Protist, Vol. 166, 599–608, December 2015

http://www.elsevier.de/protis

Published online date 19 October 2015

ORIGINAL PAPER

Phylogeny and Morphological Variability

of Trypanosomes from African

Pelomedusid Turtles with Redescription

of mocambicum Pienaar, 1962

a,b,1 c b,d e

Nela Dvorákovᡠ, Ivan Cepiˇ ckaˇ , Moneeb A. Qablan , Wendy Gibson ,

f a,b

Radim Blazekˇ , and Pavel Sirokˇ y´

a

Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology,

University of Veterinary and Pharmaceutical Sciences Brno, Palackého tr.ˇ 1/3,

612 42 Brno, Czech Republic

b

CEITEC-Central European Institute of Technology, University of Veterinary and

Pharmaceutical Sciences Brno, Palackého tr.ˇ 1/3, 612 42 Brno, Czech Republic

c

Department of Zoology, Faculty of Science, Charles University in Prague, Vinicnᡠ7,

120 44 Prague 2, Czech Republic

d

Department of Pathology and Parasitology, Faculty of Veterinary Medicine, University of

Veterinary and Pharmaceutical Sciences, Palackého tr.ˇ 1/3, 612 42 Brno, Czech Republic

e

School of Biological Sciences, University of Bristol, Bristol BS8 1TQ, United Kingdom

f

Institute of Biology, Academy of Sciences of the Czech Republic, v. v. i.,

Kvetnᡠ8, 603 65 Brno, Czech Republic

Submitted May 13, 2015; Accepted October 3, 2015

Monitoring Editor: Dmitri Maslov

Little is known about host specificity, genetic diversity and phylogenetic relationships of African

turtle trypanosomes. Using PCR targeting the SSU rRNA gene, we detected trypanosomes in 24 of

134 (17.9%) wild caught African pelomedusid turtles: Pelusios upembae (n = 14), P. bechuanicus (n = 1),

P. rhodesianus (n = 3) and P. subniger (n = 6). Mixed infection of Trypanosoma species was confirmed

by PCR in three specimens of P. upembae, and in one specimen each of P. bechuanicus, P. rhode-

sianus, and P. subniger. Microscopic examination of stained blood smears revealed two distinct forms

(broad and slender) of trypomastigotes. The broad form coincided in morphology with T. mocambicum

Pienaar, 1962. Accordingly, we have designated this form as the neotype of T. mocambicum. In phylo-

genetic analysis of the SSU rRNA gene, all the new turtle trypanosome sequences grouped in a single

clade within the strongly supported “aquatic” clade of Trypanosoma species. The turtle trypanosome

clade was further subdivided into two subclades, which did not correlate with host turtle species or

1

Corresponding author; fax +420 541 562 631

e-mail [email protected] (N. Dvoráková).ˇ

http://dx.doi.org/10.1016/j.protis.2015.10.002

1434-4610/© 2015 Elsevier GmbH. All rights reserved.

600 N. Dvorákovᡠet al.

trypanosome morphology. This study provides the first sequence data of Trypanosoma species isolated

from freshwater turtles from tropical Africa and extends knowledge on diversity of trypanosomes in

the Afrotropical zoogeographical realm.

© 2015 Elsevier GmbH. All rights reserved.

Key words: Trypanosoma; turtle; Pelusios; polymorphism; phylogeny; SSU rRNA gene.

Introduction not been reported since their original descrip-

tions were published. Paperna (1989) found a

Genus Trypanosoma Gruby, 1843 (: trypanosome in the peripheral blood of Pelusios

Kinetoplastea) infects all classes of vertebrate sinuatus and in the foregut of the leech Pla-

hosts, but most attention is directed to species that cobdella multistrigata, but did not provide full

cause serious forms of human and animal diseases morphological data. The original descriptions of

and heavy economic losses (Hoare 1972). Tr y- turtle trypanosomes often include host species,

panosomes are transmitted mostly by bloodsucking locality, prevalence, and drawings of bloodstream

and leeches (Hamilton et al. 2005). forms with inadequate morphometric data. With-

