Western Painted Turtle Monitoring at Alaksen National Wildlife Area (NWA) Final Report – March 2011 Prepared by Vanessa Kilburn and Aimee Mitchell of the The South Coast Western Painted Turtle Recovery Project For Stephen Hureau, Canadian Wildlife Service, Delta, BC
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Western Painted Turtle Monitoring at Alaksen National Wildlife Area (NWA)
Final Report – March 2011
Prepared by
Vanessa Kilburn and Aimee Mitchell
of the
The South Coast Western Painted Turtle Recovery Project
For
Stephen Hureau, Canadian Wildlife Service, Delta, BC
Western Painted Turtle Monitoring at Alaksen NWA: Summary Report December 2010
The South Coast Western Painted Turtle Recovery Project Page 2
Table of Contents Introduction ................................................................................................................................................................4
Species Description ................................................................................................................................................4
Site Description ......................................................................................................................................................5
Agriculture and pollution ....................................................................................................................................7
Invasive species ..................................................................................................................................................7
Road-related threats and collection ...................................................................................................................7
Surveying, Monitoring, Tracking, and Habitat Characterization ............................................................................8
Data Analysis of Habitat Characteristics .................................................................................................................8
Radio-Tracking and Movements .......................................................................................................................... 11
Population Status ................................................................................................................................................ 14
Habitat Use and Movement ................................................................................................................................ 15
Population Augmentation ................................................................................................................................... 15
Invasive Species Removal .................................................................................................................................... 16
Additional Research ............................................................................................................................................. 16
Outreach and education ...................................................................................................................................... 16
locations was sparse and scattered - no difference
Water visibility c - 0 / 1 / 2 30 / 0 / 0 100 / 0 / 0 29 / 0 / 0 100 / 0 / 0 N/A Water visibility in all waterbodies
was opaque - no difference
Substrated # of points # of points Sand 8 0.27 18 0.62
p < 0.0001
A greater proporation of random points were in purely sandy areas than any other category. Tracking points more frequently occurred in
sandy areas with some organics (detritus or muck) or in areas with
purely organics
Sand/detritus 7 0.23 0 0.00
Sand/muck 4 0.13 0 0.00
Muck/detritus 7 0.23 2 0.07
Muck/clay 3 0.10 7 0.24
Clay and/or mud 1 0.04 2 0.07
aTemperatures separated into summer (July-August) and fall (September-November) periods
bEmergents / Submergents: 0 - sparse, 1- scattered, 2 - common, 3 – abundant within 2 m. Categories were combined into two; 0 - sparse with 1- scattered; AND 2-common with 3-abundant for analyses b/c Chi-square suspect with more than 20% of fields with less than 5
dSubstrate - Categories were combined into three; 1) sand, 2) sand/detritus with sand/muck, AND 3) muck/detritus with muck/clay and clay and/or mud for analyses b/c Chi square suspect with more than 20% of fields with less than 5
Table 3: Habitat characteristics and analyses at Western Painted Turtle tracking and random locations at Alaksen
National Wildlife Area, sampled late summer 2010 through to winter 2011.
Western Painted Turtle Monitoring at Alaksen NWA: Summary Report December 2010
The South Coast Western Painted Turtle Recovery Project Page 14
Characterization of Overwintering Habitat
The habitat at the chosen overwintering location was very different from all other aquatic habitat at Alaksen
NWA. Firstly, there was ample aquatic vegetation, both emergent (mostly cattails) and submergent, compared
to at all other areas of the site. The wetland is small and shallow with stagnant water and has abundant
emergent and submerged woody debris (Fig. 9). The bottom substrate is organic in nature with a deep substrate
profile, in contrast to the bottom of the vast majority of the sloughs at this site, which are composed of sandy
substrate with a layer of mud and clay. The riparian area consists of deciduous trees, blackberry and spirea, with
overhanging branches. During all winter tracking events (two in November and one in March), the Western
Painted Turtle was located in shallow (ca. 50 cm) edge habitat with overhanging vegetation. A distance of 20 m
separated the November locations from the March location. Although we did not detect movement by the turtle
in November, in March the turtle appeared to be moving slightly along the bottom as evidenced by changes in
location of the strongest tracking signal. The water temperature on this day was 6 °C.
Discussion
Population Status
Only two Western Painted Turtles were observed at one time at this site. No juveniles were captured during the
trapping session, despite the fact that they tend to be easier to catch in the fish-baited traps than adults due to
their more carnivorous foraging habits (personal observations from the authors). Additionally, the tagged
Painted Turtle shows characteristics of both a Western and Southern Painted Turtle, and thus the origin of this
individual and even population cannot be determined. However, individual variations in colour patterns have
been noted in other areas for this species, so it is also possible that this turtle is a true Western Painted Turtle
with colour and pattern variation, and not a hybrid.
Figure 8: Western Painted Turtle overwintering location at Alaksen National Wildlife Area. The left photo is the wetland in
September and the right photo is the wetland in November 2010. Turtle overwintering location is in bottom left corner of
wetland where some ice on the water surface has melted.
Western Painted Turtle Monitoring at Alaksen NWA: Summary Report December 2010
The South Coast Western Painted Turtle Recovery Project Page 15
Red-eared Sliders have obviously been released into this site, so there is a possibility that the individual tagged,
and even the additional individual that has been observed, are released pets. There have been debates over
whether the Pacific Coast Western Painted Turtle population is native or introduced, however COSEWIC has
declared this Population native [15]. There is still a possibility however that some local populations are
introduced. A blood sample was taken from the tagged individual for future population genetic analysis, which
will aid in our understanding of the origin of Western Painted Turtle populations on the south coast, including
the population at Alaksen NWA.
