Non-invasive method to obtain DNA from freshwater mussels (Bivalvia: Unionidae).ABSTRACT To determine whether DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. could be isolated from tissues obtained by brash-swabbing the mantle, viscera viscera /vis·ce·ra/ (vis´er-ah) plural of viscus. vis·cer·a pl.n. 1. The soft internal organs of the body, especially those contained within the abdominal and thoracic cavities. and foot, mantle-clips and swabbed cells were obtained from eight Quadrula pustulosa (Lea, 1831). DNA yields from clips and swabbings were 447.0 and 975.3 [eta]g/[micro]L, respectively. Furthermore, comparisons of sequences from the ND-1 mitochondrial mitochondrial pertaining to mitochondria. mitochondrial RNAs a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by a mitochondrial-specific RNA polymerase, that account for about 4% of the total cell RNA that gene region showed a 100% sequence agreement of DNA from cells obtained by clips and swabs. To determine the number of swabs needed to obtain adequate yields of DNA for analyses, the visceras and feet of 5 Q. pustulosa each were successively swabbed 2, 4 and 6 times. DNA yields from the 2, 4 and 6 swabbed mussel mussel, edible freshwater or marine bivalve mollusk. Mussels are able to move slowly by means of the muscular foot. They feed and breathe by filtering water through extensible tubes called siphons; a large mussel filters 10 gal (38 liters) of water per day. groups were 399.4, 833.8 and 852.6 ng/[micro]L, respectively. ND-1 sequences from the lowest yield still provided 846-901 bp for the ND-1 region. Nevertheless, to ensure adequate DNA yield from cell samples obtained by swabbing, we recommend that 4 swab-strokes of the viscera and foot be obtained. The use of integumental Adj. 1. integumental - of or relating to the integument integumentary swabbing for collection of cells for determination of genetic relationships among freshwater mussels is noninvasive, when compared with tissue collection by mantle-clipping. Therefore, its use is recommended for freshwater mussels, especially state-protected or federally listed mussel species. KEY WORDS: Unionidae, genetics, integument integument Covering of the body, which protects it from the outside world and from drying out. In humans and other mammals it consists of the skin (including outer epidermis and inner dermis) and its related structures, including hair, nails, and sebaceous and sweat glands. , swabbing, mantle-clipping INTRODUCTION The use of mantle-clipping for biopsy has become a common technique for collection of tissues from unionoid mussels for genetic analyses (Buhay et al. 2002, Eackles & King 2002, Jones et al. 2004, Curule cu·rule adj. Privileged to sit in a curule chair; of superior rank. [Latin cur et al. 2004, Campbell et al. 2005, Geist & Kuehn 2005, Grobler et al. 2006). Berg et al. (1995) observe no significant differences in mortality rates in mantle-biopsied versus nonbiopsied muckets, Actinonaias ligamentina (Lamarck, 1819) and mapleleafs, Quadrula quadrula (Rafinesque, 1820). However, mantle-clipped snuffboxes, Epioblasma triquetra Triquetra (IPA: [tɹaɪ'kwεtɹə]) is a word derived from the Latin tri- ("three") and quetrus ("cornered"). (Rafinesque, 1820), showed a mortality rate of 56.3% (n = 16) after 1.5 y of postbiopsy observation at the Aquatic Wildlife Conservation Center (AWCC AWCC Arkansas Workers' Compensation Commission AWCC Afghan Wireless Communication Company AWCC Association of Wisconsin Cleaning Contractors AWCC Active Well Coincidence Counter AWCC Alaska Wildlife Conservation Center ) (Eckert, N., VDGIF VDGIF Virginia Department of Game and Inland Fisheries , Marion, Virginia, pers. comm.). Although mortality of mantle-clipped mussels may not be attributed directly to tissue removal, inspection of the dead E. triquetra showed regression of the nacre nacre: see mother-of-pearl. and shell deformity in the valve locations where mantle edges were removed (Fig. 1). Because mantle biopsy is an invasive procedure that may induce mortality, its use on federally endangered mussel species is a questionable procedure for genetic analyses. The goal of this study was to determine whether a procedure less invasive than mantle biopsy is available for collection of DNA for genetic analyses. Our objective was to determine whether viable DNA could be obtained by integumental swabbing from