Morphological diagnosis and identification of try- out using molecular genetic methods, estimation of

panosomes based on microscopic examination of prevalence could be misleading because of the fre-

stained blood films has many pitfalls due to overlap quently low parasitaemia (Gibson 2003). To date,

of characteristics, species polymorphism and false sequence data of turtle trypanosomes are available

negative results associated with low parasitaemia only for T. chelodinae Johnston, 1907 from Australia

(Austen et al. 2009; Gu et al. 2007; Thompson (Jakes et al. 2001).

et al. 2013). Diagnostic sensitivity increases with African side-neck turtles of the family Pelome-

the use of specific PCR-based methods, which can dusidae Cope, 1868 include more than 20 currently

also help to classify a parasite into species using recognized species of two genera – Pelomedusa

genetic barcodes, and also distinguish mixed infec- Wagler, 1830 and Pelusios Wagler, 1830 (for recent

tions, depending on the marker used. Small subunit of the family Pelomedusidae used in this

(SSU) rRNA and glycosomal glyceraldehyde 3- paper see Fritz and Havasˇ 2007, 2013; Petzold

phosphate dehydrogenase (gGAPDH) genes are et al. 2014). Members of both genera are freshwa-

currently the most widely used markers to iden- ter turtles living in a variety of water habitats from

tify Trypanosoma species and to characterize their rainforest to open savannah of the Afrotropic zoo-

genetic variability and phylogeny (Ferreira et al. geographic realm (Fritz et al. 2011; Fritz and Havasˇ

2008; McInnes et al. 2010; Paparini et al. 2014), 2007).

ensuring that many sequences are available for In this paper, we examined samples from

comparison from public databases. twelve African side-neck turtle species originat-

Based on morphology, 14 Trypanosoma species ing from sub-Saharan Africa for the presence of

from 19 chelonian species have been described, Trypanosoma species. We evaluated their host

although some of them may be synonymous specificity, genetic diversity and phylogenetic rela-

(Telford 2009). The first trypanosome reported in tionships with other Trypanosoma species on the

turtles was T. damoniae Laveran et Mesnil, 1902 basis of SSU rRNA gene sequences. Additionally,

in the Chinese pond turtle (Mauremys reevesii). morphological characteristics were compared with

However, a complete life cycle is known only for published data of known species from the Afrotropic

T. balithaensis Ray, 1987, T. chrysemydis Roud- zoogeographic region.

abush et Coatney, 1937 and T. vittata Robertson,

1908; aquatic leeches of genera Glossiphonia,

Helobdella and Placobdella were confirmed as their Results

vectors (Jefferson 1965; Ray 1987; Siddall and

Desser 1992). Five Trypanosoma species parasitiz-

Morphology of Endogenous Stages

ing chelonians were reported in the Afrotropical

realm - T. pontyi Bouet, 1909, T. leroyi Commes, A total of 49 hemoflagellates were detected by

1919, T. neitzi Travassos Santos Dias, 1952, T. microscopic examination in 11 out of 94 (11.7%)

sheppardi Travassos Santos Dias, 1952 and T. peripheral blood films. Nine adult specimens of

mocambicum Pienaar, 1962. These species have Pelusios upembae, one of P. bechuanicus and one

Neotype of Trypanosoma mocambicum 601

Figure 1. Detected broad (a, b, e-h) and slender (c, d) forms of trypomastigotes isolated from African side-neck

turtles Pelusios upembae isolates 2575 (e, f), 3182 (b), 6212 (h), 6213 (g) and 6214 (a, c, d). Trypomastigote

stages of Trypanosoma mocambicum (e–h). Nucleus (N), kinetoplast (arrowhead) and free flagellum (arrow);

stained with Giemsa. All figures are at the same scale; scale bar = 10 m.

of P. subniger were infected with trypanosomes. dense granular cytoplasm (Fig. 1e). The kineto-

Typical morphology of observed parasites in plast was weakly stained and located closer to the

Giemsa-stained specimens corresponded to the nucleus than to the posterior end. The cytoplasm

description of trypomastigote stages by Hoare at both extremities stained pale blue and consisted

(1972) and none of the observed trypanosomes of more distinct granules. Cell dimensions of both

was dividing. The observed trypanosomes were forms are shown in Supplementary Material Table

long and narrow, with a well-developed undulat- S1.

