Morphological and molecular investigations of Tubulinosema ratisbonensis gen. nov., sp. nov. (Microsporidia: Tubulinosematidae fam. nov.), a parasite infecting a laboratory colony of Drosophila melanogaster (Diptera: Drosophilidae)

A new species of microsporidia from Drosophila melanogaster was investigated by light and electron microscopy and by ribosomal RNA (rRNA) sequencing. This microsporidium and the previously described Nosema kingi and Nosema acridophagus have been transferred to the new genus Tubulinosema gen. nov. with the following characters: nuclei are in diplokaryotic arrangement during the life cycle. All stages are in direct contact with the host cell cytoplasm, slightly anisofilar polar tube with the last coils being smaller in diameter arranged in one or two rows on both sides of the diplokaryon and small tubuli on the surface of late meronts. Spores are oval or slightly pyriform. Thick endospore wall, thinner over anchoring disc. This new genus and the genus Brachiola have been placed in a new family Tubulinosematidae fam. nov. Phylogenetic analysis of small subunit rRNA sequences by different methods placed Tubulinosema spp. in one clade with the genus Brachiola forming its sister clade, which is distant from the clade containing the true Nosema spp. including Nosema bombycis.     

Apotrachelocerca arenicola (Kahl, 1933) n. g., comb. n. (Protozoa, Ciliophora, Trachelocercidae): morphology and phylogeny

During faunistic study on psammophilic ciliates along the coast of Qingdao, China, a population of Trachelocerca arenicola Kahl, 1933 was found and then investigated using silver staining and gene sequencing methods. The results indicated that it represented a new genus Apotrachelocerca characterized by uninterrupted circumoral kineties composed of two rows of dikinetids and no brosse or ciliary tuft in the oral cavity. This new genus should be assigned to the family Prototrachelocercidae Foissner, 1996. Based on the small subunit rRNA gene sequence, phylogenetic trees revealed that Apotrachelocerca arenicola occupied a basal position to other trachelocercids.     

Ichthyosporidium weissii n. sp. (Microsporidia) infecting the arrow goby (Clevelandia ios)

Gonadal infections by a novel microsporidium were discovered in 34% (13/38) of arrow gobies, Clevelandia ios, sampled over a 3‐yr period from Morro Bay Marina in Morro Bay, California. Gonadal tumors had been reported in arrow gobies from this geographic area. The infected gonads, found primarily in females, typically appeared grossly as large, white‐gray firm and lobulated masses. Histological examination revealed large, multilobate xenomas within the ovaries and no evidence of neoplasia. Typical of the genus Ichthyosporidium, the large xenomas were filled with developmental stages and pleomorphic spores. Wet mount preparations showed two general spore types: microspores with mean length of 6.2 (7.0–4.9, SD = 0.6, N = 20) μm and mean width of 4.3 (5.3–2.9, SD = 0.8) μm; and less numerous macrospores with mean length of 8.5 (10.1–7.1, SD = 1.0, N = 10) μm and mean width of 5.5 (6.2–4.8, SD = 0.5) μm. Transmission electron microscopy demonstrated stages consistent with the genus and 35–50 turns of the polar filament. Small subunit rDNA gene sequence analysis revealed that the parasite from arrow gobies was most closely related to, but distinct from Ichthyosporidium sp. based on sequences available in GenBank. We conclude that this microsporidium represents a new species of Ichthyosporidium, the first species of this genus described from a member of the family Gobiidae and from the Pacific Ocean.     

A new isolate of Nosema sp. (Microsporidia, Nosematidae) from Phyllobrotica armata Baly (Coleoptera, Chrysomelidae) from China

We studied the spore morphology and molecular systematics of a novel microsporidian isolate from Phyllobrotica armata Baly collected in China. The spores were long-oval and measured 4.7 × 2.6 μm on fresh smears. Ultrastructure of the spores was characteristic for the genus Nosema: 13-14 polar filament coils, posterior vacuole, and a diplokaryon. The complete rRNA gene sequence of the isolate was 4308 bp long. The organization of the rRNA gene was 5′-LSU rRNA-ITS-SSU rRNA-IGS-5S-3′, which corresponds to that of the Nosema species. Phylogenetic analysis based on the rRNA gene sequence indicated that this isolate, designated as Nosema sp. PA, is closely related to Nosemabombycis and is correctly assigned to the “true” Nosema group.     

