Honey Bee Midgut Research Articles

Construction of a Full-Length Transcriptome of Western Honeybee Midgut Tissue and Improved Genome Annotation.

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.

Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

Features of digestion in the middle intestine of the honey bee

The midgut of the honey bee is considered the only midgut. Processes related to digestion take place in this department. At the same time, the middle intestine takes part in the processes that indicate the immune status. The purpose of the research was to study the effect of protein feed on the morphometric parameters of the epithelium of the midgut of honeybees and the peretrophic membrane. Two groups of families participated in the study. The first group received bee pollen collected from winter rape with a protein content of 25 % as protein feed. The second group consumed dandelion meal with a protein content of 15 %. After reaching the required age, the insects were taken from the nest, and the midgut was dissected. The conducted studies indicate that the length of the enterocytes of the midgut of a honey bee at the age of 10 days ranges from 18.89 to 34.11 μm. Morphometric differences of midgut enterocytes in the experimental groups did not reveal any significant difference. It has been proven that the thickness of the pertrophic membrane in hive bees is greater than that of flying bees by 22.7 % (P < 0.001). This value depends on the area where it is placed. In particular, in the caudal part, the thickness of the pertrophic membrane is more significant on average by 25.0 % compared to the cranial part (P < 0.01). The decrease in the thickness of the midgut of flying bees is related to the nature of nutrition. Older bees consume less pollen and eat more carbohydrate feed. Along with this, it has been proven that the thickness of the pertrophic membrane depends on the composition of the diet. In hive bees that consumed protein-enriched feed, the thickness of the pertrophic membrane in the caudal part of the midgut is 22.1 % greater (P < 0.001).

Ame-miR-980-3p participates in autophagy-mediated midgut remodelling in Apis mellifera via targeting Atg2B.

Autophagy is a process that serves to degrade damaged proteins and organelles, thereby promoting cell homeostasis, differentiation, development and survival. Many miRNAs have been found to have regulatory roles in autophagy. In insects, it has been shown that autophagy is involved in hormone-regulated programmed cell death during metamorphic midgut remodelling. However, whether this is also true during the remodelling of the honey bee midgut is unclear. In the present study, we explored the relationship between autophagy and midgut remodelling and sought to identify miRNAs involved in this physiological process. We found that autophagy occurred during midgut remodelling and that the inhibition of autophagy resulted in midgut dysplasia in prepupae. Differentially expressed miRNAs enriched in the autophagy signalling pathway during midgut remodelling were identified by small RNA-seq. Ame-miR-980-3p, which targets the autophagy-related gene Atg2B, was screened out. Furthermore, abnormal expression of ame-miR-980-3p in the pupal stage led to the thinning of the midgut wall of newly emerged bees (NE). When ame-miR-980-3p expression was inhibited, the intestinal villi of NE bees became significantly shorter and sparse, and the lipid signal in the peritrophic matrix of Pb almost disappeared, indicating that the adult midgut was underdeveloped and the lipid absorption ability was weakened. Taken together, ame-miR-980-3p targeted Atg2B to participate in the regulation of midgut autophagy in the pupae, and the abnormal expression of ame-miR-980-3p would interfere with cell proliferation and death in the process of midgut remodelling, hinder the formation of adult midgut and eventually lead to adult midgut dysplasia and affect the lipid absorption function of the midgut in Apis mellifera.

