Structure of ribosomal RNA gene and phytogeny ofNosema isolates in Korea

The ribosomal RNA (rRNA) gene region of the fourNosema sp. isolates (C01, C02, C03 and C04) fromPieris rapae in Korea has been examined. Complete DNA sequence data (3779 bp) of The rRNA gene ofNosema sp. C01 are presented for the small subunit gene (SSU rRNA: 1236 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA 2506 bp). The secondary structures ofNosema sp. COI SSU and LSU rRNA genes are constructed and described. The SSU rRNA showed a hypervariable V4 region identified four additional stems including a pseudoknot. Phylogenetic analysis based on the SSU rRNA suggests that the four isolates belong to the ‘true’Nosema group. In contrast to theNosema/Vairimorpha clade, the members of the group are highly divergent.     

Chimerical nature of the ribosomal RNA gene of a Nosema species

Taxonomic resolution of the Nosema/Vairimorpha clade has been augmented with DNA sequences of the small subunit (SSU) and large subunit (LSU) ribosomal RNA (rRNA) and the arrangement of SSU and LSU. Based on the two characteristics, the clade is largely divided into two, i.e. ‘true’ Nosema sub-group and non-‘true’ Nosema sub-group within the clade. Our study shows that a novel Nosema species isolated from Pieris rapae has mixed characteristics of the ‘true’ and non-‘true’ Nosema sub-group based on the topology of SSU and LSU sequences. To our knowledge, this may be the first case of the incongruent phylogenetic placement of SSU and LSU in the Nosema/Vairimorpha clade. Additionally, the length of internal transcribed spacer (ITS) can be a diagnostic tool to distinguish ‘true’ Nosema from non-’true’ Nosema in the Nosema/Vairimorpha clade based on its nucleotide length as reported before.     

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, <Emphasis Type="Italic">Spodoptera litura</Emphasis> (Lepidoptera: Noctuidae), in Vietnam

In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.     

Prevalence of Nosema species infections in Apis cerana japonica and Apis mellifera honeybees in the Tohoku region of Japan

We investigated here, the prevalence of Nosema microsporidia infections in the honeybees, Apis cerana japonica and Apis mellifera, in the Tohoku region of Japan. We detected Nosema ceranae DNA in 14 (2.8%) of 509 A. cerana japonica and in 34 (21.9%) of 155 A. mellifera honeybees from Aomori, Iwate, Akita, Yamagata, and Fukushima prefectures. Nosema apis DNA was undetectable in A. cerana japonica and A. mellifera. The unidentifiable Nosema species that genetically differed from N. apis, N. ceranae, and N. neumanni in terms of small subunit (SSU) rDNA, large subunit rDNA, and internal transcribed spacer sequences was identified in 105 (20.6%) of 509 A. cerana japonica and in 1 (0.6%) of 155 A. mellifera honeybees, and from Iwate prefecture. A phylogenetic tree based on SSU rDNA sequences showed that the Nosema sp. belonged to the same clade as N. thomsoni detected in moth and solitary bees in North America and N. pieriae found in cabbage butterfly in Turkey, which have not hitherto been detected in honeybees. The morphological characteristics of the spores should be analyzed to enable species identification of the Nosema sp.     

α‐ and β‐Tubulin Phylogenies Support a Close Relationship Between the Microsporidia Brachiola algerae and Antonospora locustae

ABSTRACT. Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced α‐ and β‐tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein‐based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.     

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.     

Using the SSU,ITS, and Ribosomal DNA Operon Arrangement to Characterize Two Microsporidia Infecting Bruce Spanworm,Operophtera bruceata (Lepidoptera: Geometridae)

Research pertaining to the two closely‐related microsporidian genera Nosema and Vairimorpha is hindered by inconsistencies in species differentiation within and between the two clades. One proposal to better delimit these genera is to restructure the Nosema around a “True Nosema” clade, consisting of species that share a characteristic reversed ribosomal DNA operon arrangement and small subunit (SSU) ribosomal DNA sequences similar to that of the Nosema type species, N. bombycis. Using this framework, we assess two distinct microsporidia recovered from the forest insect Bruce spanworm (Operophtera bruceata) by sequencing their SSU and internal transcribed spacer regions. Phylogenetic analyses place one of our isolates within the proposed True Nosema clade close to N. furnacalis and place the other in the broader Nosema/Vairimorpha clade close to N. thomsoni. We found that 25% of Bruce spanworm cadavers collected over the four‐year study period were infected with microsporidia, but no infections were detected in cadavers of the Bruce spanworm's invasive congener, the winter moth (O. brumata), collected over the same period. We comment on these findings as they relate to the population dynamics of the Bruce spanworm‐winter moth system in this region, and more broadly, on the value of ribosomal DNA operon arrangement in Nosema systematics.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.     

