Alexis Tapanes

University of Miami, Department of Biology

P.O. Box 249118, Coral Gables, Florida 33124, U.S.A.

Abstract

The objective of this research was to estimate allozyme variability in the South Everglades apple snail, Pomacea paludosa. Protein electrophoresis, using cellulose acetate plates, was conducted on homogenized muscle tissue. Fifteen enzymes- EST, GPDH, G6PDH, GPI, HEX, LAP, MDH-1, MDH-2, ME, MPI, three PEP's (LEU-GLY-GLY, PHE-PRO, and VAL-LEU), 6PGDH, and PGM- were resolved.

Between seven and eighteen snails, depending on the system, were scored. The percent of polymorphic loci (P) was 0.067. At the GPI locus, sixteen snails were homozygous for the anodal band and two snails were heterozygous. The average heterozygousity (Hs) was 0.106. There was no statistically significant deviation from Hardy-Weinberg Equilibrium. The Hs over all loci was 0.007.

P and Hs were low compared to land and marine snails (Selander 1976). Allozyme variability was low among individuals. Genetic drift and/or selection may favor homozygotes. Small sample size and/or related individuals may have biased results. However, before any conclusions are made, a larger sample size consisting of individuals collected at greater geographical distances should be scored and more loci should be screened for polymorphism.

The information acquired from this project may be included in a future study about the population genetics of South Florida's and Cuba's apple snails. The research eventually may be used to determine if Florida's apple snail population was founded by a Cuban population.

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Figure 1. Alexis Tapanes holding an apple snail in Dr. Gaines' lab at the University of Miami.

The Florida apple snail (Figures 1 and 2), Pomacea paludosa, lives primarily in freshwater and is capable of both aquatic and aerial respiration. The species is dioecious. Females lay gelatinous eggs on emergent vegetation. Apple snails feed on vegetation, small invertebrates, and animal remains. They are a primary food source of the endangered Everglades kite, and they are part of the diet of other birds, alligators, and turtles.

Figure 2. Apple snails collected on February 1997 from State Wate Management Area 3A in South Florida's Everglades.

Apple snails are found throughout the tropics, but their distribution is not completely mapped. P. paludosa is widely distributed throughout Florida (Figure 3a and b). It is also found in some springs in southern Georgia and southern Alabama (Thompson 1984). P. paludosa or perhaps a closely related species is believed to reside in Cuba (Rich, personal communication).

Figure 3a. Apple snail habitat in the South Everglades.

The goal of this project was to estimate allozyme variability in the South Everglades apple snail population. Allozymes are different electrophoretic forms of an enzyme expressed by alleles at the same locus. Electrophoresis is the separation of proteins by an electric field. The mobility of proteins varies due to unique amino acid compositions. Cellulose acetate electrophoresis uses a cellulose acetate medium to separate proteins primarily by charge. Protein migration takes place on the buffer film on the surface of the cellulose acetate plate. The buffer used depends on the enzyme being screened. Cellulose acetate plates require less sample and stain volume and allow shorter gel running and staining time than other mediums.

Figure 3b. Apple snail habitat in the South Everglades.

Sixteen apple snails were collected on February 1997 and four more were collected on July of the same year from State Water Management Area 3A in South Florida's Everglades. Individuals were frozen alive and stored at -80 oC. They were thawed in a refrigerated ice bath for 10-25 minutes prior to being dissected. Once their muscle tissue was cut into small pieces, a homogenizing solution of approximately equal volume was added to the tissue (Mulvey, personal communication). The solution consisted of 100ml double-distilled water, 10mg NADP, and 100 ul B-mercaptoethanol (Richardson et. al., 1986). The solution helped stabilize tissue enzymes. Then the samples were centrifugedfor 8 minutes at 14,000xg, divided into aliquots, and stored at -80 oC. Enzymes were thawed and refrozen a maximum of five times before being discarded.