ing membrane and distinct axoneme. The free Both broad and slender forms were observed

part of the flagellum, situated at the anterior end in blood smears of two individuals of P. upembae

of the body, was not seen very clearly in most (samples no. 3182 and 6214). Only the broad form

cases. Blood trypomastigotes were of two forms: was observed in 6 specimens of P. upembae (2575,

larger broad form (n = 39) and smaller slender form 3184, 3188, 3190, 6212, and 6213) and one individ-

(n = 10). The more frequent broad form was curved ual of P. bechuanicus (5204). Only the slender form

into the C-shape (Fig. 1a), S-shape (Fig. 1b), was detected in a single individual of P. subniger

conch-shape or that with crossed extremities. The (5199) and in one P. upembae (2572). Overview

broad form possessed an oval or rounded nucleus, of detected forms in individual samples is in the

which was located towards the posterior from the Supplementary Material Table S2.

centre of the trypomatigotes. The small, lateral

kinetoplast was very close to the posterior end of

Molecular Characteristics and Phylogeny

the cell, often less visible because of the intensely

staining, dark purple cytoplasm with Giemsa stain- Using PCR, the overall detection rate of try-

ing. The cytoplasm was filled with coarse material panosome SSU rRNA in the 12 turtle species

that was less compact at both extremities. The was 17.9% (24/134) (Supplementary Material Table

smaller and slender form was either C- or S-shaped S2). All microscopically positive specimens were

and was less conspicuous in smears (Fig. 1c, d). also positive by PCR. Mixed infection of Tr y-

The undulating membrane exhibited 6 waves. The panosoma species was confirmed by PCR in three

pale purple nucleus was situated in the middle individuals of P. upembae (sample nos. 2572, 3182

of the posterior half of the cell and also visu- and 6214), one P. bechuanicus (5204), one P.

ally divided the speckled posterior extremity of rhodesianus (3159) and one P. subniger (5199).

602 N. Dvorákovᡠet al.

Figure 2. Phylogenetic tree of African turtle trypanosomes inferred by maximum likelihood of partial SSU

rRNA sequences from 25 trypanosome isolates. Numbers at the nodes show posterior probabilities under

BI/ bootstrap values for ML higher than 0.50 or 50%, respectively. Posterior probabilities and bootstrap that

supports lower than 0.50 or 50% are marked with asterisk (*). Taxa for which new sequences were obtained in

this study are printed in bold. Isolates with PCR confirmed mixed infection are marked with cross (†).

Neotype of Trypanosoma mocambicum 603

A total of 25 new SSU rRNA sequences were However, its original description was based only

obtained, including two different sequences from a on the light microscopy examination of a stained

single specimen of P. subniger (5199A and B). All slide without any notes on type material (Pienaar

sequences are included in the maximum likelihood 1962). Therefore, we re-describe T. mocambicum

tree of the genus Trypanosoma shown in Figure 2. with its neotype designation based on microscopic

Although the overall topology of the tree remained examination of blood smears obtained from six P.

unresolved, a large clade of trypanosomes from upembae and additional data on the hosts.

aquatic or semiaquatic hosts (e.g. Trypanosoma

chattoni, T. mega, T. murmarensis, T. cobitis and T.

chelodinae) was recovered and highly supported. Diagnosis

All of our sequences were placed into this “aquatic”

Phylum Euglenozoa Cavalier-Smith, 1981

clade as a single clade with two subclades, hence-

Class Kinetoplastea Honigberg, 1963, emend.

forth referred to as clade I and clade II. Clade I

Vickerman, 1976

comprised 8 sequences obtained from P. rhode-

Subclass Metakinetoplastina Vickerman, 2004

sianus, P. upembae, and P. subniger, while clade II

Order Kent, 1880, stat. nov.

comprised 17 sequences obtained from the same

Hollande, 1952

three host species plus P. bechuanicus. The two

Family Trypanosomatidae Doflein, 1951

different sequences obtained from the same spec-

imen of P. subniger belonged to different clades

(haplotypes T6 and M3). Although bootstrap sup-

Trypanosoma mocambicum Pienaar,

port for the relationship was low, however, clades ˇ

1962 emend. Dvorákovᡠet Siroky´

I and II were robustly monophyletic and appeared

closely related. The phylogenetic position of the lin- Species diagnosis: The species is identified by its

eages within the “aquatic” clade was not resolved unique rRNA SSU sequence (KP888901).

