Honeybee gut microbiota dysbiosis in pesticide/parasite co-exposures is mainly induced by Nosema ceranae.

Honeybees ensure a key ecosystem service by pollinating many agricultural crops and wild plants. However, in the past few decades, managed bee colonies have been declining in Europe and North America. Researchers have emphasized both parasites and pesticides as the most important factors. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to gut dysbiosis, impacting the honeybee physiology. Here, we examined and quantified the effects of N. ceranae, the neonicotinoid thiamethoxam, the phenylpyrazole fipronil and the carboxamide boscalid, alone and in combination, on the honeybee gut microbiota. Chronic exposures to fipronil and thiamethoxam alone or combined with N. ceranae infection significantly decreased honeybee survival whereas the fungicide boscalid had no effect on uninfected bees. Interestingly, increased mortality was observed in N. ceranae-infected bees after exposure to boscalid, with synergistic negative effects. Regarding gut microbiota composition, co-exposure to the parasite and each pesticide led to decreased abundance of Alphaproteobacteria, and increased abundance of Gammaproteobacteria. The parasite also induced an increase of bacterial alpha-diversity (species richness). Our findings demonstrated that exposure of honeybees to N. ceranae and/or pesticides play a significant role in colony health and is associated with the establishment of a dysbiotic gut microbiota.

A combined LC-MS and NMR approach to reveal metabolic changes in the hemolymph of honeybees infected by the gut parasite Nosema ceranae.

Nosema ceranae is an emerging and invasive gut pathogen in Apis mellifera and is considered as a factor contributing to the decline of honeybee populations. Here, we used a combined LC-MS and NMR approach to reveal the metabolomics changes in the hemolymph of honeybees infected by this obligate intracellular parasite. For metabolic profiling, hemolymph samples were collected from both uninfected and N. ceranae-infected bees at two time points, 2 days and 10 days after the experimental infection of emergent bees. Hemolymph samples were individually analyzed by LC-MS, whereas each NMR spectrum was obtained from a pool of three hemolymphs. Multivariate statistical PLS-DA models clearly showed that the age of bees was the parameter with the strongest effect on the metabolite profiles. Interestingly, a total of 15 biomarkers were accurately identified and were assigned as candidate biomarkers representative of infection alone or combined effect of age and infection. These biomarkers included carbohydrates (α/ß glucose, α/ß fructose and hexosamine), amino acids (histidine and proline), dipeptides (Glu-Thr, Cys-Cys and γ-Glu-Leu/Ile), metabolites involved in lipid metabolism (choline, glycerophosphocholine and O-phosphorylethanolamine) and a polyamine compound (spermidine). Our study demonstrated that this untargeted metabolomics-based approach may be useful for a better understanding of pathophysiological mechanisms of the honeybee infection by N. ceranae.

A Pediococcus strain to rescue honeybees by decreasing Nosema ceranae- and pesticide-induced adverse effects.

Honeybees ensure a key ecosystemic service by pollinating many agricultural crops and wild plants. However, since few decades, managed bee colonies have declined worldwide. This phenomenon is considered to be multifactorial, with a strong emphasis on both parasites and pesticides. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to adverse effects, resulting in a perturbation of the honeybee physiology. We thus hypothesized that probiotic treatment could be promising to treat or prevent these disturbances. The aim of this study was to evaluate the effects of probiotics on N. ceranae-infected and intoxicated honeybees (by the insecticide thiamethoxam and the fungicide boscalid). For this purpose, experiments were conducted with five probiotics. Among them, Pediococcus acidilactici (PA) showed the best protective effect against the parasite and pesticides. PA significantly improved the infected honeybee lifespan as prophylactic and curative treatments (respectively 2.3 fold and 1.7 fold). Furthermore, the exposure to pesticides induced an increase of honeybee mortality compared with the control group (p < .001) that was restored by the PA treatment. Despite its beneficial effect on honeybee lifespan, the PA administration did not induce changes in the gut bacterial communities (neither in abundance or diversity). N. ceranae and the pesticides were shown to deregulate genes involved in honeybee development (vitellogenin), immunity (serine protease 40, defensin) and detoxification system (glutathione peroxidase-like 2, catalase), and these effects were corrected by the PA treatment. This study highlights the promising use of PA to protect honeybees from both pathogens and pesticides.

