Genomic and metagenomic analyses of the domestic mite Tyrophagus putrescentiae identify it as a widespread environmental contaminant and a host of a basal, mite-specific Wolbachia lineage (supergroup Q).

Tyrophagus putrescentiae (mould mite) is a global, microscopic trophic generalist that commonly occurs in various human-created habitats, causing allergies and damaging stored food. Its ubiquity and extraordinary ability to penetrate research samples or cultures through air currents or by active walking through tights spaces (such as treads of screw caps) may lead to sample contamination and introduction of its DNA to research materials in the laboratory. This prompts a thorough investigation into potential sequence contamination in public genomic databases. The trophic success of T. putrescentiae is primarily attributed to the symbiotic bacteria housed in specialized internal mite structures, facilitating adaptation to varied nutritional niches. However, recent work suggests that horizontal transfer of bacterial/fungal genes related to nutritional functionality may also contribute to the mite's trophic versatility. This aspect requires independent confirmation. Additionally, T. putrescentiae harbors an uncharacterized and genetically divergent bacterium, Wolbachia, displaying blocking and microbiome-modifying effects. The phylogenomic position and supergroup assignment of this bacterium are unknown. Here, we sequenced and assembled the T. putrescentiae genome, analyzed its microbiome, and performed detailed phylogenomic analyses of the mite-specific Wolbachia. We show that T. putrescentiae DNA is a substantial source of contamination of research samples. Its DNA may inadvertently be co-extracted with the DNA of the target organism, eventually leading to sequence contamination in public databases. We identified a diversity of bacterial species associated with T. putrescentiae, including those capable of rapidly developing antibiotic resistance, such as Escherichia coli. Despite the presence of diverse bacterial communities in T. putrescentiae, we did not detect any recent horizontal gene transfers in this mite species and/or in astigmatid (domestic) mites in general. Our phylogenomic analysis of Wolbachia recovered a basal, mite-specific lineage (supergroup Q) represented by two Wolbachia spp. from the mould mite and a gall-inducing plant mite. Fluorescence in situ hybridization confirmed the presence of Wolbachia inside the mould mite. The discovery of an early derivative Wolbachia lineage (supergroup Q) in two phylogenetically unrelated and ecologically dissimilar mites suggests that this endosymbiotic bacterial lineage formed a long-term association with mites. This finding provides a unique insight into the early evolution and host associations of Wolbachia. Further discoveries of Wolbachia diversity in acariform mites are anticipated.

Understanding bacterial pathogen diversity: A proteogenomic analysis and use of an array of genome assemblies to identify novel virulence factors of the honey bee bacterial pathogen Paenibacillus larvae.

Mass spectrometry proteomics data are typically evaluated against publicly available annotated sequences, but the proteogenomics approach is a useful alternative. A single genome is commonly utilized in custom proteomic and proteogenomic data analysis. We pose the question of whether utilizing numerous different genome assemblies in a search database would be beneficial. We reanalyzed raw data from the exoprotein fraction of four reference Enterobacterial Repetitive Intergenic Consensus (ERIC) I-IV genotypes of the honey bee bacterial pathogen Paenibacillus larvae and evaluated them against three reference databases (from NCBI-protein, RefSeq, and UniProt) together with an array of protein sequences generated by six-frame direct translation of 15 genome assemblies from GenBank. The wide search yielded 453 protein hits/groups, which UpSet analysis categorized into 50 groups based on the success of protein identification by the 18 database components. Nine hits that were not identified by a unique peptide were not considered for marker selection, which discarded the only protein that was not identified by the reference databases. We propose that the variability in successful identifications between genome assemblies is useful for marker mining. The results suggest that various strains of P. larvae can exhibit specific traits that set them apart from the established genotypes ERIC I-V.

Discovery and characterization of novel DNA viruses in Apis mellifera: expanding the honey bee virome through metagenomic analysis.

