Matrix-assisted laser desorption/ionization mass spectrometry biotyping, an approach for deciphering and assessing the identity of the honeybee pathogen Nosema.

RATIONALE: The microsporidia are obligate intracellular pathogenic fungi that parasitize a wide range of invertebrate and vertebrate hosts and have important impacts on health, food security and the economy. In this paper, we focus on Nosema ceranae and N. apis, which chronically infect the digestive tract of honeybees, altering their physiology and lifespan. METHODS: We applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for rapid molecular profiling of extracts of Nosema spores in order to identify the species and the geographical origin, and assess the viability status of Nosema microsporidia in conjunction with a flow cytometric approach. Pure solutions of spores were prepared for flow cytometric analysis and MALDI-MS profiling. A mechanical extraction of viable or heat-killed Nosema spores was conducted to obtain mass fingerprints of peptides/proteins for samples of microsporidia from different geographical origins (MBO.NC01, MBO.NC02 and MBO.NA01). RESULTS: A distinction in the peptide/protein profiles between two isolates with different geographical origins was observed. Mass fingerprints of viable and experimentally killed spores were also clearly distinguishable, regardless of Nosema species. Finally, using our computational models on the different Nosema species, we were able to classify five independent isolates of Nosema microsporidia. CONCLUSIONS: We have shown that MALDI-MS is a rapid, cost-effective and simple method for identifying Nosema species. We demonstrated that MALDI Biotyping could represent a valuable surveillance tool of nosemosis in apiaries for sanitary services and beekeepers.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Identification and characterization a novel polar tube protein (NbPTP6) from the microsporidian Nosema bombycis.

BACKGROUND: Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50-500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. RESULTS: A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. CONCLUSIONS: Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

A new isolate of Nosema fumiferanae (Microsporidia: Nosematidae) from the date moth Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839 (Lepidoptera: Pyralidae).

In this study, a microsporidian pathogen of the date moth (Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839) also known as the carob moth, is described based on light microscopy, ultrastructural characteristics and comparative molecular analysis. The pathogen infects the gut and hemolymph of A. ceratoniae. All development stages are in direct contact with the host cell cytoplasm. Fresh spores with nuclei arranged in a diplokaryon are oval and measured 3.29 ± 0.23 µm (4.18-3.03 µm, n = 200) in length and 1.91 ± 0.23 µm (2.98-1.66 µm, n = 200) in width. Spores stained with Giemsa's stain measured 3.11 ± 0.31 µm (3.72-2.41 µm, n = 150) in length and 1.76 ± 0.23 µm (2.16-1.25 µm, n = 150) in width. Spores have an isofilar polar filament with 10-12 coils. An 1110 bp long alignment of the current microsporidium showed an SSU rRNA gene difference of only 0.0009, corresponding to >99.91% sequence similarity with Nosema fumiferanae, while RPB1 gene sequences were 98.03% similar within an alignment of 969 bp. All morphological, ultrastructural and molecular features indicate that the microsporidian pathogen of A. ceratoniae is the new isolate of the N. fumiferanae and is named here as Nosema fumiferanae TY61.

Assignment of Vairimorpha leptinotarsae comb. nov. on the basis of molecular characterization of Nosema leptinotarsae Lipa, 1968 (Microsporidia: Nosematidae).

Nosema leptinotarsae Lipa, 1968 is a microsporidian pathogen of the Colorado potato beetle, Leptinotarsa decemlineata Say. (Coleoptera: Chrysomelidae). To determine the phylogenetic status of N. leptinotarsae, the 16S SSU rRNA gene was sequenced (GenBank Accession No. MN841279) and compared phylogenetically against 21 microsporidian 16S SSU rRNA sequences using neighbour-joining and maximum-parsimony methods. The per cent identities of the N. leptinotarsae and other members of the Nosema-Vairimorpha clade ranged from 78.1 to 98.5%. Pairwise phylogenetic distances between the N. leptinotarsae and other species ranged from 0.009 to 0.320. Phylogenetic analysis shows clearly that N. leptinotarsae is a member of the Vairimorpha clade rather than the Nosema clade. The sequence divergence and morphological traits separated the N. leptinotarsae from other species in the Vairimorpha complex. As a result, a new assignment of Vairimorpha leptinotarsae comb. nov. has been implemented for N. leptinotarsae according to the phylogenetical positioning in the present study.

