Procedures for sampling genomic DNA from live billfishes

involve manual restraint and tissue excision that can be difficult to carry out and may produce stresses that affect fish survival. We examined the collection of surface mucous as a less invasive alternative method for sourcing genomic DNA by comparing it to autologous muscle tissue samples

from Atlantic blue marlin (Makaira nigricans), white marlin (Tetrapturus albidus), sailfish (Istiophorus platypterus), and swordfish (Xiphias gladius). Purified DNA from mucous

was comparable to muscle and was suitable for conventional polymerase chain reaction, random amplified polymorphic

DNA analysis, and mitochondrial and nuclear locus sequencing. The nondestructive and less invasive

characteristics of surface mucous collection may promote increased survival of released specimens and may be advantageous for other marine fish genetic studies, particularly those involving large live specimens destined

for release

Bernard, Andrea M.

Faugue, Ronald M.

Hoolihan, John P.

Horn, Rebekah L.

Perez, Nerida F.

Schultz, Duane R.

Snodgrass, Derke

English

Procedures for sampling genomic DNA from live billfishes involve manual restraint and tissue excision that can be difficult to carry out and may produce stresses that affect fish survival. We examined the collection of surface mucous as a less invasive alternative method for sourcing genomic DNA by comparing it to autologous muscle tissue samples from Atlantic blue marlin (Makaira nigricans), white marlin (Tetrapturus albidus), sailfish (Istiophorus platypterus), and swordfish (Xiphias gladius). Purified DNA from mucous was comparable to muscle and was suitable for conventional polymerase chain reaction, random amplified polymorphic DNA analysis, and mitochondrial and nuclear locus sequencing. The nondestructive and less invasive characteristics of surface mucous collection may promote increased survival of released specimens and may be advantageous for other marine fish genetic studies, particularly those involving large live specimens destined for release

Wilson, Nerida G.

