Trypanosoma tungarae, Bernal & Pinto, 2016, Bernal & Pinto, 2016

Bernal, Ximena E. & Pinto, C. Miguel, 2016, Sexual differences in prevalence of a new species of trypanosome infecting túngara frogs, International Journal for Parasitology: Parasites and Wildlife 5 (1), pp. 40-47 : 43-45

3.1. Species description

The trypanosomes observed in the blood smears have a unique set of morphological characters that differentiate them from previously described species. Morphology, however, often does not allow researchers to distinguish trypanosomes species and is problematic for determining species relationships. We obtained DNA sequences that revealed this lineage constitute a new species of trypanosome that we describe below.

Taxonomic summary: Phylum Euglenozoa, Cavalier-Smith, 1981; class Kinetoplastea, Honigberg, 1963; order Trypanosomatida, (Kent, 1880) Hollande, 1952; family Trypanosomatidae ,

Doflein, 1951. Trypanosoma tungarae n. sp. Bernal and Pinto (201×).

Type material: type blood smears of three infected frogs are deposited in the Smithsonian National Museum of Natural History (USNM Numbers TBD). Type Host: Vertebrate host is the túngara frogs E. pustulosus ( Amphibia: Leuperidae); putative vectors are Corethrella spp. midges ( Diptera : Corethrellidae ). Type Locality: Panaḿa, Colon Province, Gamboa (30 m. a.s.l., 9 ǫ 79 Ɩ N, 79 ǫ 42.9 Ɩ W) ( Fig. 2 ). Location on hosts: In the vertebrate hosts peripheral blood. The location in their putative vector frog-biting midges is unknown (possibilities include the digestive tract, the hemocele and the salivary glands). Distribution: Currently known only from the type locality, Gamboa, Panama. Diagnosis: Monomorphic trypanosome with an elongated body (52.13 ± 12.94 µm) and thin soma (5.41 ± 3.62 µm). Free flagellar length (FF), 13.20 ± 5.11 µm; midnucleus to anterior end (MA), 42.71 ± 13.77 µm; midnucleus to posterior end (MP), 29.67 ± 10.59 µm; midnucleus to kinetoplast, 20.31 ± 7.41 µm; posterior end to kinetoplast, 9.71 ± 3.50 µm; relative size of flagellum (FF/MA), 0.34 ± 0.14 µm; length of nucleus, 3.63 ± 1.67 µm; nuclear index (MP/MA), 0.97 ± 0.60 µm ( Fig. 3 ). In general, this species resembles other anuran trypanosomes from Central and South America ( Desser, 2001; Ferreira et al., 2007; McKenzie and Starks, 2008). This species is longer and thinner that T. rotatorium ‾ like species found in other leptodactilyd anuran host in South America ( Lemos et al., 2008). In particular, this species corresponds to the morphology of anuran trypanosomes with elongated trypomastigotes with pointed ends observed in Bufonidae , Leiuperidae and Leptodactylidae from Brazil (Group I, Ferreira et al., 2007). The morphology of this species, however, is most similar in general to Trypanosoma sp. (e) and Trypanosoma sp. (f) described from Lithobates vaillanti syn. Rana vaillanti by Desser (2001). Although the measurements of the species described here match closely some characteristics of Trypanosoma sp. (e) such as the relative length of the free flagellum, other features, including total body length and the distance from the posterior end to the kinetoplast, are closer to the morphology of Trypanosoma sp. (f). Some other features, however, are distinct from both Trypanosoma sp. (e) and (f) (e.g. distance from the center of the nucleus to the anterior end). A Trypanosoma montrealis -like species was found to be transmitted by North American frog-biting midges ( C. wirthi ) in Florida ( Johnson et al., 1993). Although the body length and width of Trypanosoma montrealis ( Fantham et al., 1942) fall within the dimensions of the species described here, that previously described species has a much shorter free flagellum than T. tungarae n. sp (3‾5.5 µm vs 13.20 ± 5.11 µm). The validity of T. montrealis , however, has been questioned ( Werner et al., 1988). More detailed morphological comparisons with previously described species of anuran trypanosomes from the same geographical area are unfeasible given that detailed morphological measurements are not often reported and recent, updated species descriptions frequently focus on the species genotypes (e.g Ferreira et al., 2007). Intraspecific morphological variation of amphibian trypanosomes, however, is so high that precludes its use for species identification. For example, amphibian trypanosomes can significantly change their morphotype when infecting different hosts ( Hysek and Zizka, 1976).

