Methods

Preservation of tissues for DNA analysis

(From: Dawson et al. 1998)

Equipment

1. Sterile syringes, forceps, dissecting scissors, or razor blades
2. A way of cleaning the sampling tools if they are not disposable (e.g. flaming metal objects after dipping in ethanol, or a good rinse and wipe dry)
3. Vials containing 1-2 ml of preservative (DMSO+NaCl or ethanol [70% to 95%; >90% preferred]; will be provided upon request).
4. Chest of ice (in the field); ice, refrigerator, or freezer (at lab/home, separate from food).
5. Protective gloves (latex, rubber)

Procedure A

Sampling gut or gonad:
1. Collect medusae.
2. Starve the medusae for as long as possible.
3. Using a clean syringe, flush the guts with tapwater. Repeat several times to displace any debris from the guts.
4. Using the same syringe (or other tool), suck up (tear or cut) about 0.1-0.2 ml of gastric and/or gonadal tissues.
5. Preserve the tissue in one vial of preservative. (Make sure there is excess preservative; guard against diluting the preservative with too much water.)
6. Label the vial and put it on ice. Store on ice, in a freezer or refrigerator until the samples are shipped or analyzed (separate from food).
7. Clean tools and gloves, repeat for the next sample.

Procedure B

(quicker alternative) - sampling oral arms or bell margin:

1. Collect medusae.
2. Flush the oral arms or bell margin with tapwater. Repeat several times to displace all debris.
3. Using clean forceps/scissors, cut a half-small-fingernail sized piece of tissue from the oral arm or bell margin. (It may be possible to do this simply by holding the tissue across an open vial of preservative and closing the lid.)
4. Preserve the tissue in one vial of preservative. (Make sure there is excess preservative; guard against diluting the preservative with too much water.)
5. Label the vial and put it on ice. Store on ice, in a freezer or refrigerator until the samples are shipped or analyzed (separate from food).
6. Clean tools and gloves, repeat for the next sample.

Notes

DMSO+NaCl solution is 20% DMSO, 0.25M disodium-EDTA, and NaCl to saturation, pH 7.5 (Seutin et al. 1991); the pH of this solution must be >8 for the EDTA to dissolve, and warming promotes dissolution of NaCl. Sterilized by autoclaving prior to use.

100% ethanol is diluted to 80% (or greater) using purified drinking water (tapwater generally will suffice).

An important concern when sampling is to avoid contaminating the sample with tissues from other species (e.g. food) or with tissues from other individuals (see steps 2, 3, 7 in procedure A; steps 2, 3, 6 in procedure B).

Both procedures A and B are designed to sample as much tissue and as little mesoglea (the 'jelly' of jellyfish) as possible. A third approach, not described above, is simply to preserve the whole animal, or a simple fraction thereof, if it has been cleared of potential contaminants and is small enough to fit easily into a vial with excess preservative (i.e. it is approximately <1 cm bell diameter). The figure below indicates the tissues best suited to sampling. The same methods also can be used to preserve coronate jellyfish and stauromedusae for DNA analyses.

Safety information

The chemicals in the DMSO+NaCl preservative are quite benign, being mostly water and table salt (see above). However, this information accompanies the two main chemicals.
1. DMSO (liquid, 20% of preservative by volume). "Causes skin irritation. Causes digestive and respiratory irritation. May cause severe eye irritation and possibly injury. May be absorbed through the skin. For eye contact, flush with water for 15 minutes and get medical aid immediately. For skin contact, flush with water and get medical aid. If ingested, do not induce vomiting and get medical aid immediately. If inhaled, remove to fresh air and get medical aid immediately."
2. EDTA (powder, @ 0.25M in preservative). "May cause irritation. For eye contact, flush with water and get medical aid. For skin contact, get medical aid if irritation occurs or persists. If ingested, give 2-4 cupfuls of milk or water and get medical aid. If inhaled, get medical aid if cough or other symptoms appear."

