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Herpetological Review 39(3), 2008
301
It is important to note that gloves have not been found to
negatively affect juvenile or adult amphibians. The use of gloves
to handle amphibians is widespread in the fi eld and lab. Changing
gloves between amphibians remains an important hygiene
measure to prevent transmission of infectious agents such as
B.
dendrobatidis
and ranaviruses between individual amphibians and
aquaria. However, given our tadpole results, it would be useful
to formally investigate potential non-lethal effects of gloves on
adult and juvenile amphibians to ensure that gloves really are
entirely non-injurious.
Acknowledgments
.—We thank Bryan Windmiller and Jamie Voyles
for providing helpful comments on an earlier draft of the manuscript.
This work was supported by the Department of the Environment and
Heritage (Australia) tender 42/2004 and 43/2004. The techniques and
experiments used here were approved by the Animal Ethics Committee
at James Cook University, Townsville (A930, A960 and A970). Permits
to collect and capture tadpoles were granted by Queensland Parks and
Wildlife Service (WISP03070205, WITK 03070205 and INN/004).
LITERATURE CITED
BOMAN
,
A., T. ESTLANDER, J. E. WAHLBERG, AND H. I. MAIBACH.
2004.
Protective Gloves for Occupational Use. 2nd ed. Routledge, USA. 343
pp.
BOYLE, D. G., D. B. BOYLE, V. OLSEN, J. A. T. MORGAN, AND A. D.
HYATT.
2004. Rapid quantitative detection of chytridiomycosis
(
Batrachochytrium dendrobatidis
) in amphibian samples using real-
time Taqman PCR assay. Dis. Aquat. Org. 60:141–148.
GUTLEB, A. C., M. BRONKHORST, J. H. J. VAN DEN BERG, AND A. J. MURK.
2001. Latex laboratory-gloves: an unexpected pitfall in amphibian
toxicity assays with tadpoles. Environ. Toxicol. Pharmacol. 10:119–
121.
KNAPP, R. A., AND J. A. T. MORGAN.
2006. Tadpole mouthpart
depigmentation as an accurate indicator of chytridiomycosis, an
emerging disease of amphibians. Copeia 2006:188–197.
MARANTELLI, G., L. BERGER, R. SPEARE, AND L. KEEGAN.
2004. Distribution
of the amphibian chytrid
Batrachochytrium dendrobatidis
and keratin
during tadpole development. Pac. Conserv. Biol. 10:173–179.
RETALLICK, R. W. R., V. MIERA, K. L. RICHARDS, K. J. FIELD, AND J. P.
COLLINS.
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1999. Lethal effect of latex gloves on
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tadpoles. J. Pharmacol. Toxicol. Methods 32:59.
TECHNIQUES
Herpetological Review
, 2008, 39(3), 301–303.
© 2008 by Society for the Study of Amphibians and Reptiles
Visible Implant Fluorescent Elastomer: A Reliable
Marking Alternative for Snakes
STAN J. HUTCHENS*
CHRISTOPHER S. DEPERNO
CHARLOTTE E. MATTHEWS
Department of Forestry and Environmental Resources
Fisheries and Wildlife Sciences Program, North Carolina State University
Raleigh, North Carolina 27695, USA
KENNETH H. POLLOCK
and
DAVID K. WOODWARD
Department of Zoology, North Carolina State University
Raleigh, North Carolina 27695, USA
*Corresponding author; e-mail: sjhutchi@ncsu.edu
Studies in population ecology require use of reliable marking
techniques to estimate various parameters (e.g., population size,
density, demographics, movement, or behavior; Penney et al.
2001; Perret and Joly 2002; Walsh and Winkelman 2004; Woods
and Martin-Smith 2004). However, it is imperative that marking
techniques meet standard assumptions: 1) marks must remain
visible for the duration of the experiment, 2) marks are correctly
recorded, 3) marks do not affect the survival of the animal,
and 4) marks do not affect the recapture probability of animals
(Goldsmith et al. 2003; Otis et al. 1978).