Ultrastructural and molecular characterization of Nosema alticae sp. nov. (Microsporidia: Nosematidae), pathogen of the flea beetle, Altica hampei Allard, 1867 (Coleoptera: Chrysomelidae).

In this study, the first microsporidian pathogen from Altica hampei (Coleoptera: Chrysomelidae) is described based on light microscopy, ultrastructural characteristics and comparative 16S SSU rDNA analysis. All developmental stages of the microsporidium are diplokaryotic and in direct contact with the host cell cytoplasm. Giemsa-stained mature spores are oval in shape and measured 3.82 ± 0.35 µm in length and 2.54 ± 0.27 µm in width. The polar filament of the binucleate spores is isofilar with 12-14 coils. Coils are 140.28 ± 4.88 nm (135.59-147.06; n = 36) in diameter and consist of six concentric layers of different electron density and thickness. The spores have a relatively thick (161.72 ± 29.19 nm) trilaminar spore wall. Morphological, ultrastructural and molecular features indicate that the described microsporidium belongs to the genus Nosema and is named Nosema alticae sp. nov.

A formal redefinition of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) and reassignment of species based on molecular phylogenetics.

The microsporidian genera Nosema and Vairimorpha comprise a clade described from insects. Currently the genus Nosema is defined as having a dimorphic life cycle characterized by diplokaryotic stages and diplosporoblastic sporogony with two functionally and morphologically distinct spore types ("early" or "primary" and "environmental"). The Vairimorpha life cycle, in addition to a Nosema-type diplokaryotic sporogony, includes an octosporoblastic sporogony producing eight uninucleate spores (octospores) within a sporophorous vesicle. Molecular phylogeny, however, has clearly demonstrated that the genera Nosema and Vairimorpha, characterized by the absence or presence of uninucleate octospores, respectively, represent two polyphyletic taxa, and that octosporogony is turned on and off frequently within taxa, depending on environmental factors such as host species and rearing temperature. In addition, recent studies have shown that both branches of the Vairimorpha-Nosema clade contain species that are uninucleate throughout their life cycle. The SSU rRNA gene sequence data reveal two distinct clades, those closely related to Vairimorpha necatrix, the type species for the genus Vairimorpha, and those closely related to Nosema bombycis, the type species for the genus Nosema. Here, we redefine the two genera, giving priority to molecular character states over those observed at the developmental, structural or ultrastructural levels and present a list of revised species designations. Using this approach, a series of species are renamed (combination novum) and members of two genera, Rugispora and Oligosporidium, are reassigned to Vairimorpha because of their phylogenetic position. Moreover, the family Nosematidae is redefined and includes the genera Nosema and Vairimorpha comprising a monophyletic lineage of Microsporidia.

Phylogenetic Analysis of the Complete rRNA Gene Sequence of Nosema sp. SE Isolated from the Beet Armyworm Spodoptera exigua.

The microsporidium Nosema sp. SE is a pathogen that infects the beet armyworm Spodoptera exigua. The complete sequence of its 4,302-base pair (bp) ribosomal ribonucleic acid (rRNA) gene region was obtained by polymerase chain reaction amplification and sequencing. The rRNA organization of Nosema sp. SE was 5'-large subunit (LSU) rRNA-internal transcribed spacer-small subunit (SSU) rRNA-intergenic spacer-5S-3', which corresponded to the pattern of Nosema bombycis. Phylogenetic analysis based on LSU rRNA and SSU rRNA both indicated that the parasite had a close relationship with other true Nosema species, confirming that Nosema sp. SE belongs to true Nosema group of the genus Nosema.

Heterologous overexpression of active hexokinases from microsporidia Nosema bombycis and Nosema ceranae confirms their ability to phosphorylate host glucose.

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and kcat 1726 min-1 for glucose, and Km 0.39 ± 0.05 mM and kcat 1976 min-1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and kcat 3293 min-1 for glucose, and Km 1.15 ± 0.11 mM and kcat 3732 min-1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.

