RESUMEN
Nosema ceranae is a microsporidian parasite that threatens current apiculture. N. ceranae-infected honey bees (Apis mellifera) exhibit morbid physiological impairments and reduced honey production, malnutrition, shorter life span, and higher mortality than healthy honey bees. In this study, we found that dimethyl sulfoxide (DMSO) could enhance the survival rate of N. ceranae-infected honey bees. Therefore, we investigated the effect of DMSO on N. ceranae-infected honey bees using comparative RNA sequencing analysis. Our results revealed that DMSO was able to affect several biochemical pathways, especially the metabolic-related pathways in N. ceranae-infected honey bees. Based on these findings, we conclude that DMSO may be a useful alternative for treating N. ceranae infection in apiculture.
Asunto(s)
Dimetilsulfóxido , Nosema , Animales , Nosema/efectos de los fármacos , Nosema/fisiología , Abejas/microbiología , Dimetilsulfóxido/farmacología , Microsporidiosis/veterinariaRESUMEN
Honeybees are an indispensable pollinator in nature with pivotal ecological, economic, and scientific value. However, a full-length transcriptome for Apis mellifera, assembled with the advanced third-generation nanopore sequencing technology, has yet to be reported. Here, nanopore sequencing of the midgut tissues of uninoculated and Nosema ceranae-inoculated A. mellifera workers was conducted, and the full-length transcriptome was then constructed and annotated based on high-quality long reads. Next followed improvement of sequences and annotations of the current reference genome of A. mellifera. A total of 5,942,745 and 6,664,923 raw reads were produced from midguts of workers at 7 days post-inoculation (dpi) with N. ceranae and 10 dpi, while 7,100,161 and 6,506,665 raw reads were generated from the midguts of corresponding uninoculated workers. After strict quality control, 6,928,170, 6,353,066, 5,745,048, and 6,416,987 clean reads were obtained, with a length distribution ranging from 1 kb to 10 kb. Additionally, 16,824, 17,708, 15,744, and 18,246 full-length transcripts were respectively detected, including 28,019 nonredundant ones. Among these, 43,666, 30,945, 41,771, 26,442, and 24,532 full-length transcripts could be annotated to the Nr, KOG, eggNOG, GO, and KEGG databases, respectively. Additionally, 501 novel genes (20,326 novel transcripts) were identified for the first time, among which 401 (20,255), 193 (13,365), 414 (19,186), 228 (12,093), and 202 (11,703) were respectively annotated to each of the aforementioned five databases. The expression and sequences of three randomly selected novel transcripts were confirmed by RT-PCR and Sanger sequencing. The 5' UTR of 2082 genes, the 3' UTR of 2029 genes, and both the 5' and 3' UTRs of 730 genes were extended. Moreover, 17,345 SSRs, 14,789 complete ORFs, 1224 long non-coding RNAs (lncRNAs), and 650 transcription factors (TFs) from 37 families were detected. Findings from this work not only refine the annotation of the A. mellifera reference genome, but also provide a valuable resource and basis for relevant molecular and -omics studies.
Asunto(s)
Anotación de Secuencia Molecular , Transcriptoma , Abejas/genética , Animales , Transcriptoma/genética , Genoma de los Insectos , Nosema/genética , Secuenciación de Nanoporos/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis.
Asunto(s)
Sistemas CRISPR-Cas , Proteínas Fúngicas , Edición Génica , Nosema , Nosema/genética , Animales , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Edición Génica/métodosRESUMEN
Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis (Nb) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene (plin1) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop. This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity.IMPORTANCELipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis (Nb) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity.