Visible implant fl uorescent elastomer (VIE; Northwest Marine
Technology, Inc., Shaw Is., Washington, USA) was initially
developed for batch marking migratory fi sh, but has recently
been used to mark amphibians and lizards (Bailey 2004; Losos
et al. 2004; Nauwelaerts et al. 2000; Nishikawa and Service
1988; Penney et al. 2001). Visible implant fl uorescent elastomer
consists of a liquid polymer added to a curing agent to create a
fl exible plastic mark. Color kits are available, capable of marking
15,000 individuals depending on the number of colors used and
marking design. Our objective was to determine if VIE was an
appropriate marking technique for snake research based on the
marking assumptions of Otis et al. (1978) and Goldsmith et al.
(2003). We hypothesized that VIE would be a reliable marking
technique for snakes. To our knowledge, our study is the fi rst to
apply VIE to snakes.
We conducted this empirical study in a laboratory setting at
North Carolina State University, Raleigh, North Carolina, USA.
We marked Red Cornsnakes (
Pantherophis guttatus
; N
= 18)
between 19 and 29 April 2006. Each snake received three doses
(1, 2, and 3 µl) of yellow VIE randomized to the general area
of three locations (neck, midbody, and pre-caudal). We injected
marks subcutaneously and dorsolaterally on left sides using a
graduated 1cc Luer-lok syringe with a 25-gauge needle (Becton-
Dickinson, Franklin Lakes, New Jersey, USA). We used 1cc
syringes to better approximate volumes, which required the 25-
gauge needle for a secure fi t. We injected additional
P. guttatus
(N
= 4) and Common Kingsnakes (
Lampropeltis getula
; N = 6) with
Herpetological Review 39(3), 2008
302
blue and red to examine VIE color, ground color, and species
effects, but did not quantify results from these 10 snakes.
All snakes were captive-raised and housed individually at
a constant 26.6
°
C with food, water, and substrate provided
regularly. We checked snakes for marks every two weeks using a
UV-B light. We collected shed skins to record shedding frequency
and expulsion of marks. Our study concluded on 4 May 2007
after 370 days.
We calculated retention time as the number of days we
detected a mark until the median date between when a mark was
last detected and the following examination. We derived mean
retention times for each mark volume (1, 2, and 3 µl). To discern
the effects of mark volume or individual variation on retention
time, we analyzed retention time as a dependent variable with
mark volume and individuals as independent variables in an
additive, 2-factor analysis of variance (ANOVA). Similarly,
we employed two 1-factor ANOVAs with retention time as the
dependent variable and shedding frequency (i.e., number of sheds/
individual) or mark location (i.e., neck, midbody, and pre-caudal)
as independent variables. We performed analyses using PROC
GLM (SAS 9.1, Cary, North Carolina, USA). We calculated the
percentage of marks retained to demonstrate mark performance
by volume.
All 18
P. guttatus
used in the experiment were of similar length
(mean = 990.39 ± 79.41 mm snout–vent length) and weight
(mean = 370.33 ± 81.93 g). After 370 days, 94, 83, and 100%
of low (mean = 354 days), medium (mean = 333 days), and
high (mean = 370 days) mark volumes were retained. A 2-factor
ANOVA revealed no differences in retention time between mark
volumes (F
2, 34
= 1.27, P = 0.2940) or individuals (F
17, 34
= 0.88, P
= 0.6045). Shedding frequency (mean = 5.05 ± 1.21 sheds/snake)
did not have a signifi cant effect on mark retention time at low
(F
1, 16
= 0.79, P = 0.3860), medium (F
1, 16
= 0.00, P = 0.9501), or
high mark volumes (100% retention). Analysis of mark location
revealed a 13% lower mean retention time for marks located pre-
caudally (mean = 321 days) compared to neck (mean = 366 days)
and midbody (mean = 370 days) mark locations. However, we
did not detect a signifi cant difference among locations (F
2, 51
=
3.00, P = 0.0588).