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.

Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

The Response of the Honey Bee Gut Microbiota to Nosema ceranae Is Modulated by the Probiotic Pediococcus acidilactici and the Neonicotinoid Thiamethoxam.

The honey bee Apis mellifera is exposed to a variety of biotic and abiotic stressors, such as the highly prevalent microsporidian parasite Nosema (Vairimorpha) ceranae and neonicotinoid insecticides. Both can affect honey bee physiology and microbial gut communities, eventually reducing its lifespan. They can also have a combined effect on the insect's survival. The use of bacterial probiotics has been proposed to improve honey bee health, but their beneficial effect remains an open question. In the present study, western honey bees were experimentally infected with N. ceranae spores, chronically exposed to the neonicotinoid thiamethoxam, and/or supplied daily with the homofermentative bacterium Pediococcus acidilactici MA18/5M thought to improve the honey bees' tolerance to the parasite. Deep shotgun metagenomic sequencing allowed the response of the gut microbiota to be investigated with a taxonomic resolution at the species level. All treatments induced significant changes in honey bee gut bacterial communities. Nosema ceranae infection increased the abundance of Proteus mirabilis, Frischella perrara, and Gilliamella apicola and reduced the abundance of Bifidobacterium asteroides, Fructobacillus fructosus, and Lactobacillus spp. Supplementation with P. acidilactici overturned some of these alterations, bringing back the abundance of some altered species close to the relative abundance found in the controls. Surprisingly, the exposure to thiamethoxam also restored the relative abundance of some species modulated by N. ceranae. This study shows that stressors and probiotics may have an antagonistic impact on honey bee gut bacterial communities and that P. acidilactici may have a protective effect against the dysbiosis induced by an infection with N. ceranae.

The intracellular parasite Anncaliia algerae induces a massive miRNA down-regulation in human cells.

Anncaliia algerae belongs to microsporidia, a group of obligate intracellular parasites related to fungi. These parasites are largely spread in water and food-webs and can infect a wide variety of hosts ranging from invertebrates to vertebrates including humans. In humans, microsporidian infections are mainly opportunistic as immunocompetent hosts can clear parasites naturally. Recent studies however have reported persistent microsporidian infections and have highlighted them as a risk factor in colon cancer. This may be a direct result of cell infection or may be an indirect effect of the infectious microenvironment and the host's response. In both cases, this raises the question of the effects of microsporidian infection at the host and host-cell levels. We aimed to address the question of human host intracellular response to microsporidian infection through a transcriptomic kinetic study of human foreskin fibroblasts (HFF) infected with A.algerae, a human infecting microsporidia with an exceptionally wide host range. We focused solely on host response studying both coding and small non-coding miRNA expression. Our study revealed a generalized down-regulation of cell miRNAs throughout infection with up to 547 different miRNAs downregulated at some timepoints and also transcriptomic dysregulations that could facilitate parasite development with immune and lipid metabolism genes modulation. We also hypothesize possible small nucleic acid expropriation explaining the miRNA downregulation. This work contributes to a better understanding of the dialogue that can occur between an intracellular parasite and its host at the cellular level, and can guide future studies on microsporidian infection biology to unravel the mode of action of these minimalist parasites at the tissue or host levels.We have also generated a kinetic and comprehensive transcriptomic data set of an infectious process that can help support comparative studies in the broader field of parasitology. Lastly, these results may warrant for caution regarding microsporidian exposure and persistent infections.

Identification and localization of polar tube proteins in the extruded polar tube of the microsporidian Anncaliia algerae.

Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae. While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae.

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies.