Asunto(s)
Bombyx , Proteínas de Insectos , Quinasas Janus , Gotas Lipídicas , Nosema , Perilipina-1 , Transducción de Señal , Bombyx/microbiología , Bombyx/metabolismo , Bombyx/genética , Animales , Nosema/metabolismo , Nosema/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Gotas Lipídicas/metabolismo , Quinasas Janus/metabolismo , Quinasas Janus/genética , Perilipina-1/metabolismo , Perilipina-1/genética , Factores de Transcripción STAT/metabolismo , Factores de Transcripción STAT/genética , Cuerpo Adiposo/metabolismo , Larva/microbiología , Larva/metabolismo , Metabolismo de los LípidosRESUMEN
Microsporidia Nosema bombycis (Nb) is a cellular parasite responsible for pébrine disease in silkworms, significantly impacting the sericulture industry. Long non-coding RNAs (lncRNAs), which are RNA fragments longer than 200 nucleotides, are pivotal in a range of cellular and physiological functions. However, the potential role of silkworm lncRNAs in response to Nb infection remains unknown. This study conducted transcriptome sequencing on both larvae and Nb-infected midguts of silkworms, identifying 1,440 lncRNAs across all examined midgut samples. Within the Nb-infected group, 42 differentially expressed lncRNAs (DElncRNAs) and 305 differentially expressed mRNAs (DEmRNAs) were detected. Functional annotation and pathway analysis showed that these DEmRNAs are mostly involved in metabolism, apoptosis, autophagy, and other key pathways. The co-expression network of DEmRNAs and DElncRNAs illustrates that 1 gene could be regulated by multiple lncRNAs and 1 lncRNA may target multiple genes, indicating that the regulation of lncRNA is intricate and networked. In addition, the DElncRNA-miRNA-mRNA network showed that some DElncRNAs may be involved in the immune response and metabolism through miRNA. Notably, the study observed an increase in lncRNA MSTRG857.1 following Nb infection, which may promote Nb proliferation. These findings offer insights into the complex interplay between insects and microsporidia.
Asunto(s)
Bombyx , Larva , Nosema , ARN Largo no Codificante , Bombyx/genética , Bombyx/microbiología , Animales , ARN Largo no Codificante/genética , Nosema/fisiología , Larva/microbiología , Larva/crecimiento & desarrollo , Larva/genética , TranscriptomaRESUMEN
The genus Serratia harbors opportunistic pathogenic species, among which Serratia marcescens is pathogenic for honeybees although little studied. Recently, virulent strains of S. marcescens colonizing the Varroa destructor mite's mouth were found vectored into the honeybee body, leading to septicemia and death. Serratia also occurs as an opportunistic pathogen in the honeybee's gut with a low absolute abundance. The Serratia population seems controlled by the host immune system, but its presence may represent a hidden threat, ready to arise when honeybees are weakened by biotic and abiotic stressors. To shed light on the Serratia pathogen, this research aims at studying Serratia's development dynamics in the honeybee body and its interactions with the co-occurring fungal pathogen Vairimorpha ceranae. Firstly, the degree of pathogenicity and the ability to permeate the gut epithelial barrier of three Serratia strains, isolated from honeybees and belonging to different species (S. marcescens, Serratia liquefaciens, and Serratia nematodiphila), were assessed by artificial inoculation of newborn honeybees with different Serratia doses (104, 106, and 108 cells/mL). The absolute abundance of Serratia in the gut and in the hemocoel was assessed in qPCR with primers targeting the luxS gene. Moreover, the absolute abundance of Serratia was assessed in the gut of honeybees infected with V. ceranae at different development stages and supplied with beneficial microorganisms and fumagillin. Our results showed that all tested Serratia strains could pass through the gut epithelial barrier and proliferate in the hemocoel, with S. marcescens being the most pathogenic. Moreover, under cage conditions, Serratia better proliferates when a V. ceranae infection is co-occurring, with a positive and significant correlation. Finally, fumagillin and some of the tested beneficial microorganisms could control both Serratia and Vairimorpha development. Our findings suggest a correlation between the two pathogens under laboratory conditions, a co-occurring infection that should be taken into consideration by researches when testing antimicrobial compounds active against V. ceranae, and the related honeybees survival rate. Moreover, our findings suggest a positive control of Serratia by the environmental microorganism Apilactobacillus kunkeei in a in vivo model, confirming the potential of this specie as beneficial bacteria for honeybees.