Habitat Use and Movement
Results indicate that Western Painted Turtles at this site are utilizing the abundance of habitat available for
foraging, basking, and overwintering. Across the North American range of the Painted Turtle, preferred habitat
consists of shallow, slow moving, stagnant water with ample emergent and floating vegetation [16, 17, 18]. In
southeastern North America, abundance of Eastern Painted Turtles is positively correlated with abundant
aquatic vegetation and bottom substrates composed of organic matter like decaying plants; organic bottom
substrates provide turtles with suitable overwintering habitat, and aquatic vegetation provides foraging
opportunities for turtles [21]. However, foraging sites are lacking at Alaksen NWA; although adult freshwater
turtles are omnivorous, they do tend toward herbivory [22], and except for the small wetland on the northern
edge of the property where the tagged individual overwintered, aquatic vegetation is almost completely lacking
at this site.
The overwintering habitat chosen by this tagged turtle is similar to overwintering habitat choice reported for
turtles in sites outside of B.C. [16, 23] and in other areas of B.C. [24, 25]. In Kikomun Creek Provincial Park in the
Interior of B.C., Western Painted Turtles usually overwintered on or near the muddy bottom of lakes and ponds,
near to the shoreline at depths of about 1 m [24]. In the Williams Lake area in north-central British Columbia,
turtles overwintered at depths of less than 1 m, and occasionally less than 0.5 m, immediately adjacent to the
shore-line and buried in organic detritus amongst cattail roots [25]. Interestingly, the tagged individual was able
to locate this preferred habitat type in the Alaksen NWA wetland complex despite the habitat type’s rarity.
Emerging in this habitat type in the spring likely presents ample foraging and basking opportunities, and the
shallow stagnant water likely heats up faster than water in the other lagoons. This Western Painted Turtle had
to travel a great distance to reach this small wetland; however the distance reported for this individual (ca. 2.0
km) is not outside of the range of travel distances reported for Painted Turtles elsewhere [26]. Indeed recent
research suggests that freshwater turtles are actually very effective dispersers, regularly moving distances of up
to 1.3 km between wetlands, and as much as 3.3 km, both overland and through connecting water bodies [26].
Management Recommendations
Population Augmentation
There is considerable evidence that only a very small number of Western Painted Turtles inhabit this site, and
there is no evidence that successful reproduction is occurring. Thus this population is most likely inviable and
would require population augmentation to persist into the future. Due to the large size and extent of suitable
habitat in this area, as well as the protected status and length of time since this designation, this site has the
potential to support a large Western Painted Turtle population. Alaksen NWA could thus be a valuable asset for
potential re-introduction or supplementation of Western Painted Turtle populations on the south coast.
Western Painted Turtle Monitoring at Alaksen NWA: Summary Report December 2010
The South Coast Western Painted Turtle Recovery Project Page 16
However, before reintroduction of this species is considered, it is highly recommended that the source of
agricultural pollution be addressed. Pollution may represent a significant impact on the suitability of this habitat
for maintaining a healthy population of Western Painted Turtles. Before any consideration of potential
augmentation of turtle populations at this site, some effort should be made to reduce pollutant levels in the
aquatic habitat. Although we did not find any abnormalities in any of the adult turtles that were captured,
exposure to pollutants are much more likely to have an effect on hatchling or juvenile turtles, and could further
explain the small, inviable Western Painted Turtle population observed at this site.
Invasive Species Removal
The base-line population data for the Red-eared Slider suggests that because they outnumber native turtles they
are likely competing for ideal basking, foraging, and nesting sites, and may be negatively affecting Western
Painted Turtles. Because it is now known through trapping efforts that Red-eared Sliders and Carp can be
successfully captured, this technique could be used as part of an invasive species management plan to remove
these species. Recent frog monitoring conducted at this site has also shown that Bullfrog and Green Frog
populations are thriving [11]. Because Alaksen NWA is a federally protected and federally managed site, invasive
species removal programs could be initiated and are recommended.
Habitat Enhancement
Although there is no evidence that Western Painted Turtles are successfully reproducing at Alaksen NWA, it
would still be recommended that enhancement take place at the potential nesting area in case there is a
possibility that turtles may reproduce successfully if nesting habitat was improved. The apparently preferred
location is the open area immediately east of the visitor parking lot, where there is an encroachment of invasive
vegetation (mostly blackberry) that is filling in the open area. This habitat could be greatly enhanced through
vegetation removal and addition of more suitable nesting substrate. The work would be neither time-consuming
nor expensive, and would result in significant enhancement of this nesting area. A closure of the visitor parking
lot would be recommended for the spring nesting season if and when Western Painted Turtles are confirmed to
nest at this site. In conjunction with this closure (mid-May) enhancement work could be undertaken.
Additional Research
Potential research into nesting habitat requirements, effect of habitat conditions on nest survival and hatchling
recruitment, and possible impacts of climate change on reproduction and recruitment of Western Painted
Turtles should be conducted at this site if Western Painted Turtles are confirmed to nest here. Although a
temperature/humidity data logger has already been installed, no nests have been confirmed. Monitoring for
Western Painted Turtle nesting should continue yearly and can be conducted by Canadian Wildlife Service
biologists. Currently, there is a lack of evidence that Red-eared Sliders successfully nest on the south coast, so
any identified Red-eared Slider nests should also be monitored.
