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.

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.

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.

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 Effect of Residual Pesticide Application on Microbiomes of the Storage Mite Tyrophagus putrescentiae.

Arthropods can host well-developed microbial communities, and such microbes can degrade pesticides and confer tolerance to most types of pests. Two cultures of the stored-product mite Tyrophagus putrescentiae, one with a symbiotic microbiome containing Wolbachia and the other without Wolbachia, were compared on pesticide residue (organophosphate: pirimiphos-methyl and pyrethroid: deltamethrin, deltamethrin + piperonyl butoxide)-containing diets. The microbiomes from mite bodies, mite feces and debris from the spent mite diet were analyzed using barcode sequencing. Pesticide tolerance was different among mite cultures and organophosphate and pyrethroid pesticides. The pesticide residues influenced the microbiome composition in both cultures but without any remarkable trend for mite cultures with and without Wolbachia. The most influenced bacterial taxa were Bartonella-like and Bacillus for both cultures and Wolbachia for the culture containing this symbiont. However, there was no direct evidence of any effect of Wolbachia on pesticide tolerance. The high pesticide concentration residues in diets reduced Wolbachia, Bartonella-like and Bacillus in mites of the symbiotic culture. This effect was low for Bartonella-like and Bacillus in the asymbiotic microbiome culture. The results showed that the microbiomes of mites are affected by pesticide residues in the diets, but the effect is not systemic. No actual detoxification effect by the microbiome was observed for the tested pesticides.

Pesticide residue exposure provides different responses of the microbiomes of distinct cultures of the stored product pest mite Acarus siro.

BACKGROUND: The contribution of the microbiome to pesticide breakdown in agricultural pests remains unclear. We analyzed the effect of pirimiphos-methyl (PM) on four geographically different cultures of the stored product pest mite Acarus siro (6 L, 6Tu, 6Tk and 6Z) under laboratory experiments. The effect of PM on mite mortality in the impregnated filter paper test was compared. RESULTS: The mite sensitivity to PM decreased in the order of 6 L, 6Tu, 6Tk, and 6Z. Then, the mites were cultured on PM residues (0.0125 and 1.25 µg·g-1), and population growth was compared to the control after 21 days of exposure. The comparison showed two situations: (i) increasing population growth for the most sensitive cultures (6 L and 6Tu), and (ii) no effect on mite population growth for tolerant cultures (6Z and 6Tk). The microbiome of mites was analyzed by quantification of 16S DNA copies based on quantitative polymerase chain reaction (qPCR) and by barcode sequencing of the V4 fragment of 16S DNA on samples of 30 individuals from the control and PM residues. The microbiome comprised primarily Solitalea-like organisms in all cultures, except for 6Z, followed by Bacillus, Staphylococcus, and Lactobacillus. The microbiomes of mite cultures did not change with increasing population density. The microbiome of cultures without any differences in population density showed differences in the microbiome composition. A Sodalis-like symbiont replaced Solitalea in the 1.25 µg·g-1 PM in the 6Tk culture. Sodalis and Bacillus prevailed in the microbiomes of PM-treated mites of 6Z culture, while Solitalea was almost absent. CONCLUSION: The results showed that the microbiome of A. siro differs in composition and in response to PM residues in the diet. The results indicate that Sodalis-like symbionts can help recover mites from pesticide-induced stress.

Estimated Total Albumin in Fresh Urine Samples Based on Correlation Between the Roche Immunoturbidimetric and an In-House HPLC Method.

Background: Many studies have reported higher values of urinary albumin measured by high performance liquid chromatography (HPLC) in comparison with immunochemical methods. The aims of our study were the implementation of the HPLC method for albuminuria, testing the hypothesis about coeluting proteins, comparison of albuminuria assessed by HPLC and immunoturbidimetric (IT) methods in diabetic and non-diabetic patient samples. Methods: We compared albuminuria assessed by HPLC with albuminuria assessed by the IT method in fresh urine samples of 636 diabetics and 456 non-diabetics. We investigated relationships between albuminuria and blood glycated hemoglobin HbA1c. Results: We found significant differences between the parameters of linear regressions between albuminuria determined using HPLC and IT among patients with and without DM, and even between patients with DM type 1 and type 2. We confirmed the underestimation of albuminuria assessed by IT. We did not reveal any significant correlation between blood glycated hemoglobin and any of the parameters derived from albuminuria. Conclusions: We excluded non-specificity of the HPLC method. Despite of a little bit lower analytical sensitivity of the HPLC method in comparison with IT method the diagnostic sensitivity of HPLC method is higher, because it measures the total albuminuria (immunoreactive plus immunounreactive). We developed three formulas (for nondiabetics, for diabetics type 1 and diabetics type 2) for the estimation of the total albuminuria from IT values. We also confirmed that albuminuria and HbA1c are independent biomarkers.