in our analysis; they showed weak affinity to a clade Morphology: The circulating flagellates are broad

formed by T. binneyi and T. chelodinae. and elongate with clearly pronounced undulating

The lowest genetic distance (uncorrected p dis- membrane and axoneme. Their dimensions aver-

tance) between haplotypes of clades I and II was age 52.3 ± 3.3 ␮m by 5.4 ± 0.9 ␮m (47.0 – 60.0 by

0.022, and the maximum distance within the clades 4.0 – 8.0; n = 22). The free flagellum is often dif-

was 0.005 and 0.015, respectively. Clade II showed ficult to discern, usually being too fine or passing

a higher genetic variability because of sequence behind the parasite body or surrounding blood cells.

5204 from P. bechuanicus – its distance from the The trypomastigotes are generally curved into a C-

other sequences of the lineage varied between shape (Fig. 1e), S-shape (Fig. 1f), or conch-shape

0.014 and 0.015, while the distances between the (Fig. 1 g) or with crossed extremities (Fig. 1 h). The

remaining sequences reached only 0.004 (Sup- kinetoplast is usually small, rounded and situated

plementary Material Table S3). Six haplotypes of on the concave side, close to the posterior end of

each clade were distinguished (see Supplementary the organism, and stains deep purple. The oval

Material Table S2). Each of the detected haplotypes nucleus measures 3.8 ± 0.7 ␮m by 3.1 ± 0.4 ␮m

was distributed in a specific area with exception to (3.0 – 5.0 by 3.0 – 4.0), and is placed posterior

M2 and M3 haplotypes, which were distributed in to the centre of the body. The densely granular

DR Congo and Angola or DR Congo and Mozam- cytoplasm is slightly speckled at the extremities.

bique, respectively. The detailed overview of overall dimensions is

Regarding trypanosomes included in clade I, recorded in the Supplementary Material Table S4.

their morphology could not be analysed since all Type host: Pelusios upembae Broadley, 1981 (Tes-

trypomastigotes were detected in samples with tudines: Pelomedusidae).

confirmed mixed infection of trypanosomes from Other hosts: Pelusios bechuanicus FitzSimons,

both clades. Thus, it is not clear to which species 1932; Pelusios rhodesianus Hewitt, 1927; Pelusios

these forms (broad and slender) belong. Single subniger (Lacépède, 1788) (all belong to Tes-

infection trypanosomes from clade II were com- tudines: Pelomedusidae).

pared with already published morphological data Type locality: Luena vicinity, Bukama region, SE

on turtle trypanosomes from the Afrotropical zoo- of Democratic Republic of the Congo.

geographic region (Supplementary Material Table Other localities: Catabola, Bié province, Angola;

S4). The morphological description of broad forms Guija town, Gaza province, Mozambique; and

from clade II matched the published descrip- Democratic Republic of the Congo without exact

tion of Trypanosoma mocambicum Pienaar, 1962. locality.

604 N. Dvorákovᡠet al.

Type material: The hapantotype represents the in size. Distance between kinetoplast and nucleus

blood film marked KO-7-12, sample of full blood (KN) is generally 6 – 8 ␮m, but in T. mocambicum

in ethanol (KO-7-12), and DNA sample No. 2575 averaged 20.9 ␮m. From turtle trypanosomes of

deposited in the collection of Department of Biol- Africa, T. mocambicum is most similar to T. neitzi,

ogy and Wildlife Diseases, University of Veterinary but differs in the frequent fine vacuoles located in

and Pharmaceutical Sciences Brno, Brno, Czech the cytoplasm, prominent nucleolus and a thick and

Republic. distinct free flagellum.

