Genetic Sampling Protocol

Protocol for the sampling and storage of painted turtle (Chrysemys picta) blood and tissue for genetic analysis.

Prepared by: Elinor Hughe, 2010


Image:Blood sampling training.JPG

Sampling blood from caudal vein on tail of turtle

Introduction

The use of genetics to study diversity has become increasingly more important, useful and achievable in the recent years. In fact, many may suggest that an investigation of within-or among-population diversity is incomplete without including genetic analysis. Genetics may be used to study questions regarding: 1) the evolutionary history of a population or species, including taxonomic relationships among sister species and phylogeography, 2) the effects of a loss of genetic diversity in small populations, for example whether a loss of diversity is associated with a loss in specific phenotypes, 3) the presence of inbreeding and its effect on fitness, 4) the effect of an isolation event, either recent (e.g. a new road) or historical (e.g. glaciation) and 5) the potential for genetic management as part of a recovery strategy for a threatened species.

The following comprises a protocol for the collection of samples from painted turtles for the purposes of genetic analysis. Topics include: sampling protocol; blood sampling, including methods and sample storage requirements; tissue sampling, including methods and sample storage requirements; and genetic testing guidelines, including possible genetic markers and testing laboratories.

Sampling protocol

Blood

Ideally, blood should be drawn from all adult turtles in a population, especially if populations are small. If time or resources are a concern, then a sample of the population may be used. When taking a sample of the population, ensure that the sample is random and that the sampling regime allows you to collect blood from the required number before you run out of catchable turtles. Determine the proportion of the population to be sampled or the absolute number of individuals to be sampled. In populations with uneven sex ratios, the sample group should reflect the sex ratio. The sampling regime may be determined either by consulting a random numbers table or by sampling turtles at a specific catch interval. Random numbers tables may be found on the Internet with instructions regarding their use, but sampling at specific intervals produces an equally random sample and is much simpler. Some examples of catch intervals that may be used are as follows: draw blood from every 2nd male and every 2nd female turtle caught, draw blood from turtles of each sex caught at (or near) 9:00 am, 12:00 pm and 3:00 pm each day or draw blood from the first turtle of each sex caught each day. The sampling regime used will depend on the how frequently turtles are caught. If either the population size or the sex ratio is unknown, it is highly recommended that blood be drawn from all adult turtles caught.

Tissue

The effect of tissue removal on long-term survival of turtles has not been studied. Serious adult injuries, however, involving a partial or complete loss of tails or limbs have been observed on numerous healthy and reproductively active turtles (pers obs.)
Because of the very low survivorship of hatchling and juvenile turtles in natural populations, the likelihood that a juvenile turtle will become a reproductively active adult is very slight. As a result, their genetic makeup may prove to be irrelevant to studies of the genetic diversity of a population. Therefore, we conservatively recommend that tissue samples should only be taken from hatchling or juvenile turtles to limit possible negative effects on the individual or on the population. Furthermore, we recommend that samples should only be taken from non-adult individuals if a paternity analysis is to be performed.
If few nests or juveniles are found in a population, e.g. fewer than 20 nests or 50 juveniles, then all hatchlings/juveniles should be sampled. If there are more than 20 nests or 50 juveniles, then a sample of the population may be used. For both juveniles and nests, a similar sampling regime to that described above for adult turtles may be used. For nests, tissue should be taken from all individuals within a single nest, and so the randomness of the sample applies to the nests, not the individual hatchlings.

A note on genetic variability in turtles

Genetic variability in turtles should not be expected to change as quickly as in other species. Avise et al (1992) found a 2-14x slower rate of mitochondrial DNA (mt-DNA) differentiation in turtle species as compared to a “conventional” mt-DNA rate. These numbers are only included as a caution, not a conversion value. Hence, in researching many of the among-population questions, researchers are advised to take the slower rate into consideration, e.g. there may not be observable genetic differences among populations isolated from one another even 100 years ago.

Blood sampling

Storage of blood samples

There are two field-convenient methods of storing blood samples for future DNA analysis: cell lysis buffer or cell lysis cards. Blood samples stored in a cell lysis buffer may be held for up to one week at room temperature, but must then be kept in a refrigerator (4C). Samples may be stored at subzero temperatures for long-term storage, but freeze-thaw cycles may fragment DNA strands so care must be taken. Blood samples stored on cell lysis cards may be kept indefinitely at room temperature, once the samples have dried, however, care must be taken to ensure that the samples remain desiccated.

Comparison of blood storage methods

Cell lysis buffer:

Pros
1) Slightly cheaper per sample (~$2.80/sample as opposed to ~$4.20/sample).
2) The extraction process may be more efficient and may result in a higher DNA yield, however, since turtle blood cells contain nuclei, only a very small amount of blood is generally needed to produce enough DNA for good results.