Blastocystis anaerobic parasites are widespread worldwide in the digestive tract of many animal species, including humans. Epidemiological Blastocystis studies are often limited by the poor sensitivity of standard parasitological assays for its detection. This report presents a highly sensitive real-time quantitative PCR (qPCR) assay developed to detect Blastocystis parasites in stool samples. The assay targets a partial sequence of the Blastocystis small ribosomal subunit (SSU) rRNA gene, allowing subtyping (ST) of Blastocystis isolates by direct sequencing of qPCR products. This qPCR method was assessed in a prospective study of 186 patients belonging to two cohorts--a group of 94 immunocompromised patients presenting hematological malignancies and a control group of 92 nonimmunocompromised patients. Direct-light microscopy and xenic in vitro stool culture analysis showed only 29% and 52% sensitivity, respectively, compared to our qPCR assay. Of the 27 (14.5%) Blastocystis-positive patients, 8 (4%) experienced digestive symptoms. No correlation was found between symptomatic patients and immune status, parasite load, or parasite subtypes, although subtyping of all isolates revealed a high (63.0%) prevalence of ST4. Two unexpected avian subtypes were found, i.e., ST6 and ST7, which are frequently isolated in Asia but rarely present in Western countries. In conclusion, this qPCR proved by far the most sensitive of the tested methods and allowed subtype determination by direct sequencing of qPCR products. New diagnostic tools such as the qPCR are essential for evaluating the clinical relevance of Blastocystis subtypes and their role in acute or chronic digestive disorders.

Molecular subtyping of Blastocystis sp. isolates from symptomatic patients in Italy.

Blastocystis sp. is the most common eukaryotic parasite in the intestinal tract of humans. Due to its potential impact in public health, we determined the Blastocystis sp. subtypes (STs) and their relative frequency in symptomatic patients living in or in the vicinity of two Italian cities (Rome and Sassari). A total of 34 Blastocystis sp. isolates corresponding to 26 single and 4 mixed infections were subtyped using partial small subunit ribosomal RNA gene sequencing. From this molecular approach, the ST distribution in the present Italian population was as follows: ST3 (47.1%), ST2 (20.6%), ST4 (17.7%), ST1 (8.8%), and ST7, and ST8 (2.9%). As in almost all countries worldwide, ST3 was the most common ST reinforcing the hypothesis of its human origin. Together with a previous preliminary report, a total of seven STs (with the addition of ST5) have been found in Italian symptomatic patients. The wide range of STs identified in the Italian population suggest that Blastocystis sp. infection is not associated with specific STs even if some STs (ST1-ST4) are predominant as reported in all other countries. Since most of the STs identified in Italian patients are zoonotic, our data raise crucial questions concerning the identification of animal reservoirs for Blastocystis sp. and the potential risks of transmission to humans.

The human microsporidian Encephalitozoon hellem synthesizes two spore wall polymorphic proteins useful for epidemiological studies.

Microsporidia are obligate intracellular fungus-related parasites considered as emerging opportunistic human pathogens. Their extracellular infective and resistance stage is a spore surrounded by a unique plasma membrane protected by a thick cell wall consisting of two layers: the electron-lucent inner endospore which contains chitin and protein components and the outer-electron-dense and mainly proteinaceous exospore. We identified the whole sequences of two spore wall proteins in the microsporidian species Encephalitozoon hellem, designated EhSWP1a and EhSWP1b. Isolation of the genes encoding these SWP1-like proteins was performed using degenerate oligonucleotides based on the amino acid sequence alignment of the previously reported Encephalitozoon cuniculi and Encephalitozoon intestinalis SWP1s. Sequences lacking the 5' and 3' ends were then identified by PCR and reverse transcription (RT)-PCR amplifications. The swp1a and swp1b genes encode proteins of 509 and 533 amino acids, respectively, which present an identical N-terminal domain of 382 residues and a variable C-terminal extension mainly characterized by a 26-amino-acid (aa) deletion/insertion containing glutamate- and lysine-rich repeats. Using polyclonal antibodies raised against recombinant polypeptides, we showed that EhSWP1a and EhSWP1b appear as dithiothreitol (DTT)-soluble bands of 55 and 60 kDa in size, respectively. Immunolocalization experiments by IFA and transmission electron microscopy (TEM) indicated that both proteins are present at the onset of sporogony and are specifically located to the spore wall exospore in mature spores. Analysis of four E. hellem human isolates revealed that the C-terminal regions of both EhSWP1a and EhSWP1b are polymorphic, which is of interest for epidemiological studies.

Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt.

