Morphological and molecular studies of a microsporidium (Nosema sp.) isolated from the thee spot grass yellow butterfly, Eurema blanda arsakia (Lepidoptera: Pieridae)

A microsporidium possessing molecular and morphological characteristics of the genus Nosema was isolated from larvae of the thee-spot grass yellow butterfly, Eurema blanda arsakia. The complete rRNA gene sequences of the E. blanda isolate contained 4,428 base pairs (GenBank Accession No. EU338534). The organization of the rRNA genes is LSU rRNA-ITS-SSU rRNA-IGS-5S, which corresponds with that of Nosema species closely related to Nosema bombycis. Phylogenetic analysis based on rRNA gene sequences show that this isolate is closely related to Nosema bombycis, Nosema plutellae, Nosema spodopterae, and Nosema antheraeae. The ultrastructure of all developmental stages of this microsporidium confirmed its placement in the genus Nosema. The isolate was successfully propagated in cell lines IPLB-LD652Y (Lymantria dispar) and NTU-LY (Lymantria xylina) and, in the in vitro system, it was frequently found to develop in the nuclei of the host cells, a circumstance that seldom occurs in other Nosema species. An extra-cellular vegetative stage of this microsporidium was also observed in the culture medium after 14 days of infection. The ECMDFs might be released from disrupted host cells.     

Phylogenetic Analysis of Complete rRNA Gene Sequence of Nosema philosamiae Isolated from the Lepidopteran Philosamia cynthia ricini

ABSTRACT. The microsporidian Nosema philosamiae is a pathogen that infects the eri‐silkworm Philosamia cynthia ricini. The complete sequence of rRNA gene (4,314 bp) was obtained by polymerase chain reaction amplification with specific primers and sequencing. The sequence analysis showed that the organization of the rRNA of N. philosamiae was similar to the pattern of Nosema bombycis. Phylogenetic analysis of rRNA gene sequences revealed that N. philosamiae had a close relationship with other Nosema species, confirming that N. philosamiae is correctly assigned to the genus Nosema.     

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.     

Nosema maddoxi sp. nov. (Microsporidia,Nosematidae), a Widespread Pathogen of the Green Stink Bug Chinavia hilaris (Say) and the Brown Marmorated Stink Bug Halyomorpha halys (Stål)

We describe a unique microsporidian species that infects the green stink bug, Chinavia hilaris; the brown marmorated stink bug, Halyomorpha halys; the brown stink bug, Euschistus servus; and the dusky stink bug, Euschistus tristigmus. All life stages are unikaryotic, but analysis of the consensus small subunit region of the ribosomal gene places this microsporidium in the genus Nosema, which historically has been characterized by diplokaryotic life stages. It is also characterized by having the reversed arrangement of the ribosomal gene (LSU –ITS‐ SSU) found in species within the “true Nosema” clade. This microsporidium is apparently Holarctic in distribution. It is present in H. halys both where it is native in Asia and where it is invasive in North America, as well as in samples of North American native C. hilaris collected prior to the introduction of H. halys from Asia. Prevalence in H. halys from mid‐Atlantic, North America in 2015–2016 ranged from 0.0% to 28.3%, while prevalence in C. hilaris collected in Illinois in 1970–1972 ranged from 14.3% to 58.8%. Oral infectivity and pathogenicity were confirmed in H. halys and C. hilaris. Morphological, ultrastructural, and ecological features of the microsporidium, together with a molecular phylogeny, establish a new species named Nosema maddoxi sp. nov.     

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.     

High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.     

Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.     

Molecular data and phylogeny of Nosema infecting lepidopteran forest defoliators in the genera Choristoneura and Malacosoma