Bernard, Andrea

NSU Works

Nova Southeastern UniversityNSUWorksMarine & Environmental Sciences Faculty Articles Department of Marine and Environmental Sciences7-1-2009Surface Mucous as a Source of Genomic DNAfrom Atlantic Billfishes (Istiophoridae) andSwordfish (Xiphiidae)John P. HoolihanNational Oceanic and Atmospheric AdministrationNerida G. WilsonUniversity of MiamiRonald M. FaugueUniversity of MiamiAndrea BernardNova Southeastern University, <<span class="elink">andrbern@nova.eduRebekah L. HornNova Southeastern University, <<span class="elink">horn.rebekah@gmail.comSee next page for additional authorsFind out more information about Nova Southeastern University and the Halmos College of Natural Sciencesand Oceanography.ollow this and additional works at: https://nsuworks.nova.e u/occ_facarticlesPart of the Marine Biology Commons, and the Oceanography and Atmospheric Sciences andMeteorology CommonsThis Article is brought to you for free and open access by the Department of Marine and Environmental Sciences at NSUWorks. It has been acceptedfor inclusion in Marine & Environmental Sciences Faculty Articles by an authorized administrator of NSUWorks. For more information, please contactnsuworks@nova.edu.NSUWorks CitationJohn P. Hoolihan, Nerida G. Wilson, Ronald M. Faugue, Andrea Bernard, Rebekah L. Horn, Derke Snodgrass, and Duane R. Schultz.2009. Surface Mucous as a Source of Genomic DNA from Atlantic Billfishes (Istiophoridae) and Swordfish (Xiphiidae) .FisheryBulletin , (3) : 339 -342. https://nsuworks.nova.edu/occ_facarticles/892.AuthorsDerke SnodgrassNational Oceanic and Atmospheric AdministrationDuane R. SchultzUniversity of MiamiThis article is available at NSUWorks: https://nsuworks.nova.edu/occ_facarticles/892339Understanding genetic variation and identifying evolutionary lineages are important considerations for conser-vation management of large pelagic ﬁshes (Palumbi, 1994; Ward, 2000). Some methods used for genetic sam-pling of billﬁshes (families Istiophori-dae and Xiphiidae) require excision of muscle, skin, or fin tissue, and excision requires manual restraint or killing of the animal. Some non-destructive sampling methods, such as ﬁn and scale collection, exist that avoid the need to kill the ﬁsh (Yue and Orban, 2001; Wasko et al., 2003; Hoolihan et al., 2004). Tissue exci-sion from very large live specimens is problematic because of personal safety concerns for handlers, and injuries to ﬁsh that may reduce survival. Most recreational billfish caught in the United States are released (Prince et al., 2007), whereas possession of billﬁsh by U.S. pelagic longline ves-sels and sales of Atlantic billﬁshes have been prohibited since 1988. This regulation eliminates opportunities to obtain DNA samples from landed spec-imens, warranting a need for alterna-tive methods. To sample genomic DNA with nondestructive techniques and with minimal handling would promote the survival of released individuals and increase the opportunities to con-duct genetic studies.One alternate potential source of genomic DNA is the epidermal cells found in billﬁsh and swordﬁsh sur-face mucous. Successful extractions of sufﬁcient quantities of genomic DNA from surface mucous of freshwater ﬁshes have been reported for Salmo trutta fario (brown trout), Esox lucius (northern pike) (Livia et al., 2006) and Scleropages formosus (Asian arowana) (Chansue, 2006), provid-ing an experimental model for large pelagic species. The present study compares DNA extractions from surface mucous and autologous skeletal muscle tissue from billﬁshes and swordﬁsh to determine the suitability of DNA extracted from surface mucous for random ampliﬁed polymorphic DNA (RAPD) and DNA sequencing analyses.Materials and methodsSurface mucous and autologous skel-etal muscle samples were collected Surface mucous as a source of genomic DNA  from Atlantic billﬁshes (Istiophoridae)  and swordﬁsh (Xiphiidae)John P. Hoolihan1Nerida F. Perez2 Rebekah L. Horn3Ronald M. Faugue2 Derke Snodgrass1Andrea M. Bernard3 Duane R. Schultz (contact author)2Email address for contact author: dschultz@med.miami.edu1 NOAA Fisheries Southeast Fisheries Science Center 75 Virginia Beach Drive Miami, Florida 331492 Department of Medicine University of Miami Miller School of Medicine Miami, Florida 331363 Guy Harvey Research Institute Nova Southeastern University Oceanographic Center 8000 North Ocean Drive Dania Beach, Florida 33004Manuscript submitted 26 November 2008.Manuscript accepted 26 March 2009.Fish. Bull. 107:339–342 (2009).The views and opinions expressed  or implied in this article are those  of the author and do not necessarily  reﬂect the position of the National  Marine Fisheries Service, NOAA.Abstract—Procedures for sampling genomic DNA from live billf ishes involve manual restraint and tissue excision that can be difficult to carry out and may produce stresses that affect fish survival. We examined the collection of surface mucous as a less invasive alternative method for sourcing genomic DNA by comparing it to autologous muscle tissue samples from Atlantic blue marlin (Makaira nigricans), white marlin (Tetraptu-rus albidus), sailf ish (Istiophorus platypterus), and swordfish (Xiphias gladius). Purified DNA from mucous was comparable to muscle and was suitable for conventional polymerase chain reaction, random amplified poly-morphic DNA analysis, and mitochon-drial and nuclear locus sequencing. The nondestructive and less invasive characteristics of surface mucous col-lection may promote increased sur-vival of released specimens and may be advantageous for other marine fish genetic studies, particularly those involving large live specimens des-tined for release.340 Fishery Bulletin 107(3)Table 1Nucleotide composition of three 10-mer primers used for random ampliﬁed polymorphic DNA (RAPD) compari-son of DNA extracted from billﬁsh surface mucous and muscle tissue, and percent content of guanine and cyto-sine (% GC).Primer no. Nucleotide composition % GC1 5ʹ-GTTGCGGGCT-3ʹ 702 5ʹ-CAGCCCGGGT-3ʹ 803 5ʹ-AGGCCACCGC-3ʹ 80from three individuals from each of the following species: Makaira nigricans (blue marlin), Tetrapturus albidus (white marlin), Istiophorus platypterus (sailﬁsh), and Xiphias gladius (swordﬁsh). We compared puriﬁed DNA extracted from mucous and muscle using RAPD, mito-chondrial DNA (mtDNA) sequencing, and nuclear DNA sequencing analyses.Sample preparation and DNA purificationSurface mucous samples were collected with a poly-urethane sponge as described by Schultz et al. (2006) and immediately