To date, many viruses have been discovered to infect honey bees. In this study, we used high-throughput sequencing to expand the known virome of the honey bee, Apis mellifera, by identifying several novel DNA viruses. While the majority of previously identified bee viruses are RNA, our study reveals nine new genomes from the Parvoviridae family, tentatively named Bee densoviruses 1 to 9. In addition, we characterized a large DNA virus, Apis mellifera filamentous-like virus (AmFLV), which shares limited protein identities with the known Apis mellifera filamentous virus. The complete sequence of AmFLV, obtained by a combination of laboratory techniques and bioinformatics, spans 152,678 bp. Linear dsDNA genome encodes for 112 proteins, of which 49 are annotated. Another large virus we discovered is Apis mellifera nudivirus, which belongs to a group of Alphanudivirus. The virus has a length of 129,467 bp and a circular dsDNA genome, and has 106 protein encoding genes. The virus contains most of the core genes of the family Nudiviridae. This research demonstrates the effectiveness of viral binning in identifying viruses in honey bee virology, showcasing its initial application in this field.IMPORTANCEHoney bees contribute significantly to food security by providing pollination services. Understanding the virome of honey bees is crucial for the health and conservation of bee populations and also for the stability of the ecosystems and economies for which they are indispensable. This study unveils previously unknown DNA viruses in the honey bee virome, expanding our knowledge of potential threats to bee health. The use of the viral binning approach we employed in this study offers a promising method to uncovering and understanding the vast viral diversity in these essential pollinators.

Varroa destructor parasitism and Deformed wing virus infection in honey bees are linked to peroxisome-induced pathways.

The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-ß signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.

Sublethal acetamiprid exposure induces immunity, suppresses pathways linked to juvenile hormone synthesis in queens and affects cycle-related signaling in emerging bees.

Acetamiprid is the only neonicotinoid registered in the European Union because the risks of neonicotinoids to honey bees and other pollinators are strictly regulated. Herein, we orally exposed honey bee colonies to sublethal concentrations of acetamiprid (20 µg/L) under isolated conditions. After one month of continuous exposure, the emerging bees and queens were collected and analyzed via high-throughput label-free quantitative proteomics using a data-independent acquisition strategy. Six and 34 significantly differentially expressed proteins (DEPs) were identified in the emerging bees and queens, respectively. Mrjp3 was the only DEP found in both sample types/castes, and its opposite regulation illustrated a differential response. The DEPs in the emerging bees (H/ACA RNP, Rap1GAP, Mrjp3, and JHE) suggested that sublethal exposure to acetamiprid affected cell cycle-related signaling, which may affect the life history of workers in the colony. The DEPs with increased levels in queens, such as Mrjps 1-4 and 6-7, hymenoptaecin, and apidaecin 22, indicated an activated immune response. Additionally, the level of farnesyl pyrophosphate synthase (FPPS), which is essential for the mevalonate pathway and juvenile hormone biosynthesis, was significantly decreased in queens. The impaired utilization of juvenile hormone in queens supported the identification of additional DEPs. Furthermore, the proteome changes suggested the existence of increased neonicotinoid detoxification by UDP-glucuronosyltransferase and increased amino acid metabolism. The results suggest that the continuous exposure of bee colonies to acetamiprid at low doses (nanograms per gram in feed) may pose a threat to the colonies. The different exposure routes and durations for the emerging bees and queens in our experiment must be considered, i.e., the emerging bees were exposed as larvae via feeding royal jelly and beebread provided by workers (nurse bees), whereas the queens were fed royal jelly throughout the experiment. The biological consequences of the proteomic changes resulting from sublethal/chronic exposure require future determination.

Mixta mediterraneensis as a novel and abundant gut symbiont of the allergen-producing domestic mite Blomia tropicalis.

