Honeybee workers undergo metamorphosis in capped cells for approximately 13 days before adult emergence. During the same period, Varroa mites prick the defenseless host many times. We sought to identify proteome differences between emerging Varroa-parasitized and parasite-free honeybees showing the presence or absence of clinical signs of deformed wing virus (DWV) in the capped cells. A label-free proteomic analysis utilizing nanoLC coupled with an Orbitrap Fusion Tribrid mass spectrometer provided a quantitative comparison of 2316 protein hits. Redundancy analysis (RDA) showed that the combination of Varroa parasitism and DWV clinical signs caused proteome changes that occurred in the same direction as those of Varroa alone and were approximately two-fold higher. Furthermore, proteome changes associated with DWV signs alone were positioned above Varroa in the RDA. Multiple markers indicate that Varroa activates TGF-β-induced pathways to suppress wound healing and the immune response and that the collective action of stressors intensifies these effects. Furthermore, we indicate JAK/STAT hyperactivation, p53-BCL-6 feedback loop disruption, Wnt pathway activation, Wnt/Hippo crosstalk disruption, and NF-κB and JAK/STAT signaling conflict in the Varroa-honeybee-DWV interaction. These results illustrate the higher effect of Varroa than of DWV at the time of emergence. Markers for future research are provided.

• MeSH
• biologické markery MeSH
• biologické modely MeSH
• histony metabolismus   MeSH
• Janus kinasy metabolismus   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• proteom * MeSH
• proteomika * MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• RNA-viry * MeSH
• signální transdukce MeSH
• symbióza * MeSH
• transformující růstový faktor beta * MeSH
• transkripční faktory STAT metabolismus   MeSH
• Varroidae * MeSH
• včely metabolismus   parazitologie   virologie   MeSH
• výpočetní biologie metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• histony MeSH
• Janus kinasy MeSH
• protein-serin-threoninkinasy MeSH
• proteiny Wnt MeSH
• proteom * MeSH
• reaktivní formy kyslíku MeSH
• transformující růstový faktor beta * MeSH
• transkripční faktory STAT MeSH

The honey bee, Apis mellifera, is a globally important species that suffers from a variety of pathogens and parasites. These parasites and pathogens may have sublethal effects on their bee hosts via an array of mechanisms, including through a change in symbiotic bacterial taxa. Our aim was to assess the influence of four globally widespread parasites and pathogens on the honey bee bacteriome. We examined the effects of the ectoparasitic mite Varroa destructor, the fungal pathogens Nosema apis and Nosema ceranae, and the trypanosome Lotmaria passim. Varroa was detected by acaricidal treatment, Nosema and L. passim by PCR, and the bacteriome using MiSeq 16S rRNA gene sequencing. Overall, the 1,858,850 obtained sequences formed 86 operational taxonomic units (OTUs) at 3 % dissimilarity. Location, time of year, and degree of infestation by Varroa had significant effects on the composition of the bacteriome of honey bee workers. Based on statistical correlations, we found varroosis more important factor than N. ceranae, N. apis, and L. passim infestation influencing the honey bee bacteriome and contributing to the changes in the composition of the bacterial community in adult bees. At the population level, Varroa appeared to modify 20 OTUs. In the colonies with high Varroa infestation levels (varroosis), the relative abundance of the bacteria Bartonella apis and Lactobacillus apis decreased. In contrast, an increase in relative abundance was observed for several taxa including Lactobacillus helsingborgensis, Lactobacillus mellis, Commensalibacter intestini, and Snodgrassella alvi. The results showed that the "normal" bacterial community is altered by eukaryotic parasites as well as displaying temporal changes and changes associated with the geographical origin of the beehive.

• Klíčová slova
• Bacteria, Lotmaria passim, Nosema apis, Nosema ceranae, Sequencing, Varroa destructor,
• MeSH
• Bartonella klasifikace   genetika   izolace a purifikace   MeSH
• infestace roztoči patologie   MeSH
• Kinetoplastida patogenita   MeSH
• Lactobacillus klasifikace   genetika   izolace a purifikace   MeSH
• mikrobiota genetika   MeSH
• Nosema patogenita   MeSH
• RNA ribozomální 16S genetika   MeSH
• symbióza MeSH
• Varroidae patogenita   MeSH
• včely mikrobiologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