Cons
1) Cell lysis buffer must be made in a laboratory. It requires common laboratory chemicals, but must be adjusted to a specific pH and then autoclaved.
2) The tubes are bulky to carry and cumbersome if sampling many individuals
3) The samples must be kept in a refrigerator. Short durations at room temperature are acceptable (up to a week), but ideally, samples should be refrigerated with 24 hours of blood-taking.

Cell lysis cards:

Pros
1) Samples may be kept at room temperature indefinitely.
2) Samples may easily be transported, including through the mail.
3) The extraction technique is fairly simple and is very fast (30 minutes to usable DNA).

Cons
1) Slightly more expensive per sample.

Appendix 1 outlines the cost comparison between the two methods and Appendix 2 suggests suppliers for the required materials. Only those materials that are unique to each method are included in the cost comparison. Additionally, both methods require materials for drawing blood, and both have associated laboratory costs. See the materials section and the laboratory analysis section for more detail. Based on this cost-benefit analysis, we would recommend the use of cell lysis cards for painted turtle blood storage. Regardless, we include descriptions of both methods below. Because many of the items are only obtainable in large quantities, we would further recommend that orders for materials be placed by a manager and dispensed to researchers.

Materials

(See Appendix 2 for suggested suppliers of specialized materials)

27 gauge bevel-tip needles
1.0 ml syringe only
Sharps disposal container
Alcohol wipes (purchased at drug store)
Dishtowels
Fine permanent black marker
Small bucket (optional)
Blood storage medium (either cell lysis cards or lysis buffer in microcentrifuge tubes)
Storage bags and desiccant (for lysis cards) or storage boxes (for tubes)

Note: 1.0ml (1cc) volume syringes are available at most drug stores, however, they have needles already attached and are generally 29 gauge or smaller (larger gauge number = smaller needle diameter). The larger diameter needles are recommended because they may better resist bending against the turtles’ tough skin. That said, 29 gauge needles with 1cc syringes attached might be used if others are not available.

Making cell lysis buffer

If using buffer to store turtle blood, we recommend Queen’s lysis buffer (Seutin et al., 1991). A buffer is simply a solution of salts and soaps that is standardized to a specific pH. The recipe for Queen’s lysis buffer is as follows: 0.01 M Tris, 0.01 M NaCl, 0.01 M sodium EDTA and 10% n-lauroylsarcosine, pH 8.0. These are all common chemicals that are readily available in many laboratories. Additionally, most laboratory workers will find the recipe easy to read. The buffer should be autoclaved before use. Once the buffer is finished, dispense 1ml of buffer into each 1.5ml microcentrifuge tube, close cap tightly and place in storage box until ready to draw blood. Multiple tubes may be prepared well in advance of blood taking and may be kept at room temperature indefinitely. Care should be taken to ensure that the caps are tightly closed as any evaporation of liquid will alter the pH of the buffer and will affect the quality of the samples.
Note: lysis buffers are available commercially, however their effectiveness in turtle blood storage has not been tested.

Turtle blood sampling method

Preparation of materials

  1. Create a data sheet to keep track of blood sampling. Column headings should include: turtle ID, sex/juvenile, population, date of sample, researcher’s name, comments regarding the blood-taking procedure (e.g. pre-sample tail condition, injuries caused by sampling, difficulties with sampling).
  2. Prepare the cell lysis card or tube. If using a cell lysis card, write the turtle’s ID, sex/juvenile, and the date on the card and on the storage bag with a fine permanent felt tip marker. Place one bag of desiccant inside the storage bag. Be sure that you have familiarized yourself with the lysis card protocol provided by the manufacturer. If using lysis buffer, write the turtle’s ID, sex/juvenile, and the date in the frosted area on the side of a tube with a fine permanent felt tip marker. Write the turtle’s ID on the cap of the tube.
  3. Prepare the needle. Take one needle and open the top of the package. Take one syringe and remove from package. Seat the syringe firmly onto the needle. Loosen the cap from the needle, but don’t remove it from the needle package. Prime the syringe plunger by pulling it out a short way. If using syringes with needles attached, open the syringe package and loosen the cap from the needle, but don’t remove it from the package. Prime the syringe plunger by pulling it out a short way.
  4. Take two alcohol wipes and open the packages, but do not remove from packages.
  5. Prepare the turtle. Wrap the turtle in a dishtowel leaving the tail exposed. Be sure to secure the hind feet and ensure that all limbs and head are retracted into the body without being pinched. Seated on a chair/stump/rock/the ground, hold the turtle firmly between your knees, tail up and carapace facing towards your dominant hand. If desired, place a small bucket under your feet so you do not need to lean over as much. Grip the tail firmly with your non-dominant hand, clean the tail with a wet dishtowel, dry it with another dishtowel and swab it thoroughly with an alcohol wipe.

