Cardinium bacteria are well known as endosymbionts that infect a wide range of arthropods and can manipulate host reproduction to promote their vertical transmission. As intracellular bacteria, Cardinium species undergo dramatic genome evolution, especially their chromosomal genome reduction. Although Cardinium plasmids have been reported to harbor important genes, the role of these plasmids in the genome evolution is yet to be fully understood. In this study, 2 genomes of Cardinium endosymbiont bacteria in astigmatic mites were de novo assembled, including the complete circular chromosomal genome of Cardinium sp. DF that was constructed in high quality using high-coverage long-read sequencing data. Intriguingly, 2 circular plasmids were assembled in Cardinium sp. DF and were identified to be endogenous for over 10 homologous genes shared with the chromosomal genome. Comparative genomics analysis illustrated an outline of the genome evolution of Cardinium bacteria, and the in-depth analysis of Cardinium sp. DF shed light on the multiple roles of endogenous plasmids in the molecular process of the chromosomal genome reduction. The endogenous plasmids of Cardinium sp. DF not only harbor massive homologous sequences that enable homologous recombination with the chromosome, but also can provide necessary functional proteins when the coding genes decayed in the chromosomal genome. IMPORTANCE As bacterial endosymbionts, Cardinium typically undergoes genome reduction, but the molecular process is still unclear, such as how plasmids get involved in chromosome reduction. Here, we de novo assembled 2 genomes of Cardinium in astigmatic mites, especially the chromosome of Cardinium sp. DF was assembled in a complete circular DNA using high-coverage long-read sequencing data. In the genome assembly of Cardinium sp. DF, 2 circular endogenous plasmids were identified to share at least 10 homologous genes with the chromosomal genome. In the comparative analysis, we identified a range of genes decayed in the chromosomal genome of Cardinium sp. DF but preserved in the 2 plasmids. Taken together with in-depth analyses, our results unveil that the endogenous plasmids harbor homologous sequences of chromosomal genome and can provide a structural basis of homologous recombination. Overall, this study reveals that endogenous plasmids participate in the ongoing chromosomal genome reduction of Cardinium sp. DF.

• Klíčová slova
• Cardinium endosymbiont, chromosomal genome reduction, endogenous plasmid, host-associated bacteria,
• MeSH
• Bacteria MeSH
• Bacteroidetes * genetika   MeSH
• chromozomy MeSH
• Dermatophagoides farinae * MeSH
• genom bakteriální MeSH
• plazmidy genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Dermatophagoides farinae is an important house dust mite species that causes allergies in humans worldwide. In houses, these mites are commonly found in actively used mattresses and pillows, which provide food (i.e. sloughed skin and microorganisms), moisture, and increased temperature for faster mite development. In mattresses, feeding mites prefer the upper sector, as close as possible to the resting human (temperature 32-36 °C, humidity between 55 and 59%). However, mites that are not actively feeding prefer staying at deeper zones of the mattress. Here, we analyzed mite responses to different temperatures (15-35 °C) and relative humidity (62-94% RH) in terms of their population size growth and respiration (CO2 production) using lab mite cultures. The intrinsic rate of population increase had a single maximum at approximately 28 °C and 85% RH. At 30 °C, there were two respiration peaks at RH 90% (smaller peak) and 65% (larger peak). Therefore, there is a mismatch between the optimal temperature/humidity for the population size increase vs. respiration. We propose preliminary hypotheses explaining the two respiration peaks and suggest that future research should be done to elucidate the nature of these peaks.