Engineered symbiotic bacteria interfering Nosema redox system inhibit microsporidia parasitism in honeybees

Nosema ceranae is an intracellular parasite invading the midgut of honeybees, which causes serious nosemosis implicated in honeybee colony losses worldwide. The core gut microbiota is involved in protecting against parasitism, and the genetically engineering of the native gut symbionts provides a novel and efficient way to fight pathogens. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibit microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response. Accordingly, N. ceranae employs the thioredoxin and glutathione systems to defend against oxidative stress and maintain a balanced redox equilibrium, which is essential for the infection process. We knock down the gene expression using nanoparticle-mediated RNA interference, which targets the γ-glutamyl-cysteine synthetase and thioredoxin reductase genes of microsporidia. It significantly reduces the spore load, confirming the importance of the antioxidant mechanism for the intracellular invasion of the N. ceranae parasite. Finally, we genetically modify the symbiotic S. alvi to deliver dsRNA corresponding to the genes involved in the redox system of the microsporidia. The engineered S. alvi induces RNA interference and represses parasite gene expression, thereby inhibits the parasitism significantly. Specifically, N. ceranae is most suppressed by the recombinant strain corresponding to the glutathione synthetase or by a mixture of bacteria expressing variable dsRNA. Our findings extend our previous understanding of the protection of gut symbionts against N. ceranae and provide a symbiont-mediated RNAi system for inhibiting microsporidia infection in honeybees.

Colonization of Honey Bee Digestive Tracts by Environmental Yeast Lachancea thermotolerans Is Naturally Occurring, Temperature Dependent, and Impacts the Microbiome of Newly Emerged Bees.

Honey bees are critical pollinators in both agricultural and ecological settings. Recent declines in honey bee colonies in the United States have put increased strain on agricultural pollination. Although there are many environmental stressors implicated in honey bee disease, there has been intensifying focus on the role of microbial attacks on honey bee health. Despite the long-standing appreciation for the association of fungi of various groups with honey bees and their broader environment, the effects of these interactions on honey bee health are incompletely understood. Here, we report the discovery of colonization of the honey bee digestive tract by the environmental yeast Lachancea thermotolerans. Experimental colonization of honey bee digestive tracts by L. thermotolerans revealed that this yeast species maintains high levels in the honey bee midgut only at temperatures below the typical colony temperature. In newly eclosed bees, L. thermotolerans colonization alters the microbiome, suggesting that environmental yeasts can impact its composition. Future studies should be undertaken to better understand the role of L. thermotolerans and other environmental yeasts in honey bee health. IMPORTANCE Although many fungal species are found in association with honey bees and their broader environment, the effects of these interactions on honey bee health are largely unknown. Here, we report the discovery that a yeast commonly found in the environment can be found at high levels in honey bee digestive tracts. Experimentally feeding this yeast to honey bees showed that the yeast's ability to maintain high levels in the digestive tract is influenced by temperature and can lead to alterations of the microbiome in young bees. These studies provide a foundation for future studies to better understand the role of environmental yeasts in honey bee health.

Ecotoxicological effects of common fungicides on the eastern honeybee Apis cerana cerana (Hymenoptera)

The widespread use of fungicides for plant protection has increased the potential for pollinator exposure. This study therefore aimed at assessing the acute and chronic effects of fungicides on pollinators. For this purpose, the acute oral toxicity of the common fungicides azoxystrobin, pyraclostrobin, and boscalid to Eastern honeybee Apis cerana cerena was first evaluated, and the chronic effects on multiple aspects were investigated after exposure to a one-tenth medium lethal dose (LD50) for 10 days. This study revealed that the LD50 values of azoxystrobin, pyraclostrobin and boscalid for adult Eastern honeybees were 12.7 μg/bee, 36.6 μg/bee, and >119 μg/bee, respectively. Midgut epithelial cells revealed that fungicide exposure caused increased intercellular spaces and varying degrees of vacuolization. Exposure to these three fungicides and their binary mixtures significantly affected glycerophospholipid, alanine, aspartate, and glutamate metabolism in Eastern honeybee midguts. Additionally, the relative composition of Lactobacillus, the dominant functional genus in Eastern honeybee guts decreased and microbial balance was disrupted. All fungicides and their mixtures induced strong transcriptional upregulation of genes associated with the immune response and encoding enzymes related to oxidative phosphorylation and metabolism, including abaecin, apidaecin, hymenotaecin, cyp4c3, cyp6a2 and hbg3. Our study provides important insight for understanding the effects of commonly used fungicides on nontarget pollinator and contributes to a more comprehensive assessment of fungicide effects on ecological and environmental safety.