Outreach and education
Because Alaksen NWA is open to the public, it would be advisable to install some interpretive signage at this site
to warn visitors of the possibility of nesting female turtles on land, as well as the importance of not releasing pet
Sliders into the wetlands. All interpretive signage should be approved and sponsored by the Canadian Wildlife
Service.
Western Painted Turtle Monitoring at Alaksen NWA: Summary Report December 2010
The South Coast Western Painted Turtle Recovery Project Page 17
References
[1] The Western Painted Turtle Recovery Team. 2010. Draft Recovery Strategy for the Western Painted Turtle (Pacific Coast
Population), Chrysemys picta bellii, in British Columbia (March 2010). Original version prepared by Vanessa Kilburn for the
B.C. Ministry of Environment, Victoria, BC. 45 pp.
[2] Blood, D.A and M. Macartney. 1998. Painted Turtle. B.C. Min. Environ., Victoria, BC.
Data Collection Form 1: Turtle Basking Survey Form
Modified: May 21, 2010
Survey Information
Location name:
Observer(s):
Date (dd/mm/yr):
Time: Start: End:
Person-Search effort: hours =
Foot Canoe KSurvey method: ayak Powerboat Other:
Temperature (ºC): (at 10 cm depth in ca. ½ m deep water)
Air : Water:
Calm LightWind speed:
Moderate Strong
Binoculars Telescope Viewing equip.:
Precipitation: None Fog Light Moderate Hard Other
%Cloud cover (%):
Clear <50 >50 Overcast
estimate or
Seen Heard No Bullfrogs seen:
Species Observation(s) Information
Map reference
Species1 # seen Activity2 Microhabitat3 If possible Sex M/F/U Size class4
Notes on observations
(mark locations with letters/numbers/WPT #s): Site sketch or attach map
l = <6cm Estimated size: Large >9cm; Medium = 6 – 9cm; Smalher (describe in comments) K = rock; RHIZ = rhizome mat; SHORE - shoreline; Ot LOG= on log; Water = in water; Land = on land; ROC
alking on land nesting , digging on land; TS = swimming; TW = w BA = basking; CO = courtship; FD = feeding; NE= RSC) or other (identify or take a picture) Western Painted Turtle (CHPI); Red-eared Slider (T
1
2
3
4
.995.2428 or email If this spreadsheet is found please contact HAT 250 [email protected] Modified: May 22, 2010
nt Data Collection Form 2: Habitat and Threat Assessme
Date: (+ camera owner’s initials):Photo #’s Location Name:
Observer(s):
(Use NAD83 datum) Northing: UTM: Zone U Easting:
Type of Ownership: Private Park Crown Federal Other: Landowner contact info: Landscape context: Urban Rural Agricultural Backcountry Other:
SITE DESCRIPTION AND ASSESSMENT (in large water bodies only for surveyed areas) :
Bottom substrateLake Creek River Other: : Marsh Slough Natural-pond Human-made pond Wetland Type
:
(10 m zone from shoreline)Aquatic vegetation cover
Describe nesting habitat/assess opportunities (indicate on map):
Assess habitat suitability (loss, condition, movement barriers etc.):
ON-SITE THREAT ASSESSMENT:
(within 50 m from water’s edge or as visible): B) Threats to aquatic habitat and travel routes
A) Threats to nesting habitat (describe):
mentRoads - paved H M L n/a Housing/industrial develop 1
Roads - unpaved H M L n/a Urban activities H M L n/a
2
Introduced species YesPets H M L n/a Agriculture H M L n/a
H M L n/a Recreation - non motorized H M L n/a Grazing H M L n/a Recreation - motorized H M L n/a Logging
H M L n/a
3 No
: Name of introduced species
n/a Water use /control H M L n/a
C) Comments on threats:
ccurately Yes-Known to be present; risk cannot be assessed aassociated with housing/urban developments Landscaping, gardening, or other human activities
In progress, planned or potential
1
2 3
Recorder Name:
ation Assessment Information Data Collection Form 3: Turtle Mark-Recapture Popul
Date:
Weather:
Recorder Name: Date:
Weather:
Location Description (in detail): Circumstances of Capture:
WYPT ID:
GPS – Easting:
GPS – Northing:
Turtle Species: Age:
Sex: Gravid?
Carapace length (mm): Carapace width (mm):
Plastron length (mm): Plastron width (mm):
Height (mm): Weight (mm):
Tail total length (mm): Tail length to cloaca (mm):
: Tail width at shell (mm): Tail width at cloaca (mm)
gth (mm): Left middle claw length (mm): Right middle claw len
Injuries or distinctive features:
Location Description (in detail): Circumstances of Capture:
WYPT ID:
GPS – Easting:
GPS – Northing:
Turtle Species: Age:
Sex: Gravid?