Additional material: Blood films (marked as

KO-36-12, KO-40-12, KO-42-12, KO-40-13 and KO-

41-13) and DNA samples (no.2569, 2571, 2574, Discussion

3160, 3184, 3185, 3187, 3188, 3190, 5185, 6212,

6213 and 6216) are deposited in the same col- Our study provides the first molecular charac-

lection as the type material. GenBank accession terization of Trypanosoma species isolated from

numbers for all used sequences are provided in the freshwater turtles of tropical Africa and also extends

Supplemental Material Table S2. our knowledge on diversity of trypanosomes in

Vector: Unknown. the Afrotropical zoogeographical realm. Until now,

Stages in the vector: Unknown. five trypanosomes were reported from African

Remarks: T. mocambicum was described from chelonians, members of two genera Pelusios

a single female specimen of Pelusios sinuatus and Kinixys (Bouet 1909; Joyeux 1913; Pienaar

by Pienaar in 1962. The trypomastigotes mea- 1962; Travassos Santos Dias 1952). We con-

␮

sured 50 – 75 by 5 – 6 m in blood smears. firmed Trypanosoma sp. in four out of 12 examined

According to the original description, the body pelomedusid species (Supplementary Material

was curved into the form of S, question mark or Table S5) and two distinct forms, broad and slen-

8-shaped figures or looped on itself. The free fla- der, were recognized among the trypomastigotes

gellum was rarely seen intact and, when it can be (Fig. 1). Both forms were sometimes found in

observed, it would be extended to a length of 12 to the same sample, suggesting polymorphism or,

␮

15 m beyond the anterior end. The nucleus was alternatively, the presence of two species each rep-

located posterior to the midbody, but could hardly resented by a different morphology. In infections

be discerned, because of the intense cytoplasmic with T. copemani, Thompson et al. (2013) identified

basophilia. The distribution of basophilic material broad trypomastigotes as a blood form responsible

was dense and more homogeneous throughout the for the reproductive phase and the slender forms

cytoplasm of the flagellate, except for the ante- as mature trypomastigotes. Karlsbakk et al. (2005)

rior extremity of the body, where it was distinctly recorded that the broad trypomastigote was char-

granular and much less compact. The kinetoplast, acterized by large disparities in morphology during

like the nucleus, was obscured by the cytoplasmic infection time. Previously, polymorphic T. nudigobii,

basophilia, but could be discerned in the periph- with three morphotypes, was detected from the tis-

ery, very near the posterior end. The axoneme was sues of the leech vector and from the blood of the

distinct and undulating membrane very well devel- host fish using PCR (Hayes et al. 2014). Addition-

oped. According to Pienaar 1962, T. mocambicum ally, during culturing of trypanosomes, large and

is comparable in size and general morphology to wide trypomastigotes predominated at the end of

T. vittatae Robertson, 1908 from Lissemys punc- the stationary phase, and the number of the small-

tata from Ceylon. Nevertheless, T. mocambicum is est forms (slender metacyclic trypomastigotes)

more slender. increased in the terminal stage of cultures (Viola

Our observed trypomastigotes are comparable et al. 2008). Thus, morphology exclusively is not

in terms of individual morphological parameters. reliable in determination of Trypanosoma species

Additionally, we found that the cytoplasm may be (Lainson et al. 2008; Ziccardi and Lourenc¸o-de-

granular and less compact at both ends of the Oliveira 1999; Zintl et al. 2000). Nevertheless, it is

organism. We assume that the trypomastigotes important to combine morphological characteristics

described by Pienaar (1962) were deeply stained together with molecular-genetic data for complete

with Romanovski probably due to a long staining species descriptions.

period. This could explain the intensely stained Determination of SSU rRNA gene sequences

posterior end. We consider T. vittatate compara- is useful to differentiate morphologically similar

ble in length and width to T. mocambicum, but trypanosome species (Botero et al. 2013; Davies

other parameters are different. Trypanosoma vit- et al. 2005; Grybchuk-Ieremenko et al. 2014),

tatae kinetoplast is rod-shaped and very variable especially in a mixed infection. According to our

Neotype of Trypanosoma mocambicum 605

phylogenetic analysis, two different Trypanosoma vector of the trypanosomes found in freshwa-

species were discovered among sampled turtles; ter turtles is a leech, because there is frequent

one of them was comparable to T. mocam- coinfection with haemogregarines that are also

bicum from Pelusios sinuatus by morphology transmitted by aquatic leeches (Johnston and

as described by Pienaar (1962). Trypanosoma Cleland 1912; Robertson 1908). Also, leeches were

species are known to be not strictly host specific often observed on examined wild turtles, but they

and so are our isolates originated from several were not available for our study.

