Hemoparasites in Oregon Spotted Frogs (Rana pretiosa) from Central Oregon, USA
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Journal of Wildlife Diseases, 44(2), 2008, pp. 464–468
# Wildlife Disease Association 2008
Hemoparasites in Oregon Spotted Frogs (Rana pretiosa) from Central
Oregon, USA
Patricia L. Stenberg1 and William J. Bowerman1,2 1 Sunriver Nature Center & Observatory, Box 3533,
Sunriver, Oregon 97707, USA; 2 Corresponding author (email: frogs@sunrivernaturecenter.org)
ABSTRACT: Between 2001 and 2003, we Haemogregarina aurora, was reported
screened blood smears of 156 Oregon spotted from Rana aurora aurora (Lehmann,
frogs (Rana pretiosa) from three populations in 1960). Less information is available for
central Oregon for blood parasites. A Lankes-
terella sp. and a Trypanosoma sp. were the R. pretiosa complex in the Pacific
detected in 31% and 35% of the frogs, Northwest. Lehmann (1959a) did not
respectively. Parasite loads were generally light, detect blood parasites in R. pretiosa from
with Lankesterella sporozoites in 1–2% of the Willamette Valley. Clark et al. (1969)
erythrocytes, and extracellular trypanosomes reported a Lankesterella sp. and a Try-
were seen at rates of about one parasite per 200
fields of view at 10003. Little work has been panosoma sp. in R. pretiosa.
published on hemoparasites of ranids in the The spotted frog complex was recently
western USA in the past 30 yr. Because of the divided into R. pretiosa sensu stricto and
recent taxonomic division of the Rana pretiosa R. luteiventris (Green et al., 1997). Al-
complex, this may be the first published report though Clark et al. (1969) did not identify
of blood parasites for R. pretiosa sensu stricto.
Both parasites reported here differed in specific collection sites, much of that
morphologic features and morphometric com- survey was done in Idaho, eastern Wa-
parisons from previous descriptions of anuran shington, and eastern Oregon, and the
hemoparasites. Much work remains to sort out reported results probably relate to R.
the taxonomy of hemoparasites among western
luteiventris rather than to R. pretiosa.
USA ranids and to determine the ecological
significance of these parasites; both tasks are Both R. pretiosa and R. luteiventris are
important steps in understanding and managing candidate species for federal listing, and
these, and related, sensitive and threatened R. pretiosa is considered a ‘‘sensitive and
species. critical’’ species throughout its range in
Key words: Hemoparasites, Lankesterella,
Oregon spotted frog, Rana pretiosa, Trypano-
Oregon (Oregon Natural Heritage Pro-
soma. gram [ONHP], 2001); many populations
currently are small and isolated. Under-
Amphibians host a variety of blood standing the role of parasites in the
parasites, but there are relatively few ecology of threatened or endangered
published surveys of the hemoparasites species is important for the long-term
of anurans of the northwestern USA management of these species. Threatened
(Lehmann, 1959a; Clark et al., 1969). and endangered species and small in-bred
Taxonomic revisions of western ranids populations may exhibit reduced parasite
(Green, 1986; Green et al., 1997), and species richness (Altizer et al., 2007) or
the apparent host specificity of anuran reduced disease resistance, resulting in
hemoparasites (Martin and Desser, 1991), higher parasite burdens (Whiteman et al.,
suggest a need for fresh investigations of 2006). This study of blood parasites of R.
hemoparasites in this group of frogs. pretiosa contributes to the overall under-
Lehmann (1959b, c, d) reported Kar- standing of the ecology of this species.
yolysus sonomae, Haemogregarina boyli, From December 2001 to December
and Trypanosoma boyli from Rana boylii. 2003, we opportunistically made blood
A probable new trypanosome species, smears from adult Oregon spotted frogs
464SHORT COMMUNICATIONS 465
FIGURE 2. Trypomastigote of Trypanosoma sp.
FIGURE 1. Intracellular Lankesterella sp. from from Rana pretiosa. Single white arrow 5 nucleus;
Rana pretiosa within frog erythrocyte. Arrow 5 single black arrow 5 kinetoplast; paired black arrows
sporozoite in frog erythrocyte. Bar510 m. 5 undulating membrane; paired white arrows 5 free
flagellum. Bar510 m.