Asunto(s)
Nosema , Serratia , Animales , Abejas/microbiología , Serratia/patogenicidad , Serratia/genética , Serratia/crecimiento & desarrollo , Nosema/patogenicidad , Nosema/crecimiento & desarrollo , Nosema/fisiología , Nosema/genética , Serratia marcescens/patogenicidad , Serratia marcescens/crecimiento & desarrollo , Serratia marcescens/genética , Tracto Gastrointestinal/microbiología , Infecciones por Serratia/microbiología , Ciclohexanos/farmacología , Serratia liquefaciens/crecimiento & desarrollo , Serratia liquefaciens/genética , Ácidos Grasos Insaturados , SesquiterpenosRESUMEN
In recent years, there has been growing concern on the potential weakening of honey bees and their increased susceptibility to pathogens due to chronic exposure to xenobiotics. The present work aimed to study the effects on bees undergoing an infection by Nosema ceranae and being exposed to a frequently used in-hive acaricide, amitraz. To achieve this, newly emerged bees were individually infected with N. ceranae spores and/or received a sublethal concentration of amitraz in their diets under laboratory conditions. Mortality, food intake, total volume excrement, body appearance, and parasite development were registered. Bees exposed to both stressors jointly had higher mortality rates compared to bees exposed separately, with no difference in the parasite development. An increase in sugar syrup consumption was observed for all treated bees while infected bees fed with amitraz also showed a diminishment in pollen intake. These results coupled with an increase in the total number of excretion events, alterations in behavior and body surface on individuals that received amitraz could evidence the detrimental action of this molecule. To corroborate these findings under semi-field conditions, worker bees were artificially infected, marked, and released into colonies. Then, they were exposed to a commercial amitraz-based product by contact. The recovered bees showed no differences in the parasite development due to amitraz exposure. This study provides evidence to which extent a honey bee infected with N. ceranae could potentially be weakened by chronic exposure to amitraz treatment.
Asunto(s)
Nosema , Toluidinas , Animales , Abejas/efectos de los fármacos , Abejas/microbiología , Abejas/parasitología , Nosema/efectos de los fármacos , Nosema/fisiología , AcaricidasRESUMEN
Congenital infection caused by vertical transmission of microsporidia N. bombycis can result in severe economic losses in the silkworm-rearing industry. Whole-transcriptome analyses have revealed non-coding RNAs and their regulatory networks in N. bombycis infected embryos and larvae. However, transcriptomic changes in the microsporidia proliferation and host responses in congenitally infected embryos and larvae remains unclear. Here, we simultaneously compared the transcriptomes of N. bombycis and its host B. mori embryos of 5-day and larvae of 1-, 5- and 10-day during congenital infection. For the transcriptome of N. bombycis, a comparison of parasite expression patterns between congenital-infected embryos and larva showed most genes related to parasite central carbon metabolism were down-regulated in larvae during infection, whereas the majority of genes involved in parasite proliferation and growth were up-regulated. Interestingly, a large number of distinct or shared differentially expressed genes (DEGs) were revealed by the Venn diagram and heat map, many of them were connected to infection related factors such as Ricin B lectin, spore wall protein, polar tube protein, and polysaccharide deacetylase. For the transcriptome of B. mori infected with N. bombycis, beyond numerous DEGs related to DNA replication and repair, mRNA surveillance pathway, RNA transport, protein biosynthesis, and proteolysis, with the progression of infection, a large number of DEGs related to immune and infection pathways, including phagocytosis, apoptosis, TNF, Toll-like receptor, NF-kappa B, Fc epsilon RI, and some diseases, were successively identified. In contrast, most genes associated with the insulin signaling pathway, 2-oxacarboxylic acid metabolism, amino acid biosynthesis, and lipid metabolisms were up-regulated in larvae compared to those in embryos. Furthermore, dozens of distinct and three shared DEGs that were involved in the epigenetic regulations, such as polycomb, histone-lysine-specific demethylases, and histone-lysine-N-methyltransferases, were identified via the Venn diagram and heat maps. Notably, many DEGs of host and parasite associated with lipid-related metabolisms were verified by RT-qPCR. Taken together, simultaneous transcriptomic analyses of both host and parasite genes lead to a better understanding of changes in the microsporidia proliferation and host responses in embryos and larvae in N. bombycis congenital infection.