Prevalence of infection by the microsporidian Nosema spp. in native bumblebees (Bombus spp.) in northern Thailand.

Bumblebees (tribe Bombini, genus Bombus Latreille) play a pivotal role as pollinators in mountain regions for both native plants and for agricultural systems. In our survey of northern Thailand, four species of bumblebees (Bombus (Megabombus) montivagus Smith, B. (Alpigenobombus) breviceps Smith, B. (Orientalibombus) haemorrhoidalis Smith and B. (Melanobombus) eximius Smith), were present in 11 localities in 4 provinces (Chiang Mai, Mae Hong Son, Chiang Rai and Nan). We collected and screened 280 foraging worker bumblebees for microsporidia (Nosema spp.) and trypanosomes (Crithidia spp.). Our study is the first to demonstrate the parasite infection in bumblebees in northern Thailand. We found N. ceranae in B. montivagus (5.35%), B. haemorrhoidalis (4.76%), and B. breviceps (14.28%) and N. bombi in B. montivagus (14.28%), B. haemorrhoidalis (11.64%), and B. breviceps (28.257%).

Interaction of Phthorimaea operculella granulovirus with a Nosema sp. microsporidium in larvae of Phthorimaea operculella.

An antagonistic effect of a microsporidium (Nosema sp.) infection on the virulence of Phthorimaea operculella granulovirus (PhopGV) was recorded in potato tuber moth (Phthorimaea operculella) larvae with mixed infections. When the P. operculella colony was infected at a high rate (42.8-100%) with the microsporidium, it was less susceptible to the isolate PhopGV-GR1.1. A virus concentration 1.89 × 105 higher was necessary to cause the same level of mortality produced in the P. operculella colony when it was uninfected or had a low level of infection with the microsporidium (0-30%). This antagonistic effect was driven by a Nosema isolate (termed Nosema sp. Phop) that was purified from microsporidian-infected P. operculella individuals. The purified microsporidium was characterised by morphological features, including size, filament coils and different developmental stages using transmission electron microscopy (TEM). On the molecular level, the partial cistron rDNA information of the small ribosomal subunit (SSU), internal transcribed spacer (ITS), and the large ribosomal subunit (LSU) were identified. Phylogenetic analyses revealed that the newly described microsporidium belongs to the "true Nosema" clade. Partial sequence information of the RNA polymerase II largest subunit (RPB1) suggested that Nosema bombycis is the closest relative (98% identity). The morphological and phylogenetic characteristics suggest that it is an isolate of N. bombycis. Interactions of microsporidia and betabaculoviruses are rarely described in the literature, although mixed infections of different pathogens seem to be rather common events, ranging from antagonistic to mutualistic interactions. The observed antagonistic relationship between the Nosema sp. and PhopGV-GR1.1 showed that pathogen interactions need to be considered when single pathogens are applied to insect populations in the context of biological control of insect pests.

Detection of two Microsporidia pathogens of the European honey bee Apis Mellifera (Insecta: Apidae) in Western Siberia.

Two species of microsporidia, Nosema apis and Nosema ceranae, occur regularly and cause significant losses in apiculture throughout the world. N. ceranae is thought to be an emerging pathogen of the European honey bee which is replacing N. apis. Microscopic analysis of honey bees collected in Tyumen region, South-Western Siberia, suggested presence of two microsporidial pathogens slightly differing in spore size and shape. PCR detection using species-specific primer sets 312APIS and 218MITOC followed by PCR product sequencing confirmed the diagnosis of N. apis and N. ceranae, respectively. Microsporidia were present in private apiaries through 2008-2010, and among 21 colonies from 7 localities, two colonies were infected with both pathogens, while infections with N. apis only were detected in 8, and with N. ceranae only in 13 colonies. These data suggest that N. ceranae is widely spread in South-Western Siberia alongside with N. apis and is able to persist in the regions with average January temperatures below -18°C.

Natural infection of the beet webworm Loxostege sticticalis L. (Lepidoptera: Crambidae) with three Microsporidia and host switching in Nosema ceranae.