pimplebacks, Quadrula pustulosa (Lea, 1831) and to confirm mDNA sequence agreement among tissues obtained from swabbing and mantle-clipping from the same mussels. MATERIALS AND METHODS Tissue Collection Tissues were obtained from Q. pustulosa at the Freshwater Mollusc mollusc members of the phylum Mollusca, which comprises about 50,000 species. Includes snails, slugs and the aquatic molluscs—oysters, mussels, clams, cockles, arkshells, scallop, abalone, cuttlefish, squid. Conservation Center, Virginia Tech, Blacksburg, Virginia ([bar.X] = 70.7 mm, s = [+ or -] 11.7). To test the feasibility of isolating DNA by swabbing of the viscera, foot and mantle, we initially sampled both mantle clips and brush swabs from 8 Q. pustulosa. Clips (approximately 3 x 5 mm) were taken from the edge of the mantle and stored in 95% ethanol. Cell samples were taken by using approximately 8 vigorous strokes with a bristle bristle 1. the thick strong animal fibers collected at commercial abattoirs for use in brushes. 2. the sharp serrated awns of grass and some cereal seeds that confer a capacity to penetrate normal skin and mucosa and to cause ulcerative stomatitis, grass seed abscess and the like. brush (CYB-1; Gentra Systems, Minneapolis, Minnesota). Strokes covered all structures within the visceral cavity, including the mantle surface, viscera and foot. Brush tips were stored in lyses ly·ses n. Plural of lysis. buffer. Then, we determined (1) the amount of DNA that can be obtained using a bristle brush, compared with conventional mantle clips; (2) whether ND-1 could be amplified from the DNA and (3) whether ND-1 sequences amplified using buccal buc·cal adj. 1. Of, relating to, adjacent to, or in the direction of the cheek. 2. Of or relating to the mouth cavity. buccal swabs was identical to results from mantle clips. Because excessive scrubbing with bristle brushes resulted in some disruption of the mantle, we also investigated a collection technique using the viscera and foot only. Furthermore, it would be informative to determine the minimum number of integumental swabs required to provide an adequate amount of mDNA for sequence analyses. For this objective, we sampled an additional 15 individuals of Q. pustulosa, using 6 passes of the brush on each of 5 individuals, 4 passes on another 5, and 2 passes on the 5 remaining mussels. Care was taken to rotate the brush between strokes and thus present a clean surface for collection at each stroke. After determining the amount of DNA obtained using each number of strokes, the ND-s gene region was sequenced from the least invasive technique (2 strokes) to test the feasibility of using this as a source of DNA for sequencing reactions. DNA Extraction DNA was extracted from all samples using the Purgene DNA extraction kit. For extraction from brushes, the protocol described by Purgene was modified by increasing the initial amount of lyses buffer used from 300 [micro]L to 450 [micro]L, to ensure complete coverage of brush-bristles in a 1.5 mL Eppendorf tube. DNA was extracted from mantle-clipped tissue following the standard Puregene protocol for solid tissue. [FIGURE 1 OMITTED] Molecular Analysis We used the NADH dehydrogenase (ND-1) mitochondrial gene region to verify that DNA obtained from swabs was indeed mussel DNA and that it was suitable for sequencing reactions. Primer sequences and polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) ) amplification conditions were as reported by Buhay et al. (2002) and Serb et al. (2003). Primer sequences were forward: 5'-TGGCAGAAAAGTGCATCAGATTAAAGC-3'; reverse 5'-CCTGCTTGGAAGGCAAGTGTACT-3'. PCR reaction mixtures contained 100 ng of genomic DNA, 1x PCR buffer, 4.0 mM MgCI2, 0.4 mM dNTPs, 1.0 [micro]M of each primer and 1.5 U AmpliTaq Gold DNA polymerase, with dd[H.sub.2]O added to a total volume of 25 [micro]L. The thermal cycler profile consisted of an initial 95[degrees]C for 8 min; followed by 35 cycles of: 94[degrees]C for 40 sec, 50[degrees]C for 60 sec and 72[degrees]C for 90 sec; with a final extension step at 72[degrees]C for 2 min; and a final hold at 4[degrees]C. PCR products were purified using a Qiagen DNA Purification kit and were sequenced using Applied Biosystems Big Dye v.3.1. Reaction mixtures consisted of 30 ng PCR product, 5-[micro]M primer, 2 uL Big Dye 3.1 (diluted 1:1), with dd[H.sub.2]O added for a final volume of 7.5 uL. The cycle reaction consisted