Our results indicated that VIE was a reliable marking technique
for snakes, with 94, 83, and 100% retention for low, medium,
and high volumes after 370 days and no mortalities recorded.
Elastomer marks were easy to identify and record due to
fl uorescent colors. We observed a signifi cantly lower retention
time for pre-caudal marks. In fact, 3 of the 4 marks lost (1-low
and 2-medium volume) were located in the pre-caudal region and
were lost through expulsion within the fi rst few examinations. If
we removed these early loses from our analyses, retention times
would be 100, 94, and 100% for low, medium, and high volumes,
respectively.
Our results demonstrated that VIE marks last at least 370 days
and satisfy the marking assumptions proposed by Otis et al. (1978)
and Goldsmith et al. (2003). Branding and scale clipping have
been reported to last ≥ 3 years (Brown and Parker 1976; Winne et
al. 2006) and elastomer marks have been reported lasting well over
a year in amphibians (Davis and Ovaska 2001) and are capable
of permanence (Kinkead et al. 2006). We acknowledge our short
study duration (370 days), but believe VIE satisfi es assumptions
for correct recording, and survival and recapture effects (Davis
and Ovaska 2001; Kinkead et al. 2006) and is a reliable marking
technique for snakes. Equipment costs were initially higher for
VIE (US $465) compared to scale clipping (i.e., scissors) or
branding (i.e., cautery units ~US $20–25; Winne et al. [2006]),
but marking costs per snake were small (~$0.10– $0.29 for marks
of 1– 3 µl). Further, our retention of all mark volumes suggested
the usefulness of VIE in snakes of any size; small marks can be
applied to small-bodied species and individuals (≤ 26 cm), which
may be too small for PIT tags or scale clipping (Spellerberg
1977). However, problems were encountered with the technique.
Pre-caudal marks had a 13% lower retention time and accounted
for 75% of marks lost. Mark losses occurred from 23 days to 310
days, and were likely due to expulsion from the site of injection.
Similarly, fragmentation of marks into several pieces could cause
detection problems. The application of a liquid bandage product
would likely deter expulsion and pathogen introduction.
We recommend future studies evaluate the use and effi cacy of
VIE in snakes, both in lab and fi eld settings. Future studies should
evaluate using VIE in different species of snakes with various
ground colors and at various mark locations. Anecdotally, we
can report that yellow, blue, and red VIE colors were detectable
in Red Cornsnakes at all volumes, but blue VIE was diffi cult to
detect in Common Kingsnakes due to the dark ground color of
this species. Mark volume should be studied in the fi eld to better
understand mark retention under natural conditions and fi eld and
laboratory research should focus on survival and recapture rates
for VIE over longer periods. Future research should evaluate the
effects of growth on mark detectability and compare stress levels
incurred by traditional and VIE marking techniques.
Acknowledgments.
—
Facilities and procedures for research regarding
captive snakes followed the guidelines for the Institutional Animal
Care and Use Committee at North Carolina State University (Approval
Number 05-036-0). Funding was provided by the North Carolina State
University Department of Forestry and Environmental Resources and
the North Carolina State University Fisheries and Wildlife Sciences
Program. We thank J. W. Tomberlin, T. J. Langer, R. A. Lancia, C.
Greenberg, and anonymous reviewers for comments on earlier drafts of
the manuscript.
L
ITERATURE
C
ITED
B
AILEY
, L. L. 2004. Evaluating elastomer marking and photo identifi cation
methods for terrestrial salamanders: Marking effects and observer
bias. Herpetol. Rev. 35:38–41.
B
ROWN
, W. S., and W. S. P
ARKER
. 1976. A ventral scale clipping system
for permanently marking snakes (Reptilia, Serpentes). J. Herpetol.
10:247–249.
D
AVIS
, T. M., and K. O
VASKA
. 2001. Individual recognition of amphibians:
Effects of toe clipping and fl uorescent tagging on the salamander
Plethodon vehiculum
. J. Herpetol. 35:217–225.
