SHAKLEE and SAMOLLOW: GENETIC VARIATION IN SPINY LOBSTER 



MATERIALS AND METHODS 



Sample Collection and Processing 



A total of 1,869 spiny lobsters from the five 

 localities was collected over a 2V2-yr period 

 (Fig. 1). Lobsters from Kure Atoll, Oahu, and 

 Hawaii were collected by hand by divers using 

 scuba. Lobsters from Maro Reef and Necker Island 

 were collected using standard, wire mesh lobster 

 traps. The sampling periods and numbers of 

 lobsters collected for each of the five localities 

 are Kure: October 1978-January 1979 (N = 21), 

 June 1979-September 1979 (N = 136), August 

 1979-June 1980 {N = 249), and June 1980- 

 January 1981 (N = 176); Maro Reef: October 1978 

 (N = 60), November 1979 (N = 213), and Sep- 

 tember 1980 (N = 145); Necker: October 1978 

 {N = 97), March 1979-June 1979 (N = 421), and 

 December 1980 (N = 148); Oahu: May 1979- 

 January 1980 (N = 53), July 1980-December 1980 

 (N = 71), and March 1981 (N = 30); and Hawaii: 

 April 1980-March 1981 (N = 49). Because not all 

 individuals were sexed and measured (in some 

 cases one or both types of data were unavailable 

 for entire collections), sex ratios and size composi- 

 tions could not be accurately calculated. However, 

 all animals included in the data analyses were 

 adults. Some samples were frozen at -20°C in the 

 field (as whole animals, as carcasses minus tails, 

 or as isolated pereiopods — walking legs) and 

 shipped frozen to the laboratory. Other samples 

 were transported live to Honolulu where they were 

 dissected and sampled. In the initial screening for 

 polymorphic loci, samples of pereiopod muscle, 

 abdominal muscle, digestive gland, green gland, 

 eye, heart, and gills were dissected from whole, 

 frozen lobsters. Each of these samples was 

 homogenized at 4°C in an equal volume of 0.1 M 

 Tris-HCl pH 7.0 buffer (containing 1 x 10" 3 M 

 EDTA and 5 x 10 5 M NADP + ) using a loose fit- 

 ting, motorized pestle. Homogenates were cen- 

 trifuged at 4°C for at least 20 min at a minimum 

 of 20,000 x g. Digestive gland supernatants 

 were routinely centrifuged a second time to mini- 

 mize lipid content. The resulting supernatants 

 were transferred to individually labeled glass 

 vials which were capped and stored at -75°C 

 until the electrophoretic analyses were completed. 

 Since all variable enzymes could be analyzed in 

 pereiopod muscle extracts (and digestive gland 

 extracts for peptidase-1 (PEP-D) only these 

 tissues were examined in the subsequent 

 analyses. 



Electrophoresis 



The supernatants were analyzed by a combina- 

 tion of vertical and horizontal starch gel elec- 

 trophoresis (Selander et al. 1971; Shaklee et al. 

 1973). Each enzyme system surveyed in the initial 

 screening for genetic variation was elec- 

 trophoresed on 2-10 different buffer systems, 

 using extracts of several different tissues. Follow- 

 ing electrophoresis, isozyme patterns were vis- 

 ualized using standard recipes (modified from 

 Shaw and Prasad 1970; Selander et al. 1971; 

 Siciliano and Shaw 1976). Esterases of the lobster 

 were detected using a-naphthyl acetate (EST) or 

 4-methylumbelliferyl acetate (Umb = Est-D) as 

 substrates, both variable peptidases were stained 

 using leucylleucylleucine as substrate. 



Gel Scoring and Data Analysis 



Patterns of enzyme variation that were consis- 

 tent with the subunit structure of presumably 

 homologous proteins of other species (when 

 known), and with simple genetic models, were 

 scored and recorded as genotypes. Names of en- 

 zymes and Enzyme Commission numbers follow 

 the recommendations of the Commission on 

 Biochemical Nomenclature (1973). For multilocus 

 enzyme systems, loci were consecutively num- 

 bered beginning with the most anodal isozyme. 

 Alleles at each locus were designated according to 

 the relative electrophoretic mobilities of the 

 homomeric isozymes they encode. The most com- 

 mon allele at a locus was designated "100" and all 

 other alleles at that locus were numbered accord- 

 ing to the electrophoretic mobilities of their pro- 

 ducts relative to that of the product of the 100 

 allele. Negative numbers were given to alleles en- 

 coding isozymes with cathodal migration. In the 

 case of the glucosephosphate isomerase (GPI) sys- 

 tem of the spiny lobster, subbanding anodal to the 

 100 isozyme was often quite pronounced, espe- 

 cially in older samples. Because we could not be 

 confident of scoring allelic variation in this region, 

 no attempt was made to score isozymes having an 

 electrophoretic mobility greater than that of the 

 most common (100) isozyme. Many samples were 

 reelectrophoresed with controls of known mobility 

 to verify the identity of rare alleles for each en- 

 zyme system. 



Despite that all alleles were initially identified 

 and assigned numerical designations as described 

 above, in many cases data summaries and statisti- 

 cal analyses employed fewer electromorph (allelic) 



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