Carapace length (mm): Carapace width (mm):
Plastron length (mm): Plastron width (mm):
Height (mm): Weight (mm):
Tail total length (mm): Tail length to cloaca (mm):
: Tail width at shell (mm): Tail width at cloaca (mm)
gth (mm): Left middle claw length (mm): Right middle claw len
Injuries or distinctive features:
TURTLE NESTING SURVEY
Data Collection Form 4: Turtle Nesting Survey
TURTLE NESTING SURVEY
Recorder Name:
Recorder Name:
Date:
Date:
Weather:
Weather:
land? spotted on Turtle
Time End
Time Start
Location Survey
on land?spotted Turtle
Time End
Time Start
Location Survey
TURTLE NESTING ATTEMPT OBSERVATIONS
TURTLE NESTING ATTEMPT OBSERVATIONS
Location Description (in detail):
Location Description (in detail):
GPS Location/coordinates:
GPS Location/coordinates:
Turtle Species: Already Marked?
Turtle Species: Already Marked?
Notch Code or Radio Tag # (new or existing):
Notch Code or Radio Tag # (new or existing):
Obs. Start Time: Obs. End Time:
Obs. Start Time: Obs. End Time:
Substrate:
Substrate:
Distance from Water: Attempt Successful?
Distance from Water: Attempt Successful?
Photos: # of Eggs:
Photos: # of Eggs:
For mapping and nest re-locating purposes
For mapping and nest re-locating purposes
installed? Predator exclosure
Nest I.D.
installed? Predator exclosure
Nest I.D.
Distance from A Distance from B
Distance from A Distance from B
Describe Turtle Activity/Comments:
Describe Turtle Activity/Comments:
: by boatI (1) or (2): Triangulation - 1 or 2 people, and BO*Track type: VIS: Visual, SH: by shore (1 person), TR
CommentsStrength
Signal Triang A Triang B Actual Triang A Triang B
type*Track
Point # Time FreqSessionTrack
Bearing (degrees)Location NAD 93 10 U
Weather:Date: Observer:
Data Collection Form 5: Turtle Radio-Tracking
Track/R
type*Plot
General habitat characteristicsTemperature (
Data Collection Form 6: Turtle Habitat Use Plots
o)
adom Pt
: TR: tracking location, RA: random location*Plot type
Comments(Y/N)
Pic vis****Water
Veg***Submerg Emerg /
**Submerg*Emerg / Woody
type**habitat Aquatic
(cm)Depth
Conduct PhbaskNear
WaterBottom
WaterColumn
WaterSurface
Date Time AirLocat
**Aquatic habitat type ear of cover ; includes Degree of Cover: SH: Shady (%) or CE: Cllassifications (Rowe et al. 2009)), and BA: Basking: OP: Open water, ED: Edge (based on microhabitat c
***Emerg / Submergs itvatis 2004)) within 2 mt (based on basking site categories (Marchand and L: 0 - sparse, 1- scattered, 2 - common, 3 - abundan****Water vis - clear: 0 - completely opaque, 1 - 50% transparent, and 2
by Elinor Hughes
nvironment Prepared for the British Columbia Ministry of the E
) blood and tissue for genetic analysis. Chrysemys pictaturtle (
f painted Appendix B: Protocol for the sampling and storage o
urtle tail ............................ 10 Some points to remember when drawing blood from a t.................... 9 ...................................................Drawing blood .....................................
............................ 3...................................................Table of Figures ..................................
2
............... 12 ...................................................relation to the yellow stripe. ....................oaca and to the end of the tail and in insertion of the needle, both in relation to the cl
pace) and the location for the turtles (female cloacas are much closer to the caraimate location of the cloaca in male Figure 2 Dorsal view of a turtle showing the approxgle from vertical. ............................ 11 and needle inserted into turtle tail at a slight an
of caudal artery within caudal vertebra Figure 1 Turtle tail cross-section showing location
Table of Figures
3
possible genetic markers and testing laboratories. c testing guidelines, including methods and sample storage requirements; and genetiirements; tissue sampling, including sampling, including methods and sample storage requ
cs include: sampling protocol; blood turtles for the purposes of genetic analysis. Topion of samples from painted The following comprises a protocol for the collecti
eatened species. management as part of a recovery strategy for a thrtion) and 5) the potential for genetic recent (e.g. a new road) or historical (e.g. glacia) the effect of an isolation event, either presence of inbreeding and its effect on fitness, 4
ith a loss in specific phenotypes, 3) the example whether a loss of diversity is associated w diversity in small populations, for phylogeography, 2) the effects of a loss of geneticships among sister species and population or species, including taxonomic relation1) the evolutionary history of a Genetics may be used to study questions regarding: te without including genetic analysis. of within-or among-population diversity is incomple
, many may suggest that an investigation useful and achievable in the recent years. In factncreasingly more important, The use of genetics to study diversity has become i
4
Blood
Sampling protocol
5
Tissue
turtles caught. that blood be drawn from all adult the sex ratio is unknown, it is highly recommended
caught. If either the population size or used will depend on the how frequently turtles are caught each day. The sampling regime day or draw blood from the first turtle of each sex9:00 am, 12:00 pm and 3:00 pm each blood from turtles of each sex caught at (or near)
female turtle caught, draw nd male and every 2ndare as follows: draw blood from every 2f catch intervals that may be used random sample and is much simpler. Some examples o
pecific intervals produces an equally instructions regarding their use, but sampling at s be found on the Internet with specific catch interval. Random numbers tables mayble or by sampling turtles at a determined either by consulting a random numbers ta
ratio. The sampling regime may be sex ratios, the sample group should reflect the sex sampled. In populations with uneven sampled or the absolute number of individuals to be
the proportion of the population to be before you run out of catchable turtles. Determineblood from the required number and that the sampling regime allows you to collect
n, ensure that the sample is random may be used. When taking a sample of the populatioconcern, then a sample of the population populations are small. If time or resources are a
es in a population, especially if Ideally, blood should be drawn from all adult turtl
the individual hatchlings. randomness of the sample applies to the nests, not als within a single nest, and so the For nests, tissue should be taken from all individuabove for adult turtles may be used. nests, a similar sampling regime to that described y be used. For both juveniles and or 50 juveniles, then a sample of the population ma