Blomia tropicalis is an allergen-producing mite in the human environment in tropical regions. The microbiome of B. tropicalis was described using the barcode sequencing region of V4 16S rDNA and genome assemblage. Mixta mediterraneensis, previously isolated from human skin swabs, was identified as a B. tropicalis gut symbiont based on genome assembly. The microbiome contains two bacteria, Staphylococcus and M. mediterraneensis. The number of M. mediterraneensis 16S DNA copies was 106 per mite and 109 per feces in the rearing chamber based on qPCR quantification. The profile of this bacterium reached 50% of reads in the mite gut and feces. Genomic analyses revealed that the bacterium has several metabolic pathways that suggest metabolic cooperation with the mite host in vitamin and amino acid synthesis, nitrogen recycling, and antimicrobial defense. Lysozyme is present in the symbiotic bacterium but absent in the mite. The B. tropicalis microbiome contained Staphylococcus, which accelerates mite population growth. Mites can digest Staphylococcus by using specific enzymes with hydrolytic functions against bacterial cell walls (chitinases and cathepsin D), leading to endocytosis of bacteria and their degradation in lysosomes and phagosomes. Gene expression analysis of B. tropicalis indicated that phagocytosis was mediated by the PI3-kinase/Akt pathway interacting with the invasins produced by M. mediterraneensis. Moreover, the symbiont had metabolic pathways that allowed it to recycle the mite metabolic waste product guanine, known as a mite attractant. The mite host symbiont enhances mite aggregation in the feces, and the fecal-oral transmission route is excepted.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens.

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Predicting Blomia tropicalis allergens using a multiomics approach.

BACKGROUND: The domestic mite Blomia tropicalis is a major source of allergens in tropical and subtropical regions. Despite its great medical importance, the allergome of this mite has not been sufficiently studied. Only 14 allergen groups have been identified in B. tropicalis thus far, even though early radioimmunoelectrophoresis techniques (27 uncharacterized allergen complexes) and comparative data based on 40 allergen groups officially recognized by the World Health Organization (WHO)/IUIS in domestic astigmatid mites suggest the presence of a large set of additional allergens. METHODS: Here, we employ a multiomics approach to assess the allergome of B. tropicalis using genomic and transcriptomic sequence data and perform highly sensitive protein abundance quantification. FINDINGS: Among the 14 known allergen groups, we confirmed 13 (one WHO/IUIS allergen, Blo t 19, was not found) and identified 16 potentially novel allergens based on sequence similarity. These data indicate that B. tropicalis shares 27 known/deduced allergen groups with pyroglyphid house dust mites (genus Dermatophagoides). Among these groups, five allergen-encoding genes are highly expressed at the transcript level: Blo t 1, Blo t 5, Blo t 21 (known), Blo t 15, and Blo t 18 (predicted). However, at the protein level, a different set of most abundant allergens was found: Blo t 2, 10, 11, 20 and 21 (mite bodies) or Blo t 3, 4, 6 and predicted Blo t 13, 14 and 36 (mite feces). INTERPRETATION: We report the use of an integrated omics method to identify and predict an array of mite allergens and advanced, label-free proteomics to determine allergen protein abundance. Our research identifies a large set of novel putative allergens and shows that the expression levels of allergen-encoding genes may not be strictly correlated with the actual allergenic protein abundance in mite bodies.

Imidacloprid increases the prevalence of the intestinal parasite Lotmaria passim in honey bee workers.

A challenge in bee protection is to assess the risks of pesticide-pathogen interactions. Lotmaria passim, a ubiquitous unicellular parasite in honey bees, is considered harmful under specific conditions. Imidacloprid causes unpredictable side effects. Research indicates that both L. passim and imidacloprid may affect the physiology, behavior, immunity, microbiome and lifespan of honey bees. We designed cage experiments to test whether the infection of L. passim is affected by a sublethal dose of imidacloprid. Workers collected at the time of emergence were exposed to L. passim and 2.5 µg/L imidacloprid in the coexposure treatment group. First, samples of bees were taken from cages since they were 5 days old and 3 days postinfection, i.e., after finishing an artificial 24 h L. passim infection. Additional bees were collected every two additional days. In addition, bees frozen at the time of emergence and collected from the unexposed group were analyzed. Abdomens were analyzed using qPCR to determine parasite load, while corresponding selected heads were subjected to a label-free proteomic analysis. Our results show that bees are free of L. passim at the time of emergence. Furthermore, imidacloprid considerably increased the prevalence as well as parasite loads in individual bees. This means that imidacloprid facilitates infection, enabling faster parasite spread in a colony and potentially to surrounding colonies. The proteomic analysis of bee heads showed that imidacloprid neutralized the increased transferrin 1 expression by L. passim. Importantly, this promising marker has been previously observed to be upregulated by infections, including gut parasites. This study contributes to understanding the side effects of imidacloprid and demonstrates that a single xenobiotic/pesticide compound can interact with the gut parasite. Our methodology can be used to assess the effects of different compounds on L. passim.

