Developing Molecular Methods to Identify and Quantify Ballast Water Organisms: A Test Case with Cnidarians

Dr. Brian Kreiser | University of Southern Mississippi

RC-1251

Objective

RC-1251 Project Graphic

Amplification of 14 clones with the Alcyonaria F, Actiniaria F and R primers. The leftmost lane is a 1kb size standard (Promega). The presence of two amplification products is diagnostic of an Alcyonaria anthozoan while one major amplification product represents an Actiniaria anthozoan. Failure to amplify using these primers is taken to mean that the clone represents either a Hydrozoa or Scyphozoa. Each lane represents a load of 5├Čl of PCR product.

To comply with federal and international regulations, the Department of Defense (DoD) needs to quantify the abundance and diversity of organisms in the ballast water of DoD vessels. Traditional identification methods based on morphology are difficult and time-consuming. Full identification of certain taxa is not always possible because many of the larval forms do not have unique morphology, which can lead to an underestimate of the diversity of organisms present. Molecular techniques that use the DNA of the organism to make an identification have been successful in estimating mixed bacterial communities in soil and water; however, this technique has not been explored fully for ballast water organisms. New and novel approaches are needed to overcome many of the obstacles encountered using traditional identification methods for ballast water organisms.

 The overall goal of this project was to adapt standard molecular methods into a novel approach for quantifying the abundance and diversity of organisms in the ballast water of DoD vessels. The objective of this exploratory research, in particular, was to describe molecular markers and refine the methods necessary to identify cnidarian taxa in ballast water samples.

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Technical Approach

Bulk DNA (from all the organisms present) was extracted from a sample, and polymerase chain reaction (PCR) was used to amplify a specific target gene from a specific group of organisms (cnidarians in this case). The PCR products were cloned as a means of isolating the contributions of each species present. Identifications were made by screening these cloned PCR products using a variety of techniques, including size variation, restriction fragment length polymorphisms (RFLP), and selective amplification by PCR. To develop the molecular protocol, markers that identify various cnidarian species were characterized by determining which genes in the genome provide the appropriate level of taxonomic resolution. Lab tests were conducted to determine the marker's ability to detect the presence of specific cnidarian taxa from a variety of concentrations in mixed samples. The developed technique was tested on actual ballast water samples as a final evaluation step.

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Benefits

The use of molecular methods to identify and quantify ballast water organisms has significant potential. This project demonstrated the practicality and utility of the developed protocol in detecting, identifying, and quantifying cnidarians, the focus of this exploratory research. Additional ballast water samples containing cnidarians are needed to fully test the molecular approach. (Project Completed - 2004)

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Points of Contact

Principal Investigator

Dr. Brian Kreiser

University of Southern Mississippi

Phone: 601-266-6556

Fax: 601-266-5797

Program Manager

Resource Conservation and Resiliency

SERDP and ESTCP

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