The Texas horned lizard (Phrynosoma cornutum) is a Texas state-threatened species and acquisition of data related to the species\u27 ecology is essential. To accomplish this task individual animals need to be marked. Many marking techniques are available for lizards, however the majority of techniques have been tested on adults only. Studies involving hatchling and juvenile horned lizards are scarce due to problems associated with marking and relocating individuals in these age classes. I demonstrated that injection of passive integrated transponders (PIT\u27s) can safely be used as a marking method in young Texas horned lizards. Thirty-two captive bred hatchling lizards were used. Hatchlings were allowed to grow to 20 mm snout-vent length (SVL) before PIT\u27s were inserted into 16 hatchlings while the other 16 hatchlings were used as control animals. Hatchlings were measured and weighed weekly for 14 weeks and blood samples obtained weekly for 3 weeks to assess if PIT\u27s affected lizard growth and health. No differences were noted in treatment effects for SVL (F1450 = 0.85, P = 0.37), weight (F1,450 = 1.60, P = 0.22), or white blood cell and differential counts (F1,30  \u3c 1.47, P \u3e 0.23). However, an interaction between treatment and week occurred (F I4.450 = 1.79, P = 0.04) for SVL. Texas horned lizard hatchlings that were PIT-tagged were larger in SVL during weeks 3, 7 to 10, and 12 than control lizards. No ditferences in SVL were noted between treatment groups during the remaining weeks. A duration effect was noted for both SVL (F 14,450 = 7654.0, P = 0.0001) and mass (F14,450 = 1595.1, P = 0.000 I). Snout-vent length for both PIT-tagged and control lizards increased weekly until week 12 when growth rate began to slow down. Mass gain for both groups of lizards was similar, with weekly spurts occurring except between weeks 0 and 1,7 and 8, 12 and 13, and 13 and 14. Passive integrated transponders did not interfere with the growth or health of young Texas horned lizards; therefore, they can be safely used as a marking tool

Henke, Scott E.

English

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2008 
Use of Passive Integrated Transponders in Hatchling Texas 
Horned Lizards 
Scott E. Henke 
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Use of Passive Integrated Transponders 
in Hatchling Texas Horned Lizards 
SCOTT E. HENKEl 
MSC 218, Caesar Kleberg Wildlife Research Institute, 
Texas A&M University-Kingsville, Kingsville, TX 78363 
ABSTRACT -- The Texas horned lizard (Phrynosoma cornutum) is a Texas state-
threatened species and acquisition of data related to the species' ecology is 
essential. To accomplish this task individual animals need to be marked. Many 
marking techniques are available for lizards, however the majority of techniques 
have been tested on adults only. Studies involving hatchling and juvenile horned 
lizards are scarce due to problems associated with marking and relocating 
individuals in these age classes. I demonstrated that injection of passive 
integrated transponders (PIT's) can safely be used as a marking method in young 
Texas horned lizards. Thirty-two captive bred hatchling lizards were used. 
Hatchlings were allowed to grow to 20 mm snout-vent length (SVL) before PIT's 
were inserted into 16 hatchlings while the other 16 hatchlings were used as control 
animals. Hatchlings were measured and weighed weekly for 14 weeks and blood 
samples obtained weekly for 3 weeks to assess if PIT's affected lizard growth and 
health. No differences were noted in treatment effects for SVL (F
1450 
= 0.85, P = 
0.37), weight (F
1
.450 = 1.60, P = 0.22), or white blood cell and differential counts (F
t
•
30 
< 1.47, P > 0.23). However, an interaction between treatment and week occurred 
(F I4.450 = 1.79, P = 0.04) for SVL. Texas horned lizard hatchlings that were PIT-
tagged were larger in SVL during weeks 3, 7 to 10, and 12 than control lizards. No 
ditferences in SVL were noted between treatment groups during the remaining 
weeks. A duration effect was noted for both SVL (F
I4
,450 = 7654.0, P = 0.0001) and 
mass (F 14.450 = 1595.1, P = 0.000 I). Snout-vent length for both PIT-tagged and 
control lizards increased weekly until week 12 when growth rate began to slow 
down. Mass gain for both groups of lizards was similar, with weekly spurts 
occurring except between weeks 0 and 1,7 and 8, 12 and 13, and 13 and 14. Passive 
integrated transponders did not interfere with the growth or health of young Texas 
horned lizards; therefore, they can be safely used as a marking tool. 
IE-mail address: scott.henke@tamuk.edu 
16 The Prairie Naturalist 40(112): March/June 2008 
Key words: hatchling, marking device, passive integrated transponder, 
Phrynosoma cornulum, reptile, Texas horned lizard. 
