Spillover of Pentastome Parasites from Invasive Burmese Pythons (Python bivittatus) to Pygmy Rattlesnakes (Sistrurus miliarius), Extending Parasite Range in Florida, USA

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Herpetological Review 50(1), 2019
AMPHIBIAN AND REPTILE DISEASES 73
Herpetological Review, 2019, 50(1), 73–76.
© 2019 by Society for the Study of Amphibians and Reptiles
Spillover of Pentastome Parasites from Invasive
Burmese Pythons (Python bivittatus) to Pygmy
Rattlesnakes (Sistrurus miliarius), Extending Parasite
Range in Florida, USA
Invasive nonindigenous species can impact native
communities through direct predation on, or competition with,
native taxa. Invasive species also may have important indirect
effects on communities, including the spread of nonindigenous
parasites to native species (parasite spillover; Dunn et al.
2012). In south Florida, USA, for example, introduced Burmese
Pythons (Python bivittatus) have had large negative impacts on
prey species (Dorcas et al. 2012), and Miller et al. (2018) found
nine native snake species infected with Raillietiella orientalis,
an Asian pentastome (a vermiform endoparasitic crustacean)
that was probably introduced into south Florida with Burmese
Pythons. In this example of parasite spillover, all native snakes
infected with nonindigenous pentastomes were found in the
southernmost 17 counties in Florida, an area in which Burmese
Pythons are well established. Miller et al. (2018) did not find
Raillietiella-infected snakes beyond the geographic range of
the Burmese Python (including northern Florida, Alabama, and
Georgia), but spillover beyond the range of the nonindigenous
host may be likely when environmental barriers are absent and
native taxa provide a viable vector for dispersal.
Adult pentastome parasites typically reside in the lungs of
reptiles or mammals where they are hematophagous and may
damage the lung parenchyma of the definitive hosts (Paré 2008).
Fishes, amphibians, and mammals often serve as intermediate
hosts for the pentastomes that infect snakes (Paré 2008). Frogs
are suspected by Kelehear et al. (2014) to be the intermediate
host for introduced R. orientalis in Australia. Several pentastome
genera (Raillietiella, Porocephalus, and Kiricephalus) occur
in snakes in North America. The native range of R. orientalis is
restricted to Asia where it commonly infects Burmese Pythons.
Here we report on three Pygmy Rattlesnakes (Sistrurus
miliarius) that were infected with R. orientalis outside of the
current distribution of P. bivittatus in Florida. We found the
infected snakes during a long-term study of a field population
of S. miliarius at Lake Woodruff National Wildlife Refuge in
Volusia County, Florida. We found all three snakes in a 10-ha
mesic hammock surrounded by freshwater marsh that supports
a dense population of Pygmy Rattlesnakes (May et al. 1996).
The refuge has a diverse herpetofauna including at least 22
snake species and at least 17 anuran species (Farrell et al. 2011).
We dissected snakes and examined their respiratory tracts. We
removed all pentastomes longer than 3 mm and determined
their sex, length, and total combined mass.
Snake 1: This infected snake was part of a study that held
snakes in field enclosures (identical to those described in Lind
et al. 2017) for several weeks. It was a nonreproductive adult
female (mass = 89.5 g, SVL = 40.8 cm) captured on 23 July 2018.
It died in the field enclosure on 22 August 2018. At that time, we
found two large pentastomes crawling out of the carcass’s mouth
and found four additional pentastomes in the trachea and
lung during dissection (Table 1, Fig. 1). The male pentastomes
were 11 mm and 15 mm long, much smaller than the females
(Table 1). We examined morphological characteristics from one
male and one female pentastome. We cleared the pentastomes
using lactophenol, and examined them under a dissecting and
compound microscope. The anterior end of the female (Fig. 2A)
contained four hooks and a central buccal cadre. We measured
hook dimensions as in Kelehear et al. (2011), and found the
posterior AB hook measured 376.6 µm and the posterior BC
hook was 438.8 µm. The buccal cadre measured 472.2 µm long.
We found the anterior AB hook and BC hook were 278.2 µm and
386.2 µm, respectively. The male copulatory spicules had a large
base and were covered with an elevated reticulum of tubes (Fig.
2B). The morphological characteristics of the male and female
specimens were consistent with R. orientalis (Ali et al. 1985).
