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450 GROSS ET AL, after hatching (Coble 1975), This behavior allows for easy collection of nest-specific tissue samples for DNA analysis, Nest-specific DNA fingerprints would provide a new source of markers that could greatly assist research conducted on smallmouth bass, One area of research that may be advanced by application of this technology is determination of factors af- fecting individual reproductive success. A major obstacle to the estimation of individual reproduc- tive success for smallmouth bass has been the in- ability to identify offspring from a particular mat- ing due to dispersal and mixing offry shortly after hatching, For this reason, past studies on individ- ual reproductive success of smallmouth bass have focused only on estimates to the swim-up stage, prior to fry dispersal (Neves 1975; Goff 1986; Raf- fetto et al. 1990; Reynolds 1990), Because a large proportion of individuals survive to these early stages, factors influencing their success to a later life stage, after which the majority of them have died, may not be apparent. Generation of nest- specific DNA fingerprints of smallmouth bass would allow fisheries biologists to monitor the re- productive success of families to any life stage in a natural environment and determine which fac- tors are important in regulating success, In addi- tion, changes in reproductive success could be monitored in response to environmental changes and managerial modifications, This paper describes the application of DNA fingerprinting methods to the smallmouth bass population in Lake Opeongo, Ontario, Individual as well as nest- (family)-specific DNA fingerprints were generated. How nest-specific DNA finger- prints are generated and their potential utility in studying various aspects of small mouth bass bi- ology are discussed, Methods Study population and sample collection, - Lake Opeongo (45042'N, 78022'W) is a 5,860-ha oli- gotrophic lake in Algonquin Park, Ontario, Small- mouth bass were introduced into the lake in the 1920s and have established a prominent, self-sus- taining population (Martin and Fry 1973). Spawn- ing sites of smallmouth bass are distributed throughout the lake but several areas, such as Jones Bay, have notably large concentrations of nest sites, Sample collection was confined to the 6 km of shoreline in Jones Bay, where approximately 40% of all small mouth bass spawning in the lake occurs (M, Ridgway, Ontario Ministry of Natural Re- sources, personal communication), Nest sites were located by snorkeling the shore- line of Jones Bay every 3 d during the spawning season, Once a nest was located, it was marked with a numbered brick, The guardian male (fa- ther) was angled from each nest and tagged with a Hoy anchor tag (Hoy Tag and Manufacturing, Inc,), and a tissue sample was obtained by clipping a portion of his pelvic fin, Upon reaching the swim- up stage, 10 offspring from each nest were col- lected, nonselectively with an aquarium net and placed separately in labeled vials, All samples col- lected in the field were kept on dry ice and stored later at -70oC until the DNA could be extracted, DNA fingerprinting, -Genomic DNA was ex- tracted from the guardian male fin tissue or whole fry by dicing the tissue and suspending it in a buffer solution (50 mM tris-HCl, pH 7,5; lOO mM EDTA, pH 8.0; and 2% sodium dodecyl sulfate, SDS), Proteinase K (200 Jlg) was added to the sus- pension, which was incubated for 2 h at 550C. Two extractions were performed, one with an equal volume of phenol : chloroform (50:50) and anoth- er with an equal volume of chloroform: isoamyl alcohol (24: I). The DNA was precipitated by ad- dition of an equal volume of isopropyl alcohol, chilled for at least 1 h at - 20oC, centrifuged, and washed with 70% ethanol. The DNA was resus- pended in TE buffer (10 mM tris-HCI, pH 7,5; 1 mM EDT A, pH 8,0) for subsequent storage at 40C. Approximately 6 Jlg of DNA was digested with a lOx excess of a restriction endonuclease (H ae III, HinfI, Alu I, Taq I, Msp I, or Pst I) and incubated for 3 h according to the manufacturer's instruc- tions, Samples were processed by electrophoresis through a 1% agarose gel (25 cm; 10-tooth-l-mm comb) at 60 V for 44 h (time at which the 1.6- kilobase [kbJ marker fragment was at the bottom of the gel) in 1 x TBE (tris-borate-EDTA) buffer. At approximately the midpoint through electro- phoresis, the buffer was exchanged to prevent it from being exhausted, After electrophoresis, the DNA in the gel was depurinated in 250 mM HCl for 5 min, denatured in 1,5 M NaCl and 500 mM NaOH for 30 min, and neutralized in 3 M NaCl with 500 mM tris- HCI (pH 7,5) for 30 min, The DNA was then transferred from the gel onto a nylon membrane (Hybond-Nfp, Amersham International) by over- night capillary Southern blotting (Sambrook et al. 1989) in 20 x standard sodium citrate (3 M NaCl, 300 mM Na3 citrate, pH 7,0), The DNA was fixed to the membrane by baking for 3 h at 80oC. Prior to hybridization (the pairing of complemen- tary, single strands of DNA), membranes were wet-