sampled. If there are more than 20 nests juveniles, then all hatchlings/juveniles should be , e.g. fewer than 20 nests or 50 If few nests or juveniles are found in a population
performed. uals if a paternity analysis is to be samples should only be taken from non-adult individ
tion. Furthermore, we recommend that negative effects on the individual or on the populaor juvenile turtles to limit possible tissue samples should only be taken from hatchling
we conservatively recommend that the genetic diversity of a population. Therefore, may prove to be irrelevant to studies of is very slight. As a result, their genetic makeup will become a reproductively active adult populations, the likelihood that a juvenile turtle
g and juvenile turtles in natural Because of the very low survivorship of hatchliny active turtles (pers obs.) been observed on numerous healthy and reproductivel
l or complete loss of tails or limbs have Serious adult injuries, however, involving a partia of turtles has not been studied. The effect of tissue removal on long-term survival
A note on genetic variability in turtles
6
0 years ago. among populations isolated from one another even 10be observable genetic differences slower rate into consideration, e.g. there may not
researchers are advised to take the researching many of the among-population questions, conversion value. Hence, in These numbers are only included as a caution, not ato a “conventional” mt-DNA rate. DNA) differentiation in turtle species as compared
ower rate of mitochondrial DNA (mt-other species. Avise et al (1992) found a 2-14x sled to change as quickly as in Genetic variability in turtles should not be expect
Storage of blood samples
Blood sampling
7
Comparison of blood storage methods
hat the samples remain desiccated. have dried, however, care must be taken to ensure t at room temperature, once the samples stored on cell lysis cards may be kept indefinitely
care must be taken. Blood samples but freeze-thaw cycles may fragment DNA strands so for long-term storage, C). Samples may be stored at subzero temperatures refrigerator (4
but must then be kept in a may be held for up to one week at room temperature,lood samples stored in a cell lysis buffer analysis: cell lysis buffer or cell lysis cards. B
lood samples for future DNA There are two field-convenient methods of storing b
°
cards for painted turtle blood storage. analysis, we would recommend the use of cell lysis e detail. Based on this cost-benefit section and the laboratory analysis section for mored laboratory costs. See the materials materials for drawing blood, and both have associatonally, both methods require method are included in the cost comparison. Additi
y those materials that are unique to each suggests suppliers for the required materials. Onl two methods and Appendix 2 Appendix 1 outlines the cost comparison between the
1) Slightly more expensive per sample. Cons
very fast (30 minutes to usable DNA). 3) The extraction technique is fairly simple and isough the mail. 2) Samples may easily be transported, including thr
tely. 1) Samples may be kept at room temperature indefiniPros
Cell lysis cards:
blood-taking. uld be refrigerated with 24 hours of acceptable (up to a week), but ideally, samples short durations at room temperature are 3) The samples must be kept in a refrigerator. Sho
ampling many individuals 2) The tubes are bulky to carry and cumbersome if sd then autoclaved. chemicals, but must be adjusted to a specific pH an
It requires common laboratory 1) Cell lysis buffer must be made in a laboratory. Cons
ults. generally needed to produce enough DNA for good resnly a very small amount of blood is however, since turtle blood cells contain nuclei, o
may result in a higher DNA yield, 2) The extraction process may be more efficient andosed to ~$4.20/sample). 1) Slightly cheaper per sample (~$2.80/sample as opp
Pros
Cell lysis buffer:
Materials
esearchers. materials be placed by a manager and dispensed to rurther recommend that orders for are only obtainable in large quantities, we would f
below. Because many of the items Regardless, we include descriptions of both methods
8
storage has not been tested. ever their effectiveness in turtle blood Note: lysis buffers are available commercially, how
samples. er and will affect the quality of the evaporation of liquid will alter the pH of the buffthe caps are tightly closed as any indefinitely. Care should be taken to ensure that
be kept at room temperature be prepared well in advance of blood taking and mayady to draw blood. Multiple tubes may close cap tightly and place in storage box until re
into each 1.5ml microcentrifuge tube, Once the buffer is finished, dispense 1ml of bufferfer should be autoclaved before use. workers will find the recipe easy to read. The buftories. Additionally, most laboratory chemicals that are readily available in many labora
-lauroylsarcosine, pH 8.0. These are all common NaCl, 0.01 M sodium EDTA and 10% s as follows: 0.01 M Tris, 0.01 M specific pH. The recipe for Queen’s lysis buffer i
and soaps that is standardized to a al., 1991). A buffer is simply a solution of salts Queen’s lysis buffer (Seutin et If using buffer to store turtle blood, we recommend
Making cell lysis buffer
ot available. 1cc syringes attached might be used if others are nh skin. That said, 29 gauge needles with may better resist bending against the turtles’ toug
les are recommended because they smaller needle diameter). The larger diameter need or smaller (larger gauge number = needles already attached and are generally 29 gauge
most drug stores, however, they have Note: 1.0ml (1cc) volume syringes are available at
rage boxes (for tubes) Storage bags and desiccant (for lysis cards) or stosis buffer in microcentrifuge tubes) Blood storage medium (either cell lysis cards or ly
Small bucket (optional) Fine permanent black marker Dishtowels Alcohol wipes (purchased at drug store) Sharps disposal container
ml syringe only 1.027 gauge bevel-tip needles
zed materials) (See Appendix 2 for suggested suppliers of speciali
n
Turtle blood sampling method
9
the needle to face the bevel in different have 0.1ml of blood. If there is no blood, rotate patiently draw on the plunger until you begin to flow into the needle. If there is blood,
n the plunger; you should see blood withdraw the needle very slightly and gently draw o. Once your hand is positioned, needle firmly in the tail--there is no need to rush
unger. Be confident and keep the 3) Reposition your hand so that you can draw the plbetween two vertebrae.