Snake 2: The second infected S. miliarius was a male that was
initially marked with a passive integrated transponder (PIT) tag
as an adult in July 2015. We found it again on 27 July 2018 and
AMPHIBIAN AND REPTILE DISEASES
AMPHIBIAN AND REPTILE DISEASES
TERENCE M. FARRELL
Stetson University, Biology Department, DeLand, Florida 33177, USA
e-mail: tfarrell@stetson.edu
JOSEPH AGUGLIARO
Department of Biological and Allied Health Sciences,
Fairleigh Dickinson University, Madison, New Jersey 07940, USA
HEATHER D. S. WALDEN
JAMES F. X. WELLEHAN
APRIL L. CHILDRESS
University of Florida, Department of Comparative, Diagnostic and
Population Medicine, College of Veterinary Medicine,
Gainesville, Florida32608, USA
CRAIG M. LIND
Stockton University, Department of Natural Sciences & Mathematics,
Galloway, New Jersey 08201, USA

Herpetological Review 50(1), 2019
74 AMPHIBIAN AND REPTILE DISEASES
immediately released it because the jaw and throat were noticeably
swollen. We found this snake in the field on 3 October 2018 soon
after its death. Its SVL at death was 47.5 cm, 9.9 cm longer than its
SVL at its initial capture 34 months earlier. Our dissection revealed
two gravid female pentastomes in the lung (Table 1).
Snake 3: We found the third infected S. miliarius dead in the
field on 5 October 2018. We could not determine the sex, mass,
SVL, or PIT tag number of this adult snake because the posterior
third of its body was missing (probably eaten by a scavenger).
There were three gravid female pentastomes in the trachea and
lung (Table 1).
We extracted DNA from one pentastome from each of the
three snakes using a commercial kit (DNeasy tissue kit, Qiagen,
Valencia, California, USA). We completed PCR amplification of the
pentastomid 18S rRNA gene using previously described methods
(Brookins et al. 2009). We electrophoresed the PCR products in
1% agarose gels, and excised and purified the band using a QIA-
quick gel extraction kit (Qiagen). We performed direct sequencing
using a Big-Dye Terminator Kit (Applied Biosystems, Foster City,
California, USA) and analyzed these data on ABI automated DNA
sequencers. We sequenced the product in both directions. The
product was 382 base pairs after we edited out primer sequences.
We compared the sequences to those in GenBank (National Cen-
ter for Biotechnology Information, Bethesda, Maryland, USA),
EMBL (Cambridge, United Kingdom), and Data Bank of Japan
(Mishima, Shizuoka, Japan) databases using BLASTN (Altschul et
al. 1990). The sequences from each of the three pentastomes were
100% identical to Raillietiella orientalis isolate 30022_B (GenBank
accession #MG559636; Miller et al. 2018). We submitted the se-
quence to GenBank under accession #MK072952.
Our observations increase the known geographic range of
R. orientalis in Florida. We found all three pentastome-infected
snakes in Volusia County, more than 160 km north of Highlands
County, Florida, the nearest locality where Miller et al. (2018)
found R. orientalis. We have intensively studied the dense popu-
lation of S. miliarius at this site for 26 years, including dissection
of the respiratory tracts of more than 10 S. miliarius, and did not
observe evidence of pentastome infection until August 2018, in-
dicating likely recent introduction to the population.
Our observations of pentastomes in Sistrurus also increase
the definitive host list for R. orientalis in Florida. Miller et al.
(2018; supplementary data table S1) recorded R. orientalis in
Agkistrodon, Coluber, Drymarchon, Lampropeltis, Nerodia,
Pantherophis, Python, and Thamnophis. Whereas some native
Fig. 2. A) Female Raillietiella orientalis anterior end with four hooks and central buccal cadre. B) Male Raillietiella orientalis copulatory spic-
ule. Note the large base and the elevated reticulum of tubes.
Fig. 1. Photograph of an adult female Raillietiella orientalis in the
lung of Sistrurus miliarius. The anterior end of the snake is on the
lower left.

Herpetological Review 50(1), 2019
AMPHIBIAN AND REPTILE DISEASES 75
Florida snake species harbor native pentastome parasites
(Porocephalus crotali and Kiricephalus coarctatus), no
pentastomes had been found in members of the genus Sistrurus
(Carbajal-Márquez et al. 2018; Miller et al. 2018), indicating that
this genus may lack a coevolutionary history with pentastomes.