of 30 cycles, each of 94[degrees]C for 30 sec, 50[degrees]C for 15 sec and 60[degrees]C for 4 min. The products were cleaned by centrifuging through hydrated hy·drat·ed adj. Chemically combined with water, especially existing in the form of a hydrate. Adj. 1. hydrated - containing combined water (especially water of crystallization as in a hydrate) hydrous Sephadex (Sigma) in Millipore filter plates, dried down and resuspended in Hi-Di Formamide (Applied Biosystems). The samples were denatured de·na·ture tr.v. de·na·tured, de·na·tur·ing, de·na·tures 1. To change the nature or natural qualities of. 2. at 95[degrees]C for 5 min, and cooled to 4[degrees]C for 2 min before being loaded on the Applied Biosystems DNA Analyzer 3730 for processing. SEQUENCHER software (ver. 4.11) was used to align and edit sequences from stored GENESCAN files. RESULTS The average DNA yields obtained varied by collection technique (Table 1). The standardized collection method from mantle clips yielded an average of 447.0 ng/[micro]L. Eight vigorous passes of the bristle brush over the viscera and mantle yielded an average of 975.3 ng/[micro]L (with high deviations in yield), whereas swabs of the viscera and foot yielded 852.6, 833.8 and 399.4 ng/[micro]L using 6, 4 and 2 strokes, respectively (and with lower standard deviations compared with strokes involving the mantle). During DNA isolation, the amount of protein in mucous-derived samples was very high, and care had to be taken to ensure full precipitation of proteins before continuing with the isolation procedure. It was necessary to repeat the protein precipitation stage of the Purgene procedure in some instances. Sequences for the ND-1 gene region obtained from mantle clips and integument swab samples from the same three individuals aligned with 100% accuracy (847-895 basepairs; Genbank accession numbers DQ640237 to DQ640239). Sequences of ND-1 for DNA isolated after 2 strokes of the bristle brush yielded a clean 846-901 bp for the ND-1 region in two random samples (Genbank accession numbers DQ640240 and DQ640241). DISCUSSION Comparison of our data shows that swabbing of the foot and viscera of Q. pustulosa is a reliable method for collection of cells for DNA analyses. The DNA obtained from the swabbing technique was pure mussel DNA; our analyses show that it is of high quality, with 100% sequence alignment with DNA obtained from mantle clips from the same mussel specimens. The DNA yield from cells collected by swabbing was equal or greater than the yield from mantle clips. Our data show that 2, 4 and 6 swab passes over the foot and viscera provided sufficient quantities of DNA for reliable analyses. Because the DNA yield remains relatively constant with 4 and 6 swabbings (852.6 and 833.8 ng/[micro]L), and then declines with 2 (399.4 ng/[micro]L), we recommend that 4 swabs of the foot and integument be used as a standard method for collection of cells. The number of swabs required to provide sufficient cell samples for analyses may vary by mussel size and species. We recommend that researchers use this swabbing protocol rather than tissue collection by mantle-clipping, because it is noninvasive and provides reliable DNA for genetic analyses. This is especially true for federally listed mussels, typically restricted from intentional sacrifice in federal collection permits. We observed a high protein yield in the swabbed samples that could possibly interfere with the results of future DNA analyses. We hypothesize hy·poth·e·size v. hy·poth·e·sized, hy·poth·e·siz·ing, hy·poth·e·siz·es v.tr. To assert as a hypothesis. v.intr. To form a hypothesis. that this protein was included in integumental mucous that was sampled inadvertently with swabbed cells. We recommend special care be exhibited during the protein-precipitation step of DNA isolations from swabbed samples. ACKNOWLEDGMENTS The authors thank Mike Pinder; Wildlife Diversity Division, VDGIF, Blacksburg, Virginia and Roberta Hylton and Shane Hanlon; Southwest Virginia Field Office, US Fish and Wildlife Service, Abingdon, Virginia, for funding this research. Shane Hanlon provided the photograph documenting nacre regression. LITERATURE CITED Berg, D. J., W. R. Hang, S. I. Guttman & J. B. Sickel. 1995. Mantle biopsy: a technique for nondestructive non·de·struc·tive adj. Of, relating to, or being a process that does not result in damage to the material under investigation or testing. non tissue-sampling of freshwater mussels. J. N. Am. Benthol. Soc. 14:577-581. Buhay, J. E., J. M. Serb, C. R. Dean, Q. Parham & C. Lydeard. 