Comparison of Microbiomes between Red Poultry Mite Populations (Dermanyssus gallinae): Predominance of Bartonella-like Bacteria.

Blood feeding red poultry mites (RPM) serve as vectors of pathogenic bacteria and viruses among vertebrate hosts including wild birds, poultry hens, mammals, and humans. The microbiome of RPM has not yet been studied by high-throughput sequencing. RPM eggs, larvae, and engorged adult/nymph samples obtained in four poultry houses in Czechia were used for microbiome analyses by Illumina amplicon sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. A laboratory RPM population was used as positive control for transcriptome analysis by pyrosequencing with identification of sequences originating from bacteria. The samples of engorged adult/nymph stages had 100-fold more copies of 16S rRNA gene copies than the samples of eggs and larvae. The microbiome composition showed differences among the four poultry houses and among observed developmental stadia. In the adults' microbiome 10 OTUs comprised 90 to 99% of all sequences. Bartonella-like bacteria covered between 30 and 70% of sequences in RPM microbiome and 25% bacterial sequences in transcriptome. The phylogenetic analyses of 16S rRNA gene sequences revealed two distinct groups of Bartonella-like bacteria forming sister groups: (i) symbionts of ants; (ii) Bartonella genus. Cardinium, Wolbachia, and Rickettsiella sp. were found in the microbiomes of all tested stadia, while Spiroplasma eriocheiris and Wolbachia were identified in the laboratory RPM transcriptome. The microbiomes from eggs, larvae, and engorged adults/nymphs differed. Bartonella-like symbionts were found in all stadia and sampling sites. Bartonella-like bacteria was the most diversified group within the RPM microbiome. The presence of identified putative pathogenic bacteria is relevant with respect to human and animal health issues while the identification of symbiontic bacteria can lead to new control methods targeting them to destabilize the arthropod host.

Urinary Neutrophil Gelatinase-Associated Lipocalin Does Not Distinguish Acute Rejection from Other Causes of Acute Kidney Injury in Pediatric Renal Transplant Recipients.

BACKGROUND: The aim of this prospective single center study was to investigate the ability of urinary neutrophil gelatinase-associated lipocalin (NGAL) to distinguish acute rejection from other causes of acute kidney injury (AKI) in children after renal transplantation. METHODS: Fifteen children fulfilled the inclusion criteria (acute kidney injury (AKI) with allograft biopsy, at least 21 days after renal transplantation, no sepsis) during 2013 - 2014 in our pediatric transplantation center. The mean age was 14.8  2.8, median time after renal transplantation was 0.4 years (range 0.1 - 3.8). Urinary NGAL was measured in spot urine by Chemiluminescent Microparticle Immunoassay technology. RESULTS: Four patients had biopsy proven acute rejection (rejection group), eleven children had AKI of other cause (non-rejection group). The median urinary NGAL concentration in the rejection group was not significantly different from NGAL in the non-rejection group (7.3 ng/mL, range 3.0 - 42.3 vs. 8.6 ng/mL, range 3.4 - 54.7, p = 0.48). There was a significant negative correlation between eGFR and urinary NGAL concentrations (r = -0.77, p < 0.001). CONCLUSIONS: Our small study suggests that in children after renal transplantation, urinary NGAL cannot be used as a specific marker for distinguishing acute rejection from other non-rejection causes of AKI. Urinary NGAL was mainly associated with graft function but not with the etiology of AKI.

Estimation of Problem Drug Users in Prague in 2011 from Low-threshold Data: Modified Capture-Recapture Method, Adjusted for Clients Avoiding Any Identification (Non-Coded Clients).

AIM: Local prevalence estimates of problem drug use (PDU) are crucial in the process of assessment of drug situation and trends and for the planning of evidence-based policy responses. The aims of our study are twofold: to estimate the number of problem drug users (PDUs) in the Czech capital city in 2011, and to examine the usability of the capture-recapture method (CRM) modified for data from low-threshold programmes (LTPs) for drug users. METHOD: Six independent LTPs provided data for analysis (SANANIM, Drop-in and Progressive, each of these providing one drop-in centre and one outreach programme). After adjustment of the standard CRM formula for cases without an individual identifier, the overlaps between programmes were calculated and the size of hidden population was estimated. RESULTS: In total, it was estimated that there were 10,754 PDUs in Prague in 2011. The current estimate is in line with estimates obtained previously using another indirect standard approach - the multiplier method. CONCLUSION: The modified version of CRM thus proved a reliable method for local PDU estimates.

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.

A novel Bartonella-like bacterium forms an interdependent mutualistic symbiosis with its host, the stored-product mite Tyrophagus putrescentiae.