Asunto(s)
Bombyx , Nosema , Animales , Transcriptoma , Larva/genética , Larva/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Nosema/fisiología , Perfilación de la Expresión Génica , Proliferación Celular , Lípidos , Bombyx/genéticaRESUMEN
Growing evidences have shown that the decline in honey bee populations is mainly caused by the combination of multiple stressors. However, the impacts of parasitic Nosema ceranae to host fitness during long-term pesticide exposure-induced stress is largely unknown. In this study, the effects of chronic exposure to a sublethal dose of dinotefuran, in the presence or absence of N. ceranae, was examined in terms of survival, food consumption, detoxification enzyme activities and gut microbial community. The interaction between dinotefuran and Nosema ceranae on the survival of honey bee was synergistic. Co-exposure to dinotefuran and N. ceranae led to less food consumption and greater changes of enzyme activities involved in defenses against oxidative stress. Particularly, N. ceranae and dinotefuran-N. ceranae co-exposure significantly impacted the gut microbiota structure and richness in adult honey bees, while dinotefuran alone did not show significant alternation of core gut microbiota compared to the control group. We herein demonstrated that chronical exposure to dinotefuran decreases honey bee's survival but is not steadily associated with the gut microbiota dysbiosis; by contrast, N. ceranae parasitism plays a dominant role in the combination in influencing the gut microbial community of the host honey bee. Our findings provide a comprehensive understanding of combinatorial effects between biotic and abiotic stressors on one of the most important pollinators, honey bees.
Asunto(s)
Microbioma Gastrointestinal , Guanidinas , Nitrocompuestos , Nosema , Abejas , Animales , Neonicotinoides/toxicidad , Nitrocompuestos/toxicidadRESUMEN
The current status of Nosema spp. infections in A. mellifera throughout Eurasia was characterized using electronic databases. Although N. ceranae was predominantly detected in southwestern and south-central regions and N. apis in northwestern and north-central areas, most studies reported the occurrence of both species in Eurasia. In addition, the occurrence of Nosema spp. and Ptp3 gene haplotypes was investigated in the Republic of Tatarstan, Russia. Most of the examined honey bees were infected with both N. apis and N. ceranae. N. apis and N. ceranae isolates were either heterozygous or belonged to different strains and showed infection with more than one strain. New haplotypes were found for N. apis and N. ceranae in the Republic of Tatarstan, Russia. This study expands the data regarding existing haplotypes of Nosema species: there are currently 9 shared and 56 unique Ptp3 nucleotide sequence haplotypes of N. ceranae, and 2 shared and 7 unique haplotypes of N. apis, respectively.
Asunto(s)
Haplotipos , Nosema , Animales , Abejas/microbiología , Nosema/genética , Federación de Rusia/epidemiologíaRESUMEN
Health of honey bees is threatened by a variety of stressors, including pesticides and parasites. Here, we investigated effects of acetamiprid, Varroa destructor, and Nosema ceranae, which act either alone or in combination. Our results suggested that interaction between the three factors was additive, with survival risk increasing as the number of stressors increased. Although exposure to 150 µg/L acetamiprid alone did not negatively impact honey bee survival, it caused severe damage to midgut tissue. Among the three stressors, V. destructor posed the greatest threat to honey bee survival, and N. ceranae exacerbated intestinal damage and increased thickness of the midgut wall. Transcriptomic analysis indicated that different combinations of stressors elicited specific gene expression responses in honey bees, and genes involved in energy metabolism, immunity, and detoxification were altered in response to multiple stressor combinations. Additionally, genes associated with Toll and Imd signalling, tyrosine metabolism, and phototransduction pathway were significantly suppressed in response to different combinations of multiple stressors. This study enhances our understanding of the adaptation mechanisms to multiple stressors and aids in development of suitable protective measures for honey bees. ENVIRONMENTAL IMPLICATION: We believe our study is environmentally relevant for the following reasons: This study investigates combined effects of pesticide, Varroa destructor, and Nosema ceranae. These stressors are known to pose a threat to long-term survival of honey bees (Apis mellifera) and stability of the ecosystems. The research provides valuable insights into the adaptive mechanisms of honey bees in response to multiple stressors and developing effective conservation strategies. Further research can identify traits that promote honey bee survival in the face of future challenges from multiple stressors to maintain the overall stability of environment.
