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.     

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.     

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.     

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.     

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.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

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.     

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.     

Nosema ceranae infection in honeybee samples from Tuscanian Archipelago (Central Italy) investigated by two qPCR methods

Nosema apis and Nosema ceranae are microsporidian parasite worldwide spread causing an emerging infectious disease of European honeybee Apis mellifera. The Nosema presence was deeply investigated in several countries but low information are presents about islands. In this investigation was evaluated the presence N. ceranae and N. apis in apiaries located in Tuscanian Archipelago islands (Central Italy). For N. ceranae detection, two different Real-Time PCR (qPCR) methods, the 16S rRNA and Hsp70 gene amplification qPCR, were performed on honey bee samples; while, for N. apis only the 16S rRNA qPCR amplification was performed. On all islands, only N. ceranae was present, while N. apis was not found in the samples. The two qPCR showed significant difference (p < 0.040) in N. ceranae spores quantification. The single-copy Hsp70 gene method qPCR assay systematically detected a lower amount of N. ceranae copies compared to the multi-copy 16S rRNA gene method.     

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.     

Asymmetrical coexistence of Nosema ceranae and Nosema apis in honey bees

Globalization has provided opportunities for parasites/pathogens to cross geographic boundaries and expand to new hosts. Recent studies showed that Nosema ceranae, originally considered a microsporidian parasite of Eastern honey bees, Apis cerana, is a disease agent of nosemosis in European honey bees, Apis mellifera, along with the resident species, Nosema apis. Further studies indicated that disease caused by N. ceranae in European honey bees is far more prevalent than that caused by N. apis. In order to gain more insight into the epidemiology of Nosema parasitism in honey bees, we conducted studies to investigate infection of Nosema in its original host, Eastern honey bees, using conventional PCR and duplex real time quantitative PCR methods. Our results showed that A. cerana was infected not only with N. ceranae as previously reported [Fries, I., Feng, F., Silva, A.D., Slemenda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol. 32, 356-365], but also with N. apis. Both microsporidia produced single and mixed infections. Overall and at each location alone, the prevalence of N. ceranae was higher than that of N. apis. In all cases of mixed infections, the number of N. ceranae gene copies (corresponding to the parasite load) significantly out numbered those of N. apis. Phylogenetic analysis based on a variable region of small subunit ribosomal RNA (SSUrRNA) showed four distinct clades of N. apis and five clades of N. ceranae and that geographical distance does not appear to influence the genetic diversity of Nosema populations. The results from this study demonstrated that duplex real-time qPCR assay developed in this study is a valuable tool for quantitative measurement of Nosema and can be used to monitor the progression of microsprodian infections of honey bees in a timely and cost efficient manner.     

Estimation of the influence of selected products on co-infection with N. apis/N. ceranae in Apis mellifera using real-time PCR

To protect the world’s honey bee population many scientific centres are searching for products and methods that control nosemosis. Real-time PCR was used to assess infection level in worker bees infected with Nosema spp. in bee colonies co-infected with Nosema apis and Nosema ceranae after the administration of three products (Nozevit, ApiHerb and ApiX) and sugar syrup. The study was conducted in the field condition therefore there was no possibility to affect the number of spores in the selected material. The study demonstrated considerable differences in the number of spores of individual Nosema spp. in the analysed samples of bees. HSD Tukey’s test showed that the statistically significant effect on limiting the N. apis invasion had ApiX (p – 0.049). Nozevit, Apiherb and syrup showed no statistically significant effect on reducing the amount of N. apis spores. The same test showed that the statistically significant effect on limiting the N. ceranae invasion had: Nozevit (p – 0.014), Apiherb (p – 0.032), ApiX (p – 0.034) and syrup (p – 0.033). There was no statistically significant decrease in the N. ceranae spores in the control group.     

Identification of a microspordium isolated from Megacopta cribraria (Hemiptera: Plataspidae) and characterization of its pathogenicity in silkworms

A new microsporidium isolated from Megacopta cribraria was characterized by both biological characteristics and phylogenetic analysis. Moreover, its pathogenicity to silkworms was also studied. The spores are oval in shape and measured 3.64 ± 0.2 × 2.20 ± 0.2 μm in size. Its ultrastructure is characteristic of the genus Nosema: a diplokaryon, 13–14 polar filament coils and posterior vacuole. Its life cycle includes meronts, sporonts, sporoblasts and mature spores, with a typical diplokaryon in each stage and propagation in a binary fission. A phylogenetic tree based on SSU rRNA and rRNA ITS gene sequence analysis further indicated that the parasite is closely related to Nosema bombycis and should be placed in the genus Nosema and sub-group ‘true’ Nosema. Furthermore, the microsporidium heavily infects lepidopteran silkworm insect and can be transmitted per os (horizontally) and transovarially (vertically). Our findings showed that the microsporidium belongs to the ‘true’ Nosema group within the genus Nosema and heavily infects silkworms. Based on the information obtained during this study, we named this new microsporidium isolated from M. cribraria as Nosema sp. MC.     

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.     

Distribution of Nosema ceranae in the European honeybee, Apis mellifera in Japan

The microsporidian species, Nosema apis and Nosema ceranae are both known to infect the European honeybee, Apis mellifera. Nosema disease has a global distribution and is responsible for considerable economic losses among apiculturists. In this study, 336 honeybee samples from 18 different prefectures in Japan were examined for the presence of N. apis and N. ceranae using a PCR technique. Although N. ceranae was not detected in most of the apiaries surveyed, the parasite was detected at three of the sites examined. Further, N. ceranae appears to be patchily distributed across Japan and no apparent geographic difference was observed among the areas surveyed. In addition, the apparent absence of N. apis suggests that N. ceranae may be displacing N. apis in A. mellifera in Japan. Partial SSU rRNA gene sequence analysis revealed the possible existence of two N. ceranae groups from different geographic regions in Japan. It seems likely that these microsporidian parasites were introduced into Japan through the importation of either contaminated honeybee-related products or infected queens. This study confirmed that PCR detection is effective for indicating the presence of this pathogen in seemingly healthy colonies. It is therefore hoped that the results presented here will improve our understanding of the epidemiology of Nosema disease so that effective controls can be implemented.     

Increased survival of the honey bee Apis mellifera infected with the microsporidian Nosema ceranae by effective gene silencing

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology.     

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.