G
OLDSMITH
, R. J., G. P. C
LOSS
, and H. S
TEEN
. 2003. Evaluation of visible
implant elastomer for individual marking of small perch and common
bully. J. Fish Biol. 63:631–636.
K
INKEAD
, K. E., J. D. L
ANHAM
, and R. R. M
ONTANUCCI
. 2006. Comparison
of anesthesia and marking techniques on stress and behavioral
responses in two
Desmognathus
salamanders. J. Herpetol. 40:323–
328.
L
OSOS
, J. B., T. W. S
CHOENER
, and D. A. S
PILLER
. 2004. Predator-induced
behaviour shifts and natural selection in fi eld-experimental lizard
populations. Nature 432:505–508.
Herpetological Review 39(3), 2008
303
N
AUWELAERTS
, S., J. C
OECK
, and P. A
ERTS
. 2000. Visible implant elastomers
as a method for marking adult anurans. Herpetol. Rev. 31:154–155.
N
ISHIKAWA
, K. C., and P. M. S
ERVICE
. 1988. A fl uorescent marking
technique for individual recognition of terrestrial salamanders. J.
Herpetol. 22:351–353.
O
TIS
, D. L., K. P. B
URNHAM
, G. C. W
HITE
, and D. R. A
NDERSON
. 1978.
Statistical inference from capture data on closed animal populations.
Wildl. Monogr. 62:135 pp.
P
ENNEY
, K. M., K. D. G
IANOPULOS
, E. D. M
CCOY
, and H. R. M
USHINSKY
.
2001. The visible implant elastomer marking technique in use for
small reptiles. Herpetol. Rev. 32:236–241.
P
ERRET
, N., and P. J
OLLY
. 2002. Impacts of tattooing and PIT-tagging
on survival and fecundity in the alpine newt (
Triturus alpestris
).
Herpetologica 58:131–138.
S
PELLERBERG
, I. F. 1977. Marking live snakes for identifi cation of
individuals in population studies. J. App. Ecol. 14:137–138.
W
ALSH
WALSHW
, M. G., and D. L. W
INKLEMAN
, M. G., and D. L. WINKLEMAN, M. G., and D. L. W
. 2004. Anchor and visible implant
elastomer tag retention by hatchery rainbow trout stocked into an
Ozark stream. N. Amer. J. Fish. Man. 24:1435–1439.
W
INNE
WINNEW
, C. T., J. D. W
ILLSON
, C. T., J. D. WILLSON, C. T., J. D. W
, K. M. A
NDREWS
, and R. N. R
EED
. 2006.
Effi cacy of marking snakes with disposable medical cautery units.
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W
OODS
WOODSW
, C. M. C., and K. M. M
ARTIN
-S
MITH
. 2004. Visible implant
fl uorescent elastomer tagging of the big-bellied seahorse,
Hippocampus
abdominalis
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Herpetological Review
, 2008, 39(3), 303–306.
© 2008 by Society for the Study of Amphibians and Reptiles
Monitoring Non-breeding Habitat Activity by
Subterranean Detection of Ambystomatid
Salamanders with Implanted Passive Integrated
Transponder (PIT) Tags and a Radio Frequency
Identifi cation (RFID) Antenna System
M. KEVIN HAMED
Virginia Highlands Community College
P.O. Box 828, Abingdon, Virginia 24210, USA
e-mail: khamed@vhcc.edu
and
DALE P. LEDFORD
THOMAS F. LAUGHLIN
East Tennessee State University, Department of Biological Sciences
Box 70703, Johnson City, Tennessee 37614-1710, USA
e-mail: laughlin@etsu.edu
Pond breeding amphibians require terrestrial habitat during
the non-breeding season in addition to aquatic reproductive
habitat. Ambystomatid salamanders such as spotted (
Ambystoma
maculatum
) and marbled salamanders (
Ambystoma opacum
) have
been previously studied to determine migration movements and
terrestrial habitat requirements. Several migration studies have
estimated the “life” or buffer zones required for terrestrial non-
breeding habitat. Estimates of habitat area that encompass 95%
of the population have ranged from 159 m to 370 m (Faccio 2003;
McDonough and Paton 2007; Rittenhouse and Semlitsch 2007;
Semlitsch 1998; Semlitsch and Bodie 2003). Few studies have
examined migration and non-breeding home range of
Ambystoma
species over multiple years because of the diffi culties of long
term tracking and monitoring of these animals. Recent studies
have shown that vertebrates with implanted passive integrated
transponder (PIT) tags are detectable subterraneously (Cabarle et
al. 2007; Kuhnz 2000). The purpose of this study was to examine
the utility of PIT tags and a multidirectional radio frequency
identifi cation (RFID) antenna system (FS 2001 Destron reader
and Biomark triangle antenna) for tracking and monitoring
ambystomatid salamanders during the non-breeding season.