Pelusios species. Trypanosomes tend to infect Analysis of our dataset shows no signifi-

more than one turtle species of the same family cant geographic pattern, when African chelonian

(Australian T. chelodinae - Johnston and Cleland trypanosomes were most closely related to Tr y-

1912; Mackerras 1961) or even of various families panosoma from Australian chelid turtles and

(North American T. chrysemydis - Jefferson 1965; to trypanosomes from platypus, whereas other

Roudabush and Coatney 1937; Woo 1969). It African reptilian trypanosomes were shown to be

is known from experimental studies that cross- more evolutionary distant. Apparently limited dis-

transmission between various hosts also exists in tribution of some haplotypes could be confirmed

fish trypanosomes (Khan 1977; Lom 1973; Woo by analyzing a larger collection of isolates. Nev-

and Black 1984). On the other hand, a single host ertheless, limited markers and rather incomplete

may be infected with more than one Trypanosoma sequence data yet did not allow searching of traits

species at the same time (Gu et al. 2007). Our of co-evolutionary history between chelonian try-

findings further confirm these observations. panosomes and their host species.

Employment of PCR-based methods reveals two

Trypanosoma species in the same host speci-

men, the case which could be easily misidentified Methods

as two morphotypes of T. mocambicum. Never-

theless, due to mixed infection, we were unable

Sample collection, microscopy and morphological mea-

to characterize the slender Trypanosoma species.

surements: Material was obtained from 134 pet-traded

Unfortunately our attempts to use different meth- freshwater turtles of family Pelomedusidae belonging to 2 gen-

era and 12 species (9 Pelomedusa neumanni and the following

ods to amplify a longer fragment of the SSU

Pelusios species: 6 P. bechuanicus, 2 P. carinatus, 10 P. cas-

rRNA and the gGAPDH genes were not suc-

tanoides, 11 P. gabonensis, 9 P. marani, 27 P. nanus, 6 P.

cessful. Trypanosoma infected animals are often

rhodesianus, 2 P. sinuatus, 28 P. subniger, 18 P. upembae and 6

microscopically diagnosed negative as a result of P. williamsi). The specimens were wild caught in Angola (n = 12),

generally low intensity of Trypanosoma infection of Central African Republic (n = 1), Democratic Republic of the

Congo (n = 67), Gabon (n = 11), Kenya (n = 16), Mozambique

naturally infected reptilian hosts (Telford 2009).

(n = 18) and Seychelles (n = 9) (Supplementary Material Table

In the phylogenetic tree, the two newly detected

S5). All the turtles were inspected and sampled during the vet-

turtle Trypanosoma species clustered within a

erinary examination in the Czech Republic during spring 2012

strongly supported “aquatic” clade (Fig. 2). Pienaar to summer 2014 and in total 94 blood smears and 134 blood

(1962) suggested that leeches are vectors of samples were obtained.

Blood was collected from each turtle by puncture of the

T. mocambicum and it seems plausible that the

dorsal coccygeal vein with insulin syringes without using anti-

ancestor of the two species identified in our

coagulant. Thin blood smears were prepared immediately from

study was originally a fish parasite that has later

a single drop. The remaining blood (∼ 0.2 ml) was stored in

◦

adapted to turtles. Trypanosomes of aquatic verte- 96% ethanol and frozen at -20 C. Blood films were immersed in

brates are transmitted by a range of blood-sucking absolute methanol, air-dried and stained with Giemsa (Sigma-

Aldrich, St. Louis, USA) (diluted 1:10 in distilled water, pH 7.0)

invertebrates: sand flies act as vectors of anuran

for 15 minutes and then rinsed in distilled water. Stained smears

trypanosomes (Ayala 1971; Ferreira et al. 2008);

were mounted with coverslip using Entellan rapid mounting

tsetse flies transmit trypanosomes of crocodiles

medium (Merck KGaA, Darmstadt, Germany). The smears

(Hoare 1931a,b; Lloyd and Johnson 1924) and were systematically screened for the presence of parasites

using an Olympus BX53 microscope with 1,000 × magnification

leeches are implicated as vectors for trypanosomes

and immersion oil.

of fish, salamanders and turtles (Karlsbakk 2004;

For distinction of parasitic species, morphological parame-

Ray 1987; Woo and Bogart 2011). Woo (1969)

ters of bloodstream forms were measured according to Telford

attempted to transfer T. chrysemydis from Chry- (2009). The images were acquired by Quick Photo Camera 3.0

×

semys picta marginata to Aedes mosquitoes, but software at 1,000 magnification.

