Journal of Fish Biology (2011) 79, 801805

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

BRIEF COMMUNICATIONS
A quick, least-invasive, inexpensive and reliable method
for sampling Gadus morhua postlarvae for genetic analysis
L. Mirimin*, D. OKeeffe, A. Ruggiero, M. Bolton-Warberg, S. Vartia
and 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, reliable
and inexpensive sampling procedure for Atlantic cod Gadus morhua. This protocol can be easily
applied to postlarval fish following a simple three-step procedure, without availing of commercial
2011 The Authors
DNA extraction kits, while ensuring survival of sampled individuals.
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 aspects
of the study of both wild and cultivated species. In finfishes, collection of samples
for 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
(Martnez 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 fragments are subsequently amplified for DNA analysis via PCR. Such a process can be
carried out successfully with a very small amount of tissue, and hence sampling techniques tend to be more or less invasive depending on the type of tissue and the size of
fish 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 of
individuals. This is a limiting factor in experimental studies of captive stocks, where
different families or strains may need to be monitored at an early life stage, and also
in studies of wild populations, especially when dealing with species of conservation
concern. A number of ad hoc methods have been developed for least-invasive finfish sample collection and subsequent DNA extraction: body mucus and epithelial
cells 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: lmirimin@gmail.com

801
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L. MIRIMIN ET AL.

subsequently isolated in combination with commercially available extraction kits (Le

Vin 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 dealing with large numbers of samples. The present study describes the designing and
testing 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 simple
three-step procedure, without requiring expensive sampling tools or commercial DNA
extraction kits. Such a method was developed in order to allow sampling and survival
of postlarval fish, for successful collection of tissue for reliable DNA analyses.
The sampling procedure consists of three main steps: (1) swabbing a fish with
sterile paper, (2) enzymatic digestion of collected cells and (3) DNA extraction by
chelating-resin protocol. First, small pieces of blotting paper (05 cm2 ) were cut and
placed in 15 ml tubes and sterilized by autoclaving at 121 C for 15 min, prior to
use. Epithelial cells and mucus were collected by gently swabbing twice a sterile
piece of blotting paper over the head, abdomen or tail (head-to-tail direction). This
process lasted a few seconds per fish and did not require anaesthesia, which tend to
be unsuitable to fish of small size (e.g. <1 g). Samples were stored at 20 C for
up to 2 months from the day of sampling. DNA was extracted from each sample
via proteinase K digestion (2 h at 56 C, total volume of 200 l, with 01 mg proteinase K) and Chelex (Bio-Rad; www.bio-rad.com) extraction procedure (10 min at
99 C, in 10% Chelex resin) (modified from Walsh et al., 1991). Additionally, the
DNA-containing aqueous solution was separated from Chelex beads by centrifuging
at 8000 g for 2 min and then by transferring the supernatant into a new clean tube
or plate.
In order to evaluate effectiveness and reliability of the method, the experiment
was divided into two parts: (1) testing efficiency and reliability of the sampling
method on postlarval fish in the presence of positive controls (i.e. fin clips from the
same fish) and (2) assessing performance of resulting molecular data in parentage
assignment analyses of fish of known pedigree, while monitoring the response of
fish to the sampling procedure. All experimental fish were kept at an approximate
tank density of one fish l1 . For part (1), a total of 15 individuals were sampled
at 113 days post hatch (dph). Fish ranged between 033 and 161 g in total body
mass and between 43 and 52 cm in total body length (LT ) (Table I). Fin clips
(02 cm 02 cm) were also obtained from the same fish to be used as positive
controls. In part (2), a total of 48 individuals (c. 100 dph, averaging 117 047 g
in total body mass) of known pedigree (i.e. originating from two half-sibling families) were sampled and subsequently monitored for adverse effects every 4 to 6 h
up 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 quantify
and compare amounts of extracted DNA from swabs and fin clips, concentration of
DNA was estimated by UV absorbance, using a NanoDrop 3300 Fluorospectrometer
(www.nanodrop.com). To test for successful and reliable DNA extraction, multi-locus
genotypes were obtained for three microsatellite loci: Gmo19 (Miller et al., 2000),
Gmo132 (Brooker et al., 1994) and PGmo76 (Skirnisdottir et al., 2008). Each amplification reaction was carried out in a total volume of 10 l, containing c. 4080 ng
of DNA, 1 Green GoTaq buffer (Promega; www.promega.com), 3 mM MgCl2 ,
1 mol of each primer, 250 M of each dNTP and 05 U of GoTaq DNA Polymerase (Promega). PCR thermal conditions included an initial denaturation step of
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S A M P L I N G M E T H O D F O R G A D U S M O R H UA

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

LT (cm)

M (g)

DNA from swab

(ng l1 )

DNA from fin

(ng l1 )

Cod 1 (head)
Cod 2 (head)
Cod 3 (head)
Cod 4 (head)
Cod 5 (head)
Cod 6 (abdomen)
Cod 7 (abdomen)
Cod 8 (abdomen)
Cod 9 (abdomen)
Cod 10 (abdomen)
Cod 11 (tail)
Cod 12 (tail)
Cod 13 (tail)
Cod 14 (tail)
Cod 15 (tail)
Mean s.d.