Three species of Microsporidia were identified from a population of the beet webworm Loxostege sticticalis at prevalence rates of 35, 4, and 3%. The most prevalent parasite (Tubulinosema sp.) was similar to Tubulinosema acridophagus (99.8% ssrDNA sequence similarity) and was also isolated from the parasitoid Lydella thompsoni (Diptera, Tachinidae) that emerged from the beet webworms. In laboratory assays, spores of this Tubulinosema sp. showed an infection rate of up to 80% for both L. sticticalis and Galleria mellonella larvae. The spores were viable after 12 months of storage in dried infected cadavers. The second most prevalent parasite was closely related to Nosema furnacalis and Nosema granulosis (98.7% similarity). Fresh spores showed a 50% infection rate under laboratory conditions. The third most abundant parasite was identified as the honeybee pathogen Nosema ceranae (100% ssrDNA and 95-100% IGS similarity). In the laboratory, fresh spores of N. ceranae isolated from beet webworm and honey bee were infective to L. sticticalis larvae at the rates of 5 and 2%, respectively.

Biological and molecular features of Nosema rachiplusiae sp. n., a microsporidium isolated from the neotropical moth Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae).

Light, electron microscopy and DNA analyses were performed to characterize a microsporidium infecting Rachiplusia nu larvae from a laboratory rearing in Argentina. Diplokaryotic spores were oval and measured 3.61 ± 0.29 × 1.61 ± 0.14 µM (fresh). The spore wall was composed of an electron-dense exospore and an electron-lucent endospore, ca. 30 nm and 100-120 nm thick, respectively. The polar filament was arranged in a single rank of 10-12 coils (typically 11). Microsporidian cells were found in the cytoplasm, next to the endoplasmic reticulum (especially the prespore stages) and generally surrounded by electron-lucent spaces. The infection was polyorganotropic; the fat body appeared as the most heavily invaded tissue, followed by tracheal matrix and epidermis. A molecular phylogeny based on the small (SSU) and large subunit (LSU) ribosomal RNA genes clearly placed the new isolate within the "Nosema bombycis clade". Considering both SSU and LSU concatenated partial sequences, the microsporidium from R. nu showed 99.5% nucleotide similarity with N. bombycis and 99.8% with its closest relative, a microsporidium isolated from Philosamia cynthia. According to its genetic and biological features, the R. nu isolate is proposed as the new species Nosema rachiplusiae sp. n., expanding the limited knowledge on microsporidia associated to endemic South-American moths.

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.

Morphological and phylogenetic analysis of a microsporidium (Nosema sp.) isolated from rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae).

A new microsporidium was isolated from Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), one of the most important rice pests in China. The morphology and molecular systematics of this novel microsporidium were described in this study. The spores were long oval and measured 3.17 × 1.64 µm on fresh smears. Ultrastructure of the spores was characteristic for the genus Nosema: a diplokaryon, 10-12 polar filament coils of the same type, and posterior vacuole. Small subunit rRNA gene sequence data and phylogenetic analysis further confirmed that the microsporidian species from C. suppressalis belong to the true Nosema sub-group of the genus Nosema. Besides, the microsporidium Nosema sp. CS could cause systemic infection of Bombyx mori and infect silkworms through vertical transmission. Therefore, mulberry field pest control should be carefully monitored, and sanitation of mulberry leaves is essential to control the pebrine disease in sericulture.

Nosema neumanni n. sp. (Microsporidia, Nosematidae), a new microsporidian parasite of honeybees, Apis mellifera in Uganda.

The microsporidium Nosema neumanni n. sp., a new parasite of the honeybee Apis mellifera is described based on its ultra-structural and molecular characteristics. Structures resembling microsporidian spores were found by microscopic examination of honeybees from Uganda. Molecular confirmation failed when PCR primers specific for Nosema apis and Nosema ceranae were used, but was successful with primers covering the whole family of Nosematidae. We performed transmission electron microscopy and found typical microsporidian spores which were smaller (length: 2.36±0.14µm and width: 1.78±0.06µm; n=6) and had fewer polar filament coils (10-12) when compared to those of known species infecting honeybees. The entire 16S SSU rRNA region was amplified, cloned and sequenced and was found to be unique with the highest resemblance (97% identity) to N. apis. The incidence of N. neumanni n. sp. in Ugandan honeybees was found to be much higher than of the two other Nosema species.