Infections of S. miliarius by R. orientalis at our study site may
result from an anuran-rich diet (unpublished data).
The pentastomes may possibly have negative health conse-
quences for these Pygmy Rattlesnakes. Although histopathologic
examination was not done, two of the dead individuals had no
obvious gross lesions observed on external examination or dur-
ing dissection other than the presence of R. orientalis in the lung
and trachea. The third dead individual, found adjacent to a foot
trail, had some wounds consistent with human-caused mortality
and/or postmortem scavenger damage. The largest female pen-
tastomes were approximately the same diameter as the trachea
of the S. miliarius in which they were found and could potential-
ly cause airway obstruction. In south Florida, Miller et al. (2016)
found that native species of snakes infected with R. orientalis
harbored both larger pentastomes and more pentastomes than
P. bivittatus, the host with which R. orientalis shares a coevolu-
tionary history. This was also true for S. miliarius in our study. In-
fected Pygmy Rattlesnakes contained a mean of 3.67 individual
R. orientalis compared to a mean of 2.3 individuals per infected
P. bivittatus from south Florida. The mean length of the nine fe-
male R. orientalis we removed from S. miliarius was 59.0 mm,
significantly longer than the mean female length of 37.7 mm re-
ported by Miller et al. (2016) for south Florida pythons (one sam-
ple t-test, t = 6.42, P = 0.0002). Several researchers have reported
(J. D. Willson; pers. comm.) major declines in Pygmy Rattlesnake
populations in the last 15 years in portions of Everglades Nation-
al Park where S. miliarius was formerly abundant (Dalrymple et
al. 1991), perhaps indicating negative impacts on S. miliarius re-
sulting from parasite spillover of R. orientalis from P. bivittatus.
The only named Raillietiella species for which gene
sequences are available in public databases are R. orientalis
and R. hebitihamata (= R. frenatus or R. frenata). Given the
size variation observed in R. orientalis, the lack of reference
sequence data for Raillietiella is concerning. Many of the
characteristics utilized in differentiation of Raillietiella are
based on size, and it has been shown that morphological
features used in pentastomid taxonomy change as the parasite
transitions through developmental stages in the definitive host
(Ali et al. 1985; Kelehear et al. 2011). As examples, morphological
identification found that 47% of Tokay Geckos (Gekko gecko)
imported into the United States were positive for R. affinis, and R.
teagueselfi was reported from introduced Mediterranean Geckos
(Hemidactylus turcicus) in Texas; both of these Rallietiella
species lack reference gene sequences (Riley et al. 1988; Reese
et al. 2004). Introduced Tokay Gecko and Mediterranean Gecko
populations are well established in Florida (Krysko and Daniels
2005). Genetic identification of Raillietiella in different host
species and resultant taxonomic revision is needed.
Observation of invasive pentastome parasites well beyond
the geographic range of the Burmese Python raises a series of
important unresolved questions for conservation biology in-
cluding: What is the current geographic range of R. orientalis in
the United States, and how rapidly is it expanding? What are the
intermediate hosts of R. orientalis in Florida, and how does this
invasive pentastome affect these hosts? What are the sublethal
effects on native snakes, and to what degree does R. orientalis
reduce snake fitness and impact snake populations?
Acknowledgments.—This research was funded by the Brown
Scholar Program at Stetson University and a Research and Profes-
sional Development Grant from Stockton University. We thank Can-
dice Stevenson at Lake Woodruff National Wildlife Refuge for logisti-
cal support. The research took place under Stetson University animal
use protocol SU-IACUC-154.
literature Cited
ali, j. h., j. riley, and j. t. selF. 1985. A review of the taxonomy and
systematics of the pentastomid genus Raillietiella Sambon, 1910
with a description of a new species. Syst. Parasitol. 7:111–123.
altsChul, s. F., w. gish, w. Miller, e. w. Meyers, and d. j. lipMan. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
brooKins, M. d., j. F. x. wellehan, j. F. roberts, K. allison, s. s. Cur-
ran, a. l. Childress, and e. C. greiner. 2009. Massive visceral pen-
tastomiasis caused by Porocephalus crotali in a dog. Vet. Pathol.
46:460–463.