Drawing blood

  1. Take the syringe in your dominant hand and ensure that the bevel of the needle is facing up. Hold the tail firmly between the thumb and forefinger of your non-dominant hand.
  2. Gently insert the needle into the dorsal side of the tail about one-finger’s width from the end of the tail just inside the yellow stripe. Aim for the centre line of the tail (the needle will be at a slight angle from vertical, see Figures 1 and 2). If you hit bone immediately remove the needle and insert it slightly closer to the carapace. Repeat this until you feel the needle go in 1/8” to 1/4”. This means that you have found a space between two vertebrae.
  3. Reposition your hand so that you can draw the plunger. Be confident and keep the needle firmly in the tail--there is no need to rush. Once your hand is positioned, withdraw the needle very slightly and gently draw on the plunger; you should see blood begin to flow into the needle. If there is blood, patiently draw on the plunger until you have 0.1ml of blood. If there is no blood, rotate the needle to face the bevel in different directions, or remove the needle and re-insert it in another spot. Keep trying until you get blood. Some turtles are harder to bleed than others. If the turtle is pulling in its tail, you will find it difficult to insert the needle between vertebrae. If the turtle is very resistant, stop trying and come back to that turtle later.
  4. Draw 0.1ml of blood. If the blood is aerated draw blood to the 0.2ml mark. If using the blood lysis cards dispense the blood onto the collection area on the card. Be sure that the blood does not go beyond the circle indicated. Allow sample to air-dry for 30 minutes and then place in storage bag with desiccant. If using tubes with buffer, dispense the blood into the tube. Ensure that the cap is tightly closed and invert the tube to mix the blood with the buffer. Put the tube back in the box. Dispose of the needle in a sharps container.
  5. Swab the tail with the alcohol wipe and release the turtle. Any bleeding should stop within about 30 seconds to1 minute.
  6. Check the injection site after 1 minute and 5 minutes for bleeding or swelling. Bactine should be applied if there is any sign of bleeding or swelling after 5 minutes, and the injection site should be checked again after 30 minutes.

Some points to remember when drawing blood from a turtle tail

  1. Familiarize yourself with the needle and syringe, especially the location of the bevel on the tip. Be sure that you have all required materials at hand and ready to use (needle seated on syringe and primed, alcohol wipes open, collection medium labelled and ready to receive the sample)
  2. Ensure that the turtle’s head and limbs are well secured in the towel. While painted turtles are small, they have fierce bites, and the head will be very near to body parts that the researcher might not want bitten. The hind limbs should also be well wrapped, as the turtle will kick at the syringe.
  3. Be sure that you are inserting the needle distal to the cloaca. This reduces the chance that you will hit any vital nerves leading from the cloaca into the turtle’s body.
  4. You are aiming for the caudal artery, which runs within the vertebral column. You will be trying to insert the needle between vertebrae.
  5. If the turtle has a severe tail injury, you may attempt to draw blood from the tail between the cloaca and the carapace. Only attempt this if you are confident in your blood-taking technique.
  6. Turtle skin is very tough. If you need to insert the needle repeatedly, the tip may become bent. Be sure to check the tip of the needle and use a new needle if necessary.
  7. It is very difficult to draw blood from smaller juvenile turtles and impossible to draw blood from hatchlings. If genetic testing is to be done on those individuals, a tissue sample must be taken.

INSERT FIGURE

Figure 1 Turtle tail cross-section showing location of caudal artery within caudal vertebra and needle inserted into turtle tail at a slight angle from vertical.

Figure 2 Dorsal view of a turtle showing the approximate location of the cloaca in male turtles (female cloacas are much closer to the carapace) and the location for the insertion of the needle, both in relation to the cloaca and to the end of the tail and in relation to the yellow stripe

Tissue Sampling

Toe clipping vs. tail clipping

Toe clipping should only be used on hatchling turtles, i.e., those sampled directly from the nest or caught in their first active season, while tail clipping may be used on hatchlings or juvenile turtles. Up to two non-adjacent toes on each foot may be sampled (ASIH 2004; we recommend fewer), whereas only one tail sample may be taken. Hence a larger amount of tissue may be collected by toe-clip than by tail-clip. Nonetheless, if samples are stored correctly, a single piece of tissue is all that is required. Which method is used for hatchling turtles depends on the researcher’s comfort level with removing a digit.

