) 5·1 1·49 83·02 63·64Cod 6 (abdomen) 4·7 1·20 39·14 52·83Cod 7 (abdomen) 5·0 1·32 40·37 88·91Cod 8 (abdomen) 4·8 1·12 41·33 189·50Cod 9 (abdomen) 5·0 1·04 32·38 117·39Cod 10 (abdomen) 5·1 1·15 51·07 142·36Cod 11 (tail) 4·7 0·80 80·08 95·73Cod 12 (tail) 5·2 1·59 58·24 100·53Cod 13 (tail) 5·0 0·93 59·78 74·87Cod 14 (tail) 4·8 1·49 81·40 90·17Cod 15 (tail) 4·3 1·12 78·76 78·76Mean ± s.d. 4·9 ± 0·2 1·23 ± 0·26 65·41 ± 23·95 85·78 ± 40·63

3 min at 95◦ C, followed by 32 cycles of 30 s at 95◦ C, 30 s at 52◦ C, 30 s at 72◦ C,followed by a final extension step of 10 min at 72◦ C. Size of PCR products wasresolved on 6·5% acrylamide gels by comparison to reference size standards usinga LI-COR 4300 DNA analyser (www.licor.com). Finally, to further test for reliabil-ity of amplified DNA fragments, parentage analyses were carried out by genotypicexclusion without allowing for mismatching loci, using VITASSIGN (Vandeputteet al., 2006).

DNA was successfully extracted from all individuals, which yielded sufficientamounts of DNA for PCR amplification. Over the 15 fish for which both swabsand fin clips were collected, the mean ± s.d. amount of DNA per swabbed sam-ple was 65·41 ± 23·95 ng μl−1, which was slightly lower than that from fin clips(85·78 ± 40·63 ng μl−1) (Table I), but higher than that obtained using commer-cial swabs in a previous study (Le Vin et al., 2010). Individual fish appeared torespond well to the sampling procedure, as no mortalities (100% survival) and noappreciable differences in swimming and feeding behaviour were observed in thesampled fish compared to the unsampled (control) fish. For part 1, successful PCRamplification of microsatellite loci was achieved for all 15 postlarvae, with size ofDNA fragments ranging between 124 and 274 bp and seven, six and eight allelesper locus. Multi-locus genotypes were obtained from all fish that were swabbedeither over the head (n = 5), abdomen (n = 5) or tail (n = 5), although two head-swabbed individuals failed to amplify one of three loci, indicating that the mostreliable body areas for swabbing appeared to be the abdomen and along the tail.Direct comparison between test genotypes (from swabbing) and their respective pos-itive controls (from fin clips from the same fish) showed a 100% match, indicatingreliability of the sampling procedure and no evidence of cross-sample contamination.This supports previous findings (though at lower tank density; Le Vin et al., 2010),


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confirming that sampling external body mucus can be carried out without riskingcross-sample contamination in fish kept at a tank density of up to one fish l−1. Forpart 2, successful microsatellite loci amplification was obtained for the 48 postlar-vae of known pedigree. Parentage analyses allowed unambiguous identification ofparental origin of 45 fish (for which all three loci were scored), while only maternalorigin could be identified for the three fish that were missing data at one of three loci(PGmo76). On the basis of previous testing of unrelated fish from the population oforigin, Celtic Sea, the markers used in the present study appear not to be affected bynull alleles or large allele dropout thus, failure to obtain one of three loci in somesamples could be due to human error or sample mishandling, and hence re-typingof the missing locus and additional loci should resolve parentage of unassignedoffspring.

The present study provides a quick, least-invasive, inexpensive and reliable methodfor sampling G. morhua postlarvae for genetic analysis. The ease and speed of collec-tion allows this procedure to be performed on large number of samples in a relativelyshort time, which can be incorporated into routine monitoring practices of captivestocks or intensive sampling of wild populations. Furthermore, the low cost of mate-rials involved in both sampling and DNA extraction procedures guarantees a cheapalternative to commercially available kits. Although post-extraction storage time andconditions were not tested in the present study, one drawback of using Chelex-based extraction methods is that the isolated genomic DNA may not be suitable forlong-term storage (e.g. >12 months). Thus, additional modifications to the protocolincluding the use of buffers (e.g. Tris-EDTA buffer) and pH control (as nucleases tendto be less active at pH 7·5–8·0) should be considered to enhance quality and extendstorage time. Because this approach offers the possibility to isolate genomic DNAfrom postlarval fish that are too small to be fin-clipped or individually tagged with-out detrimental effects, the performance of specific families or strains from early lifestages (e.g. early juvenile) can thus be evaluated and measured, without sacrificingindividuals. Equally, the procedure can be successfully applied to larger fish (e.g. >40g; unpubl. data) and, therefore, it could be used as a quick, least-invasive samplingtechnique for valuable brood stock or wild populations of conservation concern.

The EIRCOD project, the cod broodstock and breeding programme for Ireland, is fundedunder the Sea Change initiative with the support of the Marine Institute and the MarineResearch Sub-programme of the National Development Plan 2007–2013 co-funded by theEuropean Regional Development Fund. The authors would like to acknowledge all staff atCarna Research Station and anonymous reviewers for improving previous versions of themanuscript.

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