Proteomic insight into the interaction of Paenibacillus larvae with honey bee larvae before capping collected from an American foulbrood outbreak: Pathogen proteins within the host, lysis signatures and interaction markers.

American foulbrood (AFB) is a devastating disease of honey bees. There remains a gap in the understanding of the interactions between the causative agent and host, so we used shotgun proteomics to gain new insights. Nano-LC-MS/MS analysis preceded visual description and Paenibacillus larvae identification in the same individual sample. A further critical part of our methodology was that larvae before capping were used as the model stage. The identification of the virulence factors SplA, PlCBP49, enolase, and DnaK in all P. larvae-positive samples was consistent with previous studies. Furthermore, the results were consistent with the array of virulence factors identified in an in vitro study of P. larvae exoprotein fractions. Although an S-layer protein and a putative bacteriocin were highlighted as important, the microbial collagenase ColA and InhA were not found in our samples. The most important virulence factor identified was isoform of neutral metalloproteinase (UniProt: V9WB82), a major protein marker responsible for the shift in the PCA biplot. This protein is associated with larval decay and together with other virulence factors (bacteriocin) can play a key role in protection against secondary invaders. Overall, this study provides new knowledge on host-pathogen interactions and a new methodical approach to study the disease.

The Virome of Healthy Honey Bee Colonies: Ubiquitous Occurrence of Known and New Viruses in Bee Populations.

Honey bees are globally important pollinators threatened by many different pathogens, including viruses. We investigated the virome of honey bees collected at the end of the beekeeping season (August/September) in Czechia, a Central European country. Samples were examined in biological replicates to assess the homogeneity, stability, and composition of the virome inside a single hive. By choice of healthy workers from colonies, where Varroa destructor was under control, we could identify ubiquitous bee viruses. Deformed wing virus (DWV) was highly prevalent, even though the bees were healthy, without any noticeable disease signs. The overall virome composition (consisting of honey bee-, plant-, and bacterium-infecting viruses) was driven primarily by the hive and its location. However, honey bee-specific viruses showed an uneven distribution within the same hive. In addition, our results point to an unusual cooccurrence between two rhabdoviruses and reveal the presence of five distinct lineages of Lake Sinai viruses (LSVs) clustering with other LSV strains described globally. Comparison of our results with the virome of Australian honey bees, the last truly Varroa- and DWV-free population, showed a strong difference with respect to DWV and a set of diverse members of the Picornavirales, of which the latter were absent in our samples. We hypothesize that the occurrence of DWV introduced by Varroa strongly affects the virome structure despite the mite being under control. IMPORTANCE The Western honey bee, Apis mellifera, is a vital part of our ecosystem as well as cultural heritage. Annual colony losses endanger beekeeping. In this study, we examined healthy bees from the heart of Central Europe, where honey bee colonies have been commonly affected by varroosis over 5 decades. Our virome analysis showed the presence of ubiquitous viruses in colonies where the mite Varroa destructor was under control and no honey bee disease signs were observed. Compared to previous studies, an important part of our study was the analysis of multiple replicates from individual hives. Our overall results indicate that the virome structure (including bee-infecting viruses, plant-infecting viruses, and bacteriophages) is stable within hives; however, the bee-infecting viruses varied largely within interhive replicates, suggesting variation of honey bee viruses within individual bees. Of interest was the striking difference between the viromes of our 39 pools and 9 pools of honey bee viromes previously analyzed in Australia. It could be suggested that Varroa not only affects DWV spread in bee colonies but also affects diverse members of the Picornavirales, which were strongly decreased in Czech bees compared to the Varroa- and DWV-naive Australian bees.

Interactions of the Intracellular Bacterium Cardinium with Its Host, the House Dust Mite Dermatophagoides farinae, Based on Gene Expression Data.

