Impact of Sample Preservation on the Detection of Invasive Mussel Larvae (veligers) by Microscopy and PCR

Project ID: 3157
Principal Investigator: Sherri Pucherelli
Research Topic: Invasive Species
Priority Area Assignments: 2014 (Zebra and Quagga Mussels)
Funded Fiscal Years: 2014
Keywords: quagga mussels, zebra mussels, invasive mussels, early detection, pcr, microscopy, veliger, sample preservation

Research Question

Reclamation's early detection program for invasive mussels primarily focuses on the identification of the larval (veliger) stage of the life cycle. Veligers can be difficult to detect if they are not properly preserved. Sample preservation methods may be one of the most important aspects of accurate veliger identification. One of the goals of this study is to determine what preservation methods are required to maintain veliger integrity in samples containing organic/ inorganic content, which is representative of ‘real world' water sample conditions. How does the organic/ inorganic content of a water sample and the preservation method (i.e. 0 percent or 20 percent alcohol, with and without buffer) impact the pH of samples over time? And how does the organic/ inorganic content and preservation method influence veliger morphology and detectability by microscopy and PCR?

Need and Benefit

Reclamation has been successfully monitoring for the early detection of zebra and quagga mussels in the west since 2007. Microscopy has been the primary mode of analysis for detecting veligers. Samples are surveyed under a dissecting microscope with a cross polarizing filter. The veliger shell is made of calcium carbonate, and as light passes through the cross polarizing filter it causes the veliger to birefringe (glow against the dark background). Some portions of the shell fall in line with the axis of the cross polarizing filter creating a distinctive Maltese cross pattern across the top of the shell. This Maltese cross is the main identifying feature for veliger identification. Because the veliger shell is made of calcium carbonate, it is possible that an acidic pH would degrade the shell, making the Maltese cross undetectable.

Sample preservation is the most important factor in the discovery of invasive veligers in water samples. When sample collection first began in 2007, samples were preserved in 20 percent Everclear, when the samples arrived back in the lab the technicians noticed the pH would shift during transport. A pilot study advised keeping samples on ice and preserving samples in 20 percent alcohol with baking soda to raise the pH and maintain a consistent pH over time.

In 2012 another pilot study was performed, with the addition of PCR analysis. This study looked at veliger morphology in samples that were unbuffered (pH 5) and buffered (pH 8). Deionized (DI) water was used to maintain a constant pH over time. Veligers in unbuffered samples lost the characteristic birefringent Maltese cross pattern quickly, but the veliger body was still in the sample. When that sample was run by PCR, the sample came back positive, which indicates there was tissue in the non-birefringent veliger.

This proposal builds on the pilot study conducted in 2012, by including variables that are more true to water samples the Reclamation actually receives. The goal of the study is to determine how veliger birefringence loss is affected by samples containing organic and inorganic material. Buffered and unbuffered samples containing zooplankton, sediment and algae will be analyzed. Samples will be preserved in 0 and 20 percent ethanol. This study will test how pH shifts over time, and it will help determine the microscopist's ability to detect degraded veligers. Veligers will be analyzed by PCR using Reclamation's Standard Operating Procedure for PCR. This will determine the efficiency rate of PCR to determine presence/ absence of veligers.

Reclamation's mussel detection program involves several agencies who all collaborate to sample multiple water bodies across the western United States. Because this program has so many collaborators sample collection and preservation methods are not always uniform. This study will greatly benefit Reclamation's mussel detection program, by providing information on how veliger morphology and detectability is impacted by improper preservation in ‘real world' water samples over time. Reclamation needs to be able to present this data to its collaborators so that they fully understand the consequences of improper sample preservation. This data will provide updated statistics on the effectiveness of microscopy and PCR in detecting veligers for early detection in water samples. The efficacy of alcohol and baking soda as preservatives of veliger birefringence in samples containing organic/inorganic materials will also be determined.

Contributing Partners

Contact the Principal Investigator for information about partners.

Research Products

Bureau of Reclamation Review

The following documents were reviewed by experts in fields relating to this project's study and findings. The results were determined to be achieved using valid means.

Document ID 1340: this document contains protected information and it cannot be freely downloaded from Contact the Principal Investigator to request a copy of this document.

Impact of Sample Preservation on Detection of Invasive Mussels (final, PDF, 849KB)
By Jamie Carmon
Publication completed on September 30, 2015

This bulletin summarizes the research results and potential application to Reclamation's mission.

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Last Updated: 6/22/20