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported AlocSWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. AlocSWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT‐PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.     

Systematics of African lowland rainforest Praomys (Rodentia, Muridae) based on molecular and craniometrical data

Representatives of the genus Praomys occur throughout the African intertropical zone. It is unclear how many species this genus contains, nor do we know the exact distribution ranges and phylogenetic relationships of these taxa. Using molecular (16S rRNA gene sequencing) and morphological (multivariate craniometry) analyses we clarify the taxonomy and phylogenetic relationships among the Praomys occurring in Africa's lowland tropical rainforests. We studied most species known from this area, based on specimens collected in seven countries (Guinea, Ivory Coast, Cameroon, Central African Republic, Gabon, Congo Republic and Democratic Republic of Congo). In our study, Praomys appears to be monophyletic. Our results identify two species complexes: the jacksoni complex includes at least two species ( P. jacksoni and P. mutoni ) and the tullbergi complex contains at least four species ( P. tullbergi , P. rostratus , P. misonnei , P. petteri ). Although the 16S rRNA gene appears insufficient to resolve the phylogenetic relationships among all the members of the tullbergi species complex, it is suitable for the identification of most of the studied species, and its use has allowed us to redefine the geographical limits of several species.  © 2005 The Linnean Society of London, Zoological Journal of the Linnean Society , 2005, 145 , 539–553.     

Revision of Phymatolithon purpureum (Hapalidiales,Rhodophyta) based on ultrastructural and molecular data

ABSTRACT

To elucidate the taxonomy and phylogeny of Phymatolithon purpureum (P. Crouan & H. Crouan) Woelkerling & L.M. Irvine, we observed the type specimens and fresh samples using SEM (ultra-morphology) and analysed DNA sequences. Phymatolithon purpureum was originally described as Lithothamnion purpureum P. Crouan & H. Crouan from Mingant, Brittany, France. Our molecular phylogenetic analyses using psbA and COI–5P regions placed our collections from Ireland, UK and France in a clade with ‘P. borealis W.H. Adey, J.J. Hernandez-Kantun & P.W. Gabrielson (MH252286)’ from Iceland and ‘uncultured Corallinales (GQ917711 and GQ917512)’ from Roscoff, Brittany, France, near the type locality of P. purpureum. We show that P. purpureum is conspecific with P. borealis and P. polymorphum f. papillatum (Foslie) Foslie based on morphology and molecular data. Also, although P. purpureum has been often confused with P. laevigatum (Foslie) Foslie because of their similar morphology (e.g. smooth surface and sunken tetra/bisporangial conceptacles), our molecular phylogenetic analyses indicate that P. purpureum and P. laevigatum are sister taxa. Our sequences from lectotype material of P. laevigatum and syntype material of Lithothamnion emboloides Heydrich are identical.     

Transfer of the members of the genus Brachiola (microsporidia) to the genus Anncaliia based on ultrastructural and molecular data

Two microsporidian genera, AnncaliiaIssi, Krylova, & Nicolaeva 1993 and BrachiolaCali et al. 1998, possess a Nosema-type life cycle and unique cell surface ornamentations, which include precocious electron-dense coating of the plasmalemma and a variety of secretory structures deposited on the parasite surface and scattered in the host cell cytoplasm. Comparative analysis of ultrastructure of Anncaliia meligethi (the type species of the genus Anncaliia) and of B. vesicularum and B. algerae (the best-studied members of the genus Brachiola) clearly demonstrated that these microsporidia share many distinctive morphological features. The comparison of small subunit ribosomal DNA sequences showed high sequence identity of A. meligethi and B. algerae. Phylogenetic analyses indicated that the rDNA sequences of A. meligethi clustered with those of B. algerae suggesting a close relatedness of these microsporidia. The combination of molecular and morphological data provided clear evidence that these microsporidia belong to the same genus and therefore, warranted emendation of the genus Anncaliia and establishments of the following new combinations: Anncaliia vesicularum nov. comb., Anncaliia algerae nov. comb., Anncaliia connori nov. comb., and Anncaliia gambiae nov. comb. The generic name Brachiola is submerged according to the rule of priority.     