Dermatophagoides farinae is inhabited by an intracellular bacterium, Cardinium. Using correlations between host and symbiont gene expression profiles, we identified several important molecular pathways that potentially regulate/facilitate their interactions. The expression of Cardinium genes collectively explained 95% of the variation in the expression of mite genes assigned to pathways for phagocytosis, apoptosis, the MAPK signaling cascade, endocytosis, the tumor necrosis factor (TNF) pathway, the transforming growth factor beta (TGF-ß) pathway, lysozyme, and the Toll/Imd pathway. In addition, expression of mite genes explained 76% of the variability in Cardinium gene expression. In particular, the expression of the Cardinium genes encoding the signaling molecules BamD, LepA, SymE, and VirD4 was either positively or negatively correlated with the expression levels of mite genes involved in endocytosis, phagocytosis, and apoptosis. We also found that Cardinium possesses a complete biosynthetic pathway for lipoic acid and may provide lipoate, but not biotin, to mites. Cardinium gene expression collectively explained 84% of the variation in expression related to several core mite metabolic pathways, and, most notably, a negative correlation was observed between bacterial gene expression and expression of mite genes assigned to the glycolysis and citric acid cycle pathways. Furthermore, we showed that Cardinium gene expression is correlated with expression levels of genes associated with terpenoid backbone biosynthesis. This pathway is important for the synthesis of pheromones, thus providing an opportunity for Cardinium to influence mite reproductive behavior to facilitate transmission of the bacterium. Overall, our study provided correlational gene expression data that can be useful for future research on mite-Cardinium interactions. IMPORTANCE The molecular mechanisms of mite-symbiont interactions and their impacts on human health are largely unknown. Astigmatid mites, such as house dust and stored-product mites, are among the most significant allergen sources worldwide. Although mites themselves are the main allergen sources, recent studies have indicated that mite-associated microbiomes may have implications for allergen production and human health. The major medically important house dust mite, D. farinae, is known to harbor a highly abundant intracellular bacterium belonging to the genus Cardinium. Expression analysis of the mite and symbiont genes can identify key mite molecular pathways that facilitate interactions with this endosymbiont and possibly shed light on how this bacterium affects mite allergen production and physiology in general.

Label-free proteomic analysis reveals differentially expressed Wolbachia proteins in Tyrophagus putrescentiae: Mite allergens and markers reflecting population-related proteome differences.

Tyrophagus putrescentiae is an astigmatid mite of great economic, medical and veterinary importance. The microbiome, especially intracellular bacteria, may affect allergy/allergen expression. We targeted Wolbachia proteins, allergen comparisons and markers in Wolbachia-mite interactions in three mite populations. A decoy database was constructed by proteogenomics using the T. putrescentiae draft genome, Wolbachia transcriptome assembly and current T. putrescentiae-related sequences in GenBank. Among thousands of mite-derived proteins, 18 Wolbachia proteins were reliably identified. We suggest that peroxiredoxin, bacterioferritin, ankyrin repeat domain-containing protein and DegQ family serine endoprotease indicate a higher-level bacterium-bacterium-host interaction. We produced evidence that the host-Wolbachia interaction is modulated through pattern recognition receptors (PRRs), mannose-binding lectins/mannose receptors, the cholinergic anti-inflammatory pathway with TNF-α, and others. We observed Tyr p 3 suppression in mites with Wolbachia, linking trypsin to PRR modulation. Nine out of the 12 current WHO/IUIS official allergens were reliably identified, but the remaining three allergens, Tyr p 1, 8 and 35, were detected as only trace hits. This study provides numerous markers for further Wolbachia-host interaction research. For accuracy, mite allergens should be considered according to abundance in species, but mite populations/strains, as well as their microbiome structure, may be key factors. SIGNIFICANCE: The astigmatid mites occurring in homes are significant producers of allergens that are highly dangerous to humans and domesticated animals. Mites are tightly associated with microorganisms that affect their biology and consequently allergy signatures. Mite populations were found to be infected with certain intracellular bacteria, but some populations lacked an intracellular bacterium. Our previous research showed that some populations of Tyrophagus putrescentiae are infected with Wolbachia, but some populations host additional bacteria of interest. Thus, there are not only interactions between the mites and Wolbachia but also likely an additional level of interaction that can be found in the interaction between different bacteria in the mites. These "higher-level" signatures and consequences that bacteria affect, including allergen production, are not understood in mites. In this study, we identified Wolbachia-specific proteins in mites for the first time. This study provides Wolbachia- and mite-derived markers that can be clues for describing "higher-level" mite-bacterium-bacterium interactions. Indeed, the microbiome contribution to allergies can potentially be derived directly from bacterial proteins, especially if they are abundant.