Identifying and locating individual salamanders is important
in determining terrestrial home range and migration. Several
methods of identifi cation have been used including: photographs
of spot patterns (Stenhouse 1985), toe clips (Ott and Scott 1999),
radiotelemetry (Faccio 2003; Madison 1997; McDonough and
Paton 2007), PIT tags (Blackwell et al. 2004; Gibbons and Andrews
2004; Ott and Scott 1999), and subcutaneously implanted refl ector
tags (Moseley and Castleberry 2005). Drift fences have been
frequently used to monitor migrants to and from breeding pools
(Kleeberger and Werner 1983; Sexton et al. 1990). Concentric
circles of drift fences spaced at regular intervals allow researchers
to determine the approximate area in which salamanders establish
a home range. However, drift fences only capture salamanders
moving on or just below the surface. Radiotelemetry studies can
track animals underground, but transmitters have a short (< 5
months) battery life and can have high cost (> US $150 each).
Radioisotopes were used to determine the home range and summer
movements of
A. maculatum
(Kleeberger and Werner 1983),
A.
talpoideum
(Semlitsch 1983),
Plethodon jordani
(Madison and
Shoop 1970), and
Desmognathus fuscus
(Ashton 1975), but do
not allow for individual specifi c identifi cation.
Materials and Methods.
—
In this study we attempted to locate
and track ambystomatid salamanders using RFID antenna systems
with animals that had been marked during previous reproductive
seasons with PIT tags. The study area was a constructed vernal
pool on the Tennessee Valley Authority’s (TVA) South Holston
Weir Dam property (36.5239
°
N, 82.1100
°
W) in Sullivan County,
Tennessee, USA. The fl oodplain forest at this location supports
populations of both
A. maculatum
and
A. opacum
, with the
population of
A. opacum
being distinct and disjunct in eastern
Tennessee (Hamed et al. 2007). A two lane paved road (Holston
View Dam Road) bisects the property and salamanders living on
the north side of the vernal pool must cross the road to reach the
pool. The forested area (0.79 ha) north of the road consists mainly
of Virginia Pine (
Pinus virginiana
), Boxelder (
Acer negundo
),
and Sycamore (
Plantus occidentalis
). A mixed deciduous forest
composed mainly of Sweetgum (
Liquidambar stryacifl ua
),
Sycamore, White Oak (
Quercus alba
), and a 0.5 ha patch of non-
native bamboo (
Phyllostachys aureosulcata
)
border the south
side of the vernal pool.
Previous studies have reported variable detection distances
of PIT tagged vertebrates beneath the soil surface (12–22 cm)
in both
in situ
(Kuhnz 2000) and experimental (Cabarle et
al. 2007) conditions. The observed variability was a result of
antenna sensitivity and environmental conditions. We fi rst used
a preserved specimen of
A. maculatum
obtained from the East
Tennessee State University teaching collection to determine
the sensitivity of subterranean detection of PIT tags specifi c to
our study site in a series of ten location accuracy trials. A TX-
1411-SST PIT tag (Biomark, Idaho) was injected into the body
cavity of the preserved specimen anterior to the rear limbs. To