processed, or stored at –80°C for later extraction. Each sponge was cut into small pieces, mixed with 1500 μL of phosphate buffered saline, and com-pressed repeatedly to remove mucous. All ﬂuids were centrifuged through a single QIAamp® (Qiagen Inc., Valencia, CA) spin column. DNA puriﬁcation was car-ried out by using Qiagen® buccal swab spin protocol with the following modiﬁcations: 1) a ﬁnal concentration of 1 millimolar (mM) ethylenediaminetetraacetic acid (EDTA) was added to the phosphate-buffered saline extraction buffer; 2) the proteinase K treatment step was eliminated; and 3) an RNase mixture of A and T1 enzymes was used to degrade RNA after the ﬁnal DNA puriﬁcation step (Ambion Inc., Foster City, CA). The RNase cocktail enzyme mix was necessary because the Qiagen® spin columns copurify RNA and DNA in parallel when both are present in a sample. Cold ethanol precipitation and inclusion of EDTA in buffers were used to reduce nuclease degradation (Dessauer et al., 1996; Wasko et al., 2003). For muscle tissue, 25 mg were macerated in 180 μL Buffer ATL (Qiagen Inc., Valencia, CA), and in-cubated overnight in a 56°C water bath in the pres-ence of 20 μL proteinase K. Purification followed the QIAamp® manufacturer’s protocol for tissue. Aque-ous samples of nucleic acid (1.5 μL) from mucous and muscle extractions were measured for purity with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific,Wilmington, DE) and showed a range of 1.7 to 2.0 for the DNA and RNA absorbance ratios (260 nm:280 nm).RAPD analysisThree different 10-mer oligonucleotide primers (Table 1) of arbitrary sequence (IDT Integrated DNA Technolo-gies, Coralville, IA) were tested against surface mucous DNA and autologous muscle DNA from three individuals of M. nigricans, T. albidus, I. platypterus, and X. gla-dius. PCR reactions for RAPD analysis were performed in a total volume of 25 μL containing 3 μL extracted genomic DNA, 2.5 μL of 10 mM 10-mer primer, 2.5 μL of 2.5 mM deoxynucleotide triphosphate (dNTPs), 0.75 μL of 25 mM MgCl2, 0.20 μL Taq DNA polymerase, 2.5 μL 10× buffer, 5 μL 5M betaine (N, N, N-trimethylgly-cine), 2 μL HotStart-IT® binding protein (USB Corp., Cleveland, OH), and 6.55 μL distilled water. Pre-PCR incubation with the HotStart-IT™ binding protein was executed at 25°C for four hours to prevent mispriming and primer dimerization during ampliﬁcation (Chou et al., 1992). RAPD PCR was performed in an Eppen-dorf Mastercycler (Westbury, NY) starting with initial heating for 5 minutes at 94°C, followed by 34 cycles at 94°C for 5 minutes, 42°C for 30 seconds, 72°C for 90 seconds, and a ﬁnal extension of 72°C for 10 minutes. A negative control (no genomic DNA) was included in each PCR set to verify no reagent contamination. The PCR products were veriﬁed by electrophoresing 5 μL in 1.2% agarose gel (ISC BioExpress, Kayville, UT) and TAE buffer (pH 8.5) for 60 min at 100 V (60 mA), stained with ethidium bromide, and visualized through a UV transilluminator.Sequencing analysisMitochondrial and single-copy nuclear loci were sequenced from three individuals from each of the fol-lowing species: M. nigricans, T. albidus, and I. platyp-terus. Insufficient samples of mucous and muscle were available for sequencing X. gladius. Mitochondrial NADH dehydrogenase subunit4 (ND4) was ampli-fied by PCR with the primer pair 61F and 1837R and cycling parameters outlined in Shivji et al. (2006). All amplifications of the anonymous single-copy nuclear locus WM13 were performed with the primer pair WM13-F and WM13-R developed by Buonaccorsi et al. (1999). Nuclear PCR reactions were performed in a total reaction volume of 50 μL containing 1 μL of extracted genomic DNA, 10 pmol/μL of each primer, 40 μM dNTPs, 10× PCR buffer, and 1 unit of HotStar Taq™ DNA Polymerase (Qiagen Inc., Valencia, CA). The PCR thermal profile consisted of an initial heating at 95°C for 15 minutes to activate the DNA polymerase, followed by 35–40 cycles at 94°C for 1 minute, 1 minute at 50°C, and 1 minute at 72°C, with a 5-minute final extension step at 72°C. Both nuclear and mitochondrial amplifications were performed in a MJ Research PTC-200 thermal cycler (Waltham, MA). A negative control (no genomic DNA) was included in each PCR set to verify that there was no reagent contamination.All amplified products were purified by using the QIAquick® PCR puriﬁcation kit (Qiagen Inc., Valencia, CA) and sequenced with an Applied Biosystems 3130 341Hoolihan et al.: Surface mucous as a source of genomic DNA from Istiophoridae and XiphiidaeFigure 1Representative random amplified polymorphic DNA (RAPD) banding patterns from four species: A) Makaira nigricans (blue marlin), B) Tetrapturus albidus (white marlin), C) Istiophorus platypterus (sailfish), and D) Xiphias gladius (swordfish) for three 10-mer primers (Table 1) tested on each fish for the template DNA from surface mucus (SM), and autologous muscle tissue (MT). Lane contents are as fol-lows: 1, 100-bp ladder; 2, 5, and 8, negative controls (no DNA); 3 and 4, primer no. 1; 6 and 7, primer no. 2; and, 9 and 10, primer no. 3.A BC Dgenetic analyzer (Foster City, CA). Forward and reverse se-quences were assembled and edited with GeneDoc 2.6.002 (http://www.psc.edu/biomed/genedoc, accessed 1 June 2008). To control for cross-contamination with other billﬁsh or possible parasites or microbes, we compared the mitochondrial and nuclear se-quences derived from surface mucous to sequences obtained from autologous muscle tis-sue. Because autologous mus-cle tissue was not available for the T. albidus sequencing analyses, the mucous sample sequences were compared to homologous locus sequences obtained from reference T. albidus tissues available in our laboratory. Results and conclusionsRAPD analysisComparable RAPD amplicons were derived from surface FJ809991, FJ809992, and FJ809994; WM13 (279bp): FJ809986, FJ809987, and FJ809989; I. platypterus: ND4 (1009bp): FJ809990 and FJ809993; WM13 (277bp): FJ809984 and FJ809985. This study has shown that surface mucous contains sufﬁcient quantities of genomic DNA to carry out RAPD analyses of istiophorid billﬁshes and swordﬁsh, as well as sequencing applications of istiophorid billfishes. These extractions compare favorably to genomic DNA extractions from surface mucous reported for freshwater species S. formosus (Chansue, 2006), E. lucius, and S. trutta fario (Livia et al., 2006). The slight differences between some of the mucous and muscle RAPD ampliﬁcation proﬁles (Fig. 1) may be a result of contaminant DNA in the mucous (e.g., bacteria, microalgae), or other artifact variations known to occur in RAPD studies (Ellsworth et al., 1993). We found that the pre-PCR incubation of the template DNA master mix with HotStart-IT™ binding protein was crucial for preventing mispriming and primer dimer-ization that produced these artifact bands (Chou et al., 1992). In addition, the inclusion of betaine (N, N, N-tri-methylglycine) improved band visibility by eliminating the smearing attributed to the formation of secondary structure, which is caused by G-C rich regions (Henke et al., 1997). Importantly, for surface mucous RAPD analysis, the RNAase treatment of nucleic acid was a necessary step, whereas the inclusion of proteinase K was not.mucous and autologous muscle tissue of M. nigricans, T. albidus, I. platypterus, and X. gladius for each of the three 10-mer primers tested. Representative RAPD banding patterns are illustrated in Figure 1. The nega-tive control reactions (minus template DNA) observed with each primer conﬁrmed that the ampliﬁed genomic DNA from both sources was not an artifact of the primer concentration. Results indicated that sufﬁcient quanti-ties of genomic DNA are available in surface mucous from all the species tested. Sequencing analysisFor the three species screened, M. nigricans, T. albidus, and I. platypterus, the quality of nuclear and mitochon-drial PCR ampliﬁcations were comparable. For both the nuclear WM13 and the mt ND4 locus, sequences derived from a single individual from both sources of genomic DNA (mucous and autologous muscle tissue) were found to be identical when compared with respect to nucleotide base composition (5ʹ–3ʹ and 3ʹ–5ʹ directions), thereby demonstrating that the genomic DNA derived from sur-face mucous was not due to cross-contamination from other billﬁsh or microbes, and in fact originated from the ﬁsh sampled. Nuclear and mitochondrial sequences derived from both sources of genomic DNA are avail-able from GenBank under the following accession num-bers: T. albidus: ND4 (997bp): FJ809995-FJ809997; WM13 (279bp): FJ809988; M. nigricans: ND4 (966bp): 342 Fishery Bulletin 107(3)The nondestructive and less invasive procedures associated with sampling surface mucous offers many advantages when compared to f in clip and muscle tissue collection, particularly when very large fishes destined for live release are involved. Although the collection of surface mucous with sponges was ad-equate for the present study, commercially available products such as FTA® cards (Whatman Inc., Florham Park, NJ) may offer advantages. For example, Livia et al. (2006) reported that FTA® cards were a fast and reliable method of collecting, storing, and extracting genomic DNA from E. lucius and S. trutta fario. FTA® cards can be stored dry at room temperature, thus eliminating the need for laboratory freezers or special shipping considerations. We tested FTA® cards on a limited number of I. platypterus and T. albidus surface mucous samples (data not shown). Preliminary results were successful for PCR amplification, and further analyses are planned.We report the ﬁrst use of surface mucous from ma-rine ﬁshes as an alternative method of DNA sampling. As such, the method offers advantages that warrant consideration when planning genetic studies on other marine species, particularly those where live-release is desirable.AcknowledgmentsThis study was supported by National Oceanic and Atmospheric Administration grants to E. D. Prince and M. S. Shivji. Appreciation is extended to the captains and crew members who assisted in the ﬁeld sampling. Literature citedBuonaccorsi, V. P., K. S. Reece, L. W. Morgan, and J. E. Graves.1999. Geographic distribution of molecular variance within the blue marlin (Makaira nigricans): a hierarchi-cal analysis of allozyme, single-copy nuclear DNA, and mitochondrial DNA markers. Evolution 53:568–579.Chansue, N.2006. Rapid isolation of DNA from the mucus of Asian arowana (Scleropages formosus, Osteoglossidae). Thai J. Vet. Med. 36:55–59.Chou, Q., M. Russell, D. E. Birch, J. Raymond, and W. Bloch.1992. Prevention of pre-PCR mispriming and primer dimer-ization improves low-copy-number amplifications. Nu-cleic Acids Res. 20:1717–1723.Dessauer, H. C., C. J. Cole, and M. S. Hafner.1996. Collection and storage of tissues. In Molecular systematics, 2nd ed. (D. M. Hillis, C. Moritz, and B. K. Mable, eds.), p. 29–47. Sinauer, Sunderland, MA.Ellsworth, D. L., K. D. Rittenhouse, and R. L. Honeycutt.1993. Artifactual variation in randomly amplif ied polymorhic DNA banding patterns. Biotechniques 14:214–217.Henke, W., K. Herdel, K. Jung, D. Schnorr, and S. A. Loening.1997. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25:3957–3958.Hoolihan, J. P., J. Premanandh, M. A. D’Aloia-Palmieri, and J. A. H. Benzie.2004. Intraspecific phylogeographic isolation of Arabian Gulf sailfish Istiophorus platypterus inferred from mito-chondrial DNA. Mar. Biol. 145:465–475.Livia, L., P. Antonella, L. Hovirag, N. Mauro, and F. Panara.2006. A nondestructive, rapid, reliable and inexpensive method to sample, store and extract high-quality DNA from fish body mucus and buccal cells. Mol. Ecol. Notes 6:257–260.Palumbi, S. R.1994. Genetic divergence, reproductive isolation, and marine speciation. Annu. Rev. Ecol. Systemat. 25:547–572.Prince, E. D., D. Snodgrass, E. S. Orbesen, J. P. Hoolihan, and J. E. Serafy.2007. Circle hooks, “J” hooks, and “drop-back” time: a hook performance study of the south Florida recreational live bait fishery for sailfish, Istiophorus platypterus (Shaw). Fish. Manag. Ecol. 14:173–182.Schultz, D. R., N. F. Perez, D. Snodgrass, J. E. Serafy, E. D. Prince, F. Arocha, T. R. Capo, and C. K. Tan.2006. Enzyme-linked immunosorbent assays and billfish gender: testing muscle tissue and surface mucus in tag-ging studies. Bull. Mar. Sci. 79:859–864.Shivji, M. S., J. E. Magnussen, L. R. Beerkircher, G. Hinteregger, D. W. Lee, J. E. Serafy, and E. D. Prince.2006. Validity, identification, and distribution of the roundscale spearfish, Tetrapturus georgii (Teleostei: Istio-phoridae): Morphological and molecular evidence. Bull. Mar. Sci. 79:483–491.Ward, R. D.2000. Genetics in fisheries management. Hydrobiologia 420:191–201.Wasko, A. P., C. Martins, C. Oliveira, and F. Foresti.2003. Non-destructive genetic sampling in f ish. An improved method for DNA extraction from fish fins and scales. Hereditas 138:161–165.Yue, G. H., and L. Orban.2001. Rapid isolation of DNA from fresh and preserved fish scales for polymerase chain reaction. Mar. Bio-technol. 3:199–204.