Glyphosate Interaction with eEF1α1 Indicates Altered Protein Synthesis: Evidence for Reduced Spermatogenesis and Cytostatic Effect.

The broad-spectrum herbicide, glyphosate, is considered safe for animals because it selectively affects the shikimate pathway that is specific to plants and microorganisms. We sought a previously unknown mechanism to explain the concerns that glyphosate exposure can negatively affect animals, including humans. Computer modeling showed a probable interaction between glyphosate and eukaryotic translation elongation factor 1 subunit alpha 1 (eEF1α1), which was confirmed by microcalorimetry. Only restricted, nondisrupted spermatogenesis in rats was observed after chronic glyphosate treatments (0.7 and 7 mg/L). Cytostatic and antiproliferative effects of glyphosate in GC-1 and SUP-B15 cells were indicated. Meta-analysis of public health data suggested a possible effect of glyphosate use on sperm count. The in silico, in vitro, and in vivo experimental results as well as the metastatistics indicate side effects of chronic glyphosate exposure. Together, these findings indicate that glyphosate delays protein synthesis through an interaction with eEF1α1, thereby suppressing spermatogenesis and cell growth.

Microbiome variation during culture growth of the European house dust mite, Dermatophagoides pteronyssinus.

In culture, the house dust mite, Dermatophagoides pteronyssinus, shows different growth patterns, but the composition and changes in the associated microbial community during mite culture growth are poorly known. In this study, we analyzed temporal changes in microbial communities including 'internal' communities (inside mites, ingested) and 'environmental' communities (from culture environment). Microbial community structure was correlated with guanine content (a nitrogenous waste product of mites) and mite population density. Both internal and environmental microbial communities were remarkably consistent between biological replicates from the same culture age group and were composed of relatively few dominant taxa-11 bacterial and 3 fungal operational taxonomic units (OTUs). Significant changes over time in microbial community structure in the bulk culture environment and in internal mite samples were observed. The yeast, Saccharomyces cerevisiae, a main component of the mite diet, gradually disappeared during mite culture growth and was replaced by fungi from the genera Aspergillus and Candida in both 'internal' and 'environmental' samples. In environmental samples, bacteria from the genus Lactobacillus and S. cerevisiae were negatively correlated, and Aspergillus and Candida positively correlated, with guanine content. The relative abundance of bacteria from the genus Kocuria increased with mite density but declined with increasing guanine content. The relative abundance of bacteria from the genus Virgibacillus was negatively correlated with mite density in 'internal' samples. Gram-positive bacteria dominated bacterial microbiomes at all time points in our experiments, indicating a more limited possibility for vaccine contamination by bacterial endotoxins (heat-stable lipopolysaccharides produced mostly by Gram-negative bacteria) in our experimental cultures.

A single honey proteome dataset for identifying adulteration by foreign amylases and mining various protein markers natural to honey.