2002. Conservation genetics of two endangered unionid bivalve bivalve, aquatic mollusk of the class Pelecypoda ("hatchet-foot") or Bivalvia, with a laterally compressed body and a shell consisting of two valves, or movable pieces, hinged by an elastic ligament. species, Epioblasma florentina walkeri and E. capsaeformis (Unionidae: Lampsilini). J. Moll. Stud. 68:385-391. Campbell, D. C., J. M. Serb, J. D. Buhay, K. J. Roe, R. L. Minton & C. Lydeard. 2005. Phylogeny of North American amblemines (Bivalvia, Unionoida): prodigious polyphyly proves pervasive across genera. Invert in·vert v. 1. To turn inside out or upside down. 2. To reverse the position, order, or condition of. 3. To subject to inversion. n. Something inverted. . Biol. 124:131-164. Curole, J. P., D. W. Foltz & D. M. Brown. 2004. Extensive allozyme monomorphism For other uses, see Dimorphism (disambiguation) or Polymorphism (disambiguation). In the context of abstract algebra or universal algebra, a monomorphism is simply an injective homomorphism. in a threatened species of freshwater mussel, Margaritifera hembeli Conrad (Bivalvia: Margaritiferidae). Conserv. Genet genet: see civet. . 5:271-278. Eackles, M. S. & T. L. King. 2002. Isolation and characterization of microsatellite See miniaturized satellite. loci in Lampsilis abrupta (Bivalvia: Unionidae) and crossspecies amplification within the genus. Mol. Ecol. Notes 2:559-562. Geist, J. & R. Kuehn. 2005. Genetic diversity and differentiation of central European freshwater pearl mussel The freshwater pearl mussel, Margaritifera margaritifera, is an endangered species of freshwater mussel. They are used in the production of freshwater pearls. Subspecies:
Grobler, P. J., J. W. Jones, N. A. Johnson, B. Beaty, J. Struthers, R. J. Neves & E. M. Hallerman. 2006. Patterns of genetic differentiation and conservation of the slabside pearlymussel, Lexingtonia dolabelloides (Lea, 1840) in the Tennessee River drainage. J. Moll, Stud. 72:65-75. Jones, J. W., M. Culver, V. David, J. Struthers, N. A. Johnson, R. J. Neves, S. J. O'Brien & E. M. Hallerman. 2004. Development and characterization of microsatellite loci in the endangered mussel Epioblasma capsaeformis (Bivalvia: Unionidae). Mol. Ecol. Notes 4:649-652. Serb, J. M., J. E. Buhay & C. Lydeard. 2003. Molecular systematics systematics: see classification. of the North American freshwater bivalve genus Quadrula (Unionidae: Ambleminae) based on mitochondrial ND1 sequences. Mol. Phylogenet. Evol. 28:1-11. WILLIAM F. HENLEY, (1) PAUL J. GROBLER (2) AND RICHARD J. NEVES (3) * (1) Freshwater Mollusk mollusk: see Mollusca. mollusk or mollusc Any of some 75,000 species of soft-bodied invertebrate animals (phylum Mollusca), many of which are wholly or partly enclosed in a calcium carbonate shell secreted by the mantle, a soft Conservation Center, Department of Fisheries and Wildlife Sciences, Virginia Tech, Blacksburg, Virginia, 24061; (2) Department of Fisheries and Wildlife Sciences, Virginia Tech, and Department of Biodiversity, School of Molecular and Life Sciences, University of Limpopo History The University of the North was established in 1959 under the apartheid regime's policy of separate ethnically-based institutions of higher learning policy. The university was sited at Turfloop farm about 40 km east of Pietersburg. , P/Bag X1106, Sovenga, South Africa; (3) Virginia Cooperative Fish and Wildlife Research Unit, United States Geological Survey The United States Geological Survey (USGS) is a scientific agency of the United States government. The scientists of the USGS study the landscape of the United States, its natural resources, and the natural hazards that threaten it. , Virginia Tech, Virginia, 24061 * Corresponding author. E-mail: mussel@vt.edu
TABLE 1.
Results of DNA extraction from mantle and integument samples.
n = number of mussels tested.
Number of n
Cell Collection Tissue Swab Mean DNA
Technique Source Strokes Yield (ng/[micro]l)
Mantle Mantle 8 447.0 [[+ or -] 143.3
clipping
Integument Viscera and 8 8 975.6 [+ or -] 779.5
swabbing mantle
Viscera and 6 5 852.6 [+ or -] 237.4
foot
Viscera and 4 5 833.8 [+ or -] 317.0
foot
Viscera and 2 5 399.4 [+ or -] 83.5
foot
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