ABSTRACT. Nosema isolates from five lepidopteran forest defoliators, Nosema fumiferanae from spruce budworm, Choristoneura fumiferana ; a Nosema sp. from jack pine budworm, Choristoneura pinus pinus and western spruce budworm, Choristoneura occidentalis ( Nosema sp. CPP and Nosema sp. CO, respectively); Nosema thomsoni from large aspen tortrix, Choristoneura conflictana ; and Nosema disstriae , from the forest tent caterpillar, Malacosoma disstria were compared based on their small subunit (SSU) ribosomal RNA (rRNA) gene sequences. Four of the species sequenced, N. fumiferanae , Nosema sp. CPP, Nosema sp. CO, and N . disstriae have a high SSU rDNA sequence identity (0.6%–1.5%) and are members of the "true Nosema " clade. They all showed the reverse arrangement of the (large subunit [LSU]–internal transcribed spacer [ITS]–SSU) of the rRNA gene. The fifth species, N. thomsoni has the usual (SSU–ITS–LSU) arrangement and is not a member of this clade showing only an 82% sequence similarity. We speculate, therefore, that a genetic reversal may have occurred in the common ancestor to the "true Nosema " clade. Although, the mechanism for rearrangement of the rRNA gene subunits is not known we provide a possible explanation for the localization. N. fumiferanae , Nosema sp. CPP, and Nosema sp. CO clustered together on the inferred phylogenetic tree. The high sequence similarities, the reverse arrangement in the rRNA gene subunits, and the phylogenetic clustering suggest that these three species are closely related but separate species.     

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.     

Honeybee glands as possible infection reservoirs of Nosema ceranae and Nosema apis in naturally infected forager bees

Aims: To determine whether Nosema ceranae and Nosema apis are present in different gland tissues of honeybee, Apis mellifera L. and to monitor spore presence and quantity in these glands in naturally infected hives from July 2009 to July 2010 in Quebec, Canada. Methods and Results: Nosema spp. were quantified using duplex quantitative real‐time PCR in the thoracic salivary, hypopharyngeal, mandibular glands, and venom sac and glands of A. mellifera over a period of 8 months. Both Nosema species were present in all the glands as single or mixed species; however, N. apis was not present as single‐species detections in the salivary glands (see Table 2). Nosema ceranae was more prevalent throughout the 8 months. Significant correlative relationships were established for N. ceranae and N. apis levels in the honeybee glands and those found within the intestines of forager honeybees. Overall, the seasonality of N. ceranae and N. apis in the different glands tightly followed the seasonal patterns in the honeybee guts. Conclusions: Nosema ceranae and N. apis are not tissue specific, and honeybee glands have potential to become a useful indicator of the extent of disease in the colony and may represent a potential infection reservoir. Significance and Impact of the Study: First report of spore load quantification of Nosema spp. in different honeybee glands.     

Prevalence,transmission and mortality associated with Nosema fumiferanae infections in field populations of spruce budworm Choristoneura fumiferana

• 1 The prevalence, intensity and transmission of Nosema fumiferanae (Thomson) (Microsporidae) infections and potential impacts on the survival of field populations of spruce budworm Choristoneura fumiferana (Clem.) were examined in three plots in New Brunswick, Canada, from 1983 to 1992.
• 2 The highest prevalence of N. fumiferanae infection in post‐hibernation second‐instar larvae occurred in the plot where prevalence in female pupae was the highest in the previous generation, suggesting higher rates of vertical transmission. There was little change in the prevalence of N. fumiferanae infections between the second and sixth instars in the later generations. In the two other plots, N. fumiferanae prevalence increased by approximately 25% from the second to sixth larval stadia. Coincident with the changes in N. fumiferanae prevalence were substantial declines in the populations of spruce budworms, making it difficult to determine rates of horizontal transfer of the disease.
• 3 In all plots and in all years, there were progressive increases in the intensity of N. fumiferanae infections (spore loads/individual) from the second to sixth instars and pupae.
• 4 Annual spruce budworm mortality associated with N. fumiferanae was ≤15% of all mortality in reared specimens and was positively correlated with but generally less than 30% of annual N. fumiferanae prevalence.

     

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.     

Morphological,Molecular, and Phylogenetic Characterization of Nosema ceranae,a Microsporidian Parasite Isolated from the European Honey Bee,Apis mellifera1

ABSTRACT. Nosema ceranae, a microsporidian parasite originally described from Apis cerana, has been found to infect Apis melllifera and is highly pathogenic to its new host. In the present study, data on the ultrastructure of N. ceranae, presence of N. ceranae-specific nucleic acid in host tissues, and phylogenetic relationships with other microsporidia species are described. The ultrastructural features indicate that N. ceranae possesses all of the characteristics of the genus Nosema. Spores of N. ceranae measured approximately 4.4 × 2.2 μm on fresh smears. The number of coils of the polar filament inside spores was 18–21. Polymerase chain reaction (PCR) signals specific for N. ceranae were detected not only in the primary infection site, the midgut, but also in the tissues of hypopharyngeal glands, salivary glands, Malpighian tubules, and fat body. The detection rate and intensity of PCR signals in the fat body were relatively low compared with other examined tissues. Maximum parsimony analysis of the small subunit rRNA gene sequences showed that N. ceranae appeared to be more closely related to the wasp parasite, Nosema vespula, than to N. apis, a parasite infecting the same host.     