Storage of tissue samples

  • Tissue samples must be stored either in a lysis buffer or at extremely low temperatures. The DNA yield is slightly higher when tissue is cryogenically frozen, but a significant amount of good quality DNA can be obtained from samples in lysis buffer (Seutin et al., 1991). If samples are to be frozen, sample collection must be performed in a field laboratory setting since access to liquid nitrogen is necessary and carboys are cumbersome to take into the field. Frozen samples should be stored in cryotubes in liquid nitrogen until tubes can be transferred to a -80C freezer. Liquid nitrogen will sublimate, even in a closed carboy and so tubes should be transferred to a -80C freezer within one week.
  • Tissue samples may also be stored in Queen’s lysis buffer and kept at room temperature for up to a week. Following that, the samples may be refrigerated at 4C indefinitely. To use Queen’s lysis buffer, follow the protocol for making and dispensing the buffer described in the above section on blood sampling.

Materials

(See Appendix 2 for suggested suppliers of specialized materials)

  • Fine permanent black marker
  • Dishtowels
  • Sharp dissection scissors (new, unused nail scissors also work well)
  • Fine-tipped forceps or tweezers
  • 70% isopropyl alcohol in squirt bottle
  • Bunsen burner (or other flame source)
  • Bactine antibiotic spray
  • 2 small dishes, one for waste isopropyl alcohol and one for Bactine
  • Liquid nitrogen (if using)
  • Cryostore tubes (if using liquid nitrogen)
  • 1.5 ml microcentrifuge tubes each containing 1.0ml Queen’s lysis buffer (if using)

Toe clipping

This method should only be used for hatchling turtles (i.e., those sampled directly from the nest or caught in their first active season). The American Society of Ichthyologists and Herpetologists (ASIH 2004) recommends that not more than two adjacent digits be removed. Male and female painted turtles use the digits on their forelimbs as part of the courtship ritual. While many turtles have missing digits due to depredation attempts (pers obs) it is not known how these missing digits influence courtship and mating success. Furthermore, female painted turtles dig their nests using the hind limbs. Hence, we recommend that toes be removed only from the hind limbs of painted turtles and that a maximum of one toe be removed from each hind limb.

Method

  1. Create a data sheet to track tissue collection. Columns should include: turtle ID, life stage (e.g. hatchling), date of collection, researcher name, recovery time of turtle and comments.
  2. Write the turtle ID and date of collection in the frosted area on the side of the storage tube with fine permanent black marker. Write the turtle ID on the cap of the tube. Open tube. Dispense small dish of Bactine. Thoroughly rinse scissor blades and forceps in alcohol and briefly hold in flame to sterilize. Store scissors and forceps on clean piece of paper after sterilizing.
  3. Dry hatchling thoroughly with dishtowel. Hold hatchling in non-dominant hand with hind feet towards your fingers and head towards your palm. Gently, but firmly, grip one of the hind feet between your thumb and forefinger with toes splayed over your forefinger. Dip the foot in Bactine. Using scissors, isolate a single toe and gently move down the toe until the scissor blades are past the second knuckle (the toe webbing will stop you from cutting the toe any closer to the turtle’s foot). Cut the toe and place it immediately into the storage tube using scissors and forceps (if necessary). Hold the foot over a clean piece of paper as the toe will often stick to the scissors after clipping, and may fall. If the toe should fall on an unclean surface, it is still usable.
  4. Dip the foot in Bactine. Place the hatchling in a secure container. Tightly cap the storage tube and place in liquid nitrogen (if using) or in storage box.
  5. Repeat procedure for the other hind foot if necessary.
  6. Observe the turtle at intervals for 5 minutes to ensure that there is no bleeding or swelling at the incision site and that normal movement is regained in the limb. If bleeding or swelling is observed or if normal movement is not regained, observe the turtle for a further 30 minutes. Most turtles begin moving within 2 minutes at temperatures above 20 C.
  7. Rinse scissors and forceps in isopropyl alcohol and heat sterilize between each cut.