Blastocystis sp. has been described as the most common intestinal parasite in humans and has an increased impact in public health. To improve our understanding of the molecular epidemiology of this human-emerging parasite, we determined the Blastocystis subtypes (STs) and their relative frequency in Egyptian patients living in or in the vicinity of Cairo and presenting gastrointestinal symptoms. We obtained a total of 20 stool samples identified as positive for Blastocystis by microscopic examination of smears. Genotyping using partial small subunit ribosomal RNA gene analysis identified a total of 21 Blastocystis isolates corresponding to 19 single infections and one mixed infection (ST1 and ST3). Three STs were identified: ST3 was the most common ST in the present Egyptian population (61.90%) followed by ST1 (19.05%) and ST2 (19.05%). Together with previous studies carried out in different areas in Egypt, a total of five STs (ST1, ST2, ST3, ST4, and ST6) have been found in symptomatic patients. These data were compared to those available in the literature, and we underlined variations observed in the number and relative proportions of STs between and within countries. On the whole, it seemed that Blastocystis infection is likely not associated with specific STs even if some STs are predominant in the epidemiologic studies, but rather with a conjunction of factors in the course of infection including environmental risk and parasite and host factors.

Relationships between Plant Defense Inducer Activities and Molecular Structure of Gallomolecules.

Plant defense inducers (PDIs) are booming and attractive protection agents designed to immunostimulate the plant to reduce subsequent pathogen colonization. The structure-PDI activity relationships of four flavan-3-ols: Epicatechin (EC), Epigallocatechin (EGC), Epicatechin gallate (ECG), Epigallocatechin gallate (EGCG) and Gallotannic acid (GTA) were investigated in both whole plant and suspension cell systems. ECG, EGCG, and GTA displayed elicitor activities. Their infiltration into tobacco leaves induced hypersensitive reaction-like lesions with topical scopoletin and PR-target transcript accumulations. On the contrary, EC and EGC infiltrations fail to trigger the biochemical changes in tobacco tissues. The tobacco BY-2 cells challenged with ECG, EGCG, or GTA led to alkalinization of the BY-2 extracellular medium while EC and EGC did not trigger any pH variation. This work provides evidence that the esterified gallate pattern is as an essential flavonoid entity to induce plant defense reactions in tobacco. The phytoprotective properties of the esterified gallate-free EC and the esterified gallate-rich GTA were evaluated on the tobacco/Phytophthora parasitica var. nicotianae (Ppn) pathosystem. Tobacco treatment with EC did not induce significant protection against Ppn compared to GTA which shows antimicrobial properties on Ppn and decreases the infection on GTA-infiltrated and -sprayed wild-type leaves. GTA protection was impaired in the transgenic NahG tobacco plants, suggesting that protection was mediated by salicylic acid.

Effects of the gut parasite Nosema ceranae on honey bee physiology and behavior.

The common and widespread parasite Nosema ceranae is considered a major threat to the Western honey bee at both the individual and colony levels. Several studies demonstrated that infection by this parasite may affect physiology, behavior, and survival of honey bees. N. ceranae infection impairs midgut integrity and alters the energy demand in honey bees. The infection can also significantly suppress the bee immune response and modify pheromone production in worker and queen honey bees leading to precocious foraging. However, the presence of N. ceranae is not systematically associated with colony weakening and honey bee mortality. This variability depends upon parasite or host genetics, nutrition, climate or interactions with other stressors such as environmental contaminants or other parasites.

Occurrence of the microsporidian parasite Nucleospora salmonis in four species of salmonids from the Massif Central of France.

Nucleospora salmonis (Hedrick, Groff et Baxa, 1991), an intranuclear microsporidian parasite of marine and freshwater fish, causes diseases mainly in salmonid species. Losses have been reported in stocks of salmonid fish reared in the region of Auvergne (France). The cause of chronic mortalities in the local host species raised in aquaculture and destined for supplementation of the river system Loire-Allier was examined. The presence of N. salmonis was confirmed by PCR and histology in Salmo salar L. previously and in newly investigated salmonid species, Salmo salar, Salmo trutta fario L., Thymallus thymallus (L.) and Salvelinus alpinus (L.), present in European streams. The infection by N. salmonis was consistent in all cases with characteristic symptoms of the disease in deceased or moribund fish. The small subunit ribosomal DNA from N. salmonis was partially sequenced and compared to previously characterised N. salmonis isolates. As a result, a genotype, or clonal entity, was attributed to N. salmonis among Atlantic salmon found along the Northern Atlantic coastal lines and other salmonid species co-inhabiting or co-cultivated in the Auvergne region.

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.