Asunto(s)
Neonicotinoides , Nosema , Varroidae , Animales , Abejas/efectos de los fármacos , Nosema/efectos de los fármacos , Neonicotinoides/toxicidad , Varroidae/efectos de los fármacos , Insecticidas/toxicidadRESUMEN
The interspecific transmission of pathogens can occur frequently in the environment. Among wild bees, the main spillover cases are caused by pathogens associated with Apis mellifera, whose colonies can act as reservoirs. Due to the limited availability of data in Italy, it is challenging to accurately assess the impact and implications of this phenomenon on the wild bee populations. In this study, a total of 3372 bees were sampled from 11 Italian regions within the BeeNet project, evaluating the prevalence and the abundance of the major honey bee pathogens (DWV, BQCV, ABPV, CBPV, KBV, Nosema ceranae, Ascosphaera apis, Crithidia mellificae, Lotmaria passim, Crithidia bombi). The 68.4% of samples were positive for at least one pathogen. DWV, BQCV, N. ceranae and CBPV showed the highest prevalence and abundance values, confirming them as the most prevalent pathogens spread in the environment. For these pathogens, Andrena, Bombus, Eucera and Seladonia showed the highest mean prevalence and abundance values. Generally, time trends showed a prevalence and abundance decrease from April to July. In order to predict the risk of infection among wild bees, statistical models were developed. A low influence of apiary density on pathogen occurrence was observed, while meteorological conditions and agricultural management showed a greater impact on pathogen persistence in the environment. Social and biological traits of wild bees also contributed to defining a higher risk of infection for bivoltine, communal, mining and oligolectic bees. Out of all the samples tested, 40.5% were co-infected with two or more pathogens. In some cases, individuals were simultaneously infected with up to five different pathogens. It is essential to increase knowledge about the transmission of pathogens among wild bees to understand dynamics, impact and effects on pollinator populations. Implementing concrete plans for the conservation of wild bee species is important to ensure the health of wild and human-managed bees within a One-Health perspective.
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Nosema , Onygenales , Trypanosomatina , Humanos , Animales , Abejas , Factores Sociales , Crithidia , Italia/epidemiologíaRESUMEN
Most honey bee pathogens, such as Vairimorpha (Nosema), cannot be rapidly and definitively diagnosed in a natural setting, consequently there is typically the spread of these diseases through shared and re-use of beekeeping equipment. Furthermore, there are no viable treatment options available for Nosema spores to aid in managing the spread of this bee disease. We therefore aimed to develop a new method using novel Zinc Phthalocyanine (ZnPc) as a photosensitizer for the photodynamic inactivation of Nosema spores that could be used for the decontamination of beekeeping equipment. Nosema spores were propagated for in vitro testing using four caged Apis mellifera honey bees. The ZnPc treatment was characterized, encapsulated with a liposome, and then used as either a 10 or 100 µM treatment for the freshly harvested Nosema spores, for either a 30 and or 60-minute time period, under either light or dark conditions, in-vitro, in 96-well plates. In the dark treatment, after 30-min, the ZnPc 100 µM treatment, caused a 30 % Nosema mortality, while this increased to 80 % at the same concentration after the light treatment. The high rate of anti-spore effects, in a short period of time, supports the notion that this could be an effective treatment for managing honey bee Nosema infections in the future. Our results also suggest that the photo activation of the treatment could be applied in the field setting and this would increase the sterilization of beekeeping equipment against Nosema.