This species does not resemble in morphology Trypanosoma chattoni , the closest related species known to date (see under Phylogenetic relationships below), that has a characteristic round to oval body ( Lemos et al., 2008). Trypomastigotes of both species, however, have large size and this new species thus becomes a new member of the giant trypanosomes that includes species such as Trypanosoma mega , T. ranarum and T. rotatorium ( Martin et al., 2002) . Despite the widespread distribution of T. chattoni including Asia ( China, Werner, 1993; Kyushu and Ryukyu Islands, Miyata, 1978; Thailand, Sailasuta et al., 2011), North America (United Sates, Diamond, 1965; Canada, Jones and Woo, 1986) and South America ( Brazil, Lemos et al., 2008), this species is monomorphic with little geographic variation. Both T. chattoni and T. tungarae n. sp. have heavily stained cytoplasms that often obscure the nucleus and kinetoplast. When visible, the kinetoplast lays towards the anterior end at about a fourth of the total length of the cell. Glass slides of Giemsa-stained smears from túngara frog blood samples and DNA samples are kept at the Smithsonian National Museum of Natural History, Washington, DC. To comply with the regulations of the International Code of Zoological Nomenclature (ICZN), details of this species have been submitted to ZooBank with the Life Science Identifier (LSID) zoobank.org:pub:TBD.

Etymology: Túngara (English pronunciation: toon-gah-rra) is the common name of the frog E. pustulosus , the vertebrate host of this new species of trypanosome. Túngara is a feminine Spanish onomatopoeic word resembling part of the singing repertoire of the E. pustulosus males. We treat tungarae as a feminine noun in the genitive case.

3.2. Host prevalence

Consistent with our prediction, we found sexual differences in trypanosomes infection in túngara frogs (Z-test, Z = 2.28, p = 0.022).While 40% of male túngara frogs sampled were infected with this blood parasite, only 6.6% of the females were infected (males: 10/25; females: 1/15). We were, however, expecting that no females would be infected since female túngara frogs do not vocalize. Frog-biting midges are attracted to the mating calls produced by males ( Bernal et al., 2006; Borkent, 2008; McKeever and Hartberg, 1980), so our results beg the question, if frog-biting midges are the vectors, how did a female become infected with this new species of trypanosomes? Careful inspections of our records confirmed this result and field observations revealed a potential path of transmission for female frogs to be infected. When túngara frog are in amplexus, frog-biting midges attempting to feed on the calling male have an opportunity to move directly from their original victim, the male, to the female and obtain a blood meal ( Fig. 1b,c ).

3.3. Phylogenetic relationships

The maximum likelihood and the Bayesian inference phylogenies of the 18S rRNA gene are highly concordant, and show strong support for the placement of the new species, Trypanosoma tungarae , in the clade with aquatic trypanosomes; however, several internal branches are poorly supported for both methods. Trypanosoma tungarae n. sp. is sister to T. chattoni , and both form a highly supported clade sister to other trypanosomes of South American frogs ( Fig. 4 ).

Both, the maximum likelihood and Bayesian solutions of the putative species delimitation analysis in PTP indicate that T. tungarae n. sp. is a different species from other trypanosomes for which molecular data is available. Also, the PTP analyses indicate that it might be some over splitting of species in fish trypanosomes, and several unrecognized species of frog trypanosomes ( Fig. 4 ). The two sequences of T. tungarae n. sp. diverge in eight nucleotides, and it is likely that additional genetic variation can be found within the study area. Despite that the 18S rRNA gene is a slowly evolving marker, the variation that we found is not surprising given the complex patterns of intra and inter specific trypanosome diversity found in this geographic region ( Pinto et al., 2012; Cottontail et al., 2014).

Figures

Fig. 2. Map of the Republic of Panaḿa indicating with a star the location of Gamboa, the type locality of Trypanosoma tungarae n. sp. Insert shows the location of Panamáin the New World.

Fig. 3. Light microscopy of Trypanosoma tungarae n. sp. (Giemasa-staining). (a e) Trypomastigotes stained using Hemacolor ® Giemsa stain kit (Voigt Global Distribution Inc, USA); ‾ (f‾i) Trypomastigotes stained using Giemsa stain following Mohr (1981). Scale bars: 10 µm.

Fig.1. Photographs of túngara frogs (Engystomops pustulosus) and frog-biting midges (Corethrella spp). (a) Calling male túngara frog preyed upon by frog-biting midges; (b) female (bottom) in amplexus with a male (top) covered with biting midges; (c) female (bottom) with a biting midge on her nostril that was passed from the male during amplexus.Túngara frogs are about 30 mm long while the frog-biting midges are only about 1.5 mm. Photos taken by Alexander Baugh (a) and Ximena E Bernal (b,c).

Fig. 4. Phylogeny of the aquatic clade, and PTP species delimitation results. Best maximum likelihood tree of the18S rRNA gene of member of the aquatic clade and selected outgroups. Numbers on the branches represent support values corresponding to ±70% bootstrap replicates (left) and ±0.9 Bayesian posterior probabilities (right). Subclades are highlighted with colored boxes to indicate host associations. Color of the branches indicate the PTP species delimitation results; monophyletic groups in red indicate members of a single species, blue terminal branches indicate that only one sample is included in such species. Names of the terminals indicate the GenBank accession numbers, scientific name, and sample or isolate code.Star indicates the position of T.tungarae n. sp. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)