The Texas horned lizard (Phrynosoma cornutum) was once widespread and 
abundant throughout Texas (Donaldson et al. 1994), however its population has 
experienced a dramatic decline (Henke 2003). Because of this, the Texas horned 
lizard is listed as a threatened species by the state of Texas (Texas Parks and 
Wildlife Code 1987) and as a Species of Concern by the United States Fish and 
Wildlife Service, federal category C2 (http://www.tpwd.state.tx.us/huntwild/wild/ 
species/thlizard/) . 
Knowledge of individual movements is essential to understand a species' 
ecology, however animals must be individually marked to obtain such informa-
tion. Marking methods for horned lizards have included hot branding (Clark 
1971), toe clipping (Ferner 1979, Henke and Montemayor 1998), body tags 
(Fisher and Muth 1989), fluorescent powder (Stark and Fox 2000), radioactive 
markers (O'Brien et al. 1965), and transponders (Camper and Dixon 1988, Henke 
and Montemayor 1998). Problefns associated with each marking method have 
been espoused (Ferner 1979). For example, hot branding can cause abnormal 
behavior and greater mortality due to infection (N ietfeld et al. 1994), toe 
clipping is not favored by many Institutional Animal Care and Use committees 
because it is a mutilation technique (S. E. Henke, Texas A&M University-
Kingsville, personal observation), body tags can get caught in vegetation and 
debris entangling the animal (N ietfeld et al. 1994), radioactive markers can harm 
or kill animals carrying them (Nietfeld et al. 1994), and fluorescent powder and 
dyes potentially can make lizards visible to predators thus increasing their 
mortality, however to my knowledge this has not been quantified for lizards. 
Passive integrated transponders (PITs) have been used safely and successfully 
in adult Texas horned lizards (Camper and Dixon 1988), but have yet to be 
evaluated for safe use in hatchling and juvenile horned lizards. 1 evaluate the 
use of PIT's in hatchling (i.e., 3 to 14 week old) Texas horned lizards. 
METHODS 
Five adult Texas horned lizards (2M:3F) were obtained from Texas Parks and 
Wildlife Department biologists, who confiscated them from individuals who 
illegally collected them in Texas. Because the collection location was unknown, 
lizards were not released. Instead the adults were allowed to be maintained in 
captivity for educational purposes via permit No. SPR0890-274, which is held by 
the Horned Lizard Conservation Society. 
Henke: Marking hatchling horned lizards 17 
Adults were held in a 227-liter aquarium equipped with a 13-cm layer of sandy 
soil, heat lamp, and refugia. They were given food (i.e., harvester ants 
(Pogonomrl'mex sp.) and crickets (Family Gryllidae)) and water ad libitum. 
From these adults, two clutches of hatchlings were born in captivity. Clutch I, 
which consisted of 14 hatchlings (7M:7F), emerged on 13 July 200 I, while Clutch 2, 
which consisted of 18 hatchlings (8M: I OF), emerged on 17 July 200 I. Upon emergence, 
hatchlings were measured for snout-vent length (SYL), weighed, and sexed. Sex 
determination was verified after they were greater than I year old. Sex determination of 
individuals at hatching was accurate. Because hatchlings at emergence measured 10 to 
11 mm SVL and weighed less than I gram, they were allowed three weeks growth to be 
at least 20 mm SVL. This growth time was needed because many hatchlings at 
emergence were smaller than the length of a PIT. 
Hatchlings were divided into two groups. Group I received a PIT and group 
2 was the control group. Group 1 consisted of seven hatchlings from clutch I 
(4M:3F) and nine hatchlings from clutch 2 (SM:4F). Group 2 consisted of seven 
hatchlings from clutch I (3M:4F) and nine hatchlings from clutch 2 (3M:6F). All 
hatchlings were marked individually by toe-clipping (Cagle 1939) and in addition, 
members of group I received a PIT (AVID Microchip ID Systems, Mandeville, 
Louisiana). Passive integrated ,transponders, which measured II x 2 mm and 
weighed O.OX g, were injected intraperitoneally in the abdominal region via a 16-
gauge needle. Hatchlings were maintained in eight 76-liter aquaria, which were set 
up as previously described. Four hatchlings, two from groups I and 2, 
respectively, were maintained per aquarium. Food and water were provided ad 
libitum. Hatchlings were monitored weekly to assess their health for three weeks 
after implanted with a PIT. About 0.2 mL of blood were collected in heparinized 
syringes via ventral tail caudal vein puncture (Powell and Knesel 1992). White 
blood cells (WBC) were counted by using Isoton II solution and lytic reagent in a 
ZI particle counter (Beckman-Coulter, Inc., Fullerton, California). Thin blood 
smears were prepared at the time of collection and stained with a Wrights-Giemsa 
stain within 24 hr. Differential WBC counts were conducted by counting 200 
leukocytes at I,OOOX magnification. Hatchlings also were weighed to the nearest 
0.1 g and measured (SYL) weekly until they entered hibernation. Individuals again 
were monitored at time of spring emergence. In addition, hatchlings were observed 
throughout the study to subjectively determine if PIT's adversely affected 
movements or caused obvious abnormal behaviors. 