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.     

Blastodinium galatheanum sp. nov. (Dinophyceae) a parasite of the planktonic copepod Acartia negligens (Crustacea,Calanoida) in the central Atlantic Ocean

A new dinoflagellate species, Blastodinium galatheanum sp. nov., was found parasitizing the planktonic copepods Acartia negligens and Acartia sp. in the Atlantic Ocean between the Azores and the Cape Verde Islands. These copepods have not previously been reported hosting a Blastodinium species. Characters that distinguish the new species are the shape and size of the trophic stage, its host species, and its predominantly solitary existence. Dinospores of Blastodinium galatheanum sp. nov. are peridinioid in nature and morphologically indistinguishable from dinospores of two other previously investigated Blastodinium species. SSU rRNA gene sequences from two isolates of this new species were almost identical and showed similarities to SSU rRNA sequences of other species of Blastodinium. A phylogenetic analysis based on SSU rRNA gene sequences suggested monophyly for all existing sequences of Blastodinium spp., including a sequence from the type species B. pruvoti, presented here for the first time.     

Cloning and expression profiles of tumor suppressor QM homologue in response to granulovirus in Pieris rapae

The tumor suppressor, QM, has been cloned and characterized from various model organisms such as human, plant and invertebrates. Yet, it has not been seriously investigated for its role in conjunction with antiviral mechanisms involving innate insect immunity. From the expressed sequence tag (ESTs) project, conducted with larval cDNA library of cabbage butterfly, Pieris rapae, a partial fragment (718 bp) of QM homologue, termed PrQM containing 660 bp long open reading frame (ORF) encoding protein of 219 amino acids was identified. In silico analysis of PrQM ORF revealed the presence of ribosomal protein L10a/L10e type domain. Phylogenetic analysis of the P. rapae QM‐like protein showed high amino acid sequence similarity with other PrQM polypeptides identified from Heliothis virescenes (95%), Plutella rapae (92%), Bombyx mori (92%), Drosophila melanogaster (89%), and Polyrhachis vicina (85%). The butterfly QM has the closest phylogenetic relationship to a moth (Hv) QM homologue. Further investigations revealed the expression of PrQM at all developmental stages, with pronounced presence at the egg stage. In addition, spatial pattern analysis indicated its high expression in the head, salivary gland, integument and fat body with visible presence in Malpighian tubule and gut. Time course expression studies conducted after immune‐challenge with lipoteichoic acid (LTA) showed the induction of PrQM mRNA at 12 h and 24 h after challenge and also in response to granulovirus (GV). Results of this investigation therefore suggest possible role of QM‐like proteins from Pieris rapae to be involved in innate antiviral immune responses. Further elucidation on the precise function of PrQM during antiviral immune responses by using RNA interference remains a viable research front.     

Complete rRNA sequence, arrangement of tandem repeated units and phylogeny of Nosema fumiferanae from spruce budworm, Choristoneura fumiferana (Clemens)

We provide molecular systematics of a microporidian species, Nosema fumiferanae, one of the most common natural enemies of spruce budworm, Choristoneura fumiferana. The uncharacterized flanking region upstream of the large subunit (LSU) rRNA and the complete rRNA cistron of N. fumiferanae was 4,769 bp long. The organization of the rRNA gene was 5′‐LSU rRNA‐ITS‐SSU rRNA‐IGS‐5S‐3′ and corresponded primarily to most insect (i.e. lepidopteran) Nosema species identified and classified to date. Phylogenetic analysis based on the complete rRNA cistron indicated that N. fumiferanae is closely related to Nosema plutellae and is correctly assigned to the “true” Nosema group. Suggestions were provided on a criterion to delineate the “true” Nosema from other microsporidian species.     