Gels were soaked for at least 20 minutes in one of four buffers: 0.01M citrate-phosphate pH 6.4, 0.02M phosphate pH 7.0, 0.015M Tris-EDTA-borate-MgCl2 pH 7.8 (Richardson et. al., 1986), or Tris-Glycine (Hebert and Beaton, 1993). The buffer used varied according to which enzyme was being scored. Samples were loaded onto cellulose acetate plates, placed in an electrophoresis chamber containing the same buffer used to soak the plate, and ran at 200V unless otherwise stated. Optimal electrophoresis conditions varied among enzymes (Tables 1 and 2). Plates were stained with the recipes provided by Hebert and Beaton (1993). Recipes were slightly modified depending on the age of staining ingredients, number of times a sample was thawed, and enzyme activity at the locus being stained. Each staining mixture contained substrate and diazonium salt that made a specific enzymatic reaction visible.

Table 1. Optimal Electrophoresis Conditions

Electrophoresis was conducted at 200 V unless otherwise stated.

Table 2. Buffers

Fifteen loci-EST, GPDH, G6PDH, GPI, HEX, LAP, MDH-1, MDH-2, ME, MPI, three PEP's (LEU-GLY-GLY, PHE-PRO, and VAL-LEU), 6PGDH, and PGM-were resolved. Between seven and eighteen snails, depending on the system, were scored (Figure 4). The percent of polymorphic loci (P) was 1/15 or 0.067 (Figure 5). At the GPI locus, sixteen snails were homozygous for the anodal band and two were heterozygous (Figure 6). GPI is a dimer; each enzyme consists of two polypeptide subunits. Thus, heterozygotes have three bands because they make three different forms of the same enzyme. The individuals homozygous for the anodal band have alleles that only make anodal polypeptide chains. The fainter, faster anodal bands are probably due to post-translational modifications. Genotypic frequencies at the GPI locus were p=0.944 and q=0.056. Average heterozygousity (Hs) was 0.106 (Figure 7). There was no statistically significant deviation from Hardy-Weinberg Equilibrium. The Hs over all loci was 0.007.

Figure 4. Snails scored per locus.

Figure 5. Percent polymorphic vs. monomorphic loci.

Figure 6. Snails stained on a cellulose acetate plate for GPI. Snails one and three are heterozygotes. The other snails are homozygotes.

Figure 7. Genotypic frequencies vs. genotype at the GPI locus.

Discussion

Percent polymorphic loci and average heterozygousity were low compared to mean P and Hs of land and marine snails. Genic variation was estimated among land snails as P=0.437 and Hs over all loci=0.150; it was estimated among marine snails as P=0.175 and Hs over all loci=0.083 (Selander 1976). Allozyme variability was low among the individuals sampled. This may be a result of genetic drift and/or natural selection favoring homozygotes. The most likely explanation is small sample size. However, before any conclusions are drawn, a larger sample size consisting of individuals collected at greater geographical distances should be scored and more loci should be screened for polymorphism.

The data acquired from this project may be included in a future study about the population genetics of South Florida's and Cuba's apple snails. The research eventually may be used to determine the likelihood that Florida's apple snail population was founded by a Cuban population.

Acknowledgements

I would like to thank Dr. Earl Rich, Dr. Michael Gaines, Howard Hughes Medical Institute, Brady Barr, Mei Len Sanchez, Dr. Ken Spitze, Marcos Ottone, Maria Rimoldi, Margaret Mulvey, Betty Blanco, Xiao-Yuan Kong, Dr. Peter Luykx, Robb Wright, M. Chiappone, and my family and friends for all of their support.

Hebert, P.D.N. and M.J. Beaton. 1993. Methodologies for Allozyme Analysis Using Cellulose Acetate Electrophoresis: A Practical Handbook. Helena Laboratories, 32 pp.

Richardson, B.J., P.R. Bavenstock, M. Adams. 1986. Allozyme Electrophoresis: A Handbook for Animal Systematics and Population Studies. New York: Academic Press, 410 pp.

Selander, R.K. 1976. Genic Variation in Natural Populations. In: Molecular Evolution (Ayala, F.D. ed). Sunderland: Sinauer Associates, pp. 21-46.

Thompson, F.G. 1984. Freshwater Snails of Florida: A Manual for Identification. Gainesville: University Presses of Florida.