CarbaJal-Mrquez, r. a., d. gonzlez-solís, and J. r. Cedeño-Vzquez.
2018. Endoparasites of Crotalus tzabcan (Serpentes: Viperidae),
with a checklist in rattlesnakes. J. Parasit. Dis. 42:303–314.
dalryMple, g. h., F. s. bernardino, t. M. steiner, and r. j. nodell. 1991.
Patterns of species diversity of snake community assemblages,
with data on two Everglades snake assemblages. Copeia 1991:517–
521.
dorCas, M. e., j. d. willson, r. n. reed, r. w. snow, M. r. roChFord, M.
a. Miller, w. e. MeshaKa, p. t. andreadis, F. j. MaZZotti, C. M. roMa-
gosa, and K. M. hart. 2012. Severe mammal declines coincide with
proliferation of invasive Burmese Pythons in Everglades National
Park. Proc. Nat. Acad. Sci. USA 109:2418–2422.
dunn, a. M., M. e. torChin, M. j. hatCher, p. M. Kotanen, d. M. bluMen-
thal, j. e. byers, C. a. Coon, v. M. FranKel, r. d. holt, r. a. huFbauer,
and a. r. KanareK. 2012. Indirect effects of parasites in invasions.
Funct. Ecol. 26:1262–1274.
Farrell, t. M., M. a. pilgriM, p. g. May, and w. b. blihovde. 2011. The
herpetofauna of Lake Woodruff National Wildlife Refuge, Florida.
Southeast. Nat. 10:647–658.
Kelehear C., d. M. spratt, s. dubey, g. p. brown, and r. shine. 2011. Using
combined morphological, allometric and molecular approaches
to identify species of the genus Raillietiella (Pentastomida). PLoS
ONE 6:e24936.
table 1. Summary of three Raillietiella orientalis infections in Sistrurus miliarius from central Florida, USA. Pentastome
mass is the combined mass of all pentastomes in a snake.
Snake ID No. Pentastomes Mean female Sex ratio Pentastome mass
length ± SD (mm) (% female) (g)
1 6 65.0 ± 4.1 66.6 1.34
2 2 47.5 ± 6.4 100 0.45
3 3 60.0 ± 9.8 100 0.53

Herpetological Review 50(1), 2019
76 AMPHIBIAN AND REPTILE DISEASES
Kelehear, C., d. M. spratt, d. o’Meally, and r. shine. 2014. Pentastomids
of wild snakes in the Australian tropics. Internat. J. Parasitol. Paras.
Wildl. 3:20–31.
KrysKo, K. l., and K. j. daniels. 2005. A key to the geckos (Sauria: Gek-
konidae) of Florida. Caribb. J. Sci. 41:28–36.
lind, C. M., F. raMis, d. j. g. paredes, i. t. Moore, b. j. vernasCo, and
t. M. Farrell. 2017. Mechanisms underlying maternal care in
a viviparous snake, Sistrurus miliarius: Does maternal body
condition, corticosterone, or arginine vasotocin mediate postō
birth maternal investment? J. Exp. Zool. Part A 327:458–465.
May, p. g., t. M. Farrell, s. t. heulett, M. a. pilgriM, l. a. bishop, d.
j. spenCe, a. M. rabatsKy, M. g. CaMpbell, a. d. ayCrigg, and w. e.
riChardson. 1996. Seasonal abundance and activity of a rattlesnake
(Sistrurus miliarius barbouri) in central Florida. Copeia 1996:389–
401.
Miller, M. a., j. M. Kinsella, r. w. snow, b. FalK, r. n. reed, s. M.
goetZ, l. garner, F. MaZZotti, C. guyer, and C. M. roMagosa. 2016.
Parasite spillover: Invasive Burmese Pythons introduce a non-
native parasite to native snakes in southern Florida. Ecological
Society of America Abstract (https://eco.confex.com/eco/2016/
webprogram/Paper61493.html).
Miller, M. a., j. M. Kinsella, r. w. snow, M. M. hayes, b. g. FalK, r.
n. reed, F. j. MaZZotti, C. guyer, and C. M. roMagosa. 2018. Parasite
spillover: Indirect effects of invasive Burmese Pythons. Ecol. Evol.
8:830–840.
Par, J. a. 2008. An overview of pentastomiasis in reptiles and other
vertebrates. J. Exot. Pet Med. 17:285–294.
reese, d. j., j. M. Kinsella, j. M. ZdZiarsKi, q. y. Zeng, and e. C. greiner.