The ectoparasitic mite Varroa destructor is a major pest of the honeybee Apis mellifera. In a previous study, bacteria were found in the guts of mites collected from winter beehive debris and were identified using Sanger sequencing of their 16S rRNA genes. In this study, community comparison and diversity analyses were performed to examine the microbiota of honeybees and mites at the population level. The microbiota of the mites and honeybees in 26 colonies in seven apiaries in Czechia was studied. Between 10 and 50 Varroa females were collected from the bottom board, and 10 worker bees were removed from the peripheral comb of the same beehive. Both bees and mites were surface sterilized. Analysis of the 16S rRNA gene libraries revealed significant differences in the Varroa and honeybee microbiota. The Varroa microbiota was less diverse than was the honeybee microbiota, and the relative abundances of bacterial taxa in the mite and bee microbiota differed. The Varroa mites, but not the honeybees, were found to be inhabited by Diplorickettsia. The relative abundance of Arsenophonus, Morganella, Spiroplasma, Enterococcus, and Pseudomonas was higher in Varroa than in honeybees, and the Diplorickettsia symbiont detected in this study is specific to Varroa mites. The results demonstrated that there are shared bacteria between Varroa and honeybee populations but that these bacteria occur in different relative proportions in the honeybee and mite bacteriomes. These results support the suggestion of bacterial transfer via mites, although only some of the transferred bacteria may be harmful.

• Klíčová slova
• Apis mellifera, Arsenophonus, Diplorickettsia, Spiroplasma, Symbiosis, Varroa destructor,
• MeSH
• biodiverzita MeSH
• DNA bakterií genetika   MeSH
• mikrobiota * MeSH
• RNA ribozomální 16S genetika   MeSH
• roční období MeSH
• sekvenční analýza DNA MeSH
• Spiroplasma klasifikace   izolace a purifikace   MeSH
• symbióza MeSH
• Varroidae mikrobiologie   MeSH
• včely mikrobiologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

The parasitic mite Varroa destructor is a major pest of the western honeybee, Apis mellifera. The development of acaricide resistance in Varroa populations is a global issue. Discriminating concentrations of acaricides are widely used to detect pest resistance. Two methods, using either glass vials or paraffin capsules, are used to screen for Varroa resistance to various acaricides. We found the glass vial method to be useless for testing Varroa resistance to acaridices, so we developed a polypropylene vial bioassay. This method was tested on tau-fluvalinate-, acrinathrin-, and amitraz-resistant mite populations from three apiaries in Czechia. Acetone was used as a control and technical grade acaricide compounds diluted in acetone were applied to the polypropylene vials. The solutions were spread on the vial surface by rolling the vial, and were then evaporated. Freshly collected Varroa females were placed in the vials and the mortality of the exposed mites was measured after 24 h. The Varroa populations differed in mortality between the apiaries and the tested compounds. Mites from the Kyvalka site were resistant to acrinathrin, tau-fluvalinate, and amitraz, while mites from the Postrizin site were susceptible to all three acaricides. In Prelovice apiary, the mites were susceptible to acrinathrin and amitraz, but not to tau-fluvalinate. The calculated discriminating concentrations for tau-fluvalinate, acrinathrin, and amitraz were 0.66, 0.26 and 0.19 µg/mL, respectively. These results indicate that polyproplyne vial tests can be used to determine discriminating concentrations for the early detection of acaricide resistant Varroa. Finally, multiple-resistance in Kyvalka may indicate metabolic resistance.

• Klíčová slova
• Acaricide, Apiculture, Discriminating concentrations, Multiple-resistance, Varroa,
• MeSH
• akaricidy * MeSH
• fixní kombinace léků MeSH
• kontrola klíšťat * MeSH
• nitrily * MeSH
• polylysin analogy a deriváty   MeSH
• pyrethriny * MeSH
• toluidiny * MeSH
• Varroidae * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• acrinathrin MeSH Prohlížeč
• akaricidy * MeSH
• amitraz MeSH Prohlížeč
• fixní kombinace léků MeSH
• fluvalinate MeSH Prohlížeč
• gemals MeSH Prohlížeč
• nitrily * MeSH
• polylysin MeSH
• pyrethriny * MeSH
• toluidiny * MeSH

Tyrophagus putrescentiae (Schrank, 1781) is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida). In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities, and mite-bacterial interaction in dry dog food (DDF). Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30) and (poly)ubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases) of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (DDF) and low-fat, low-protein (flour) diets to 1 and 5% (w/w), and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist with the mite in balance to be beneficial for the mite. The mite-B. cereus symbiosis can be beneficial-suppressive at some level. The results increase the veterinary and medical importance of the allergens detected in feces. The B. cereus enzymes/toxins are important components of mite allergens. The strong symbiotic association of T. putrescentiae with B. cereus in DDF was indicated.

• Klíčová slova
• Bacillus cereus, Tyrophagus putrescentiae, allergen, bacillolysin, exochitinase, nutrition, protease, symbiosis,
• Publikační typ
• časopisecké články MeSH