I used a repeated measures design with treatment (PIT-tagged and control), sex 
(male and female), and week (0 = initial through 14) as main effects and hatchlings (N = 
32) as replications. The distribution of residual errors was tested to verify normality 
with the Shapiro-Wilk test (PROC UNIY ARIA TE procedure; SAS 1989). Homogeneity 
of variances among treatments was evaluated with Bartlett's test (Steel and Torrie 
19XO). A general linear analysis of variance (PROC GLM; SAS 1989) was used to test 
the main and interactive effects on the growth and health of the hatchlings. Because 
18 The Prairie Naturalist 40(112): MarchlJune 2008 
the effect of sex and its corresponding interactive effects were not significant (FI.II < 
1.48; P> 0.44), hatchlings were pooled irrespective of sex and re-analyzed with the main 
and interactive effects of treatment and weeks. Multiple comparisons were made by 
using Tukey's studentized range (HSD) test when a significant F-test was noted 
(Cochran and Cox 1957). Single variants of the interaction were analyzed separately 
within each grouping of the other main effects when a significant interaction was 
noted. Statistical significance was inferred at P < 0.05. 
RESULTS and DISCUSSION 
Passive integrated transponders did not affect the growth (Table 1) or 
health (Table 2) of hatchlings. No mortalities or abnormal behaviors and 
movements were noted for any individual. White blood cell and differential 
counts were similar (F I.3o <1.47, P > 0.23) between PIT-tagged and control 
lizards (Table 2). Also, differences were not observed (F
2
.
60 
< 0.78, P > 0.46) in 
week or interactive effects for blood counts. No differences were noted in 
treatment effects for SVL (F I,450 = 0.85, P = 0.37), mass (F 1450 = 1.60, P = 0.22), or 
in the interactive effect fO{ mass (F I4,450 = 1.26, P = 0.23). However, an 
interaction between treatment and week occurred (F 14.450 = 1.79, P = 0.04) for 
SVL. Hatchlings that were PIT-tagged were larger in SVL during weeks 3, 7, 8, 
9, 10, and 12 than control hatchlings (Table 1). No differences in SVL were 
noted between treatment groups during the remaining weeks. A duration effect 
was noted for both SVL (F 14.450 = 7654.0, P = 0.0001) and mass (F 14.450 = 1595.1, P 
= 0.0001). Snout-vent length for both PIT-tagged and control hatchlings 
increased weekly until week 12 when growth rate began to slow down (Table 1). 
Mass gain for both treatment groups was similar (Table 1), with weekly gains 
occurring except between weeks 0 and 1,7 and 8, 12 and 13, and 13 and 14 
(Table 1 ). Average growth rate for hatchlings was 0.518 ± 0.0 I mmld during the 
first 11 weeks; whereas average growth rate declined to 0.171 ± 0.0 I mmld 
during the 3 weeks prior to hibernation. Hibernation occurred between 16 
November and 1 December 2001 and re-emergence after hibernation occurred 
between 15 and 21 March 2002. Average hatchling growth during hibernation 
was 0.011 ± 0.001 mm/d. Mass loss after re-emergence was similar (F 1.30 = 1.3, P 
= 0.27) between hatchlings that received a PIT (5.6 ± 0.2 g; x ± SE) and the 
control hatchlings (5.1 ± 0.3 g). 
Passive integrated transponders can be safely used to individually mark 
Texas horned lizards that are at least 20 mm SVL. Transponders did not cause 
mortality, affect health, obstruct movements, or affect the growth of the hatchlings. 
Mean growth of hatchlings with and without PIT's was simi lar to that reported for 
juvenile Texas horned lizards (Henke and Montemayor 1997). In addition, PIT's did 
not appear to alter hatchling behavior such as feeding, digging, and basking. 
Table 1. Weekly growth of PIT-tagged (treatment; N = 16) and non-tagged (control; N = 16) hatchling Texas horned lizards :c 
r'> 
maintained in captivity from July 2001 to March 2002. = ~ 
r'> 
Snout vent length (mm) Mass (g) 
~ 
Control Treatment Control Treatment ... :>:-
Weeks x SE x SE x SE x SE ~. 