• Klíčová slova
• Dermatophagoides farinae, House dust mites, Humidity, Physiology, Population growth, Respiration, Temperature,
• MeSH
• alergeny MeSH
• antigeny roztočů domácího prachu MeSH
• Dermatophagoides farinae * fyziologie   MeSH
• lidé MeSH
• populační růst * MeSH
• prach MeSH
• teplota MeSH
• vlhkost MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alergeny MeSH
• antigeny roztočů domácího prachu MeSH
• prach MeSH

House dust mites inhabit bed mattresses contaminating them with allergens. A strong temperature/moisture gradient exists in mattresses when it is used by humans daily. Here, we studied migration patterns of the mite Dermatophagoides farinae in continuous and time-discontinuous temperature gradients consisting of five sectors with 19-23, 23-28, 28-32, 32-36 and 36-41 °C, containing dye-labeled diets as an indicator of mite presence and feeding. The mites migrated through the sectors and fed on the labeled diets or stayed unfed. The numbers of mites with the same coloration in their guts and the numbers of unfed mites in the sectors were recorded. Unfed mites provided information on short-term temperature preferences. Apart from a control trial, two experiments were performed: (i) a constant 19-41 °C gradient for 24 h, and (ii) alternating cycles of the same temperature gradient (19-41 °C, 8 h) and room temperature (16 h) for 5 days to model the typical daily occupancy of bed by humans. In both experiments, fed mites preferred a sector with 32-36 °C, suggesting that in mattresses, house dust mites prefer to stay as close as possible to the resting human, thus maximizing allergen exposure. However, the number of unfed mites decreased with increased temperatures in the gradient. Experiment (ii) showed that the fed mites remained at the same optimal distance from the heat source, suggesting that they stay at the upper surface of the regularly used mattress, even when human was temporarily absent during the day. Unfed mites apparently hide deeper in mattresses as suggested by their avoidance of increased temperatures.

• Klíčová slova
• Allergens, Feeding, House dust mites, Temperature, Temperature preferences,
• MeSH
• alergeny MeSH
• antigeny roztočů domácího prachu MeSH
• Dermatophagoides farinae MeSH
• lidé MeSH
• prach analýza   MeSH
• Pyroglyphidae * MeSH
• roztoči * MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alergeny MeSH
• antigeny roztočů domácího prachu MeSH
• prach MeSH

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.

• Klíčová slova
• Acaridida, Allergen production, Astigmata, Cardinium, Dermatophagoides pteronyssinus, Microbiome,
• MeSH
• Bacteroidetes izolace a purifikace   MeSH
• Dermatophagoides farinae mikrobiologie   MeSH
• Dermatophagoides pteronyssinus mikrobiologie   MeSH
• genom bakteriální * MeSH
• mikrobiota MeSH
• sekvenování celého genomu MeSH
• symbióza MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Čína MeSH
• Evropa MeSH
• Spojené státy americké MeSH

The allergen repertoire of the house dust mite, Dermatophagoides farinae, is incomplete despite most mite allergens having been described in this species. Using proteogenomics, we aimed to compare proteins and allergens between sexes and provide a foundation for the identification of novel allergens. Overall, 6297 protein hits were identified, and 2899 and 886 were male- and female-specific, respectively. Removal of trace results narrowed the dataset to 3478 hits, including 275 and 157 male- and female-specific hits, respectively. All 34 WHO/IUIS-approved D. farinae allergens (omitting Der f 17) were identified, and we also identified homologs of the yet undescribed Der f 9 and 38. Der f 27/serpin exhibited the largest sex-dependent difference and was dominant in females. Using official protein sequences, Der f 11, 14, 23, 28 and 30 were identified with low success. However, identification success of Der f 11 and 14 was greatly increased by using longer/complete sequences. Der f 30 is characterized by the same tryptic digests as the more abundant Der f 30 (isoform) identified here. Der f 23 appears to be of low abundance in mite bodies. Der f 28.0101 and Der f 28.0201 were detected at low abundance and in trace amounts, respectively. SIGNIFICANCE: In this work, we performed a proteogenomic annotation of the house dust mite, Dermatophagoides farinae, which is the most important source of house dust allergens. The proteogenomic analysis performed here provides a foundation for not only understanding the biology of the mite but also the identification of novel allergens. This study generated a robust proteomic dataset for D. farinae and reviewed existing and candidate allergens in this species. We stress some pitfalls of high-throughput analyses, especially that improper headers of allergen protein records provided in databases can lead to confusion. Using partial sequences in proteomic identification and quantification can lead to low identification success (low signal intensity or MS/MS counts). Thus, we individually curated the protein sequences for proper identification and quantification. The discovered sex differences can be one factor affecting allergen/immunogen variations in mite extracts. Overall, this work provides a benchmark for accurate identification of mite immunogenic proteins using proteomics.

