title: A method for the determination of genetic sex in the fathead minnow, Pimephales promelas, to support testing of endocrine-active chemicals.
abstract: Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.
authors.affiliation: Office of Research and Development, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, U.S. Environmental Protection Agency, 6201 Congdon Blvd., Duluth, Minnesota 55804, United States. olmstead.allen@epa.gov
authors.forename: Allen W
authors.initials: AW
authors.lastname: Olmstead
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Villeneuve
authors.forename: Gerald T
authors.initials: GT
authors.lastname: Ankley
authors.forename: Jenna E
authors.initials: JE
authors.lastname: Cavallin
authors.forename: Annelie
authors.initials: A
authors.lastname: Lindberg-Livingston
authors.forename: Leah C
authors.initials: LC
authors.lastname: Wehmas
authors.forename: Sigmund J
authors.initials: SJ
authors.lastname: Degitz
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D000818:Animals; D003362:Cost-Benefit Analysis; D003530:Cyprinidae / Q000235:genetics* / Q000502:physiology; D052244:Endocrine Disruptors / Q000633:toxicity*; D005260:Female; D005838:Genotype; D008297:Male; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics; D012732:Sex Determination Analysis / Q000191:economics / Q000379:methods*; D018675:Toxicity Tests / Q000379:methods
chemical_list: D052244:Endocrine Disruptors