Ocean Process Analysis Laboratory

Institute for the Study of Earth, Oceans, and Space

142 Morse Hall, 39 College Road

University of New Hampshire

Durham, NH 03824-3525

 

SUBJECT: ZooGene: zooplankton collections for molecular analysis

 

FROM: Ann Bucklin, University of New Hampshire

 

The ZooGene project, including four lead investigators and thirteen expert taxonomic consultants from seven countries, has created a database of DNA type sequences for calanoid copepods and euphausiids (see http://www.ZooGene.org). Following taxonomic identification of specimens, a DNA type sequence is determined for a 660-base pair portion of the mitochondrial cytochrome oxidase I (mtCOI) gene; multiple mtCOI sequences are included as necessary to reflect intraspecific variation.

 

The ZooGene project seeks zooplankton collections containing calanoid copepods and euphausiids from any region of the world oceans; samples must be preserved especially for molecular analysis following the instructions provided here.

 

In some cases, we would greatly appreciate your assistance in confirming the identification of a targeted species. In this case, please remove 20 30 individuals (adult females are preferred) from the sample and place in a small glass vial (5 - 20 ml) with a plastic top that will not leak or allow evaporation. Please include a label inside the vial; use a label without ink markings and write all collection information in pencil.

 

PROTOCOL FOR COLLECTION AND PRESERVATION OF ZOOGENE SAMPLES

 

Please follow these instructions carefully. Samples that are not carefully preserved will yield no DNA for our analyses. This protocol is also available on the project website at http://www.ZooGene.org.

 

1)      Samples collected with minimum damage to plankton are preferred (i.e., short tows taken in good weather). Samples must be preserved immediately upon collection. Only those individuals that are ALIVE up to the moment of preservation should be used. DNA is destroyed by enzymes immediately upon the death of the organism.

 

2)      Samples should be collected using nets with 100 um to 333 um mesh for copepods, and 333 to 550 um mesh for euphausiids. Samples need not be quantitative; non-quantitative portions of samples are acceptable.

 

3)      Immediately after collection, drain samples of excess seawater (using a sieve with mesh of same size - or smaller - as the net).

 

4)      Wash the sample into a glass jar using 95% un-denatured (i.e., drinkable) ethyl alcohol. Add additional 95% ethyl alcohol to fill the jar. NOTE: there must be 3 to 4 times more alcohol than plankton volume. Samples can be split to keep plankton biovolume to one-third or one-fourth of the jar volume. We recommend removing fish from the samples.

 

5)      NOTE: ONLY 95% UNDENATURED ETHYL ALCOHOL CAN BE USED TO PRESERVE ZOOGENE SAMPLES. PLEASE DO NOT USE 100% ETHANOL. DO NOT USE DENATURED ALCOHOL. If you are not sure which alcohol to use, please ask us for specific information and suggestions for vendors in your region.

 

6)      Place a label inside the jar or vial, writing in pencil. Unprinted labels are preferred since the ink may dissolve in the alcohol. Use small labels made from acid-free paper. (We have discovered that some labels change the sample pH significantly, especially in small volumes). Sample pH should remain close to pH 8.0. Please note collection information desired: cruise (ship and cruise name or number); collection date and local time; georeference coordinates (latitude and longitude); station or tow number; net and mesh size.

 

7)      After 24 hours, drain off alcohol and replace with fresh alcohol. Continue to change the alcohol every 24 to 48 hours, until the fluid remains clear and free of debris.

 

8)      Ship samples to Ann Bucklin. Please use airmail if possible, and ensure that samples will not be exposed to extreme heat (over 30 degrees C) at any time during shipment. Costs of sample shipment will be paid or reimbursed upon request.

 

MICROSCOPIC EXAMINATION OF SPECIMENS FOR MOLECULAR ANALYSIS

 

When microscopic examination is required for species identification of copepods and euphausiids, please take special care in order to allow later molecular analysis of these individuals. View the specimens in 95% ethyl alcohol; do not move them to water or other fluid. Do not use stains or other treatments. Avoid dissection; if necessary, use sterilized tools that have never been exposed to formalin; do not use the same tools for multiple individuals. Do not allow the alcohol to evaporate or become warm; minimize light exposure by limiting both duration and intensity.

 

If such handling is not possible to allow identification, consider examining only some individuals of each sample carefully. Keep these and send the others individuals of that sample to us for molecular analysis.

 

THANK YOU VERY MUCH!!!

 

 

Ann Bucklin

Professor of Zoology / EOS

Ocean Process Analysis Laboratory

University of New Hampshire

Durham, NH 03824 USA

Tel. (603) 862-0122; Fax (603) 862-0243; Email ann.bucklin@unh.edu