Development of two species of the Trypanosoma theileri complex in tabanids

Abstract

Background: Trypanosoma theileri species complex includes parasites of Bovidae (cattle, sheep, goat, etc.) and Cervidae (deer) transmitted mainly by Tabanidae (horse flies and deerflies) and keds (Hippoboscidae). While morphological discrimination of species is challenging, two big clades, TthI and TthII, each containing parasites isolated from bovids and cervids, have been identified phylogenetically. To date, the development in the vector has been studied in detail only for the ked-transmitted sheep parasite T. melophagium (TthII), while the fate of trypanosomes in tabanids was described only briefly by light microscopy.

Methods: We collected infected tabanids of various species and identified trypanosomes by molecular phylogenetic analysis. The morphology and development of trypanosomes was studied using the combination of statistical analyses as well as light and electron microscopy.

Results: Two trypanosome species belonging to both TthI and TthII clades of the T. theileri complex were identified. The phylogenetic position of these two trypanosomes suggests that they parasitize deer. Both species were indiscernible by morphology in the vector and showed the same development in its intestine. In contrast to the previously described development of T. melophagium, both trypanosomes of tabanids only transiently infected midgut and settled mainly in the ileum, while pylorus and rectum were neglected. Meanwhile, the flagellates developing in the tabanid ileum (pyriform epimastigotes and metacyclic trypomastigotes) showed similarities to the corresponding stages in T. melophagium by morphology, mode of attachment to the host cuticle and formation of the fibrillar matrix surrounding the mass of developing parasites. In addition, for the first time to our knowledge we documented extraintestinal stages in these trypanosomes, located in the space between the epithelium and circular muscles.

Conclusions: The development of different species of flagellates of the T. theileri complex in their insect vectors shows many similarities, which can be explained not only by their common origin, but also the same transmission mode, i.e. contamination of the oral mucosa with the gut content released after squashing the insect either by tongue or teeth. The observed differences (concerning primarily the distribution of developmental stages in the intestine) are associated rather with the identity of vectors than the phylogenetic position of parasites.

Keywords: Deerflies; Horseflies; Life cycle; Trypanosomes; Vector.

Fig. 1

Generalized structure of the digestive system in Tabanidae

Fig. 2

Unrooted maximum likelihood tree based on 18S rRNA gene sequences of the Trypanosoma theileri complex. Pictograms show the source of isolates from which sequences have been obtained. Two-letter codes indicate countries of origin following the scheme of national internet domains: AR Argentina, BR Brazil, CF Central African Republic, DE Germany, HR Croatia, IT Italy, JP Japan, PL Poland, RU Russia, UK United Kingdom, US United States, VE Venezuela. Scale corresponds to the number of substitutions per site. Ultrafast bootstrap supports are shown by branch coloring only for values > 70. The borders of the TthI and TthII clades are marked according to [6]. GenBank accession numbers of the sequences used are available in Additional file 2: Fig. S1, representing the same tree in a traditional rectangular format with labels

Fig. 3

Maximum likelihood phylogenetic tree of Trypanosoma (Megatrypanum) spp. based on gGAPDH gene sequences. Bayesian posterior probabilities and bootstraps are shown at branches for values > 0.5 and 50, respectively. Two-letter codes indicate countries of origin following the scheme of national internet domains (BR Brazil, CM Cameroon, DE Germany, JP Japan, RU Russia, UG Uganda, UK United Kingdom, US United States, VE Venezuela). Scale corresponds to the number of substitutions per site. The borders of the TthI and TthII clades are marked according to [6]. Three sequences of trypanosomes not belonging to the T. theileri complex are used as outgroups (not highlighted)

Fig. 4

Semithin cross sections of the hindgut (isolate 194Tab). Richardson stain. a, b Trypanosomes on the ileum cuticle. c, d Posterior portion of ileum, containing only symbiotic bacteria. e Rectal valve. f Rectal ampulla with one of the six rectal glands. bs bacterial symbionts, cf cuticle folds, cu cuticle, ep intestinal epithelium, he hemocoel, lu intestinal lumen, mu muscles of the intestine, rg rectal gland, tr trypanosome cells. Arrows in a and c show trypanosomes and bacteria, respectively. Scale: a 50 µm; b, d 10 µm; c, e 40 µm; f 100 µm

Fig. 5

Morphotypes of trypanosomes observed in the hindgut of tabanids (Giemsa-stained smears). a, b Non-dividing elongated epimastigotes. c Dividing elongated epimastigotes. d, e Non-dividing pyriform epimastigotes. f, g Dividing pyriform epimastigotes. h–k Metacyclics. l Dead cell from the rectum. a, d, i 513SL (Tthβ); b, c F1206 (Tthα); e, g—KrSL7 (Tthα); f, h—KrSL1 (Tthβ); j, k, l—194Tab (Tthα). Scale: a–c 10 µm; d–l 5 µm

Fig. 6

Scheme of the distribution of observed cell types in the intestine of tabanids

Fig. 7

Scatterplot of the principal component analysis showing a two-dimensional space of morphometric data in three cell morphotypes of two trypanosome species: Tthα (isolates F1206, D1017, KrSL7, and 194Tab) and Tthβ (isolates 513SL and KrSL1). PC1 and PC2: principal components 1 and 2, respectively; numbers in parentheses show the percentage of variance explained by a particular component. ee elongated epimastigotes, pe pyriform epimastigotes, m metacyclics

Fig. 8

Trypanosomes occupying the cuticular lining of ileum. a, b SEM; c, d TEM. a, d General view. b Lateral view of a single cell with broken flagellum. c Cross section of a trypanosome cell. Arrows: longitudinal ridges; arrowheads: long flagella of cells from distal rows; cu cuticle, fl flagellum, fm fibrillar matrix surrounding trypanosomes, fp flagellar pocket, lu intestinal lumen, tr trypanosomes. Scale: a 10 µm, b 1 µm, c 0.4 µm, d 4 µm

Fig. 9

Fine structure of trypanosomes in the ileum. a–c Pyriform epimastigotes. d Metacyclic. Cells are attached using widened tips of short (a, b, c) or long (b) flagella with a zonal hemidesmosome and surrounded by fibrillar matrix. ap autophagosome, cu cuticle, fl flagellum, fm fibrillar matrix, fp flagellar pocket, hd hemidesmosome, kp kinetoplast, li lipid droplet, nu nucleus, r ribosomes. Scale: a 0.6 µm; b, c 2 µm; d 0.8 µm

Fig. 10

Extraintestinal trypanosome cells. a Trypanosomes from both sides of the intestinal wall. b, c Single cells, cross and longitudinal sections, respectively. d Cell coverings structure. ac acidocalcisome, cu cuticle, ep intestinal epithelium, fl flagellum, gc glycocalyx, gl glycosome, kp kinetoplast, mt microtubules, mu muscles, nu nucleus, pm plasmalemma, tc tracheoles, tr trypanosomes in the gut lumen. Arrows point to extraintestinal trypanosomes. Scale: a 6 µm, b 0.4 µm; c 1 µm; d 0.2 µm