Outcome of Colonization of Apis mellifera by Nosema ceranae

A multiplex PCR-based method, in which two small-subunit rRNA regions are simultaneously amplified in a single reaction, was designed for parallel detection of honeybee microsporidians (Nosema apis and Nosema ceranae). Each of two pairs of primers exclusively amplified the 16S rRNA targeted gene of a specific microsporidian. The multiplex PCR assay was useful for specific detection of the two species of microsporidians related to bee nosemosis, not only in purified spores but also in honeybee homogenates and in naturally infected bees. The multiplex PCR assay was also able to detect coinfections by the two species. Screening of bee samples from Spain, Switzerland, France, and Germany using the PCR technique revealed a greater presence of N. ceranae than of N. apis in Europe, although both species are widely distributed. From the year 2000 onward, statistically significant differences have been found in the proportions of Nosema spp. spore-positive samples collected between and within years. In the first period examined (1999 to 2002), the smallest number of samples diagnosed as Nosema positive was found during the summer months, showing clear seasonality in the diagnosis, which is characteristic of N. apis. From 2003 onward a change in the tendency resulted in an increase in Nosema-positive samples in all months until 2005, when a total absence of seasonality was detected. A significant causative association between the presence of N. ceranae and hive depopulation clearly indicates that the colonization of Apis mellifera by N. ceranae is related to bee losses.     

BIOGEOGRAPHIC ANALYSIS OF THE GLOBALLY DISTRIBUTED HARMFUL ALGAL BLOOM SPECIES ALEXANDRIUM MINUTUM (DINOPHYCEAE) BASED ON rRNA GENE SEQUENCES AND MICROSATELLITE MARKERS1

The toxic dinoflagellate Alexandrium minutum Halim is one of three species that comprise the “minutum” species complex. This complex is notable due to its role in the etiology of paralytic shellfish poisoning (PSP). Recent increases in PSP incidence and the geographic expansion of toxin‐producing Alexandrium dinoflagellates have prompted the intensive examination of genetic relationships among globally distributed strains to address questions regarding their present distribution and reasons for their apparent increase. The biogeography of A. minutum was studied using large subunit ribosomal DNA gene (LSU rRNA) and internal transcribed spacer (ITS) sequences and genotypic data from 12 microsatellite loci. rRNA gene and ITS sequencing data distinguished between two clades, herein termed the “Global” and the “Pacific”; however, little to no resolution was seen within each clade. Genotypic data from 12 microsatellite loci provided additional information regarding genetic relationships within the Global clade, but it was not possible to amplify DNA from the Pacific clade using these markers. With the exception of isolates from Italy and Spain, strains generally clustered according to origin, revealing geographic structuring within the Global clade. Additionally, no evidence supported the separation of A. lusitanicum and A. minutum as different species. With the use of microsatellites, it is now possible to initiate studies on the origin, history, and genetic heterogeneity of A. minutum that were not previously possible using only rRNA gene sequence data. This study demonstrates the power of combining a marker with intermediate resolution (rRNA sequences) with finer‐scale markers (microsatellites) to examine intraspecies variability among globally distributed isolates and represents the first effort to employ this technique in A. minutum.     

Occurrence and effects of Nosema fumiferanae infections on adult spruce budworm caught above and within the forest canopy

1 Nosema fumiferanae infections in populations of both sexes of spruce budworm Choristoneura fumiferana moths, collected live above the forest canopy (canopy moths), within the tree crown (crown moths) and in drop trays (dead moths), were examined over a 5‐year period in New Brunswick, Canada. 2 The incidence of infection and of moderate–heavy infections in canopy and crown moths of both sexes increased concomitantly with moth eclosion, indicating that N. fumiferanae retards larval/pupal development, with infected moths, particularly those having higher disease loads, emerging later in the season. 3 Infection rates differed among canopy, crown, and dead female, but not male, moths. Canopy (i.e. emigrating) females had a lower incidence of infection, lower incidence of moderate–heavy infections, and had longer forewings and higher dry weights, than crown females. These results suggest that N. fumiferanae infections negatively affect aspects of female, but not male, flight performance. Regardless of infection, forewing length and dry weight of both canopy and crown females declined over the moth flight period, but infected females in both moth types were smaller than their uninfected counterparts. Forewing lengths and dry weights of moderately–heavily infected females were most severely affected. 4 Despite high annual infection rates in parents, only a small percentage of offspring (second‐instar larvae) that established feeding sites each spring were infected, indicating that high rates of horizontal transmission occurred annually throughout the larval period. 5 The present study indicates that whether N. fumiferanae infections are a debilitating sublethal factor in spruce budworm populations depends more on the disease load than on the overall incidence of infection. The potential importance of N. fumiferanae infections on various fitness parameters related to host dispersal is discussed.     