Surface Mucous as a Source of Genomic DNA from Atlantic Billfishes (Istiophoridae) and Swordfish (Xiphiidae)

Aquatic Commons

339
Understanding genetic variation and 
identifying evolutionary lineages are 
important considerations for conser-
vation management of large pelagic 
fishes (Palumbi, 1994; Ward, 2000). 
Some methods used for genetic sam-
pling of billfishes (families Istiophori-
dae and Xiphiidae) require excision 
of muscle, skin, or fin tissue, and 
excision requires manual restraint 
or killing of the animal. Some non-
destructive sampling methods, such 
as fin and scale collection, exist that 
avoid the need to kill the fish (Yue 
and Orban, 2001; Wasko et al., 2003; 
Hoolihan et al., 2004). Tissue exci-
sion from very large live specimens is 
problematic because of personal safety 
concerns for handlers, and injuries to 
fish that may reduce survival. Most 
recreational billfish caught in the 
United States are released (Prince 
et al., 2007), whereas possession of 
billfish by U.S. pelagic longline ves-
sels and sales of Atlantic billfishes 
have been prohibited since 1988. This 
regulation eliminates opportunities to 
obtain DNA samples from landed spec-
imens, warranting a need for alterna-
tive methods. To sample genomic DNA 
with nondestructive techniques and 
with minimal handling would promote 
the survival of released individuals 
and increase the opportunities to con-
duct genetic studies.
One alternate potential source of 
genomic DNA is the epidermal cells 
found in billfish and swordfish sur-
face mucous. Successful extractions of 
sufficient quantities of genomic DNA 
from surface mucous of freshwater 
fishes have been reported for Salmo 
trutta fario (brown trout), Esox lucius 
(northern pike) (Livia et al., 2006) 
and Scleropages formosus (Asian 
arowana) (Chansue, 2006), provid-
ing an experimental model for large 
pelagic species. 
The present study compares DNA 
extractions from surface mucous and 
autologous skeletal muscle tissue from 
billfishes and swordfish to determine 
the suitability of DNA extracted from 
surface mucous for random amplified 
polymorphic DNA (RAPD) and DNA 
sequencing analyses.
Materials and methods
Surface mucous and autologous skel-
etal muscle samples were collected 
Surface mucous as a source of genomic DNA  
from Atlantic billfishes (Istiophoridae)  
and swordfish (Xiphiidae)
John P. Hoolihan1
Nerida F. Perez2 Rebekah L. Horn3
Ronald M. Faugue2 Derke Snodgrass1
Andrea M. Bernard3 Duane R. Schultz (contact author)2
Email address for contact author: dschultz@med.miami.edu
1 NOAA Fisheries
 Southeast Fisheries Science Center
 75 Virginia Beach Drive
 Miami, Florida 33149
2 Department of Medicine
 University of Miami
 Miller School of Medicine
 Miami, Florida 33136
3 Guy Harvey Research Institute
 Nova Southeastern University
 Oceanographic Center
 8000 North Ocean Drive
 Dania Beach, Florida 33004
Manuscript submitted 26 November 2008.
Manuscript accepted 26 March 2009.
Fish. Bull. 107:339–342 (2009).
The views and opinions expressed  
or implied in this article are those  
of the author and do not necessarily  
reflect the position of the National  
Marine Fisheries Service, NOAA.
Abstract—Procedures for sampling 
genomic DNA from live billf ishes 
involve manual restraint and tissue 
excision that can be difficult to carry 
out and may produce stresses that 
affect fish survival. We examined 
the collection of surface mucous as 
a less invasive alternative method for 
sourcing genomic DNA by comparing 
it to autologous muscle tissue samples 
from Atlantic blue marlin (Makaira 
nigricans), white marlin (Tetraptu-
rus albidus), sailf ish (Istiophorus 
platypterus), and swordfish (Xiphias 
gladius). Purified DNA from mucous 
was comparable to muscle and was 
suitable for conventional polymerase 
chain reaction, random amplified poly-
morphic DNA analysis, and mitochon-
drial and nuclear locus sequencing. 
The nondestructive and less invasive 
characteristics of surface mucous col-
lection may promote increased sur-
vival of released specimens and may 
be advantageous for other marine fish 
genetic studies, particularly those 
involving large live specimens des-
tined for release.
340 Fishery Bulletin 107(3)
Table 1
Nucleotide composition of three 10-mer primers used for 
random amplified polymorphic DNA (RAPD) compari-
son of DNA extracted from billfish surface mucous and 
muscle tissue, and percent content of guanine and cyto-
sine (% GC).
Primer no. Nucleotide composition % GC
1 5ʹ-GTTGCGGGCT-3ʹ 70
2 5ʹ-CAGCCCGGGT-3ʹ 80
3 5ʹ-AGGCCACCGC-3ʹ 80
from three individuals from each of the following species: 
Makaira nigricans (blue marlin), Tetrapturus albidus 
(white marlin), Istiophorus platypterus (sailfish), and 