means that you have found a space until you feel the needle go in 1/8” to 1/4” . Thisy closer to the carapace. Repeat this immediately remove the needle and insert it slightl
Figures 1 and 2). If you hit bone needle will be at a slight angle from vertical, see Aim for the centre line of the tail (the the end of the tail just inside the yellow stripe.
the tail about one-finger’s width from 2) Gently insert the needle into the dorsal side ofhand.
and forefinger of your non-dominant facing up. Hold the tail firmly between the thumb e that the bevel of the needle is 1) Take the syringe in your dominant hand and ensur
Drawing blood
cohol wipe. another dishtowel and swab it thoroughly with an alwith a wet dishtowel, dry it with firmly with your non-dominant hand, clean the tail lean over as much. Grip the tail small bucket under your feet so you do not need to r dominant hand. If desired, place a your knees, tail up and carapace facing towards youound, hold the turtle firmly between being pinched. Seated on a chair/stump/rock/the gr
head are retracted into the body without secure the hind feet and ensure that all limbs and wel leaving the tail exposed. Be sure to 5) Prepare the turtle. Wrap the turtle in a dishto
t do not remove from packages. 4) Take two alcohol wipes and open the packages, buut a short way. package. Prime the syringe plunger by pulling it o
but don’ t remove it from the syringe package and loosen the cap from the needle,ringes with needles attached, open the plunger by pulling it out a short way. If using sy
eedle package. Prime the syringe cap from the needle, but don’ t remove it from the nfirmly onto the needle. Loosen the syringe and remove from package. Seat the syringe
e top of the package. Take one 3) Prepare the needle. Take one needle and open thof the tube.
arker. Write the turtle’s ID on the cap the side of a tube with a fine permanent felt tip mjuvenile, and the date in the frosted area on If using lysis buffer, write the turtle’s ID, sex/
provided by the manufacturer. familiarized yourself with the lysis card protocol age bag. Be sure that you have marker. Place one bag of desiccant inside the stortorage bag with a fine permanent felt tip sex/juvenile, and the date on the card and on the s
cell lysis card, write the turtle’s ID, 2) Prepare the cell lysis card or tube. If using asampling, difficulties with sampling).
le tail condition, injuries caused by regarding the blood-taking procedure (e.g. pre-samp, researcher’s name, comments turtle ID, sex/juvenile, population, date of sampleing. Column headings should include: 1) Create a data sheet to keep track of blood sampl
Preparation of materials
sample must be taken. done on those individuals, a tissue blood from hatchlings. If genetic testing is to bejuvenile turtles and impossible to draw 7) It is very difficult to draw blood from smaller
e and use a new needle if necessary. become bent. Be sure to check the tip of the needlt the needle repeatedly, the tip may 6) Turtle skin is very tough. If you need to inser
blood-taking technique. this if you are confident in your between the cloaca and the carapace. Only attempt
attempt to draw blood from the tail 5) If the turtle has a severe tail injury, you may ae. will be trying to insert the needle between vertebr
within the vertebral column. You 4) You are aiming for the caudal artery, which runs cloaca into the turtle’s body. that you will hit any vital nerves leading from the to the cloaca. This reduces the chance 3) Be sure that you are inserting the needle distal
turtle will kick at the syringe. bs should also be well wrapped, as the the researcher might not want bitten. The hind lim
head will be very near to body parts that turtles are small, they have fierce bites, and the secured in the towel. While painted 2) Ensure that the turtle’s head and limbs are well
to receive the sample) ollection medium labelled and ready seated on syringe and primed, alcohol wipes open, c
erials at hand and ready to use (needle on the tip. Be sure that you have all required mat, especially the location of the bevel 1) Familiarize yourself with the needle and syringe
urtle tail Some points to remember when drawing blood from a t
utes. injection site should be checked again after 30 minor swelling after 5 minutes, and the should be applied if there is any sign of bleeding nutes for bleeding or swelling. Bactine 6) Check the injection site after 1 minute and 5 mi
within about 30 seconds to1 minute. the turtle. Any bleeding should stop 5) Swab the tail with the alcohol wipe and release
container. x. Dispose of the needle in a sharps blood with the buffer. Put the tube back in the bo
ghtly closed and invert the tube to mix the the blood into the tube. Ensure that the cap is tit. If using tubes with buffer, dispense minutes and then place in storage bag with desiccan
Allow sample to air-dry for 30 the blood does not go beyond the circle indicated. ollection area on the card. Be sure that the blood lysis cards dispense the blood onto the caw blood to the 0.2ml mark. If using 4) Draw 0.1ml of blood. If the blood is aerated dr