Molecular characterization of the obligate endosymbiont "Caedibacter macronucleorum"Fokin and Görtz, 1993 and of its host Paramecium duboscqui strain Ku4-8

ABSTRACT. Bacterial endosymbionts of protozoa were often described as new species by protozoologists mainly on the basis of few morphological characters and partly by host specificity. Many of these species have never been validated by prokaryotic microbiologists whose taxonomic rules are quite different from those of protozoologists, who use the Zoological Code of Nomenclature. “Caedibacter macronucleorum” Fokin and Görtz 1993 , an endosymbiont of Paramecium duboscqui, belongs to this category. Here we provide the molecular characterization of this organism and of its host P. duboscqui strain Ku4‐8. Bacterial 16S rRNA gene sequence analysis proved that “C. macronucleorum” belongs to the Alphaproteobacteria. It is closely related to Caedibacter caryophilus but not to Caedibacter taeniospiralis, which belongs to the Gammaproteobacteria. “Caedibacter macronucleorum” and C. caryophilus 16S rRNA genes show a similarity value of 99%. This high 16S rRNA sequence similarity and the lack of a specific oligonucleotide probe for distinguishing the two endosymbionts do not allow validating “C. macronucleorum” as a provisional taxon (Candidatus). Nevertheless, “C. macronucleorum” and C. caryophilus can be easily discriminated on the basis of a highly variable stretch of nucleotides that interrupts the 16S rRNA genes of both organisms.     

Twenty-five-year study of Nosema spp. in honey bees (Apis mellifera) in Serbia

A total of 7386 samples of adult honey bees from different areas of Serbia (fifteen regions and 79 municipalities) were selected for light microscopy analysis for Nosema species during 1992–2017. A selection of honey bee samples from colonies positive for microsporidian spores during 2009–2011, 2015 and 2017 were then subjected to molecular diagnosis by multiplex PCR using specific primers for a region of the 16S rRNA gene of Nosema species. The prevalence of microsporidian spore-positive bee colonies ranged between 14.4% in 2013 and 65.4% in 1992. PCR results show that Nosema ceranae is not the only Nosema species to infect honey bees in Serbia. Mixed N. apis/N. ceranae infections were detected in the two honey bee samples examined by mPCR during 2017. The beekeeping management of disease prevention, such as replacement of combs and queens and hygienic handling of colonies are useful in the prevention of Nosema infection.     

TAXONOMIC STUDY OF TWO NEW GENERA OF FUSIFORM GREEN FLAGELLATES,TABRIS GEN. NOV. AND HAMAKKO GEN. NOV. (VOLVOCALES,CHLOROPHYCEAE)1

On the basis of LM, we isolated strains of two species of fusiform green flagellates that could be assigned to former Chlorogonium (Cg.) Ehrenb. One species, “Cg.”heimii Bourr., lacked a pyrenoid in its vegetative cells and required organic compounds for growth. The other was similar to Cg. elongatum (P. A. Dang.) Francé and “Cg.”acus Nayal, but with slightly smaller vegetative cells. Their molecular phylogeny was also studied based on combined 18S rRNA, RUBISCO LSU (rbcL), and P700 chl a‐apoprotein A2 (psaB) gene sequences. Both species were separated from Chlorogonium emend., Gungnir Nakada and Rusalka Nakada, which were formerly assigned to Chlorogonium. They were accordingly assigned to new genera, Tabris Nakada gen. nov. and Hamakko (Hk.) Nakada gen. nov. as T. heimii (Bourr.) Nakada comb. nov. and Hk. caudatus Nakada sp. nov., respectively. Tabris is differentiated from other genera of fusiform green flagellates by its vegetative cells, which only have two apical contractile vacuoles and lack a pyrenoid in the chloroplast. Hamakko, on the other hand, is distinguishable by the fact that its pyrenoids in vegetative cells are penetrated by flattened thylakoid lamellae.