Asunto(s)
Isoindoles , Nosema , Compuestos Organometálicos , Compuestos de Zinc , Abejas , Animales , Nosema/fisiología , ApiculturaRESUMEN
Nosema disease, caused by Nosema ceranae, one of the single-celled fungal microsporidian parasites, is one of the most important and common diseases of adult honey bees. Since fumagillin, which has been used for decades in the control of Nosema disease in honey bees (Apis mellifera), poses a toxic threat and its efficacy against N. ceranae is uncertain, there is an urgent need to develop alternative prophylactic and curative strategies for the treatment of this disease. The main aim of this study was to investigate the therapeutic potential of specific egg yolk immunoglobulins (IgY) on Nosema disease. For this purpose, the presence of N. ceranae was determined by microscopic and PCR methods in honey bees collected from Nosema suspicious colonies by conducting a field survey. Layered Ataks chickens, divided into four groups each containing 20 animals, were vaccinated with live and inactivated vaccines prepared from field isolates of N. ceranae. Eggs were collected weekly for 10 weeks following the last vaccination. IgY extraction was performed using the PEG precipitation method from egg yolks collected from each group, and the purity of the antibodies was determined by SDS-PAGE and Western Blot. The presence of N. ceranae-specific IgYs was investigated by Western Blot and indirect ELISA methods. It was determined that specific IgYs showed high therapeutic efficacy on Nosema disease in naturally infected bee colonies. In addition, honey bees collected from infected colonies were brought to the laboratory and placed in cages with 30 bees each, and the effectiveness of IgYs was investigated under controlled conditions. It was detected that specific IgY reduced the Nosema spore load and the number of infected bees significantly in both the field and experimental study groups treated for seven days. It was concluded that chicken IgYs, an innovative and eco-friendly method, had a significant potential for use as an alternative to antifungal drugs.
Asunto(s)
Nosema , Animales , Abejas , Yema de Huevo , Pollos , AnticuerposRESUMEN
Pollinators are vital for food security and the maintenance of terrestrial ecosystems. Bumblebees are important pollinators across northern temperate, arctic, and alpine ecosystems, yet are in decline across the globe. Vairimorpha bombi is a parasite belonging to the fungal class Microsporidia that has been implicated in the rapid decline of bumblebees in North America, where it may be an emerging infectious disease. To investigate the evolutionary basis of pathogenicity of V. bombi, we sequenced and assembled its genome using Oxford Nanopore and Illumina technologies and performed phylogenetic and genomic evolutionary analyses. The genome assembly for V. bombi is 4.73 Mb, from which we predicted 1,870 protein-coding genes and 179 tRNA genes. The genome assembly has low repetitive content and low GC content. V. bombi's genome assembly is the smallest of the Vairimorpha and closely related Nosema genera, but larger than those found in the Encephalitozoon and Ordospora sister clades. Orthology and phylogenetic analysis revealed 18 core conserved single-copy microsporidian genes including the histone acetyltransferase (HAT) GCN5. Surprisingly, V. bombi was unique to the microsporidia in not encoding the second predicted HAT ESA1. The V. bombi genome assembly annotation included 265 unique genes (i.e. not predicted in other microsporidia genome assemblies), 20% of which encode a secretion signal, which is a significant enrichment. Intriguingly, of the 36 microsporidian genomes we analyzed, 26 also had a significant enrichment of secreted signals encoded by unique genes, ranging from 6 to 71% of those predicted genes. These results suggest that microsporidia are under selection to generate and purge diverse and unique genes encoding secreted proteins, potentially contributing to or facilitating infection of their diverse hosts. Furthermore, V. bombi has 5/7 conserved spore wall proteins (SWPs) with its closest relative V. ceranae (that primarily infects honeybees), while also uniquely encoding four additional SWPs. This gene class is thought to be essential for infection, providing both environmental protection and recognition and uptake into the host cell. Together, our results show that SWPs and unique genes encoding a secretion signal are rapidly evolving in the microsporidia, suggesting that they underpin key pathobiological traits including host specificity and pathogenicity.