Morphology and phylogeny of a new urostylid ciliate, Monocoronella carnea n. g., n. sp. (Ciliophora, Hypotricha) from Daya Bay, Southern China

The morphology, infraciliature and small subunit ribosomal RNA gene-based phylogeny of an urostylid ciliate, Monocoronella carnea n. g., n. sp., found in coastal areas off Daya Bay, Southern China, were investigated. The new genus Monocoronella n. g. is recognized by the following features: having conspicuous frontal cirri forming a long and single corona; buccal and frontoterminal cirri present; single marginal row on each side; adoral zone, midventral complex and transverse cirri in Pseudokeronopsis mode. The type species M. carnea n. sp. is diagnosed by the combination of marine habitat and brown-reddish color of the cortical granules. Phylogenetic analyses for the new taxon indicate that Monocoronella n. g. is most closely related to Bergeriella, and is located within the core Urostylida clade. A misidentification in previous literature was recognized and a new species, Monocoronella dragescoi n. sp. [Basionym: Holosticha (Keronopsis) monilata (Kahl 1928) sensu Dragesco (1970) et sensu Dragesco and Dragesco-Kernéis (1986), non sensu Kahl (1928)], was suggested.     

Establishment of Liebermannia dichroplusae n. comb. on the basis of molecular characterization of Perezia dichroplusae Lange, 1987 (Microsporidia)

Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania-Orthosomella branch forms a clade with the Endoreticulatus-Cystosporogenus-Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia.     

Hymenidium pilosum (Apiaceae) is a synonym of H. apiolens based on morphology and molecular data

Hymenidium apiolens and H. pilosum have been recorded to co-occur in the Himalayas, and it was found difficult to distinguish between them in the field. In this study, we reconstructed the phylogeny and estimated the genetic distance between individuals of H. apiolens, H. pilosum and related species based on nuclear ITS sequences, and rpl16 and rps16 introns, to determine the relationship between H. apiolens and H. pilosum. The analyses suggest that H. apiolens and H. pilosum are nested together, and that the ratio of genetic distance between them is smaller than that between individuals of Chamaesium spatuliferum, Hansenia forbesii or Ligusticum acuminatum. Furthermore, we found similar morphological characteristics of bracteoles, rays, petals and mericarps in H. apiolens and H. pilosum. Consequently, both molecular and morphological evidence supports that Hymenidium pilosum is a synonym of H. apiolens.     

The phylogenetic position of Ovavesicula popilliae (Microsporidia) and its relationship to Antonospora and Paranosema based on small subunit rDNA analysis

Comparative small subunit rDNA sequence analyses, indicate that Ovavesicula popilliae, a microsporidian parasite of the Japanese beetle, Popillia japonica, represents a distant sister group to Paranosema and Antonospora. These three genera represent a second major group (the Nosema/Vairimorpha clade representing the first) of Microsopridia which infect terrestrial insects, suggesting independent origins for both groups. Phylogenetic analyses of Ovavesicula and other Microsporidia having a multi-sporous sporogony reveal that this condition is found in several unrelated taxa implying either that multi-sporous sporogony is the ancestral condition for Microsporidia or that it has multiple origins.     

Description and Phylogenetic Relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida)

ABSTRACT. Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys ), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata ). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.     

Jirovecia branchilis n. sp. (Microsporidia) from glands of Branchiura sowerbyi (Oligochaeta: Tubificidae) in China

Jirovecia species primarily infect oligochaetes and are typically characterized by large rod-shaped spores with a tail-like posterior prolongation. Presently, seven Jirovecia spp. are reported worldwide with only one described in China. Here, a new species, Jirovecia branchilis n. sp. was discovered in glands of oligochaetes Branchiura sowerybi Beddard, 1892 in China. Jirovecia branchilis n. sp. elicited the formation of numerous opaque xenomas of 0.12 to 0.20 mm (n = 30) in diameter. Electron microscopic observations demonstrated that the earliest developmental stages observed were uninucleate meronts residing directly with the host cytoplasm. Mature spores were rod-shaped with blunt ends and possessed a collar-like anchoring disk, a manubrium-type polar filament, a bipartite polarplast, and a three-layered spore wall. A tail-like prolongation was distinctly observed in the posterior of spores and measured 13.2–28.6 μm long (n = 30). Jirovecia branchilis n. sp. showed 98.54% sequence similarity with Janacekia tainunus isolated from the fat body of chironomidae larvae Kiefferulus tainanus based on obtained partial SSU rDNA gene sequence, but was significantly different in morphology, host, and infection sites. SSU rDNA-based phylogenetic analysis indicated Jirovecia branchilis n. sp. clustered with Janacekia tainanus within the Jirovecia-Bacillidium-Janacekia clade. In conclusion, a new species within Jirovecia, Jirovecia branchilis n. sp. is erected herein based mainly on its morphological, ecological, and to a lesser degree on its molecular characteristics. The whole relationship between Jirovecia spp., Janacekia spp., and Bacillidium spp. is in need of revision and could potentially be elucidated by using additional makers and sequencing a broader diversity of the already described species.     