(R. pretiosa) during mark-and-recapture measured the width of our Lankesterella
projects at three sites in Deschutes at the widest point, while width of our
County, Oregon (Sunriver, NAD 27, Trypanosoma was measured at the nucle-
UTM Zone 10, 624632 E, 4860501 N; us. We determined the level of Lankester-
Crosswater, 624358 E, 4856498 N; and ella infection by counting the number of
Dilman Meadow, 607464 E, 4839143 N). infected erythrocytes in 100 erythrocytes
Animals were toe-clipped and blood was from successive fields of view. Ten total
applied directly to a glass microscope replicates were counted and averaged.
slide; a thin film was made using the We found a Lankesterella sp. (Lankes-
trailing edge of a second glass slide. All terellidae) (Fig. 1), a hemococcidium, in 48
frogs appeared normal and healthy and of 156 (31%) smears and a Trypanosoma
were released at the site of capture. sp. (Trypanosomatidae) (Fig. 2) in 54 of
Slides were air dried, fixed in absolute 156 (35%) smears (Table 1). Fourteen
methanol for 1 min, then air dried and smears (9%) contained both parasites.
stored at room temperature. Slides were Stages of Lankesterella sp. were gener-
immersed in freshly prepared Giemsa ally found as intraerythrocytic sporozoites,
stain (LabChem Inc., Pittsburg, Pennsyl- although 14 smears also exhibited extra-
vania, USA), diluted 1:30 with well water cellular sporozoites. Intracellular sporozo-
(pH 7) for 50 min at room temperature, ites appeared lightly stained within para-
rinsed under gently running well water, sitophorous vacuoles in erythrocytes, but
and air dried prior to examination. We did not displace the erythrocyte nucleus.
initially examined slides at 1003 (total The vacuole was ‘‘banana-shaped’’ with
magnification) and made final examination one end usually broader. The parasite
with oil immersion at 10003. For mea- within the vacuole generally appeared
surement, we selected only parasites with elongated and slightly flexed, with a light
intact cellular membranes. All measure- reddish-staining, centrally located nucle-
ments were made with an ocular micro- us. There was often a bulge on the
meter (Wards 24 V 0230, Kyowa, Kana- concave side of the vacuole opposite the
gawa, Japan) and were calibrated using a nucleus. Extracellular sporozoites were
stage micrometer (Wards 24 V 0250, elongate in form, stained lightly, and often
Kyowa). Length measurements were per- showed greater flexion than the intracel-
formed as described by Diamond (1965), lular form. Parasites were generally found
without the use of a camera lucida. We in fewer than 1–2% of the erythrocytes of466 JOURNAL OF WILDLIFE DISEASES, VOL. 44, NO. 2, APRIL 2008
TABLE 1. Prevalence of hemoparasites in three populations of Rana pretiosa in Oregon.
Site n Lankesterella sp. Trypanosoma sp. Both
Sunriver 96 43 (45%) 25 (26%) 12 (13%)
Crosswater 51 5 (10%) 26 (51%) 2 (4%)
Dilman 9 0 3 (33%) 0
Total 156 48 (31%) 54 (35%) 14 (9%)
infected frogs. The most heavily infected in about 43% of the parasites. Approxi-
frog had parasites in approximately 10% of mately 25% of the trypanosomes exhibited
the erythrocytes. We found no sporozoites the ‘‘cornucopia’’ form reported for T.
within leukocytes. ranarum (Diamond, 1965). Trypanosome
The mean length of 245 intracellular infections were light: At 10003 magnifi-
sporozoites was 15.2 m (range 11–21, SD cation, numbers of parasites seen ranged
1.5) and the mean width was 3.4 m (range from a low of one or two total parasites,
1.5–5, SD 0.6) (Table 2). found in 200 fields of view, to a high of
The Trypanosoma sp., circulating in the approximately 10 per 100 fields of view.
blood plasma, were primarily seen in Our finding of both Lankesterella and
flexed positions with a well-defined undu- Trypanosoma species in Oregon spotted
lating membrane and free flagellum of frogs is similar to the results of Clark et al.
varying lengths and were morphologically (1969). However, Clark et al. probably
variable (Table 3). Most trypanosomes sampled R. luteveintris, the Columbia
had a large oval nucleus (mean dimensions spotted frog. The Lankesterella sp. and
6.935.2 m) and a large central purple- Trypanosoma sp. described in this study
staining endosome. Generally, the long differed substantially from other pub-
axis of the nucleus oriented parallel to the lished descriptions of North American
longitudinal axis of the trypanosome. The hemoparasites. We report a mean length
nucleus was located just anterior to the for our Lankesterella sp. of 15.2 m, com-
middle of the body. The kinetoplast was pared to the 12.7 m for L. minima (Barta
small (,1 m in diameter), circular, red- and Desser, 1984) and the 10.1 m reported
dish-staining, and situated posterior to the by Clark et al. (1969). Indeed, the mean
nucleus by about 6.5 m, with a clear space length we report exceeds the maximum
extending anteriorly from the kinetoplast. length reported by Barta and Desser
Buffered formalin was not used to fix the (1984); Clark et al. (1969) did not provide
smears; this may have contributed to range or SD for their measurements.
length variability of the flagella (Lehmann, The Trypanosoma sp. described in this
1964). Myonemes appeared prominently study also differed from T. boyli (Leh-
TABLE 2. Mean dimensions of intracellular sporozoites of Lankesterella from selected North American
ranid frogs.