MOLECULAR ANALYSIS REVEALS CRYPTIC DIVERSITY IN PORPHYRA (RHODOPHYTA)1

The small subunit ribosomal RNA (SSU rRNA) gene was amplified from 15 species of the red alga Porphyra and digested with restriction enzymes to generate data for species identification. The subset of species selected for phylogenetic analysis was P. cuneiforms (Setchell et Hus) Krishnamurthy, P. nereocystis anderson, P. schizophylla Hollenberg et Abbott, P. thuretii Setchell et Dawson and Porphyra 1674. Bangia sp. was used as an out-group. Restriction sites were mapped and used as characters in parsimony and maximum likelihood analysis. The phylogenetic hypotheses generated were compared statistically to possible alternative phylogenies based on traditional morphological taxonomic characters. The results indicate that the current subgenera in Porphyra do not represent monophyletic groups and that traditional morphological and ecological taxonomic characters alone may not be adequate for definitive species identification and cannot be relied on as an indication of Porphyra have large insertions in the SSU gene that are apparently splicesd from the final SSU rRNA molecule. The possible character, distribution and potential significance of these putative introns are discussed.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

Molecular cloning and expression patterns of FK506‐binding protein 12, an immunophilin from the cabbage butterfly,Pieris rapae

FK506‐binding protein (FK506BP) class belonging to immunophilin protein family has been known to play key roles in modulating T‐cell activation, regulation of cell cycle and protein folding. However, little is known about the involvement of FK506BP during viral pathogenesis in insect host. In this study, an attempt has been made to focus on the involvement of FK506BP in antiviral innate immunity, by cloning the full‐length cDNA of FK506BP12 (PrFK506BP12) from the cabbage butterfly, Pieris rapae. It comprised of 532 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 327 bp encoding 108 amino acids. In silico analysis of PrFK506BP12 ORF revealed a highly conserved FK506‐binding domain (FKBD). As expected, it showed high homology to other FK506BPs identified from Bombyx mori (92%), Manduca sexta (91%), Suberites domuncula (82%), Tribolium castaneum (81%) and Aedes aegypti (74%) . Expression of PrFK506BP12 was observed during developmental stages of P. rapae, but was pronounced in late pupal and adult stage. In addition, spatial expression pattern analysis indicated its high expression in the head and fat body. Furthermore, PrFK506BP12 mRNA was induced 12 h after LTA, Poly I:C treatment and 3h after Pieris rapae granulovirus (PrGV) treatment in carcass. It suggests that PrFK506BP12 appears to be involved in immune responses and also play an important role in the fat body, although it remains to be clarified about their precise role in response to granulovirus.     

Conservation de virus et de bactéries entomopathogènes sous forme de comprimés

Zusammenfassung Polyederviren vonLymantria dispar undBombyx mori, Granuloseviren vonPieris brassicae, sowieBacillus thuringiensis (alesti) — Sporen mit Toxinkristallen wurden in Tablettenform aufbewahrt. Ihre Infektiosit?t hat in 5 Jahren nicht abgenommen. Verwendungsm?glichkeiten in der biologischen Sch?dlingsbek?mpfung werden besprochen.      

The complete nucleotide sequence of the mitochondrial genome of <Emphasis Type="Italic">Phthonandria atrilineata</Emphasis> (Lepidoptera: Geometridae)

Using long-polymerase chain reaction (Long-PCR) method, we determined the complete nucleotide sequence of the mitochondrial genome (mitogenome) of Phthonandria atrilineata. The complete mtDNA from P. atrilineata was 15,499 base pairs in length and contained 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The P. atrilineata genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species. The nucleotide composition of P. atrilineata mitogenome was biased toward A + T nucleotides (81.02%), and the 13 PCGs show different A + T contents that range from 73.25% (cox1) to 92.12% (atp8). Phthonandria had the canonical set of 22 tRNA genes, that fold in the typical cloverleaf structure described for metazoan mt tRNAs, with the unique exception of trnS(AGN). The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, which confirmed that P. atrilineata is most closely related to the superfamily Bombycoidea.