Systenostrema alba Larsson 1988 (Microsporidia, Thelohaniidae) in the Dragonfly Aeshna viridis (Odonata, Aeshnidae) from South Siberia: morphology and molecular characterization

An octospore microsporidium was found in the nymphs of Aeshna viridis, collected in intermittent streams near Novosibirsk, Siberia, Russia in 2003. Spores were uninucleate and measured 6.1+/-0.07 x 3.0+/-0.04 microm on fresh smears. The polar filament was anisofilar having 10-11 anterior coils (thicker filament diam.) and 10-11 posterior (thinner filament diam.) coils. Sporophorous vesicles were persistent and measured 12.3+/-0.23 x 11.9+/-0.20 microm. The infection was restricted to the adipose tissue and caused the formation of whitish "cysts" containing mature octospores. Based on ultrastructural similarity we consider this Siberian isolate to be Systenostrema alba, a species described from Aeshna grandis collected in Sweden (Larsson 1988). Maximum likelihood, neighbor joining, and maximum parsimony analyses of the small subunit rDNA all placed Systenostrema alba (Accession no. AY953292) as the sister taxon to a clade consisting of Thelohania solenopsae, Tubulinosema ratisbonensis, and Tubulinosema acridophagus.     

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in ciliophora

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.     

Molecular and Morphological Characterization of Anncaliia azovica sp. n. (Microsporidia) Infecting Niphargogammarus intermedius (Crustacea,Amphipoda) from the Azov Sea

Five out of one hundred adults of Niphargogammarus intermedius caught at the Azov sea shore were found to be infected with microsporidia. The infection was found in the subcuticular fat body and myocytes. Parasites developed in direct contact with host cells, displayed a disporoblastic sporogony and diplokaryotic arrangement of nuclei at all stages. Spores were oval, 4.6–5.8 × 2.6–3.0 μm. Exospore appendages, vesicular–tubular secretions, and the anisofilar polar filament indicated a similarity to Anncaliia species. Sporont surfaces displayed ridges of amorphous material. Meronts and sporonts formed protoplastic extensions, similar to A. vesicularum and A. meligheti. Mature spores possessed a bipartite polaroplast. The polar tube was arranged in one row of 13–18 coils including 0–3 distal coils of lesser diameter. Partial sequencing of SSU, ITS, and LSU regions of rRNA gene (GenBank accessions: KY288064 – KY288065 ) confirmed this new species to be congeneric with A. algerae (# AF069063 ) and A. meligheti (# AY894423 ). The SSU gene of this novel microsporidium shared 99.4% sequence similarity to A. algerae and 98.9% to A. meligheti. Genes for HSP70 and RPB1 amplified with primers designed for A. algerae orthologs displayed 99.7% and 97.4% similarity, respectively, between A. algerae and the novel microsporidium. A new species, Anncaliia azovica, is described based on morphological and molecular characterization.     

Morphological and molecular characterization of two Trichodina (Ciliophora,Peritrichia) species from freshwater fishes in China

In the present study, we provide morphological and molecular characterization of two Trichodina species, T. acuta Lom, 1970 and T. funduli Wellborn, 1967, isolated from koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), respectively. Morphological characters of the two Trichodina species were mainly investigated on the basis of dry silver nitrate-impregnated specimens. Both species are medium-sized and possess well-developed denticles comprising strongly sickle-shaped blades, well-developed central parts, and straight rays. Trichodina acuta can be easily distinguished from the other Trichodina species that possess a clear central circle by the well-developed sharp blade apophysis, and the gap between ray tip and central circle. Trichodina funduli is a poorly known species that is easily confused with T. heterodentata Duncan, 1977, however the latter species has thinner denticles. The small subunit ribosomal RNA gene sequences of Trichodina acuta and T. funduli were incorporated into phylogenetic analyses. Our findings suggest that the phylogenetic lineage of trichodinids might not correspond with their living environments, host species or even some morphological characteristics.     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.