USE OF MICROSATELLITE AND AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS TO STUDY EARLY EMBRYONIC MORTALITY IN FLORIDA ALLIGATORS

B. S. Arnold¹, M. S. Sepúlveda^1,4, D. S. Rotstein², T. S. Gross^1,4,
L. M. Davis³, T. C. Glenn³, and G. Clark⁴

 ¹Florida Integrated Science Center, USGS, Gainesville, FL, USA;
²College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA;
³Savannah River Ecology Lab, University of GA, Aiken, SC, USA;
⁴University of Florida, Gainesville, FL, USA.

Presented at the 40th annual meeting of the Society of Toxicology (SOT), San Francisco, CA. March 2001.
 

ABSTRACT

Reduced viability of American Alligator (Alligator mississippiensis) eggs in Florida lakes have been associated with a decline of alligator populations.  Although these lakes have measurable concentrations of organochlorine pesticides and PCBs, the relationship between egg viability and contaminants is unclear.  In order to determine if undeveloped (unbanded) eggs are the result of non-fertilization or early embryonic mortality DNA from blastodisks of unbanded eggs was compared to DNA of siblings and females captured at the nest to determine if the blastodisks contain alleles other than those from the maternal contribution. If the blastodisks contain paternal alleles (i.e., alleles present in siblings but not present in the females), they must have been from fertilized embryos and would represent embryonic death.  In contrast, eggs without these alleles would have been unfertilized. A preliminary study conducted in 1999 using microsatellite analysis, found that 4 of 5 nonviable eggs/clutches from Lake Apopka had evidence of fertilization. In a subsequent study in 2000, 157 blastodisks from 24 clutches were collected from Florida lakes (Apopka, and Griffin) and from Emeralda Marsh along with blood from females captured at each nest. Samples were split and submitted for microsatellite and amplified fragment length polymorphism analysis.  Preliminary microsatellite analysis of nonviable blastodisks indicate that the eggs are fertilized suggesting that developmental failure is the result of early embryonic mortality. Early embryonic mortality for fertilized eggs could be the result of direct contaminant embryotoxic effects or alterations in egg structure or composition. Funded by NIEHS-SFBRP.

INTRODUCTION

Ø Reduced viability of eggs and population decline of American Alligators in Central Florida lakes have produced concerns about the potential health effects of endocrine disrupting contaminants such as organochlorine pesticides and PCBs.

Ø Determining the effects of these contaminants on reproductive success, involves evaluating the roles of fertilization failure and early embryonic mortality in decreased egg viability.

Ø Fertilization failure could result from defective viability or structure of sperm and/or oocytes, anatomical or physiological alterations of male and female reproductive systems, or from behavioral changes that decrease mating success.

Ø Alternatively, early embryonic mortality could be the result of direct embryotoxic effects or alterations in egg structure or composition due to oviduct dysfunction.

OBJECTIVE

To determine if undeveloped (unbanded) eggs are the result of non-fertilization or of early embryonic mortality.

MATERIALS AND METHODS

Ø Eggs and blood samples were collected in June, 1999 from Lake Apopka and from Emeralda, Apopka, and Griffin in June, 2000.

Ø Twenty ml of blood for DNA analysis was taken from the cranial sinus of females snared at the nest, transferred to sodium heparin tubes, and kept on ice pending delivery to the laboratory.

Ø Eggs were candled with a commercial candler (Lyon Electric Company, Chula Vista, Ca) and numbers of banded and unbanded, viable and nonviable eggs were recorded.

Ø Unbanded eggs (Figure 1) usually contain a 1 to 4 mm blastodisk (Figure 2) containing 80 to 200 cells. The blastodisks were located, removed aseptically, and stored in cryovials at -80 C for DNA analysis.

Ø Blood was taken from hatchlings from each clutch for DNA analysis.

Ø A subsample of eggs from each clutch was analyzed for organochlorine pesticides (Figure 3).

Ø In 1999 DNA from blastodisks and maternal blood from four clutches were analyzed by PCR (polymerase chain reaction) using primers for alligator microsatellite loci (Table 3).

Ø Blastodisks and the associated maternal and hatchling blood collected in 2000 was analyzed by amplified fragment length polymorphism (AFLP) and microsatellite analysis (Table 4).

Ø Qiagen Kits or Phenyl Chloroform (PCI) were used for extractions (Table 1).

Ø Bead capture enrichment were used to isolate additional microsatellite loci.

Ø The complete protocols for microsatellite development are available at: http://www.uga.edu/srel/DNA_Lab/protocols.htm and http://gator.biol.sc.edu/.

Figure 2. Egg with blastodisk.

RESULTS AND DISCUSSION

Ø Data on some blastodisks tested so far show the presence of paternal DNA (Tables 3 & 4).

Ø Contaminant analysis on unbanded eggs show significant concentrations of toxaphene, DDE,  and Dieldrin as well as lower concentrations of several other organochlorines (Fig. 3).

Ø Microsatellite DNA loci (Table 2) have been described from American Alligators (Glenn et al. 1998). These loci have been used to investigate population genetic structure (Davis et al. 2001a) and mating strategies (Davis et al. 2001b) in alligators.  Although these loci are reasonably polymorphic, they are not well suited to amplifying minute amounts of DNA (i.e., the PCR efficiency is low). Thus, loci with higher PCR efficiency are needed for best use of microsatellites in this study.

Ø Data from amplified fragment length polymorphism analysis (AFLP) has not been completed,  however preliminary data from microsatellite analysis indicates that it can be used successfully to study the fertilization status of nonviable alligator eggs.  This data indicates that early embryonic mortality occurs in nonviable eggs from viable clutches. Early embryonic mortality from fertilized eggs could be the result of direct embryotoxic effects or of alterations in egg structure or composition.  Further studies to assess occurrence of early embryonic mortality will provide a better understanding of causes of reproductive failure in alligators and its relationship to previously reported effects of endocrine-disrupting contaminants.
 

CONCLUSION

Preliminary microsatellite analysis of nonviable blastodisks suggest that the eggs were fertilized indicating that developmental failure was the result of early embryonic mortality.
 

REFERENCES

1. Glenn, TC, HC Dessauer, and MJ Braun. 1998. Characterization of Microsatellite DNA Loci in American Alligators. Copeia 3: 591-601.
2. Davis, LM, TC Glenn, RM Elsey, IL Brisbin Jr, WE Rhodes, HC Dessauer, and RH Sawyer. 2001a. Genetic structure of six populations of American alligators: a microsatellite analysis.
3. Pp 38-50 In Crocodilian Biology and Evolution. GC Grigg.
4. F Seebacher, and CE Franklin eds.  Surrey Beatty & Sons, Chipping Norton. Australia.
5. Davis, LM, TC Glenn, RM Elsey, HC Dessauer, and RH Sawyer. 2001b. Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis.  Molecular Ecology 10:In press.

ACKNOWLEDGEMENTS

• NIEHS, "Superfund project: Chlorinated pesticides and developmental mortality in wildlife",
• USGS - Florida Integrated Science Center, University of Florida, Department of Physiological Sciences,
• US Fish and Wildlife Service, Department of Energy, Lucy Dueck – SREL,
• Denise Strickland, University of South Carolina, Mandy Schable, SREL