A novel Bartonella-like symbiont (BLS) of Tyrophagus putrescentiae was characterized. BLS formed a separate cluster from the Bartonella clade together with an ant symbiont. BLS was present in mite bodies (103 16S DNA copies/mite) and feces but was absent in eggs. This indicated the presence of the BLS in mite guts. The BLS showed a reduction in genome size (1.6 Mb) and indicates gene loss compared to Bartonella apis. The BLS can be interacted with its host by using host metabolic pathways (e.g., the histidine and arginine metabolic pathways) as well as by providing its own metabolic pathways (pantothenate and lipoic acid) to the host, suggesting the existence of a mutualistic association. Our experimental data further confirmed these potential mutualistic nutritional associations, as cultures of T. putrescentiae with low BLS abundance showed the strongest response after the addition of vitamins. Despite developing an arguably tight dependency on its host, the BLS has probably retained flagellar mobility, as evidenced by the 32 proteins enriched in KEGG pathways associated with flagellar assembly or chemotaxis (e.g., fliC, flgE, and flgK, as highly expressed genes). Some of these proteins probably also facilitate adhesion to host gut cells. The microcin C transporter was identified in the BLS, suggesting that microcin C may be used in competition with other gut bacteria. The 16S DNA sequence comparison indicated a mite clade of BLSs with a broad host range, including house dust and stored-product mites. Our phylogenomic analyses identified a unique lineage of arachnid specific BLSs in mites and scorpions.IMPORTANCEA Bartonella-like symbiont was found in an astigmatid mite of allergenic importance. We assembled the genome of the bacterium from metagenomes of different stored-product mite (T. putrescentiae) cultures. The bacterium provides pantothenate and lipoic acid to the mite host. The vitamin supply explains the changes in the relative abundance of BLSs in T. putrescentiae as the microbiome response to nutritional or pesticide stress, as observed previously. The phylogenomic analyses of available 16S DNA sequences originating from mite, scorpion, and insect samples identified a unique lineage of arachnid specific forming large Bartonella clade. BLSs associated with mites and a scorpion. The Bartonella clade included the previously described Ca. Tokpelaia symbionts of ants.

The Development of a Specific Nanofiber Bioreceptor for Detection of Escherichia coli and Staphylococcus aureus from Air.

Polluted air and the presence of numerous airborne pathogens affect our daily lives. The sensitive and fast detection of pollutants and pathogens is crucial for environmental monitoring and effective medical diagnostics. Compared to conventional detection methods (PCR, ELISA, metabolic tests, etc.), biosensors bring a very attractive possibility to detect chemicals and organic particles with the mentioned reliability and sensitivity in real time. Moreover, by integrating nanomaterials into the biosensor structure, it is possible to increase the sensitivity and specificity of the device significantly. However, air quality monitoring could be more problematic even with such devices. The greatest challenge with conservative and sensing methods for detecting organic matter such as bacteria is the need to use liquid samples, which slows down the detection procedure and makes it more difficult. In this work, we present the development of a polyacrylonitrile nanofiber bioreceptor functionalized with antibodies against bacterial antigens for the specific interception of bacterial cells directly from the air. We tested the presented novel nanofiber bioreceptor using a unique air filtration system we had previously created. The prepared antibody-functionalized nanofiber membranes for air filtration and pathogen detection (with model organisms E. coli and S. aureus) show a statistically significant increase in bacterial interception compared to unmodified nanofibers. Creating such a bioreceptor could lead to the development of an inexpensive, fast, sensitive, and incredibly selective bionanosensor for detecting bacterial polluted air in commercial premises or medical facilities.

Six-year monitoring of pesticide resistance in the Colorado potato beetle (Leptinotarsa decemlineata Say) during a neonicotinoid restriction period.

The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is an important potato pest with known resistance to pyrethroids and organophosphates in Czechia. Decreased efficacy of neonicotinoids has been observed in last decade. After the restriction of using chlorpyrifos, thiacloprid and thiamethoxam by EU regulation, growers seek for information about the resistance of CPB to used insecticides and recommended antiresistant strategies. The development of CPB resistance to selected insecticides was evaluated in bioassays in 69 local populations from Czechia in 2017-2022 and in 2007-2022 in small plot experiments in Zabcice in South Moravia. The mortality in each subpopulation in the bioassays was evaluated at the field-recommended rates of insecticides to estimate the 50% and 90% lethal concentrations (LC50 and LC90, respectively). High levels of CPB resistance to lambda-cyhalothrin and chlorpyrifos were demonstrated throughout Czechia, without significant changes between years and regions. The average mortality after application of the field-recommended rate of lambda-cyhalothrin was influenced by temperature before larvae were sampled for bioassays and decreased with increasing temperature in June. Downwards trends in the LC90 values of chlorpyrifos and the average mortality after application of the field-recommended rate of acetamiprid in the bioassay were recorded over a 6-year period. The baseline LC50 value (with 95% confidence limit) of 0.04 mg/L of chlorantraniliprole was established for Czech populations of CPBs for the purpose of resistance monitoring in the next years. Widespread resistance to pyrethroids, organophosphates and neonicotinoids was demonstrated, and changes in anti-resistant strategies to control CPBs were discussed.

