Four new species of monoxenous kinetoplastid parasites are described from Brachycera flies, namely Wallaceina raviniae Votýpka et Lukeš, 2014 and Crithidia otongatchiensis Votýpka et Lukeš, 2014 from Ecuador, Leptomonas moramango Votýpka et Lukeš, 2014 from Madagascar, and Crithidia pragensis Votýpka, Klepetková et Lukeš, 2014 from the Czech Republic. The new species are described here based on sequence analysis of their spliced leader (SL) RNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and small subunit (SSU) rRNA genes, as well as their morphology and ultrastructure. High-pressure freezing and Bernhard's EDTA regressive staining, used for the first time for monoxenous (one host) trypanosomatids, revealed the presence of viral particles with cytosolic localization in one and unique mitochondrial localization in another species. In accordance with previous observations, our results emphasize a discrepancy between morphology and molecular taxonomy of the family Trypanosomatidae. All four newly described species are represented by typical morphotypes (mainly choano- and promastigotes) and are virtually indistinguishable from other monoxenous trypanosomatids by morphology. Nevertheless, they all differ in their phylogenetic affinities. Whereas three of them grouped within the recently defined subfamily Leishmaniinae, which includes numerous representatives of the genera Leishmania Ross, 1903, Crithidia Léger, 1902 and Leptomonas Kent, 1880, the fourth species clusters together with the ''collosoma'' clade (named after ''Leptomonas'' collosoma Wallace, Clark, Dyer et Collins, 1960). Here we demonstrate that the ''collosoma'' group represents the elusive genus Wallaceina Podlipaev, Frolov et Kolesnikov, 1999. We redefine this genus in molecular terms based on similarities of the respective molecular markers and propose to use this taxon name for the group of species of the ''collosoma'' clade.
Using scanning and transmission electron microscopy, ultrastructure of the anterior organ and posterior funnel-shaped canal of Gyrocotyle urna Wagener, 1852 (Cestoda: Gyrocotylidea) from ratfish, Chimaera monstrosa (Holocephali), was studied for the first time. The proper anterior organ is localised at a short distance (about 170 µm) from an apical pore surrounded by a receptor field, whereas its distal end is marked by a muscular sphincter. The tegumental surface of this organ is covered with short filitriches of irregular length; large area of muscle layers traverse beneath the tegumental layer. The funnel-shaped canal of G. urna (2.5-3.0 mm long) is a specialised, muscular part of the posterior attachment organ; it opens on the rounded elevation on the dorsal body surface. The tegumental layer bears conical sclerite-like structures (up to 1.5 µm long). It produces electron-dense bodies that are transported into a canal lumen and surrounded thick muscle area mixed with numerous nerve fibres. The present ultrastructural study of G. urna indicates that gyrocotylideans share some ultrastructural characters of the anterior organ with spathebothriidean cestodes with a single anterior attachment sucker-like organ. In contrast, the unique posterior rosette attachment organ with funnel-shaped canal of the Gyrocotylidea resembles the haptor of polyopisthocotylean monogeneans in its position at the posterior end of the body and presumed origin. The above-mentioned features add more clarity to support the basal position of the Gyrocotylidea Poche, 1926 among cestodes. In addition, they also indicate a possible relationship of gyrocotylidean ancestors with monogeneans., Larisa G. Poddubnaya, Roman Kuchta, Glenn A. Bristow, Tomáš Scholz., and Obsahuje bibliografii
This study investigates the identity of hookworms parasitising the Australian sea lion, Neophoca cinerea (Péron), from three colonies in South Australia, Australia. The Australian sea lion is at risk of extinction because its population is small and genetically fragmented. Using morphological and molecular techniques, we describe a single novel species, Uncinaria sanguinis sp. n. (Nematoda: Ancylostomatidae). The new species is most similar to hookworms also parasitic in otariid hosts, Uncinaria lucasi Stiles, 1901 and Uncinaria hamiltoni Baylis, 1933. Comparative morphometrics offered limited utility for distinguishing between species within this genus whilst morphological features and differences in nuclear ribosomal DNA sequences delineated U. sanguinis sp. n. from named congeners. Male specimens of U. sanguinis sp. n. differ from U. lucasi and U. hamiltoni by relatively shorter anterolateral and externodorsal rays, respectively, and from other congeners by the relative lengths and angulations of bursal rays, and in the shape of the spicules. Female specimens of U. sanguinis sp. n. are differentiated from Uncinaria spp. parasitic in terrestrial mammals by differences in vulval anatomy and the larger size of their eggs, although are morphologically indistinguishable from U. lucasi and U. hamiltoni. Molecular techniques clearly delimited U. sanguinis sp. n. as a distinct novel species. Obtaining baseline data on the parasites of wildlife hosts is important for the investigation of disease and the effective implementation and monitoring of conservation management.
This paper summarizes work done in this laboratory over the last two years on the cloning of microsporidian rRNA by homology PCR and its subsequent use in diagnostic tests and phylogenetic studies. Using highly conserved primers in the 16S or small subunit rRNA (SSU-rRNA) these genes were cloned from human intestinal biopsies with transmission electron microscopy proven Enterocytozoon bieneusi and Septata intestinalis. The SSU-rRNA genes were then used to design and test several primer pairs for the diagnosis of microsporidian infection. Utilizing the polymerase chain reaction and primers V1 and EB45Ü Ent. bieneusi infected duodenal aspirates or intestinal biopsies could be detected. Using V I and SI500 infection with S. intestinalis could be detected. In addition to diagnostic tests, phylogenetic relationships were examined using sequence data from the fragment amplified by PCR by primer 530f in the SSU-rRNA and primer 580r in the large subunit rRNA. This data supported the placement of S. intestinalis in the family Encephalitozoonidae. In addition, it confirmed that Encephalitozoon cuniculi, E. hellem and S. intestinalis are distinct organisms. These techniques have broad applications to the study of other microsporidia and the development of a molecular phylogeny.