2004. Parasites in 30 captive Tokay Geckos, Gekko gecko. J.
Herpetol. Med. Surg. 14:21–25.
riley, j., C. MCallister, and p. Freed. 1988. Raillietiella teagueselfi n. sp.
(Pentastomida: Cephalobaenida) from the Mediterranean Gecko,
Hemidactylus turcicus (Sauria: Gekkonidae), in Texas. J. Parasitol.
74:481–486.
Herpetological Review, 2019, 50(1), 76–78.
© 2019 by Society for the Study of Amphibians and Reptiles
Surveillance of Ranavirus in False Map Turtles (Graptemys
pseudogeographica) along the Lower Missouri River, USA
Ranavirosis, the disease caused by pathogenic viruses of the
genus Ranavirus, is a highly virulent systemic infection that can
affect fish, amphibians, and reptiles (Daszak et al. 1999; Duffus et
al. 2015). Ranaviruses are multi-host pathogens that have been
reported to have the ability to transfer between amphibian and
reptile species and are known to be the causative agent of mass-
mortality events in many species, including turtles (Johnson
et al. 2008; Brenes et al. 2014). Though Ranavirus has not been
extensively screened for in turtles, Ranavirus-caused mass-
mortality events have been reported in both terrestrial turtles,
such as Eastern Box Turtles (Terrapene carolina; Allender et al.
2011; Kimble et al. 2017), and aquatic turtles, including Painted
Turtles (Chrysemys picta; Goodman et al. 2013) and European
Pond Turtles (Emys orbicularis; Blahak and Uhlenbrok 2010).
Generally, Ranavirus infection in turtles is characterized externally
by the presence of cutaneous abscesses, nasal or ocular discharge,
oral plaque, or unenergetic behavior (Allender 2012). Internally,
Ranavirus infection in turtles can cause systemic fibrin thrombi
and hemorrhagic necrosis of kidneys, liver, heart, spleen, and the
alimentary tract, ultimately leading to death (Johnson et al. 2007).
Though ranaviruses have been detected across the
United States, little is known about their geographic and host
distribution in the Midwestern United States (Duffus et al. 2015).
To our knowledge, no turtles in the region have been screened
for ranaviruses, though recent efforts have detected ranaviruses
in amphibians along the Missouri River in Nebraska (Davis
and Kerby 2016) and in South Dakota (Davis 2018) as well as in
sturgeon in the Missouri River at the Gavin’s Point National Fish
Hatchery in Yankton, South Dakota (Kurobe et al. 2011). Despite
the presence of ranaviruses in the region, nothing is known
about whether they occur locally in turtle species.
In South Dakota, conservation efforts are focused on
maintaining populations of the state-threatened False Map
Turtle (Graptemys pseudogeographica) in the Missouri River. The
False Map Turtle was once considered the most abundant turtle
in the Missouri River in South Dakota (Timken 1968); however,
river modification due to the creation of large reservoirs and
hydroelectric dams has resulted in the decline and extirpation
of historical populations (DRD, unpubl. data). As a result of
these declines, it is important to ensure the health of remaining
populations of False Map Turtles. Further, given the detection of
ranaviruses in amphibians from Nebraska and South Dakota and
the ability for pathogen transmission to occur among vertebrate
classes, there is concern over the potential transmission of
ranaviruses to False Map Turtles. Here, we investigated the
prevalence and infection load of ranaviruses in False Map
Turtles from the lower Missouri River between South Dakota and
Nebraska.
False Map Turtles were collected along the 59-mile stretch
of the Missouri National Recreation River (MNRR) between
Yankton and Elk Point, South Dakota, USA from 2015–2017 (Fig.
1). Turtles were primarily collected using partially submerged
MADELINE M. BUTTERFIELD
DREW R. DAVIS*
JOSEPH D. MADISON
JACOB L. KERBY
Department of Biology, University of South Dakota, 414 East Clark Street,
Vermillion, South Dakota 57069, USA
*Corresponding author; e-mail: drew.davis@utrgv.edu
Present address: School of Earth, Environmental, and Marine Sciences,
University of Texas Rio Grande Valley, 100 Marine Lab Drive,
South Padre Island, Texas 78597, USA