Disinfectant dodecyl dimethyl benzyl ammonium chloride (DDBAC) disrupts gut microbiota, phospholipids, and calcium signaling in honeybees (Apis mellifera) at an environmentally relevant level

One of the impacts of the Coronavirus disease 2019 (COVID-19) pandemic has been a profound increase in the application amounts of disinfectants. Dodecyl dimethyl benzyl ammonium chloride (DDBAC) is a widely used disinfectant, yet its hazards to non-target species remain largely unknown. We are unaware of any studies assessing DDBAC’s impacts on honeybee, a pollinator species that is a useful indicator of environmental pollution essential for many forms of agricultural production. Here, we assessed the potentially negative effects of DDBAC on honeybees. After conducting a formal toxicity evaluation of DDBAC on honeybee mortality, we detected an accumulation of DDBAC in the honeybee midgut. We subsequently studied the midgut tissues of honeybees exposed to sub-lethal concentrations of DDBAC: histopathological examination revealed damage to midgut tissue upon DDBAC exposure, microbiome analysis showed a decreased abundance of beneficial midgut microbiota, lipidomics analysis revealed a significant reduction in cell membrane phospholipids with known functions in signal transduction, and a transcriptome analysis detected altered expression of genes involved in calcium signaling pathways (that variously function in calcium absorption, muscle contraction, and neurotransmission). Thus, our study establishes that DDBAC impacts honeybee midgut functions at multiple levels. Our study represents an early warning about the hazards of DDBAC and appeals for the proper stewardship of DDBAC to ensure the protection of our ecological environment.

Antifungal activity of “HO21-F”, a formulation based on Olea europaea plant extract, in honey bees infected with Nosema ceranae

Nosema ceranae is a microsporidium parasite that silently affects honey bees, causing a disease called nosemosis. This parasite produces resistant spores and germinates in the midgut of honey bees, extrudes a polar tubule that injects an infective sporoplasm in the host cell epithelium, proliferates, and produces intestinal disorders that shorten honey bee lifespan. The rapid extension of this disease has been reported to be widespread among adult bees, and treatments are less effective and counterproductive weakening colonies. This work aimed to evaluate the antifungal activity of a prototype formulation based on a non-toxic plant extract (HO21-F) against N. ceranae. In laboratory, honey bees were infected artificially, kept in cages for 17 days and samples were taken at 7 and 14 days post infection (dpi). At the same time, in field conditions we evaluated the therapeutic effect of HO21-F for 28 days in naturally infected colonies. The effectiveness of the treatment has been demonstrated by a reduction of 83.6 % of the infection levels observed in laboratory conditions at concentrations of 0.5 and 1 g/L without affecting the survival rate. Besides, in-field conditions we reported a reduction of 88 % of the infection level at a concentration of 2.5 g/L, obtaining better antifungal effectiveness in comparison to other commercially available treatments.As a result, we observed that the use of HO21-F led to an increase in population size and honey production, both parameters associated with colony strength. The reported antifungal activity of HO21-F against N. ceranae, with a significant control of spore proliferation in worker bees, suggests the promising commercial application use of this product against nosemosis, and it will encourage new research studies to understand the mechanism of action, whether related to the spore-inhibition effect and/or a stimulating effect in natural response of colonies to counteract the disease.