Xiphias gladius (swordfish). We compared purified DNA 
extracted from mucous and muscle using RAPD, mito-
chondrial DNA (mtDNA) sequencing, and nuclear DNA 
sequencing analyses.
Sample preparation and DNA purification
Surface mucous samples were collected with a poly-
urethane sponge as described by Schultz et al. (2006) 
and immediately processed, or stored at –80°C for later 
extraction. Each sponge was cut into small pieces, mixed 
with 1500 μL of phosphate buffered saline, and com-
pressed repeatedly to remove mucous. All fluids were 
centrifuged through a single QIAamp® (Qiagen Inc., 
Valencia, CA) spin column. DNA purification was car-
ried out by using Qiagen® buccal swab spin protocol 
with the following modifications: 1) a final concentration 
of 1 millimolar (mM) ethylenediaminetetraacetic acid 
(EDTA) was added to the phosphate-buffered saline 
extraction buffer; 2) the proteinase K treatment step 
was eliminated; and 3) an RNase mixture of A and 
T1 enzymes was used to degrade RNA after the final 
DNA purification step (Ambion Inc., Foster City, CA). 
The RNase cocktail enzyme mix was necessary because 
the Qiagen® spin columns copurify RNA and DNA in 
parallel when both are present in a sample. Cold ethanol 
precipitation and inclusion of EDTA in buffers were used 
to reduce nuclease degradation (Dessauer et al., 1996; 
Wasko et al., 2003). 
For muscle tissue, 25 mg were macerated in 180 
μL Buffer ATL (Qiagen Inc., Valencia, CA), and in-
cubated overnight in a 56°C water bath in the pres-
ence of 20 μL proteinase K. Purification followed the 
QIAamp® manufacturer’s protocol for tissue. Aque-
ous samples of nucleic acid (1.5 μL) from mucous and 
muscle extractions were measured for purity with a 
NanoDrop 1000 Spectrophotometer (Thermo Fisher 
Scientific,Wilmington, DE) and showed a range of 1.7 
to 2.0 for the DNA and RNA absorbance ratios (260 
nm:280 nm).
RAPD analysis
Three different 10-mer oligonucleotide primers (Table 1) 
of arbitrary sequence (IDT Integrated DNA Technolo-
gies, Coralville, IA) were tested against surface mucous 
DNA and autologous muscle DNA from three individuals 
of M. nigricans, T. albidus, I. platypterus, and X. gla-
dius. PCR reactions for RAPD analysis were performed 
in a total volume of 25 μL containing 3 μL extracted 
genomic DNA, 2.5 μL of 10 mM 10-mer primer, 2.5 μL 
of 2.5 mM deoxynucleotide triphosphate (dNTPs), 0.75 
μL of 25 mM MgCl2, 0.20 μL Taq DNA polymerase, 2.5 
μL 10× buffer, 5 μL 5M betaine (N, N, N-trimethylgly-
cine), 2 μL HotStart-IT® binding protein (USB Corp., 
Cleveland, OH), and 6.55 μL distilled water. Pre-PCR 
incubation with the HotStart-IT™ binding protein was 
executed at 25°C for four hours to prevent mispriming 
and primer dimerization during amplification (Chou 
et al., 1992). RAPD PCR was performed in an Eppen-
dorf Mastercycler (Westbury, NY) starting with initial 
heating for 5 minutes at 94°C, followed by 34 cycles at 
94°C for 5 minutes, 42°C for 30 seconds, 72°C for 90 
seconds, and a final extension of 72°C for 10 minutes. 
A negative control (no genomic DNA) was included in 
each PCR set to verify no reagent contamination. The 
PCR products were verified by electrophoresing 5 μL 
in 1.2% agarose gel (ISC BioExpress, Kayville, UT) 
and TAE buffer (pH 8.5) for 60 min at 100 V (60 mA), 
stained with ethidium bromide, and visualized through 
a UV transilluminator.
Sequencing analysis
Mitochondrial and single-copy nuclear loci were 
sequenced from three individuals from each of the fol-
lowing species: M. nigricans, T. albidus, and I. platyp-
terus. Insufficient samples of mucous and muscle were 
available for sequencing X. gladius. Mitochondrial 
NADH dehydrogenase subunit4 (ND4) was ampli-
fied by PCR with the primer pair 61F and 1837R and 
cycling parameters outlined in Shivji et al. (2006). All 
amplifications of the anonymous single-copy nuclear 
locus WM13 were performed with the primer pair 
WM13-F and WM13-R developed by Buonaccorsi et 
al. (1999). Nuclear PCR reactions were performed in 
a total reaction volume of 50 μL containing 1 μL of 
extracted genomic DNA, 10 pmol/μL of each primer, 
40 μM dNTPs, 10× PCR buffer, and 1 unit of HotStar 
Taq™ DNA Polymerase (Qiagen Inc., Valencia, CA). 
The PCR thermal profile consisted of an initial heating 
at 95°C for 15 minutes to activate the DNA polymerase, 
followed by 35–40 cycles at 94°C for 1 minute, 1 minute 
at 50°C, and 1 minute at 72°C, with a 5-minute final 
extension step at 72°C. Both nuclear and mitochondrial 
amplifications were performed in a MJ Research PTC-
200 thermal cycler (Waltham, MA). A negative control 
(no genomic DNA) was included in each PCR set to 