0 21.3 Aa1.2 0.2 21A Aa 0.2 1.6 Aa 0.1 1.8 Aa 0.1 ;::-::::. 
25.2 Ab 2A Aa 0.2 2A Aa 0.1 
.... 
25A Ab 0.2 0.2 '"" ;::-
2 29A Ae 0.3 29.2 Ae 0.3 4.9Ab 0.2 4A Ab 0.2 -~. 
3 33.1 Ad OA 33.8 Bd 0.2 7A Ac 0.2 7A Ae 0.2 ;::-
c 
4 36.6 Ae OA 37.5 Ae 0.2 • 10.6 Ad 0.2 lOA Ad 0.2 ... ;::: 
~ 
5 40.2 Af OA 40.9 Af 0.3 13.8 Ae 0.3 13.7 Ae 0.2 ::::.. -6 43.8 Ag OA 44.6 Ag 0.3 17A Af 0.3 17.0Af 0.3 ;:;. ::::. ... 
7 47.2 Ah OA 48A Bh 0.2 20.8 Ag 0.3 21.5 Ag OA a-
8 50.7 Ai OA 51.8 Bi 0.2 21.9 Ag 0.3 22.8 Ag OA 
9 54.0Aj 0.2 55.2 Bj 0.2 23.9 Ah 0.3 24.5 Ah OA 
10 57.8 Ak 0.2 58A Bk 0.2 26.1 Ai 0.3 25.8 Ai 0.3 
11 61.2 Al 0.3 61.3 Al 0.2 27.9 Aj OA 27.8 Aj 0.3 
12 63.1 Am 0.3 63.7 Bm 0.2 29.5 Ak 0.5 30.1 Ak 0.3 
13 64.0 Amn 0.3 64.5 Amn 0.2 30.2 Akl 0.5 31.4 Akl 0.3 
14 64.6 An 0.3 65.1 An 0.2 30A Al 0.7 32.2 Al 0.3 
REl 66.1 Ao OA 66.2 Ao 0.1 25.3 A4 0.7 26.6A 0.5 
'Means with the same upper case letter are not different (P > 0.05) between treatments. 
'Means with the same lower case letter are not different (P > 0.05) between weeks within the same treatment. 
'RI = Re-emergence after hibernation occurred between 15 to 21 March 2002. 
"Mass loss after re-emergence was not analyzed within weekly growth. -\0 
Table 2. Weekly white blood cell (WSC) and differential counts of PIT-tagged (treatment) and non-tagged (control) hatchling 
Texas horned lizards maintained in captivity from July 200 I to March 2002. 
Control (N= 16) Treatment (N=16) 
Week 1 Week 2 Week 3 Week I Week 2 Week 3 
Blood x SE x SE x SE x SE x SE x SE 
WBC (xI0 9/1) 27.5 10.3 29.3 9.7 29.9 6.7 33.4 11.7 29.4 6.8 31.9 2.8 
Lymphocyte (%) 25.3 7.8 28.2 8.3 24.4 6.1 26.2 5.7 28.5 3.3 29.3 1.6 
Heterophils (%) 62.2 12.2 58.1 11.7 54.4 5.8 64.5 4.7 61.3 2.8 58.1 5.4 
Eosinophils (%) 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 1.7 0.2 1.0 0.3 
Basophils ('Yo) 1.0 0.3 0.5 0.1 1.8 0.2 0.0 0.0 0.5 0.1 0.5 0.1 
Monocytes (%) 11.5 1.2 13.2 1.7 19.4 2.5 9.3 2.0 8.0 0.3 11.1 2.1 
N 
<:> 
~ 
~ 
~ ;::, ::;. 
;;,. 
~ 
E' 
;:: -a" 
.j;o. 
<:> ,.-. -N 
'-' 
~ 
~ 
""I 
n 
=-~ = = ~ 
N 
<:> 
<:> 
00 
Henke: Marking hatchling horned lizards 21 
Passive integrated transponders during my study did not fail. Camper and Dixon 
(1988) reported a I % failure of PIT's implanted in reptiles and amphibians. My 
study can be used to satisfy animal welfare organizations (i.e., Institutional Animal 
Care and Use Committees) as to the safety of PIT's for use in hatchling and 
juvenile lizards. 