Diet-derived transmission of MicroRNAs from host plant into honey bee Midgut

BackgroundMicroRNA (miRNA) is a class of small noncoding RNAs, which targets on thousands of mRNA and thus plays important roles in many biological processes. It has been reported that miRNA has cross-species regulation functions between parasitoid-host, or plant-animal, etc. For example, several plant miRNAs enter into the honey bees and regulate gene expression. However, whether cross-species regulation function of miRNAs is a universal mechanism remains a debate question.ResultsWe have evaluated transmission of miRNAs from sunflower and sedr plants into the midgut of honey bee using RNA-Seq analyses complemented with confirmation by RT-qPCR. The results showed that at least 11 plant miRNAs were found in the midgut of honey bee feeding by sunflower and sedr pollen. Among which, nine miRNAs, including miR-30d, miR-143, miR-148a, miR-21, let-7 g, miR-26a, miR-126, miR-27a, and miR-203, were shared between the sunflower- and sedr-fed honey bees, suggesting they might have essential roles in plant-insect interactions. Moreover, existence of these co-shared miRNAs presents a strong evidence to support the successful transmission of miRNAs into the midgut of the insect. In total, 121 honeybee mRNAs were predicted to be the target of these 11 plant-derived miRNAs. Interestingly, a sedr-derived miRNA, miR-206, targets on 53 honeybee genes. Kyoto Encyclopedia of Genes and Genome (KEGG) analyses showed that these target genes are significantly involved in hippo signaling pathway-fly, Wnt signaling pathway, and N-Glycan biosynthesis.ConclusionsIn summary, these results provide evidence of cross-species regulation function of miRNA between honeybee and flowering host plants, extending our understanding of the molecular interactions between plants and animals.

Ex Vivo Effect of Ibogaine on the Transcriptional Level of Antioxidant Defense Related Genes in Honey Bee (Apis mellifera, L.) Midgut

HIGHLIGHTS The ibogain action has not been completely clarified. Honey bee midgut is suitable model system for testing the antioxidative mechanisms. The ex vivo ibogaine treatment induced an up-regulation of Sod1 gene. Ibogaine shows pro-antioxidant action.

A combination of Tropilaelaps mercedesae and imidacloprid negatively affects survival, pollen consumption and midgut bacterial composition of honey bee

Tropilaelaps mercedesae is not only a major threat to honey bees in Asia but also a potential risk to global apiculture due to trade. Imidacloprid is a systemic insecticide that negatively affects individual bees. Moreover, the health of honey bees may be threatened by imidacloprid exposure and T. mercedesae infestation. We studied the effects of T. mercedesae and imidacloprid on the survival, food consumption and midgut bacterial diversity of Apis mellifera in the laboratory. Illumina 16S rRNA gene sequencing was used to determine the bacterial composition in the honey bee midgut. T. mercedesae decreased survival in parasitized honey bees compared with nonparasitized honey bees, but there was no significant difference in food consumption. The imidacloprid 50 μg/L diet significantly decreased syrup consumption of A. mellifera compared with the control diet. The combination of T. mercedesae infestation and imidacloprid 50 μg/L exposure reduced survival and increased pollen consumption of A. mellifera. T. mercedesae infestation or a combination of T. mercedesae infestation and exposure to 25 μg/L imidacloprid affected the midgut bacterial composition of honey bees. T. mercedesae infestation and imidacloprid exposure may reduce the survival and affect honey bee health.

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1pg vs 10pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

Changes in the gut microbiota of honey bees associated with jujube flower disease.

Honeybees are prone to poisoning after collecting jujube nectar during the jujube flowering period ('honeybee's jujube flower disease'). To explore the mechanism of honeybee poisoning, the gut microbiota of honeybees undergoing the disease were characterised based on amplicon sequencing of the 16S rRNA gene. Our results showed that the composition and diversity of the gut microbiota were significantly altered in diseased honeybees. We observed a decrease in the relative abundance of Proteobacteria and increased abundances of Firmicutes and Actinobacteria in the midgut and hindgut of diseased honeybees. Moreover, linear discriminant analysis (LDA) effect size revealed significantly selected enrichment of Fructobacillus and Snodgrassella in the midguts from diseased honeybees and Lactobacillus, Bifidobacterium, and Snodgrassella in the hindguts from diseased honeybees. Tax4Fun anylasis indicated that the functional potential of the diseased honeybee gut bacterial community was significantly changed relative to the healthy honeybee. Carbohydrate metabolism, nucleotides metabolism, amino acid synthesis metabolism, coenzyme and vitamins metabolism were increased, while energy metabolism and xenobiotic biodegradation and metabolism were decreased in the diseased honeybees. These results provide a new perspective for evaluating the response of honeybees to jujube flower disease based on changes in the intestinal microflora.