stop trying and come back to that turtle later. vertebrae. If the turtle is very resistant, will find it difficult to insert the needle between
s. If the turtle is pulling in its tail, you blood. Some turtles are harder to bleed than othern another spot. Keep trying until you get directions, or remove the needle and re-insert it i
10
11
gle from vertical. and needle inserted into turtle tail at a slight an of caudal artery within caudal vertebra Figure 1 Turtle tail cross-section showing location
12
relation to the yellow stripe. oaca and to the end of the tail and in insertion of the needle, both in relation to the cl
pace) and the location for the turtles (female cloacas are much closer to the caraimate location of the cloaca in male Figure 2 Dorsal view of a turtle showing the approx
Toe clipping vs. tail clippingTissue Sampling
13
Storage of tissue samples
digit. cher’s comfort level with removing a is used for hatchling turtles depends on the resear
sue is all that is required. Which method samples are stored correctly, a single piece of tislip than by tail-clip. Nonetheless, if a larger amount of tissue may be collected by toe-c
ail sample may be taken. Hence (ASIH 2004; we recommend fewer), whereas only one tcent toes on each foot may be sampled hatchlings or juvenile turtles. Up to two non-adja
n, while tail clipping may be used on from the nest or caught in their first active seasoles, i.e., those sampled directly Toe clipping should only be used on hatchling turt
Materials
sampling. the buffer described in the above section on blood the protocol for making and dispensing indefinitely. To use Queen’s lysis buffer, follow
C samples may be refrigerated at 4temperature for up to a week. Following that, the buffer and kept at room Tissue samples may also be stored in Queen’s lysis
week. C freezer within one sferred to a -80even in a closed carboy and so tubes should be tran
C freezer. Liquid nitrogen will sublimate, nitrogen until tubes can be transferred to a -80should be stored in cryotubes in liquid cumbersome to take into the field. Frozen samples
itrogen is necessary and carboys are a field laboratory setting since access to liquid n, sample collection must be performed in (Seutin et al., 1991). If samples are to be frozen
ned from samples in lysis buffer significant amount of good quality DNA can be obtain tissue is cryogenically frozen, but a temperatures. The DNA yield is slightly higher whe
ffer or at extremely low Tissue samples must be stored either in a lysis bu
°°
°
Queen’s lysis buffer (if using) 1.5 ml microcentrifuge tubes each containing 1.0ml
Cryostore tubes (if using liquid nitrogen) Liquid nitrogen (if using)
one for Bactine 2 small dishes, one for waste isopropyl alcohol andBactine antibiotic spray Bunsen burner (or other flame source) 70% isopropyl alcohol in squirt bottle Fine-tipped forceps or tweezers
s also work well) Sharp dissection scissors (new, unused nail scissorDishtowels Fine permanent black marker
zed materials) (See Appendix 2 for suggested suppliers of speciali
Toe clipping
14
and heat sterilize between each cut. 7) Rinse scissors and forceps in isopropyl alcohol C. above 20
g within 2 minutes at temperatures for a further 30 minutes. Most turtles begin movinent is not regained, observe the turtle bleeding or swelling is observed or if normal movem
ent is regained in the limb. If swelling at the incision site and that normal movem ensure that there is no bleeding or 6) Observe the turtle at intervals for 5 minutes to
ssary. 5) Repeat procedure for the other hind foot if nece) or in storage box. storage tube and place in liquid nitrogen (if using a secure container. Tightly cap the 4) Dip the foot in Bactine. Place the hatchling inface, it is still usable. may fall. If the toe should fall on an unclean surtick to the scissors after clipping, and over a clean piece of paper as the toe will often s
d forceps (if necessary). Hold the foot immediately into the storage tube using scissors antle’s foot). Cut the toe and place it stop you from cutting the toe any closer to the tursecond knuckle (the toe webbing will down the toe until the scissor blades are past the rs, isolate a single toe and gently move forefinger. Dip the foot in Bactine. Using scisso
with toes splayed over your of the hind feet between your thumb and forefinger r palm. Gently, but firmly, grip one hind feet towards your fingers and head towards youatchling in non-dominant hand with 3) Dry hatchling thoroughly with dishtowel. Hold h
paper after sterilizing. ore scissors and forceps on clean piece of alcohol and briefly hold in flame to sterilize. St
rinse scissor blades and forceps in tube. Dispense small dish of Bactine. Thoroughly urtle ID on the cap of the tube. Open tube with fine permanent black marker. Write the t
e frosted area on the side of the storage 2) Write the turtle ID and date of collection in thcomments.
her name, recovery time of turtle and stage (e.g. hatchling), date of collection, researc Columns should include: turtle ID, life 1) Create a data sheet to track tissue collection.