[Prevalence estimates of problem drug users in the Czech Republic in 2006 and 2007 using the capture-recapture method]. / Prevalencní odhad problémových uzivatelu drog v CR v letech 2006 a 2007 metodou zpetného záchytu.

BACKGROUND: An estimate of problem drug use prevalence is a substantial part of the monitoring of the epidemiological situation in drug use and its consequences and an important indicator for drug policy implementation. The capture-recapture method (CRM) is one of the most commonly used standard methods for this purpose worldwide. METHODS AND MATERIAL: The CRM was used to estimate the numbers of problem drug users (PDU) and problem users of opiates/opioids (PUO) in the Czech Republic in 2006 and 2007. The following data sources were used: the General Health Insurance Company records of payments to out- and in-patient psychiatric care providers, records of admissions to psychiatric hospitals, replacement therapy register, and reports of newly diagnosed cases of viral hepatitis. Cases were defined as diagnoses F11, F15, and F19 according to the International Classification of Diseases, Tenth Revision (ICD-10) from the health insurance and admission records, the replacement therapy register covers PUO by definition, and injecting drug users were selected from reported cases of viral hepatitis. Log-linear analysis in Rcapture (R) was performed and the Akaike information criterion was used for model selection. RESULTS: Altogether 12,882 and 13,505 individuals entered into analysis of PDUs and 5146 and 5409 individuals entered into analysis of PUO in 2006 and 2007, respectively. The estimates of PDUs were 23,900 (95% CI: 20,700-28,500) in 2006 and 31,000 (25,500-39,400) in 2007. The estimates of PUO were 6,864 (6,641-7,113) in 2006 and 7,096 (6,871-7,346) in 2007. The male/female ratio was 2.2/1. In PDU estimates, 83% were in the age range 15-34 and 17% were aged above 34; in PUO estimates, the respective rates were 80% and 20%. The prevalence rates of PDU in the age range 15-64 were 2.28 and 2.95 per 1,000 population in 2006 and 2007, respectively. The prevalence rates of PUO in the age range 15-64 were 0.65 and 0.67 per 1,000 in 2006 and 2007, respectively. The highest prevalence of both PDUs and PUO was observed in the Usti Region and Prague, followed by the Karlovy Vary, Plzen, and Central Bohemia Regions. CONCLUSION: PDU estimates in the Czech Republic obtained using the CRM are consistent with those derived by other methods. The major drawbacks are the quality of potential data sources and hassles in their acquisition and preparation. The interconnection of the available data sources also limits the validity and representativeness of estimates and therefore, another independent data source(s) should be sought in the future.

Diet modulation of the microbiome of the pest storage mite Tyrophagus putrescentiae.

Storage mites colonize a wide spectrum of food commodities and adaptations to diets have been suggested as mechanisms enabling successful colonization. We characterized the response of seven unique Tyrophagus putrescentiae cultures (5K, 5L, 5N, 5P, 5Pi, 5S, and 5Tk) with different baseline microbiomes to different diets. The offered diets included a rearing diet, protein-enriched diet, oat flakes, and sunflower seeds. Microbiome characterization was performed using 16S ribosomal RNA (rRNA) gene amplicon sequencing and 16S rRNA gene quantitative PCR. The mite culture microbiomes were classified into four groups: (i) Sodalis-dominated (5Pi), (ii) Wolbachia-dominated (5N and 5P), (iii) Cardinium-dominated (5L and 5S), and (iv) asymbiontic (5K and 5Tk) mites dominated by Bacillus and Bartonella. Mite growth rates were most strongly affected by nutrients in the diet, while respiration and microbial community profiles were largely influenced by mite culture. While growth rate was not directly explained by microbiome composition, microbiomes strongly influenced mite fitness as measured by respiration. While diet significantly influenced microbial profiles in all cultures, the effect of diet differed in impact between cultures (5Pi > 5S > 5N > 5K > 5Tk > 5L > 5P). Furthermore, no new bacterial taxa were acquired by mites after dietary changes. Bacteria from the taxa Bacillus, Bartonella-like, Solitalea-like, Kocuria, and Sodalis-like contributed most strongly to differentiating mite-associated microbiomes.

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.

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.

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.