47
51
48
52
51
47
50
48
50
51
47
52
50
48
43
49 02

091
161
118
147
149
120
132
112
104
115
080
159
093
149
112
123 026

9555
10992
8037
4356
8302
3914
4037
4133
3238
5107
8008
5824
5978
8140
7876
6541 2395

3405
5950
5332
4519
6364
5283
8891
18950
11739
14236
9573
10053
7487
9017
7876
8578 4063

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 was
resolved on 65% acrylamide gels by comparison to reference size standards using
a LI-COR 4300 DNA analyser (www.licor.com). Finally, to further test for reliability of amplified DNA fragments, parentage analyses were carried out by genotypic
exclusion without allowing for mismatching loci, using VITASSIGN (Vandeputte
et al., 2006).
DNA was successfully extracted from all individuals, which yielded sufficient
amounts of DNA for PCR amplification. Over the 15 fish for which both swabs
and fin clips were collected, the mean s.d. amount of DNA per swabbed sample was 6541 2395 ng l1 , which was slightly lower than that from fin clips
(8578 4063 ng l1 ) (Table I), but higher than that obtained using commercial swabs in a previous study (Le Vin et al., 2010). Individual fish appeared to
respond well to the sampling procedure, as no mortalities (100% survival) and no
appreciable differences in swimming and feeding behaviour were observed in the
sampled fish compared to the unsampled (control) fish. For part 1, successful PCR
amplification of microsatellite loci was achieved for all 15 postlarvae, with size of
DNA fragments ranging between 124 and 274 bp and seven, six and eight alleles
per locus. Multi-locus genotypes were obtained from all fish that were swabbed
either over the head (n = 5), abdomen (n = 5) or tail (n = 5), although two headswabbed individuals failed to amplify one of three loci, indicating that the most
reliable body areas for swabbing appeared to be the abdomen and along the tail.
Direct comparison between test genotypes (from swabbing) and their respective positive controls (from fin clips from the same fish) showed a 100% match, indicating
reliability 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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L. MIRIMIN ET AL.

confirming that sampling external body mucus can be carried out without risking
cross-sample contamination in fish kept at a tank density of up to one fish l1 . For
part 2, successful microsatellite loci amplification was obtained for the 48 postlarvae of known pedigree. Parentage analyses allowed unambiguous identification of
parental origin of 45 fish (for which all three loci were scored), while only maternal
origin 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 of
origin, Celtic Sea, the markers used in the present study appear not to be affected by
null alleles or large allele dropout thus, failure to obtain one of three loci in some
samples could be due to human error or sample mishandling, and hence re-typing
of the missing locus and additional loci should resolve parentage of unassigned
offspring.
The present study provides a quick, least-invasive, inexpensive and reliable method
for sampling G. morhua postlarvae for genetic analysis. The ease and speed of collection allows this procedure to be performed on large number of samples in a relatively
short time, which can be incorporated into routine monitoring practices of captive
stocks or intensive sampling of wild populations. Furthermore, the low cost of materials involved in both sampling and DNA extraction procedures guarantees a cheap
alternative to commercially available kits. Although post-extraction storage time and
conditions were not tested in the present study, one drawback of using Chelexbased extraction methods is that the isolated genomic DNA may not be suitable for
long-term storage (e.g. >12 months). Thus, additional modifications to the protocol
including the use of buffers (e.g. Tris-EDTA buffer) and pH control (as nucleases tend
to be less active at pH 7580) should be considered to enhance quality and extend
storage time. Because this approach offers the possibility to isolate genomic DNA
from postlarval fish that are too small to be fin-clipped or individually tagged without detrimental effects, the performance of specific families or strains from early life
stages (e.g. early juvenile) can thus be evaluated and measured, without sacrificing
individuals. Equally, the procedure can be successfully applied to larger fish (e.g. >40
g; unpubl. data) and, therefore, it could be used as a quick, least-invasive sampling
technique for valuable brood stock or wild populations of conservation concern.
The EIRCOD project, the cod broodstock and breeding programme for Ireland, is funded
under the Sea Change initiative with the support of the Marine Institute and the Marine
Research Sub-programme of the National Development Plan 20072013 co-funded by the
European Regional Development Fund. The authors would like to acknowledge all staff at
Carna Research Station and anonymous reviewers for improving previous versions of the
manuscript.

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Journal of Fish Biology 2011 The Fisheries Society of the British Isles, Journal of Fish Biology 2011, 79, 801805