Long-Term Temporal Trends of Nosema spp. Infection Prevalence in Northeast Germany: Continuous Spread of Nosema ceranae, an Emerging Pathogen of Honey Bees (Apis mellifera), but No General Replacement of Nosema apis.

The Western honey bee (Apis mellifera) is widely used as commercial pollinator in worldwide agriculture and, therefore, plays an important role in global food security. Among the parasites and pathogens threatening health and survival of honey bees are two species of microsporidia, Nosema apis and Nosema ceranae. Nosema ceranae is considered an emerging pathogen of the Western honey bee. Reports on the spread of N. ceranae suggested that this presumably highly virulent species is replacing its more benign congener N. apis in the global A. mellifera population. We here present a 12 year longitudinal cohort study on the prevalence of N. apis and N. ceranae in Northeast Germany. Between 2005 and 2016, a cohort of about 230 honey bee colonies originating from 23 apiaries was sampled twice a year (spring and autumn) resulting in a total of 5,600 bee samples which were subjected to microscopic and molecular analysis for determining the presence of infections with N. apis or/and N. ceranae. Throughout the entire study period, both N. apis- and N. ceranae-infections could be diagnosed within the cohort. Logistic regression analysis of the prevalence data demonstrated a significant increase of N. ceranae-infections over the last 12 years, both in autumn (reflecting the development during the summer) and in spring (reflecting the development over winter) samples. Cell culture experiments confirmed that N. ceranae has a higher proliferative potential than N. apis at 27° and 33°C potentially explaining the increase in N. ceranae prevalence during summer. In autumn, characterized by generally low infection prevalence, this increase was accompanied by a significant decrease in N. apis-infection prevalence. In contrast, in spring, the season with a higher prevalence of infection, no significant decrease of N. apis infections despite a significant increase in N. ceranae infections could be observed. Therefore, our data do not support a general advantage of N. ceranae over N. apis and an overall replacement of N. apis by N. ceranae in the studied honey bee population.

Microsporidia: An Emerging Threat to Bumblebees?

Microsporidia may cause emerging infectious diseases (EIDs) in bumblebees. Two drivers - commercial bumblebees and managed honey bees - have been identified as possible sources of pathogen spillover. In addition, declines in bumblebee populations may have led to lower genetic diversity and subsequent higher susceptibility to infection, enabling microsporidia to increase in prevalence. There is strong evidence for relatively recent increases in the prevalence of Nosema bombi in North America. However, the lack of definitive data on spillover by microsporidia, in North America or elsewhere, makes it difficult to identify the causes of such increases. Phylogenomic studies are urgently needed to identify the global population structure of microsporidia in bumblebees, and thus identify the source of current and future epidemics.

Characterization of Nosema ceranae Genetic Variants from Different Geographic Origins.

In recent years, large-scale colony losses of honey bees (Apis mellifera) have been reported and the infection with the microsporidia Nosema ceranae has been involved. However, the effect of N. ceranae at the colony level and its role in colony losses vary in different geographic areas. This difference may be related to the presence of multiple N. ceranae genetic variants resulting in different biological consequences. In this study, we analyzed the genetic diversity of 75 N. ceranae samples obtained from 13 countries and Hawaii through inter-sequence single repetition (ISSR) and evaluated if two of these genetic variants triggered different immune responses when infecting Apis mellifera iberiensis. The genetic diversity analysis showed that 41% of the samples had the same DNA amplification pattern, including samples from most European countries except Spain, while the remaining samples showed high variability. Infection assays were performed to analyze the infection levels and the immune response of bees infected with N. ceranae from Spain and Uruguay. The infected bees presented similar infection levels, and both isolates downregulated the expression of abaecin, confirming the ability of the microsporidia to depress the immune response. Only N. ceranae from Uruguay downregulated the expression level of imd compared to control bees. On the other hand, both genetic variants triggered different expression levels of lysozyme. As imd and lysozyme play important roles in the response to pathogens, these results could reflect differences in the biological consequences of N. ceranae variants in A. mellifera infection.