Ethanol is highly flammable. It should be kept away from naked flame and sources of heat at all times. It should not be transported except in small quantities packaged appropriately (check protocols for transporting hazardous substances).

Jellyfish stings can be at best uncomfortable and at worst lethal. Jellyfish must be handled with the utmost care, and protective clothing and gloves worn at all times. If you have any doubts, do not touch them. Ask for expert advice.

FREE DNA Sequencing

Identify and sequence any scyphozoan for free! If you find a jellyfish and would like to know what it is and about it's DNA, then send pictures and a sample to us! We will identify the animals to the best of our ability, as well as sequence molecular markers including "genetic barcodes" from the mitochondrial (16S rDNA, cytochrome c oxidase subunit I) and nuclear (18S, 28S, and other markers in the future) genomes, then send the information to you. 

Genetic Barcodes

Like the UPC system of barcodes employed to uniquely identify manufactured goods, a single genetic marker could provide an universal barcode system that uniquely identifies all animals on Earth (Hebert et al. 2003). Cytochrome Oxidase c subunit I (COI) has been adopted by the Census of Marine Life (COML) as the preferred genetic barcode. COML is recommending the small subunit nuclear ribosomal RNA (SSU rRNA, also known as 18S), which forms the basis of the Tree of Life project, as a second elective barcode

Although there are limitations to the use of genetic barcodes—for example COI and 18S may not reflect important ecological, morphological, or adaptive genetic variation, and COI is not useful for phylogenetic reconstruction of anciently diverged species—a standardized genetic toolkit for helping to identify species is an important step toward a comprehensive global inventory of biodiversity.

See COML's DNA barcoding protocol and Barcoding Life for detailed explanations and additional resources.

To send in your samples follow the steps below:

1. Photograph the jellyfish

• See http://www.jellieszone.com/photography.htm Jellies Zone for tips on jelly photography.
  

1. Preserve the DNA

• See descriptions under "Preservation of DNA" for more detailed information.
  

1. Preserve the remainder of the specimen for morphological analysis (optional, but highly preferred)

• See descriptions under "Preservation of Jelly" for more detailed information.
  

1. Provide information about the collection

When sending samples, please include the following information for each specimen:

Geographic location (place name; GPS/latitude+longitude; brief description of the site; if possible, include a copy of a map showing the location

Depth (in metres)

Date (of collection) <u

Collector (e.g. your name)<u

Photograph<u

Whole jellyfish preserved? (yes/no; where)

Conditions

While we will endeavor to provide sequence data to you in a timely manner, turnaround will depend upon resources at the time. We expect that samples will be processed in less than one month in only exceptional circumstances. A more realistic expectation is 2-6 months, but sometimes longer as laboratory facilities may change from time to time. As resources are established and subsequently increased we hope to provide a more efficient service. Depending on your needs, arrangements can be made on a case by case basis. Please contact us if you have any questions.

We will attempt to sequence all species submitted, but success cannot be guaranteed. Our ability to generate sequence data will depend on the quality of the sample and whether existing techniques work for that species. If techniques don't work initially, we will attempt to modify them appropriately, but this will take time and sometimes may not be successful if resources (e.g. samples, time) are limited. Also, due to financial restrictions, we will only be able to sequence one or two jellyfish per species per location or region. If you would like to know whether we will sequence your samples before you send them, please contact us here. It may be possible to arrange a subset of analysis.

Submission of samples implicitly gives us the right to use the sequences, including, but not limited to, publication on this site and in the scientific literature. Our intention is to advance knowledge of the scyphozoans as broadly as possible. If this might conflict with your immediate interests, please do not hesitate to discuss the matter with us. We welcome the opportunity to collaborate with anyone interested in these animals and may forgo this condition in the interests of progress. All contributors to all studies will be acknowledged.

Shipping

Please send all samples through airmail at room temperature to:

Dr. Michael Dawson
School of Natural Sciences
University of California at Merced
5200 North Lake Road
Merced, CA 95340 USA

See Shipping information and details for more information.