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