Method
moved from each hind limb. painted turtles and that a maximum of one toe be reemoved only from the hind limbs of the hind limbs. Hence, we recommend that toes be rpainted turtles dig their nests using courtship and mating success. Furthermore, female these missing digits influence depredation attempts (pers obs) it is not known how
any turtles have missing digits due to forelimbs as part of the courtship ritual. While md turtles use the digits on their adjacent digits be removed. Male and female painte
mends that not more than two Ichthyologists and Herpetologists (ASIH 2004) recomn). The American Society of from the nest or caught in their first active seaso
les (i.e., those sampled directly This method should only be used for hatchling turt
°
Tail clipping
15
individual.
and heat sterilize between each 6) Rinse scissors and forceps in isopropyl alcohol ement is regained. turtle for a further 30 minutes or until normal mov
ains retracted into the shell, observe the bleeding or swelling is observed or if the tail remno longer retracted into the shell. If swelling at the incision site and that the tail is
ensure that there is no bleeding or 5) Observe the turtle at intervals of 5 minutes to ) or in storage box. storage tube and place in liquid nitrogen (if using
a secure container. Tightly cap the 4) Dip the tail in Bactine. Place the hatchling inon an unclean surface, it is still usable.
ipping, and may fall. If the tail should fall tail clip will often stick to the scissors after clhling over a clean piece of paper as the scissors and forceps (if necessary). Hold the hatc immediately into the storage tube using Bactine. Cut a ½ cm piece of the tail and place it
from the tip. Dip the tail in between your thumb and forefinger approximately 1cmm. Gently, but firmly, grip the tail feet towards your fingers and head towards your palatchling in non-dominant hand with 3) Dry hatchling thoroughly with dishtowel. Hold h
clean piece of paper after sterilizing. rilize. Store scissors and forceps on forceps in alcohol and briefly hold in flame to ste Thoroughly rinse scissor blades and tube. Open tube. Dispense small dish of Bactine. urtle ID on the cap of the tube. Open tube with fine permanent black marker. Write the t
frosted area on the side of the storage 2 Write the turtle ID and date of collection in thecomments.
her name, recovery time of turtle and stage (e.g. hatchling), date of collection, researc Columns should include: turtle ID, life 1) Create a data sheet to track tissue collection.
Method
Contact: Carol Ritland, versity of British Columbia. 1) Genetic Data Centre, Department of Forestry, Uni
Possible laboratories
ablished laboratory. We recommend that this work be contracted to an estized equipment and skills. DNA extraction and genetic analyses require special
Website: Fax (604) 822-9102 Telephone (604) 822-3908 or (604) 822-1543
http://www.forestry.ubc.ca/gdc/
s. are highly polymorphic (variable) in painted turtleon painted turtle DNA and some loci terrapins (Hauswaldt and Glenn, 2003) amplify well
d one set developed for diamondback (Osentoski et al., 2002 and Libants et al, 2004) anites developed for Blanding’s turtles especially painted turtles. Two set of microsatell
d genetic markers for turtles, through the maternal line). There are few publisheDNA (mitochondrial DNA, inherited (single nucleotide polymorphisms or repeats) and mt
peated fragments of DNA), SNPs investigated using microsatellites (small highly re among species may be Relatedness among individuals, among populations or
one day). NA may be obtained from the kit in chemicals and takes up to three days to complete (D
for tissue samples, however it uses toxic extraction is an alternative, very reliable method om tissue. Phenol-chloroform Qiagen DNeasy kit also produces high-quality DNA fr
DNA in a relatively short time. The to use and produce reliable amounts of high-qualityy kit and the GenElute kit are simple commercially available kits. Both the Qiagen DNeas
g any number of DNA may be extracted from turtle blood samples usin
communicating with the contracted laboratories: oses of effectively The following information is included for the purpNote:
work. ave a laboratory that does genetic 3) The Ministry of Agriculture and Lands may also h
am not a government worker. e information, which I cannot since I have to gain access to their website to provide mor
ey may be a good option. I would among branches of the provincial government, but th work or the nature of the relationship I am not sure whether they are set up to do genetic
nalytical Laboratory 2) Government of British Columbia Research Branch A
mtDNA and SNPs, among other genetic markers. ic testing using microsatellites, The genetic data centre accepts contracts for genet
: 82-90. 69gy samples for DNA analysis. Canadian Journal of Zoolorvation of avian blood and tissue Seutin, G., White, B.N., and Boag, P.T. 1991. Prese
atellite loci from the C.R. 2002. Isolation and characterization of microser, M., Herman, T.B., and Hughes, Osentoski, M.F., Mockford, S.W., Wright, J.M., Snyd
: 300-302. 2004. Molecular Ecology Notes blandingiiEmydoidea loci from cross-species amplification of seven microsatellite
Congdon, J.D. 2004. Isolation and Libants, S., Kamarainen, A.M., Scribner, K.T., and
: 174-176. ). Molecular Ecology Notes Malaclemys terrapinterrapin (e DNA loci from the Diamondback Hauswaldt, J.S. and Glenn, T.C. 2003. Microsatellit
: 457-473. Biology and Evolution he Testudines. Molecular variability and reduced microevolutionary rate in t
dence for low genetic Mitochondrial DNA evolution at a turtle’s pace: evi. d Bermingham, E. 1992Avise, J.C., Bowen, B.W., Lamb, T., Meylan, A.B. an
http://www.asih.org/files/hacc-final.pdf mudio. Members: Jacobson, E.R., Lillywhite, H.B. and K. Za
Chair: Beaupre, S.J., Herpetological Animal Care and Use Committee (HACC) ed, Rev by ndearch, 2amphibians and reptiles in field and laboratory res
ts. 2004. Guidelines for use of live American Society of Ichthyologists and Herpetologis
References
17
9
3
Blood lysis cards Blood lysis buffer
s Appendix 1: Cost comparison of blood storage method