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Ecosistema , Microsporidios , Nosema , Abejas/genética , Animales , Filogenia , Nosema/genética , América del NorteRESUMEN
USDA-ARS Bee Research Laboratory received symptomatic honey bee (Apis mellifera L.) samples across the United States for disease diagnosis. Here, we present a retrospective study and cartography of ectoparasite Varroa destructor and intracellular microsporidia parasite Nosema spp. These two major parasites were identified in the diseased honey bee samples between 2015 and 2022. Varroa infestation level (VIL) was examined by a wash technique (Mites/100 bees) and calculated as a percentage, while Nosema infection was quantified by microscopical spore count (Million Spores/Bee). Data were analyzed by month, year, state, and by nine geographical climate regions described in the U.S. Of adult bee samples (n = 4039) that were analyzed for Varroa mite infestation, the overall VIL in the U.S. ranged between 0.4 and 30.85%, with an overall national VIL and Varroa prevalence of 8.21% and 85.14%, respectively. Overall monthly data showed VIL constantly exceeded the critical level of 4% except from June to September and reached a maximum of 15% in January and December. Nationwide, VIL significantly (p < 0.001) increased from 2015 to 2018 (1.1-4.7%), plateaued from 2018 to 2021 (4.7-4.5%), followed by a significant decrease in 2022 (3.6%). Significant VIL differences (p < 0.001) were recorded among climate regions, with the highest mite infestation levels in the Upper Midwest region (13.9%) and the lowest in the West region (5.1%). Of adult bee samples (n = 2,994) that were analyzed for Nosema infection, Nosema spore count ranged between (1-16.8) million spores per bee among states, with a national average of 6.8 and a prevalence of 99.7%. The lowest and highest Nosema loads were respectively recorded in the South region (3.1) and Upper Midwest (10.5), a significant difference (p < 0.001). No statistical differences were recorded among the six other climate regions. Overall, VIL and Nosema infection correlated significantly (p < 0.001) with a regression coefficient of (R2 = 0.6). Our data, which originated from ailing bee colonies, showed significantly higher rates of maladies compared to data from healthy colonies obtained by the USDA-APHIS National Honey Bee Survey, demonstrating the role of bee diseases caused by Varroa mite and Nosema in honey bee population declines.
Asunto(s)
Nosema , Escabiosis , Varroidae , Abejas , Animales , Estudios Retrospectivos , PrevalenciaRESUMEN
Microsporidia cause disease in many beneficial insects, including honey bees, yet few pathogen control tools are available for protecting these important organisms against infection. Some evidence suggests that microsporidia possess a reduced number of genes encoding DNA repair proteins. We hypothesized that microsporidia would thus be susceptible to treatment with DNA-damaging agents and tested this hypothesis using a novel, rapid method for achieving robust and homogenous experimental infection of large numbers of newly emerged honey bees with one of its microsporidia pathogens, Vairimorpha (Nosema) ceranae. In carrying out these experiments, we found this novel V. ceranae inoculation method to have similar efficacy as other traditional methods. We show that the DNA-damaging agent bleomycin reduces V. ceranae levels, with minimal but measurable effects on honey bee survival and increased expression of midgut cellular stress genes, including those encoding SHSP. Increased expression of UpdlC suggests the occurrence of epithelial regeneration, which may contribute to host resistance to bleomycin treatment. While bleomycin does reduce infection levels, host toxicity issues may preclude its use in the field. However, with further work, bleomycin may provide a useful tool in the research setting as a potential selection agent for genetic modification of microsporidia.IMPORTANCEMicrosporidia cause disease in many beneficial insects, yet there are few tools available for control in the field or laboratory. Based on the reported paucity of DNA repair enzymes found in microsporidia genomes, we hypothesized that these obligate intracellular parasites would be sensitive to DNA damage. In support of this, we observed that the well-characterized DNA damage agent bleomycin can reduce levels of the microsporidia Vairimorpha (Nosema) ceranae in experimental infections in honey bees. Observation of slightly reduced honey bee survival and evidence of sublethal toxicity likely preclude the use of bleomycin in the field. However, this work identifies bleomycin as a compound that merits further exploration for use in research laboratories as a potential selection agent for generating genetically modified microsporidia.