The different dietary sugars modulate the composition of the gut microbiota in honeybee during overwintering

BackgroundThe health of honeybee colonies is critical for bee products and agricultural production, and colony health is closely associated with the bacteria in the guts of honeybees. Although colony loss in winter is now the primary restriction in beekeeping, the effects of different sugars as winter food on the health of honeybee colonies are not well understood. Therefore, in this study, the influence of different sugar diets on honeybee gut bacteria during overwintering was examined.ResultsThe bacterial communities in honeybee midguts and hindguts before winter and after bees were fed honey, sucrose, and high-fructose syrup as winter-food were determined by targeting the V3-V4 region of 16S rDNA using the Illumina MiSeq platform.The dominant microbiota in honeybee guts were the phyla Proteobacteria (63.17%), Firmicutes (17.61%; Lactobacillus, 15.91%), Actinobacteria (4.06%; Bifidobacterium, 3.34%), and Bacteroidetes (1.72%). The dominant taxa were conserved and not affected by season, type of overwintering sugar, or spatial position in the gut. However, the relative abundance of the dominant taxa was affected by those factors. In the midgut, microbial diversity of the sucrose group was higher than that of the honey and high-fructose syrup groups, but in the hindgut, microbial diversity of the honey and high-fructose groups was higher than that in the sucrose group. Sucrose increased the relative abundance of Actinobacteria (Bifidobacteriales Bifidobacteriaceae) and Alphaproteobacteria (Rhizobiales and Mitochondria) of honeybee midgut, and honey enriched the Bacteroidetes and Gammaproteobacteria (Pasteurellales) in honeybee hindgut. High-fructose syrup increased the relative abundance of Betaproteobacteria (Neisseriales: Neisseriaceae) of the midgut.ConclusionThe type of sugar used as winter food affected the relative abundance of the dominant bacterial communities in honeybee guts, not the taxa, which could affect the health and safety of honeybee colonies during overwintering. The presence of the supernal Alphaproteobacteria, Bifidobacteriales, and Lactobacillaceae in the gut of honeybees fed sucrose and cheaper than honey both indicate that sucrose is very suitable as the overwintering food for honeybees.

Hydroxymethylfurfural Affects Caged Honey Bees (Apis mellifera carnica)

A high concentration of hydroxymethylfurfural (HMF) (e.g., 15 mg HMF per kg honey) indicates quality deterioration for a wide range of foods. In honey bee colonies, HMF in stored honey can negatively affect bee health and survival. Therefore, in the laboratory, we experimentally determined the effects of HMF on the longevity and midgut integrity of worker Apis mellifera carnica by feeding bees standard diets containing five concentrations of HMF (100, 500, 1000, and 1500 ppm). Simultaneously, we also examined HMF’s effect on Nosema ceranae spore counts within infected honey bees. We performed an immunohistochemical analysis of the honey bee midgut to determine possible changes at the cellular level. No correlation was established between HMF concentration and N. ceranae spore counts. Negative effects of HMF on bees were not observed in the first 15 days of exposure. However, after 15 to 30 days of exposure, HMF caused midgut cells to die and an increased mortality of honey bee workers across treatment groups.

Effects of a Resident Yeast from the Honeybee Gut on Immunity, Microbiota, and Nosema Disease.