DNA extraction, PCR amplification, cloning and

without success. Freshwater turtles of the genus

sequencing: Approximately 30 ␮l of each blood sample

Pelusios share a similar ecological niche, and

were incubated overnight with proteinase K before the DNA

hence are assumed to be accessible to the same

isolation. Whole genomic DNA was extracted using the

(unknown) vectors. It seems most likely that the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) as

606 N. Dvorákovᡠet al.

per the manufacturer’s instructions. DNA was resuspended in

◦ Acknowledgements

100 ␮l of PCR water and then stored at –20 C until processing

for molecular analyses.

Thank Hynek Prokop and Angolan team (Jana,

A nested PCR was performed to amplify an approximately

Hynek, Martin, and Jakub) for the help with samp-

900 bp fragment of the small subunit (SSU) rRNA gene. A

pair of external primers SLF and S-762 was used for the pri- ling. Thank Petr Táborsky´ and Eliskaˇ Zadrobílková

mary PCR and internal primers SLIR and S-825 were used at Charles University in Prague for the help with

for the secondary reaction. The external forward primer SLF

cloning samples. We also thank Patrick B. Hamil-

and internal reverse primer SLIR were sourced from McInnes

ton, University of Exeter, for helpful comments and

et al. (2009). Specific primers S-762 and S-825 were pre-

anonymous reviewers for detailed and constructive

viously described (Maslov et al. 1996). PCR reactions were

carried out in a 25 ␮l volume; reaction mixture consisted of criticism of the manuscript. This work was sup-

␮

12.5 l of Combi PPP Master Mix (Top-Bio, Prague, Czech ported by the project “CEITEC – Central European

Republic), 1 ␮l of each 10 ␮M PCR primer, 8.5 ␮l of PCR water

Institute of Technology” (CZ.1.05/1.1.00/02.0068)

and 1 ␮l of purified DNA. 1 ␮l of the first-step PCR reaction

from the European Regional Development Fund.

was used as template for the second-step PCR. The reac-

tion conditions according to McInnes et al. (2009) were as

◦ ◦

follows: pre-PCR step with 95 C for 5 min, 50 C for 2 min and

◦ ◦

an extension of 72 C for 4 min followed by 35 cycles of 94 C

◦ ◦

for 30 s, 52 C for 30 s and a final extension step of 72 C for Appendix A. Supplementary Data

7 min. Two pairs of primers were designed for SSU rRNA to

distinguish mixed infection of Trypanosoma species. Primers

  Supplementary data associated with this arti-

IIF1 (5 -CCGGTGTCCAGGGTGAGAAGGGTTA-3 ) and IIR1

  cle can be found, in the online version, at

(5 -AATCCCGCAGAGAAGGATACAAAT-3 ) amplified 774 bp

http://dx.doi.org/10.1016/j.protis.2015.10.002.

DNA fragment of trypanosomes forming clade I. Primers

  

IF2 (5 -CAGAATGGCTTCGGCCACTTCCTGT-3 ) and IR2 (5 -



GAAAAGAATATGCACGTAAATTG-3 ) were used to amplify

331 bp DNA fragment of trypanosomes included in clade II.

We also tested other methods for amplification of longer frag- References

ments of the SSU rRNA gene (Jakes et al. 2001; McInnes

et al. 2009) or more variable gGAPDH (McInnes et al.

Austen JM, Jefferies R, Friend JA, Ryan U, Adams P, Reid

2009).

SA (2009) Morphological and molecular characterization of

All positive PCR products were purified using the Gel/PCR

Trypanosoma copemani n. sp. (Trypanosomatidae) isolated

DNA Fragments Extraction Kit (Geneaid Biotech Ltd., New

from Gilbert’s potoroo (Potorous gilbertii) and quokka (Setonix

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