Tail clipping

Method

  1. Create a data sheet to track tissue collection. Columns should include: turtle ID, life stage (e.g. hatchling), date of collection, researcher name, recovery time of turtle and comments.
  2. Write the turtle ID and date of collection in the frosted area on the side of the storage tube with fine permanent black marker. Write the turtle ID on the cap of the tube. Open tube. Open tube. Dispense small dish of Bactine. Thoroughly rinse scissor blades and forceps in alcohol and briefly hold in flame to sterilize. Store scissors and forceps on clean piece of paper after sterilizing.
  3. Dry hatchling thoroughly with dishtowel. Hold hatchling in non-dominant hand with feet towards your fingers and head towards your palm. Gently, but firmly, grip the tail between your thumb and forefinger approximately 1cm from the tip. Dip the tail in Bactine. Cut a ½ cm piece of the tail and place it immediately into the storage tube using scissors and forceps (if necessary). Hold the hatchling over a clean piece of paper as the tail clip will often stick to the scissors after clipping, and may fall. If the tail should fall on an unclean surface, it is still usable.
  4. Dip the tail in Bactine. Place the hatchling in a secure container. Tightly cap the storage tube and place in liquid nitrogen (if using) or in storage box.
  5. Observe the turtle at intervals of 5 minutes to ensure that there is no bleeding or swelling at the incision site and that the tail is no longer retracted into the shell. If bleeding or swelling is observed or if the tail remains retracted into the shell, observe the turtle for a further 30 minutes or until normal movement is regained.
  6. Rinse scissors and forceps in isopropyl alcohol and heat sterilize between each individual.

Testing guidelines

DNA extraction and genetic analyses require specialized equipment and skills. We recommend that this work be contracted to an established laboratory.

Possible laboratories
1) Genetic Data Centre, Department of Forestry, University of British Columbia.
Contact: Carol Ritland, This email address is being protected from spambots. You need JavaScript enabled to view it.
Telephone (604) 822-3908 or (604) 822-1543
Fax (604) 822-9102
Website: http://www.forestry.ubc.ca/gdc/
The genetic data centre accepts contracts for genetic testing using microsatellites, mtDNA and SNPs, among other genetic markers.

2) Government of British Columbia Research Branch Analytical Laboratory
I am not sure whether they are set up to do genetic work or the nature of the relationship among branches of the provincial government, but they may be a good option. I would have to gain access to their website to provide more information, which I cannot since I am not a government worker.

3) The Ministry of Agriculture and Lands may also have a laboratory that does genetic work.


Note: The following information is included for the purposes of effectively communicating with the contracted laboratories:

DNA may be extracted from turtle blood samples using any number of commercially available kits. Both the Qiagen DNeasy kit and the GenElute kit are simple to use and produce reliable amounts of high-quality DNA in a relatively short time. The Qiagen DNeasy kit also produces high-quality DNA from tissue. Phenol-chloroform extraction is an alternative, very reliable method for tissue samples, however it uses toxic chemicals and takes up to three days to complete (DNA may be obtained from the kit in one day).

Relatedness among individuals, among populations or among species may be investigated using microsatellites (small highly repeated fragments of DNA), SNPs (single nucleotide polymorphisms or repeats) and mtDNA (mitochondrial DNA, inherited through the maternal line). There are few published genetic markers for turtles, especially painted turtles. Two set of microsatellites developed for Blanding’s turtles (Osentoski et al., 2002 and Libants et al, 2004) and one set developed for diamondback terrapins (Hauswaldt and Glenn, 2003) amplify well on painted turtle DNA and some loci are highly polymorphic (variable) in painted turtles.

References

American Society of Ichthyologists and Herpetologists. 2004. Guidelines for use of live amphibians and reptiles in field and laboratory research, 2nd ed, Rev by Herpetological Animal Care and Use Committee (HACC) Chair: Beaupre, S.J., Members: Jacobson, E.R., Lillywhite, H.B. and K. Zamudio. http://www.asih.org/files/hacc-final.pdf

Avise, J.C., Bowen, B.W., Lamb, T., Meylan, A.B. and Bermingham, E. 1992. Mitochondrial DNA evolution at a turtle’s pace: evidence for low genetic variability and reduced microevolutionary rate in the Testudines. Molecular Biology and Evolution 9: 457-473.

Hauswaldt, J.S. and Glenn, T.C. 2003. Microsatellite DNA loci from the Diamondback terrapin (Malaclemys terrapin). Molecular Ecology Notes 3: 174-176.

Libants, S., Kamarainen, A.M., Scribner, K.T., and Congdon, J.D. 2004. Isolation and cross-species amplification of seven microsatellite loci from Emydoidea blandingii. Molecular Ecology Notes 2004: 300-302.

Osentoski, M.F., Mockford, S.W., Wright, J.M., Snyder, M., Herman, T.B., and Hughes, C.R. 2002. Isolation and characterization of microsatellite loci from the Blanding's turtle, Emydoidea blandingii. Molecular Ecology Notes 2002: 147-149.

Seutin, G., White, B.N., and Boag, P.T. 1991. Preservation of avian blood and tissue samples for DNA analysis. Canadian Journal of Zoology 69: 82-90.