The western honeybee (Apis mellifera) has a core bacterial microbiota that is well described and important for health. Honeybees also host a yeast community that is poorly understood with respect to host nutrition and immunity, and also the symbiotic bacterial microbiota. In this work, we present two studies focusing on the consequences of dysbiosis when honeybees were control-fed a yeast that was isolated from a honeybee midgut, Wickerhamomyces anomalus. Yeast augmentation for bees with developed microbiota appeared immunomodulatory (lowered immunity and hormone-related gene expression) and affected the microbial community, while yeast augmentation for newly emerged bees without an established bacterial background did not lead to decreased immunity— and hormone—related gene expression. In newly emerged bees that had a naturally occurring baseline level of W. anomalus, we observed that the addition of N. ceranae led to a decrease in yeast levels. Overall, we show that yeasts can affect the microbiome, immunity, and physiology.

Dicer regulates Nosema ceranae proliferation in honeybees

Nosema ceranae is a microsporidian parasite that infects the honeybee midgut epithelium. The protein-coding gene Dicer is lost in most microsporidian genomes but is present in N.ceranae. By feeding infected honeybees with small interfering RNA targeting the N.ceranae gene coding Dicer (siRNA-Dicer), we found that N.ceranae spore loads were significantly reduced. In addition, over 10% of total parasite protein-coding genes showed significantly divergent expression profiles after siRNA-Dicer treatment. Parasite genes for cell proliferation, ABC transporters and hexokinase were downregulated at 3days postinfection, a key point in the middle of parasite replication cycles. In addition, genes involved in metabolic pathways of honeybees and N.ceranae showed significant co-expression. Furthermore, the siRNA-Dicer treatment partly reversed the expression patterns of honeybee genes. The honeybee gene mucin-2-like showed significantly upregulation in the siRNA-Dicer group compared with the infection group continually at 4, 5 and 6days postinfection, suggesting that the siRNA-Dicer feeding promoted the strength of the mucus barrier resulted from interrupted parasite proliferation. As the gene Dicer broadly regulates N.ceranae proliferation and honeybee metabolism, our data suggest the RNA interference pathway is an important infection strategy for N.ceranae.

Impact of the microsporidian Nosema ceranae on the gut epithelium renewal of the honeybee, Apis mellifera.

The invasive microsporidian species, Nosema ceranae, causes nosemosis in honeybees and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the site of infection; the microsporidium can disturb the functioning of this organ and, thus, the bee physiology. Host defense against pathogens is not limited to resistance (i.e. the immune response) but also involves resilience. This process implies that the host can tolerate and repair damage inflicted by the infection- by the pathogen itself or by an excessive host immune response. Enterocyte damage caused by N. ceranae can be compensated by proliferation of intestinal stem cells (ISCs) that are under the control of multiple pathways. In the present study, we investigated the impact of N. ceranae on honeybee epithelium renewal by following the mitotic index of midgut stem cells during a 22-day N. ceranae infection. Fluorescence in situ hybridization (FISH) and immunostaining experiments were performed to follow the parasite proliferation/progression in the intestinal tissue, especially in the ISCs as they are key cells for the midgut homeostasis. We also monitored the transcriptomic profile of 7 genes coding for key proteins involved in pathways implicated in the gut epithelium renewal and homeostasis. We have shown for the first time that N. ceranae can negatively alter the gut epithelium renewal rate and disrupt some signaling pathways involved in the gut homeostasis. This alteration is correlated to a reduced longevity of N. ceranae-infected honeybees and we can assume that honeybee susceptibility to N. ceranae could be due to an impaired ability to repair gut damage.

Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis.

Nosema ceranae is an intracellular microsporidian parasite that infects epithelial cells of the honey bee (Apis mellifera) midgut. Previous studies have shown that Nosema may alter cell renewal and apoptosis in honey bees. We found that the amount of apoptotic cells progressively declines from the anterior towards posterior regions of the midgut in Nosema-infected sensitive bees. There was no such pattern in the infected Nosema tolerant honey bees and controls. These data provide additional evidence that N. ceranae appears to alter apoptosis in its host cells for its own advantage.