Honey adulteration is a common practice that deceives consumers and devalues the unique curative and food properties of honey. For marketing, each honey must satisfy an internationally valid Codex standard. One of the quality parameters is diastase/amylase activity, which, if lowered, may be compensated for by the addition of foreign amylases. However, the estimation of enzyme activity does not enable identification of artificially added amylases. 45 honey samples were analyzed using label-free nanoLC-MS/MS proteomics. Four honeys were found to contain the foreign amylases from Aspergillus niger, Bacillus amyloliquefaciens and/or Bacillus licheniformis. This result was confirmed via proof of specificity at multiple levels. Furthermore, we identified a series of plant-related protein groups. Despite plant-related proteins constituting a significant portion of honey proteins, they were minor components compared to the major honey bee-derived proteins. Bioinformatic analysis also provided evidence for aphid and catalase proteins in honey, but the limited specificity of the MS/MS identified peptides must be considered. Overall, we demonstrate a proteomics approach employing LC-MS/MS that is useful for proving adulteration and assessing honey quality. As an resource useful for reference, we provide curated sequence databases. In addition, we provide many markers that are naturally found in honey for future studies. SIGNIFICANCE: Honey is unique natural product used since ancient times as a food and natural medicine. Humans strive to understand honey components because they can characterize different types of honey and be used for authentication and origin assessment. One of the important honey components are proteins. The proteins present in honey can naturally occur in honey, but some of them can be used to mask deficiencies in some honey quality properties. Diastases/amylases are such proteins, and their activity, a measure of honey freshness, can decrease in time or due to processing. To our knowledge, we for the first time specifically identify foreign amylases in honey. However, this study provided new information on other non-honey bee proteins in honey. Thus, this study is also of importance due to its identification of plant and aphid proteins and catalase-related proteins. This study provides a clue explaining the controversial presence of catalase in honey, since catalases can be identified and their origin determined via proteomics.

Proteogenomic insight into the basis of the insecticide tolerance/resistance of the pollen beetle Brassicogethes (Meligethes) aeneus.

The pollen beetle is a major pest of oilseed rape. Although various resistance mechanisms have been identified, such as kdr (mutation in the sodium channel) and metabolic resistance (CYP overexpression), other "hidden" factors also exist. Some studies have stressed the importance of epistasis as a genetic background. The combination of kdr and metabolic resistance appears to be unfavorable under field conditions in the absence of pesticide selection. The regulation of detoxification enzymes can play an important role, but we highlight different detoxification markers compared to those emphasized in other studies. We also stress the importance of studying the role of markers identified as pathogenesis-related protein 5-like (PR5; upregulated by insecticides) and highlight the role of RNA (DEAD-box) helicases (downregulated by insecticides). Thus, we suggest the importance of epigenetic drivers of resistance/tolerance to pesticides. The key results are similar to those of our previous study, in which deltamethrin treatment of the pollen beetle was also investigated by a proteogenomic approach. Indeed, the mechanism leading to resistance of the pollen beetle may be an innate mechanism that the pollen beetle can also employ in natural habitats, but under field conditions (pesticide exposure), this mechanism is used to survive in response to insecticides. SIGNIFICANCE: Pesticide resistance is a serious problem that hampers the successful production of crops. Understanding the mechanisms of insecticide resistance is highly important for successful pest control, especially when considering integrated pest management. Here, using a proteogenomic approach, we identified novel markers for understanding pollen beetle resistance to pesticides. In addition, future studies will reveal the role of these markers in the multiresistance of pollen beetle populations. We highlight that the proteins identified as PR5, which are known to occur in beetles and are similar to those in plants, may be responsible for tolerance to multiple stresses. In addition, our results indicate that the RNA helicases that exhibited changes in expression may be the epigenetic drivers of multiresistance. The nature of these changes remains an open question, and their relevance in different situations (responses to different stresses) in natural habitats in the absence of pesticides can be proposed.

The investigation of honey bee pesticide poisoning incidents in Czechia.

Honey bees are major pollinators of crops with high economic value. Thus, bees are considered to be the most important nontarget organisms exposed to adverse effects of plant protection product use. The side effects of pesticides are one of the major factors often linked to colony losses. Fewer studies have researched acute poisoning incidents in comparison to the study of the sublethal effects of pesticides. Here, we compared pesticides in dead/dying bees from suspected poisoning incidents and the suspected crop source according to government protocols. Additionally, we analyzed live bees and bee bread collected from the brood comb to determine recent in-hive contamination. We used sites with no reports of poisoning for reference. Our analysis confirmed that not all of the suspected poisonings correlated with the suspected crop. The most important pesticides related to the poisoning incidents were highly toxic chlorpyrifos, deltamethrin, cypermethrin and imidacloprid and slightly toxic prochloraz and thiacloprid. Importantly, poisoning was associated with pesticide cocktail application. Almost all poisoning incidents were investigated in relation to rapeseed. Some sites were found to be heavily contaminated with several pesticides, including a reference site. However, other sites were moderately contaminated despite agricultural use, including rapeseed cultivation sites, which can influence the extent of pesticide use, including tank mixes and other factors. We suggest that the analysis of pesticides in bee bread and in bees from the brood comb is a useful addition to dead bee and suspected crop analysis in poisoning incidents to inform the extent of recent in-hive contamination.