verify that there was no reagent contamination.
All amplified products were purified by using the 
QIAquick® PCR purification kit (Qiagen Inc., Valencia, 
CA) and sequenced with an Applied Biosystems 3130 
341Hoolihan et al.: Surface mucous as a source of genomic DNA from Istiophoridae and Xiphiidae
Figure 1
Representative random amplified polymorphic DNA (RAPD) banding patterns from 
four species: A) Makaira nigricans (blue marlin), B) Tetrapturus albidus (white 
marlin), C) Istiophorus platypterus (sailfish), and D) Xiphias gladius (swordfish) 
for three 10-mer primers (Table 1) tested on each fish for the template DNA from 
surface mucus (SM), and autologous muscle tissue (MT). Lane contents are as fol-
lows: 1, 100-bp ladder; 2, 5, and 8, negative controls (no DNA); 3 and 4, primer no. 
1; 6 and 7, primer no. 2; and, 9 and 10, primer no. 3.
A B
C D
genetic analyzer (Foster City, 
CA). Forward and reverse se-
quences were assembled and 
edited with GeneDoc 2.6.002 
(http://www.psc.edu/biomed/
genedoc, accessed 1 June 
2008). To control for cross-
contamination with other 
billfish or possible parasites 
or microbes, we compared the 
mitochondrial and nuclear se-
quences derived from surface 
mucous to sequences obtained 
from autologous muscle tis-
sue. Because autologous mus-
cle tissue was not available 
for the T. albidus sequencing 
analyses, the mucous sample 
sequences were compared to 
homologous locus sequences 
obtained from reference T. 
albidus tissues available in 
our laboratory. 
Results and conclusions
RAPD analysis
Comparable RAPD amplicons 
were derived from surface 
FJ809991, FJ809992, and FJ809994; WM13 (279bp): 
FJ809986, FJ809987, and FJ809989; I. platypterus: 
ND4 (1009bp): FJ809990 and FJ809993; WM13 (277bp): 
FJ809984 and FJ809985. 
This study has shown that surface mucous contains 
sufficient quantities of genomic DNA to carry out RAPD 
analyses of istiophorid billfishes and swordfish, as well 
as sequencing applications of istiophorid billfishes. 
These extractions compare favorably to genomic DNA 
extractions from surface mucous reported for freshwater 
species S. formosus (Chansue, 2006), E. lucius, and S. 
trutta fario (Livia et al., 2006). 
The slight differences between some of the mucous 
and muscle RAPD amplification profiles (Fig. 1) may 
be a result of contaminant DNA in the mucous (e.g., 
bacteria, microalgae), or other artifact variations known 
to occur in RAPD studies (Ellsworth et al., 1993). We 
found that the pre-PCR incubation of the template DNA 
master mix with HotStart-IT™ binding protein was 
crucial for preventing mispriming and primer dimer-
ization that produced these artifact bands (Chou et al., 
1992). In addition, the inclusion of betaine (N, N, N-tri-
methylglycine) improved band visibility by eliminating 
the smearing attributed to the formation of secondary 
structure, which is caused by G-C rich regions (Henke 
et al., 1997). Importantly, for surface mucous RAPD 
analysis, the RNAase treatment of nucleic acid was a 
necessary step, whereas the inclusion of proteinase K 
was not.
mucous and autologous muscle tissue of M. nigricans, 
T. albidus, I. platypterus, and X. gladius for each of 
the three 10-mer primers tested. Representative RAPD 
banding patterns are illustrated in Figure 1. The nega-
tive control reactions (minus template DNA) observed 
with each primer confirmed that the amplified genomic 
DNA from both sources was not an artifact of the primer 
concentration. Results indicated that sufficient quanti-
ties of genomic DNA are available in surface mucous 
from all the species tested. 
Sequencing analysis
For the three species screened, M. nigricans, T. albidus, 
and I. platypterus, the quality of nuclear and mitochon-
drial PCR amplifications were comparable. For both the 
nuclear WM13 and the mt ND4 locus, sequences derived 
from a single individual from both sources of genomic 
DNA (mucous and autologous muscle tissue) were found 
to be identical when compared with respect to nucleotide 
base composition (5ʹ–3ʹ and 3ʹ–5ʹ directions), thereby 
demonstrating that the genomic DNA derived from sur-
face mucous was not due to cross-contamination from 
other billfish or microbes, and in fact originated from 
the fish sampled. Nuclear and mitochondrial sequences 
derived from both sources of genomic DNA are avail-
able from GenBank under the following accession num-
bers: T. albidus: ND4 (997bp): FJ809995-FJ809997; 
WM13 (279bp): FJ809988; M. nigricans: ND4 (966bp): 
342 Fishery Bulletin 107(3)
The nondestructive and less invasive procedures 
associated with sampling surface mucous offers many 
advantages when compared to f in clip and muscle 
tissue collection, particularly when very large fishes 