Species n Length (6SD) Range Width (6SD) Range
a
Lankesterella sp. 245 15.2 (61.5) 11–21 3.4 (60.6) 1.5–5
Lankesterella minimab 10 12.7 (–)d 11.5–14.8 2.1 (–) 1.6–3.2
Lankesterella sp.c (–) 10.1 (–) (–) 2.2 (–) (–)
a
Obtained from R. pretiosa (this study).
b
Obtained from R. septentrionalis, R. clamitans, and R. catesbeiana (Barta and Desser, 1984).
c
Obtained from R. cascadae and R. luteiventris? (Clark et al., 1969).
d
(–) 5 not available.SHORT COMMUNICATIONS 467
TABLE 3. Morphologic and morphometric comparison among Trypanosoma from selected North American
ranid frogs.
Size (mm) or ratio
a b c
Parasite structure Trypanosoma sp. T. boyli T. ranarumd T. ranarum Ie T. ranarum IIe
PA 55.5 48.2 49.8 60.6 57.9
FF 8.1 0 14.3 13.5 13.2
PK 26.1 21.6 15.6 37.7 23.4
PN 32.5 28.2 21.8 41.8 28.6
NA 22.9 20.0 27.9 18.8 29.3
BW 13.4 10.3 18.2 4.7 8.2
KN 6.5 6.6 6.2 4.1 5.2
NL 6.9 4.7 n/a 3.0 3.6
NW 5.2 4.7 n/a 2.5 2.9
PK/PA 0.46 0.44 0.31 0.62 0.40
PN/PA 0.59 0.59 0.43 0.70 0.49
KN/PN 0.20 0.23 0.28 0.10 0.18
PK/PN 0.80 0.77 0.71 0.90 0.82
BW/PA 0.24 0.21 0.36 0.80 0.14
Kinetoplast Circular Rod-shaped Square or Rectangular Large square
rectangular or elliptical
a
Abbreviations: FF 5 length of the free flagellum; PA 5 body length excluding the free flagellum; PK 5 distance from
posterior end to the kinetoplast; PN 5 distance from the posterior end to the center of the nucleus; NA 5 distance from
the center of the nucleus to the anterior end; BW 5 width of body at the level of the nucleus excluding the undulating
membrane; KN 5 distance from the kinetoplast to the center of the nucleus; NL 5 length of the nucleus; NW 5 width
of the nucleus.
b
From R. pretiosa (this study).
c
From R. boylii (Lehmann, 1959).
d
From R. catesbeiana, R. clamitans, and R. pipiens (Woo, 1969).
e
From R. clamitans, R. esculenta, R. mugiens and R. pipiens (Diamond, 1965).
mann, 1959d), which lacked a free flagel- investigation of anuran blood parasites in
lum but was similar in general size and the northwestern USA; such studies have
shape to T. ranarum as described by received little attention over the past
Diamond (1965) and Woo (1969). How- 30 yr. Description of other life stages of
ever, the kinetoplast was small and circu- these parasites, identification of vectors
lar and thus distinct from other published (Desser et al., 1990), and molecular
descriptions in which the kinetoplasts comparisons with previously described
were square, rectangular, or rod-shaped species are needed to provide accurate
(Table 3). species designations (Desser, 2001). De-
Frogs carrying either or both infections termining ecological roles of these hemo-
appeared vigorous and healthy. We ob- parasites and their vectors will contribute
served no external physical or behavioral to a better understanding of the basic
abnormalities associated with infected biology of the Oregon spotted frog, a
frogs. Considering the current status of federal candidate for listing as an endan-
this frog species, additional investigation gered species and currently state listed
of the impacts of infection are warranted, as a critical species by the Oregon
including analysis of weight:length ratios Department of Fish and Wildlife (ONHP,
and the long-term survival of infected and 2001).
uninfected individuals. Voucher photographs of both parasites
These findings, including careful mea- have been filed with the U.S. National
surements and organelle descriptions, may Parasite Collection, as described by Ban-
provide an important step in renewing the doni (Bandoni and Duszynski, 1988):468 JOURNAL OF WILDLIFE DISEASES, VOL. 44, NO. 2, APRIL 2008
USNPC #100099.00 (Lankesterella sp.); aurora and Rana boylii: Karyological evidence.
Systematic Zoology 35: 273–282.
USNPC # 100100.00 (Trypanosoma sp.).
———, H. KAISER, T. F. SHARBEL, J. KEARSLEY, AND
We thank S. R. Telford, Jr. for assis- K. MCALLISTER. 1997. Cryptic species of spotted
tance with parasite identification and frogs, Rana pretiosa complex, in western North
manuscript review; M. Siddal for parasite America. Copeia 1997: 1–8.
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———. 1959c. The description of Haemogregarina
conducted under a scientific take permit boyli n. sp. from the yellow-legged frog, Rana b.
issued by Oregon Department of Fish and boyli. Journal of Parasitology 45: 198–203.
Wildlife. ———. 1959d. Trypanosoma boyli n. sp. from the
California yellow-legged frog, Rana b. boyli.
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