Whole genomic sequencing and sex-dependent abundance estimation of Cardinium sp., a common and hyperabundant bacterial endosymbiont of the American house dust mite, Dermatophagoides farinae.

The two common species of house dust mites (HDMs), Dermatophagoides farinae and D. pteronyssinus, are major sources of allergens in human dwellings worldwide. Many allergens from HDMs have been described, but their extracts vary in immunogens. Mite strains may differ in their microbiomes, which affect mite allergen expression and contents of bacterial endotoxins. Some bacteria, such as the intracellular symbiont Cardinium, can affect both the sex ratio and biochemical pathways of mites, resulting in abundance variations of mite allergens/immunogens. Here, we investigated the bacterial microbiomes of D. farinae and D. pteronyssinus males and females using barcode 16S rDNA sequencing, qPCR, and genomic data analysis. We found a single species of Cardinium associated with D. farinae strains from the USA, China and Europe. Cardinium had high abundance relative to other bacterial taxa and represented 99% of all bacterial DNA reads from female mites from the USA. Cardinium was also abundant with respect to the number of host cells-we estimated 10.4-11.8 cells of Cardinium per single female mite cell. In a European D. farinae strain, Cardinium was more prevalent in females than in males (representing 92 and 67% of all bacterial taxa in females and males, respectively). In contrast, D. pteronyssinus lacked any Cardinium species, and the microbiomes of male and female mites were similar. We produced a Cardinium genome assembly (1.48 Mb; GenBank: PRJNA555788, GCA_007559345.1) associated with D. farinae. The ascertained ubiquity and abundance of Cardinium strongly suggest that this intracellular bacterium plays an important biological role in D. farinae.

Pesticide comparison of Phylloneta impressa (Araneae: Theridiidae) females, cocoons and webs with prey remnants collected from a rape field before the harvest.

BACKGROUND: Pesticides or plant protection products (PPPs) are risky for spiders in or near agricultural landscapes. However, the risks posed by pesticides to spiders are largely understudied compared with the risks to pollinators. Here, we investigated the distribution of PPPs in adult females, cocoons and webs with prey remnants of Phylloneta impressa. RESULTS: Three sample types were collected from the tops of rapeseed on 18 July (before the harvest). Three different ultraperformance liquid chromatograph coupled with triple-quadrupole tandem mass spectrometer (UHPLC-QqQ-MS/MS) analyses were performed: (i) pesticides and selected metabolites; (ii) quaternary ammonium pesticides (quats); and (iii) pyrethroids. Overall, 23 compounds, 22 pesticides and the metabolite imidacloprid-urea were detected. The array of pesticides was largest in webs with prey remnants, and according to evaluation via redundancy analysis (RDA), pesticides were similar in spiders and cocoons; however, data inspection revealed differences in pesticide distribution among these samples. Clothianidin was detected in only female spiders, whereas thiamethoxam prevailed in webs with remnants of prey, and acetamiprid, thiacloprid and imidacloprid were found in all three matrices. One of the most abundant compounds was chlormequat, indicating that quats should be considered a possible risk for these spiders. None of the pyrethroids were detected despite being applied in the sampling area, indicating rapid biodegradation. By contrast, some pesticides were detected despite not being applied in the field, indicating that the source of contamination is prey or particles carried by wind and attached to webs. CONCLUSION: Overall, the results indicate the different distribution or behavior of several pesticides in the spider matrices. © 2019 Society of Chemical Industry.