destined for live release are involved. Although the 
collection of surface mucous with sponges was ad-
equate for the present study, commercially available 
products such as FTA® cards (Whatman Inc., Florham 
Park, NJ) may offer advantages. For example, Livia et 
al. (2006) reported that FTA® cards were a fast and 
reliable method of collecting, storing, and extracting 
genomic DNA from E. lucius and S. trutta fario. FTA® 
cards can be stored dry at room temperature, thus 
eliminating the need for laboratory freezers or special 
shipping considerations. We tested FTA® cards on a 
limited number of I. platypterus and T. albidus surface 
mucous samples (data not shown). Preliminary results 
were successful for PCR amplification, and further 
analyses are planned.
We report the first use of surface mucous from ma-
rine fishes as an alternative method of DNA sampling. 
As such, the method offers advantages that warrant 
consideration when planning genetic studies on other 
marine species, particularly those where live-release 
is desirable.
Acknowledgments
This study was supported by National Oceanic and 
Atmospheric Administration grants to E. D. Prince and 
M. S. Shivji. Appreciation is extended to the captains 
and crew members who assisted in the field sampling. 
Literature cited
Buonaccorsi, V. P., K. S. Reece, L. W. Morgan, and J. E. Graves.
1999. Geographic distribution of molecular variance 
within the blue marlin (Makaira nigricans): a hierarchi-
cal analysis of allozyme, single-copy nuclear DNA, and 
mitochondrial DNA markers. Evolution 53:568–579.
Chansue, N.
2006. Rapid isolation of DNA from the mucus of Asian 
arowana (Scleropages formosus, Osteoglossidae). Thai 
J. Vet. Med. 36:55–59.
Chou, Q., M. Russell, D. E. Birch, J. Raymond, and W. Bloch.
1992. Prevention of pre-PCR mispriming and primer dimer-
ization improves low-copy-number amplifications. Nu-
cleic Acids Res. 20:1717–1723.
Dessauer, H. C., C. J. Cole, and M. S. Hafner.
1996. Collection and storage of tissues. In Molecular 
systematics, 2nd ed. (D. M. Hillis, C. Moritz, and B. K. 
Mable, eds.), p. 29–47. Sinauer, Sunderland, MA.
Ellsworth, D. L., K. D. Rittenhouse, and R. L. Honeycutt.
1993. Artifactual variation in randomly amplif ied 
polymorhic DNA banding patterns. Biotechniques 
14:214–217.
Henke, W., K. Herdel, K. Jung, D. Schnorr, and S. A. Loening.
1997. Betaine improves the PCR amplification of GC-rich 
DNA sequences. Nucleic Acids Res. 25:3957–3958.
Hoolihan, J. P., J. Premanandh, M. A. D’Aloia-Palmieri, and 
J. A. H. Benzie.
2004. Intraspecific phylogeographic isolation of Arabian 
Gulf sailfish Istiophorus platypterus inferred from mito-
chondrial DNA. Mar. Biol. 145:465–475.
Livia, L., P. Antonella, L. Hovirag, N. Mauro, and F. Panara.
2006. A nondestructive, rapid, reliable and inexpensive 
method to sample, store and extract high-quality DNA 
from fish body mucus and buccal cells. Mol. Ecol. Notes 
6:257–260.
Palumbi, S. R.
1994. Genetic divergence, reproductive isolation, and 
marine speciation. Annu. Rev. Ecol. Systemat. 25:547–
572.
Prince, E. D., D. Snodgrass, E. S. Orbesen, J. P. Hoolihan, and 
J. E. Serafy.
2007. Circle hooks, “J” hooks, and “drop-back” time: a 
hook performance study of the south Florida recreational 
live bait fishery for sailfish, Istiophorus platypterus 
(Shaw). Fish. Manag. Ecol. 14:173–182.
Schultz, D. R., N. F. Perez, D. Snodgrass, J. E. Serafy, E. D. 
Prince, F. Arocha, T. R. Capo, and C. K. Tan.
2006. Enzyme-linked immunosorbent assays and billfish 
gender: testing muscle tissue and surface mucus in tag-
ging studies. Bull. Mar. Sci. 79:859–864.
Shivji, M. S., J. E. Magnussen, L. R. Beerkircher, G. Hinteregger, 
D. W. Lee, J. E. Serafy, and E. D. Prince.
2006. Validity, identification, and distribution of the 
roundscale spearfish, Tetrapturus georgii (Teleostei: Istio-
phoridae): Morphological and molecular evidence. Bull. 
Mar. Sci. 79:483–491.
Ward, R. D.
2000. Genetics in fisheries management. Hydrobiologia 
420:191–201.
Wasko, A. P., C. Martins, C. Oliveira, and F. Foresti.
2003. Non-destructive genetic sampling in f ish. An 
improved method for DNA extraction from fish fins 
and scales. Hereditas 138:161–165.
Yue, G. H., and L. Orban.
2001. Rapid isolation of DNA from fresh and preserved 
fish scales for polymerase chain reaction. Mar. Bio-
technol. 3:199–204.


Surface mucous as a source of genomic DNA from Atlantic billfishes (Istiophoridae) and swordfish (Xiphiidae)

http://aquaticcommons.org/8790/1/hoolihan_Fish_Bull_2009.pdf

Surface mucous as a source of genomic DNA from Atlantic billfishes (Istiophoridae) and swordfish (Xiphiidae)

Abstract

Similar works

Full text

Available Versions

NSU Works

Aquatic Commons