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Baseline Study for the Identification of Intestinal

Parasites
in Gopher
Tortoises
Found
in Sciences,
Blazing
and
Introduction
to Honors, Department
of Biological
CharlesStar
E. Schmidt
College Pine
of
science
JogBoca Raton, Florida, 33431
Florida Atlantic University,
1

Rachel Shanker1,2, Kent Haizlett2, Jessica Huffman2, Evelyn Frazier, Ph.D.2, Joseph Caruso,
Abstract
Objectives
Discussion
2
Ph.D.
Gopher tortoise (Gopherus polyphemus) populations are
Fecal samples from 15 Blazing Star tortoises and
1. To determine the extent of parasitic
infection in gopher tortoise populations at
two different South Florida locations.

diminishing in South Florida due to fragmented habitats in


urban sites. As a keystone species, gopher tortoises have
important ecological roles. The goal of this study was to
establish a baseline of intestinal parasites in two South
Florida populations. One population resides in a fragmented
and non-fire maintained site, Blazing Star, and the other in a
fire maintained site, Pine Jog. Fire is a natural factor in
these habitats reducing canopy cover and killing parasite
eggs. The lack of fire in Blazing Star can lead to overgrowth
of vegetation, forcing tortoises to crowd where lower
vegetation is found. We hypothesize that tortoises from
Blazing Star will have a higher degree of intestinal
parasites, when compared to those from Pine Jog due
to potential crowding. Data is currently being collected,
and may provide insight to improve future management and
conservation practices.

2. To identify different species of intestinal


parasites within gopher tortoise populations
at two different South Florida locations.

Methods

Capture Tortoise &


Collect Feces

Research Sites
Blazing Star

Plastic Bag
Fresh Feces

Collection
Tube with SAF

Boca Raton,
Florida
26 acres

No prescribed fire
implemented.

Snap Freeze
in PCR Tube

Google Images

Introduction

Flotations

Cultivations

PCR

Larvae in EtOH

Pine Jog

two Pine Jog tortoises were floated for helminth


eggs; results showed three distinct nematode
species: Chapiniella spp. (Small Strongyles,
Cyathostominae), True Hookworms, and Pinworms
(Oxyuridae).
Egg numbers seen thus far suggest fairly low worm
burdens, with Chapiniella spp. most frequent.
Intestinal parasite eggs are shed in feces unevenly
and can lead to false negatives. Testing fecal
samples from the same animal more than once will
help better determine infection rates.
PCR confirms the presence of hookworm and
Chapiniella spp. in these two populations.
Cyathostomes show great diversity in this gene;
horse spp. amplicons range from 595-1750 bp. [6]
Results from this study will provide additional
information on intestinal parasite species in G.
polyphemus in SF habitats and may shed light on
future habitat management practices.

West Palm Beach,


Florida

G. polyphemus is a
threatened keystone
species, essential for
survival of 300+
commensal species [1]

Fecal
Flotations

150 acres
Prescribed fire is
implemented.

Problem

https://www.floridastateparks.org

Gopher tortoise populations


are declining in South
Florida [2]

Preliminary Results

Why?
Less food
available

Effects

Restricted to
fragmented [3], urban
sites

Increased
crowding

Effects

2) Intestinal parasite species confirmation via PCR

1)

(polymerase chain reaction)

Why?

Fire opens up the environment


by killing plants, reducing
canopy cover [4,5]

Age
ce
an
ev
el
R

R
el
ev
an
ce

Fire is more difficult to


implement.

Relevance

Allows fresh, new


grass to grow [4,5]

Mean

SD

P value

37

+/- 4.25

.001

Weight

165 lbs.

+/- 35

n/a

Height

67 in.

+/- 12

n/a
Figure 2A: Hookworm PCR product (Lane 2 [L2]; ~310 bp)
obtained after re-amplification under identical conditions.
Hookworm primers used were human hookworm NC1 and
NC2.
L1
L3

400x
Strongyle

400x
Hookworm

Chapiniella spp. (A),


True Hookworm (B),
Oxyuridae (C)

400x
Pinworm

Our study is the continuation of previous work in order to fully


establish a baseline of intestinal parasites within South Florida
gopher tortoise populations.

2000bp
100x

Strongyle

100x
Hookworm

100x
Pinworm

100x
Strongyle

Sedimentations for identification of heavier


parasites potentially missed from flotations.
Various stains (i.e. Kohns stain, Modified Acid Fast
stain) to identify other parasite species of bacterial
and protozoan origin.
Examine morphological features of preserved
larvae from cultivations to further assist in species
identification.
DNA sequencing.
Include more study sites.

References

Less parasites [4,5]

Previous comparative study of G. polyphemus intestinal parasites


in two South Florida populations (Huffman 2014)
Jonathan Dickinson State Park: 11,500 acres, fire maintained habitat
FAU Preserve: 90 acres, poorly managed; lacking prescribed fire

~310bp

300bp

Benefits

More food

L2

2000bp

Increase habitat suitability

L1

1000bp

Exposes parasite eggs to


UV radiation, killing them

Benefits

Benefits

Future Work

100x
Hookworm

100x
Pinworm

Fgure 1) A, B,C, represent Blazing Star Preserve, D,E,F represent Pine Jog

Figure 1: Preliminary Blazing star and Pine Jog flotation results reveal three distinct
nematode species within the population, based on egg morphology; Chapiniella spp.
(Small Strongyles, Cyathostominae) (A,D), True Hookworms (B,E), and Pinworms
(Oxyuridae) (C,F). Egg numbers observed thus far suggest fairly low worm burdens, with
Chapiniella spp. most frequent.

1000bp
300bp
~120bp

1. Schwartz, Tonia S., Karl, Stephen A. (2005). Population and Conservation genetics of the
gopher tortoise (Gopherus polyphemus). Conservation Genetics. 6: 917-928.
2. Mushinksy, H.R., E.D. McCoy, J.E. Berish, R.E.J. Ashton & D.S. Wilson (2006) Gopherus
polyphemus Gopher Tortoise. Biology and Conservation of Florida Turtles 3: 350-375.
3.
Ashton, R.E. and P.S. Ashton. (2008). Natural History and Management of the Gopher
Tortoise Gopherus polyphemus (Daudin). Original Edition edition. Krieger Publishing
Company, Malabar, Florida
4. Cully, J.R., J. F. 1999. Lone star tick abundance, fire, and bison grazing in tall-grass prairie.
Journal of Range Management 52: 139-144.
5. Hoch, A. L., Semtner P. J., Barker R. W. , J. A. Hair. (1972). Preliminary observations on
controlled burning for Lone Star tick (Acarina: Ixodidae) control in woodlots. Journal of
Medical Entomology 9: 446-451.
6. Huffman , J. (2014) Identification and comparison of intestinal parasites in two differing
South Florida habitats. Thesis manuscript submitted to the Biology Honors Thesis Program
7. Kaye J. N., Love S., Lichtenfels J.R., McKeand J.B. (1998). Comparative sequence analysis
of the intergenic spacer region of cyathostome species. International Journal for
Parasitology 28: 831-836

Acknowledgements
Figure 2B: Cyathostome PCR product (Lane 3 [L3]; ~120 bp);
Cyathostome primers used were CY0 and CY3.

Chelsea Bennice, Ramon Garcia, Dr. Kate Detwiler, Terrestrial Ecology Lab mates, Florida Fish
and Wild life (FFWCC #: LSSC-13-00047), Blazing Star Preserve (City of Boca Raton Permit),
Pine Jog, IACUC (A13-15, A13-14), FAU Honors Program and Honors students, FAU
Undergraduate Research Grant