Asunto(s)
Microsporidios , Nosema , Abejas , Animales , Nosema/genética , ADNRESUMEN
The research aims to give new insights on the effect of administering selected bacterial strains, isolated from honey bee gut, and/or a commercial plant extract blend (HiveAlive®) on Nosema ceranae. Analyses were first performed under laboratory conditions such as different infective doses of N. ceranae, the effect of single strains and their mixture and the influence of pollen administration. Daily survival and feed consumption rate were recorded and pathogen development was analysed using qPCR and microscope counts. Biomarkers of immunity and physiological status were also evaluated for the different treatments tested using one bacterial strain, a mixture of all the bacteria and/or a plant extract blend as treatments. The results showed an increase of abaecin transcript levels in the midgut of the honey bees treated with the bacterial mixture and an increased expression of the protein vitellogenin in the haemolymph of honey bees treated with two separate bacterial strains (Bifidobacterium coryneforme and Apilactobacillus kunkeei). A significant effectiveness in reducing N. ceranae was shown by the bacterial mixture and the plant extract blend regardless of the composition of the diet. This bioactivity was seasonally linked. Quantitative PCR and microscope counts showed the reduction of N. ceranae under different experimental conditions. The antiparasitic efficacy of the treatments at field conditions was studied using a semi-field approach which was adapted from research on insecticides for the first time, to analyse antiparasitic activity against N. ceranae. The approach proved to be reliable and effective in validating data obtained in the laboratory. Both the mixture of beneficial bacteria and its association with Hive Alive® are effective in controlling the natural infection of N. ceranae in honey bee colonies.
Asunto(s)
Nosema , Extractos Vegetales , Abejas , Animales , Vitelogeninas , AntiparasitariosRESUMEN
IMPORTANCE: The multiplex-crRNA CRISPR/Cas12a detection method saves hands-on time, reduces the risk of aerosol pollution, and can be directly applied to detecting silkworms infected with Nosema bombycis. This study provides a new approach for the inspection and quarantine of silkworm pébrine disease in sericulture and provides a new method for the detection of other pathogens.
Asunto(s)
Bombyx , Microsporidiosis , Nosema , Animales , Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Nosema/genéticaRESUMEN
Honey bees are essential pollinators for several economically important crops. In temperate countries, honey bee colonies face multiple threats during the overwintering period, such as food availability, diseases, and confinement. Beekeepers commonly use chemicals to improve colony health during winter, but these products can have a negative impact on bee health and pathogens can develop resistance to them. Thus, there is a need for further development of alternative treatments. The aim of this study was to evaluate the impact of one endogenic bacterium (Bombella apis) and 2 commercial probiotic formulas (Bactocell and Levucell) on colony survival, spring development, and Vairimorpha (formerly Nosema) spp. spore count. Probiotic treatments were given in 1: 1 sugar syrup in October 2017 and April 2018, once a week for 2 wk. One experimental group was given Fumagilin-B, the only product approved in Canada to prevent nosemosis, once in October. The administration of 2 commercial probiotics, Bactocell (Pediococcus acidilactici) and Levucell (Saccharomyces cerevisiae boulardii), led to a significant increase in the number of sealed brood cells in spring. None of the probiotic treatments impacted the honey bee gut load of Vairimorpha spp. spores. The results suggest that beneficial microorganisms can improve spring development and performance of honey bee colonies.