Long-term population and ecological studies are needed in field herpetology, 
however herpetologists might be reluctant to use permanent marking techniques 
until such techniques are shown to be successful. I illustrated that a permanent 
marking technique can be used for rare and conservation-sensitive species without 
fear of harming individuals in a population. Permanently marking Texas horned 
lizards has allowed us to gather information such as dispersal from natal areas, 
movements, longevity, and potential causes of mortality. By conducting repeated 
searches during several years, we have been able to map Texas horned lizard 
locations through time, calculate survival rates, and growth rates (Henke and 
Montemayor 1997). Also, I have surmised Texas horned lizard mortality from PIT's 
found inside domestic cat scats, owl pellets, and raptor casts. Such information 
could be collected via radio telemetry, however, telemetry studies can be expensive 
and transmitters would require replacement for long-term studies. If budget is a 
concern and researchers are i\vailable, conducting searches for permanently 
marked Texas horned lizards can yield valuable data (Fair and Henke 1997, Henke 
and Montemayor 1998). 
ACKNOWLEDGMENTS 
I thank the Horned Lizard Conservation Society and Texas Parks and Wildlife 
Department for allowing me to raise Texas horned lizards in captivity. I thank A. M. 
Fedynich, B. Ballard, D. Fogell, and two anonymous reviewers for reviewing an 
earlier draft of this manuscript. Funding was provided by the Caesar Kleberg 
Wildlife Research Institute. This paper is publication number 03-123 of the Caesar 
Kleberg Wildlife Research Institute. 
LITERATURE CITED 
Cagle, F. R. 1939. A system of marking turtles for future identification. Copeia 
1939: 170-173. 
Camper, J. D., and J. R. Dixon. 1988. Evaluation of a microchip marking system for 
amphibians and reptiles. Texas Parks Wildlife Department Research Publica-
tion 71 00-159. Austin, Texas. 
Clark, D. R., Jr. 1971. Branding as a marking technique for amphibians and reptiles. 
Copeia 1971:148-151. 
22 The Prairie Naturalist 40(1/2): March/June 2008 
Cochran, W. G., and G. M. Cox. 1957. Experimental designs, Second edition. John 
Wiley & Sons, New York, New York. 
Donaldson, W., A. H. Price, and J. Morse. 1994. The current status and future 
prospects of the Texas horned lizard (Phrynosoma cornutum) in Texas. Texas 
Journal of Science 46:97-1 13. 
Fair, W. S., and S. E. Henke. 1997. Efficacy of capture methods for a low density 
population of Phrynosoma cornutum. Herpetological Review 28: 135-137. 
Ferner, J. W. 1979. A review of marking techniques for amphibians and reptiles. 
Society for Studies of Amphibians and Reptiles, Herpetological Circulation 9. 
Fisher, M., and A. Muth. 1989. A technique for permanently marking lizards. 
Herpetological Review 20:45-46. 
Henke, S. E. 2003. Baseline survey of Texas horned lizards. Phrynosoma 
cornutum, in Texas. Southwestern Naturalist 48:278-282. 
Henke, S. E., and M. Montemayor. 1997. Phrynosoma cornutum (Texas horned 
lizard) growth. Herpetological Review 28: 152. 
Henke, S. E., and M. Montemayor. 1998. Diel and monthly variations in capture 
success of Phrynosoma cornutum via road cruising in southern Texas. 
Herpetological Review 29: 148-150. 
Nietfeld, M. T., M. W. Barre1ot:, and N. Silvy. 1994. Wildlife marking techniques. 
Pp. 140-168 in Research and management techniques for wildlife and habitats 
(T. A. Bookhout, editor). The Wildlife Society, Bethesda, Maryland. 
O'Brien, G. P., H. K. Smith, and J. R. Meyers. 1965. An activity study of a 
radioisotope-tagged lizard, Sceloporus undulata hyacinthimus (Sauria, 
Iguanidae). Southwestern Naturalist 10: 179-187. 
Powell, S. c., and J. A. Knesel. 1992. Blood collection from Macroclemys 
temmincki (Troost). Herpetological Review 23: 19. 
SAS Institute, Inc. 1989. SAS/ST AT User's Guide, version 6. Fourth edition. 
Volume 2. SAS Institute Inc., Cary, North Carolina. 
Stark, R. c., and S. F. Fox. 2000. Use oft1uorescent powder to track horned lizards. 
Herpetological Review 31 :230-231. 
Steel, R. G. D., and J. H. Torrie. 1980. Principles and procedures of statistics. 
Second edition. McGraw-Hill, New York, New York. 
Texas Parks and Wildlife Code. 1987. 31 T. A. C. Section 65.171-65.177. 
Received: 18 August 2007 Accepted: 4 Januarv 2009 
Associate Editor for Herpetology: Dan F ogell 


Use of Passive Integrated Transponders in Hatchling Texas Horned Lizards

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Use of Passive Integrated Transponders in Hatchling Texas Horned Lizards

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