Development of Molecular Techniques to Ascertain Gender of White Sturgeon

The Problem

Knowledge of the ratios of male and female fish among populations is crucial for effective management of fisheries. Field assessments of the abundance of white sturgeon populations often use surgical techniques to attempt to determine gender of captured fish by direct observation of gonads because the gender of these long-lived fish cannot be determined by external examination. However, because gonads are not discernable until the fish are several years old and mature fish do not spawn every year, the

Are these immature fish males or females?

identification of gender by the observation of gonads is not reliable. Artificial propagation is also being used to recover and supplement populations of white sturgeon in areas with reduced abundance. Hatchery practices can skew ratios of males and females for many species of fish. Hatchery personnel need a method to determine gender of immature progeny because the release of predominately same-sex fish would have negative consequences to recovery efforts. Of concern to white sturgeon fishery managers is the recent finding of sex reversal in fall chinook salmon from the Columbia River. Tests on fish from the Hanford Reach of the Columbia River showed that 84% of the apparent females began life as genetic males. While the trigger for the chinook sex reversal is unknown, fishery managers are concerned because no method currently exists to test for sex reversal in white sturgeon. The implications are great; white sturgeon populations have a high risk of extinction if environmental factors or exposure to environmental contaminants are altering functional sex of fish.

Objectives

This research seeks to develop rapid assessment technologies using DNA markers to differentiate male and female white sturgeon, Acipenser transmontanus. The non-lethal, non-surgical techniques will allow field and hatchery workers to readily discern gender of white sturgeon with a minimum of laboratory resources. A successful technique will work on all age classes and sizes of fish regardless of state of maturity.

Methodology

Samples of tissue from ovaries and testes from unequivocally sexed fish will be collected from the Columbia River and transported to the Leetown Science Center where cDNA and genomic libraries will be produced. Differential subtraction cloning using Ligantion-Mediated Subtraction (Limes) and Differential Subtraction Chain (DSC) will be used to create genomic libraries containing sequences that are gender specific. Additional cDNA libraries will be created that contain genetic messages that are differentially expressed in ovaries and testes of white sturgeon. Representative clones from these libraries will be sequenced to characterize or identify the gene product. We expect that some of the genes identified may be suitable candidates for sex identification probes. The libraries that will be created and the genes that will be characterized will greatly enhance the body of knowledge regarding reproduction in sturgeon, and will be of great value to the scientific community for the study of this animal's physiology and genomic organization.

Highlights and Key Findings

This work was funded in 2001 by the US Fish and Wildlife Service through reverted DJ funds. Five gonadal cell lines were developed from two females and three males. Additionally, we have cDNA libraries from pooled male and pooled female RNA.

Where Are We Headed In 2003

During FY2003, putative gender specific probes will be tested for their utility by first testing the known sex cell lines currently in hand. Successful probes will then be incorporated into a polymerase chain reaction (PCR) method for field-testing. New samples will then be tested to validate the assay. Additionally, the gonadal cell lines developed in FY2002 will be evaluated for their utility in viral testing and endocrine disruption studies.

Project Contact

Michael J. Parsley
U.S. Geological Survey
Western Fisheries Research Center
Columbia River Research Laboratory
5501-A Cook-Underwood Rd.
Cook, WA 98605-9717

Email: mike_parsley@usgs.gov
Phone: 509-538-2299
Fax: 509-538-2843

William B. Schill
U.S. Geological Survey
Leetown Science Center
11700 Leetown Road
Kearneysville, WV 25430

Email: bane_schill@usgs.gov
Phone: 304-724-4438
Fax: 304-724-4435

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