• Klíčová slova
• Der f 28, Der f 38, Der f 9, Ferritin Der f 30, Label-free proteomics, Serpin Der f 27,
• MeSH
• alergeny genetika   imunologie   metabolismus   MeSH
• Dermatophagoides farinae genetika   imunologie   metabolismus   MeSH
• proteiny členovců genetika   imunologie   metabolismus   MeSH
• proteogenomika metody   MeSH
• proteom metabolismus   MeSH
• Pyroglyphidae genetika   imunologie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• sexuální faktory MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• alergeny MeSH
• proteiny členovců MeSH
• proteom MeSH

The variation in house dust mite microbial communities is important because various microorganisms modulate the production of allergens by their mite hosts and/or contaminate immunotherapeutic extracts. Temporal changes in mite microbiomes and the mite culture environment occurring at different stages of mite culture development are particularly understudied in this system. Here, we analyzed the dynamics of microbial communities during the culture growth of Dermatophagoides farinae. Changes in microbiomes were related to three key variables: the mite population density, microbial microcosm respiration and concentration of guanine (the mite nitrogenous waste metabolite). Mite populations exhibited the following phases: exponential growth, plateau and exponential decline. The intracellular bacterium Cardinium and the yeast Saccharomyces cerevisiae prevailed in the internal mite microbiomes, and the bacterium Lactobacillus fermentum was prevalent in the mite diet. The reduction in the mite population size during the late phases of culture development was related to the changes in their microbial profiles: the intracellular bacterium Cardinium was replaced by Staphylococcus, Oceanobacillus and Virgibacillus, and S. cerevisiae was replaced by the antagonistic fungi Aspergillus penicillioides and Candida. Increases in the guanine content were positively correlated with increases in the Staphylococcus and A. penicillioides profiles in the culture environment. Our results show that the mite microbiome exhibits strong, dynamic alterations in its profiles across different mite culture growth stages.

• Klíčová slova
• Dermatophagoides farinae, allergens, bacteria, guanine, microbiome, mite culture, yeasts,
• MeSH
• alergeny MeSH
• Bacteroidetes izolace a purifikace   MeSH
• Dermatophagoides farinae růst a vývoj   mikrobiologie   MeSH
• Limosilactobacillus fermentum izolace a purifikace   MeSH
• mikrobiota * MeSH
• Saccharomyces cerevisiae izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alergeny MeSH

The feeding interactions between house dust mites (HDM) and microorganisms are key factors in the survival of mites in human environments. The suitability of different microbes for HDM is not known. Here, microbial species isolated from HDM cultures were offered to HDM in food preference tests under laboratory conditions. The microbial species were added to the rearing diet of mites to reach 7% of Saccharomyces cerevisiae and 10% of each tested microorganism. The suitability of each microbe-supplemented diet for Dermatophagoides pteronyssinus and Dermatophagoides farinae was compared in terms of mite population growth and mite preference in a cafeteria test. The effect of mite feeding on the respiration of microorganisms in the diet was observed in microcosms. HDM were able to feed and reproduce on some bacterial and fungal species, but the suitability of microbial species differed. Increasing the yeast Sa. cerevisiae in the diet from 7 to 17% appeared the most suitable for both mite species. Staphylococcus spp. bacteria were preferred for D. farinae and were suitable for reproduction. The population growth and feeding preferences of D. pteronyssinus and D. farinae with respect to microorganisms indicate that D. farinae can develop on a diet with bacterial (Staphylococcus nepalensis and Staphylococcus kloosii) additions, whereas D. pteronyssinus was successful on a diet with fungal (Aspergillus jensenii and Aspergillus ruber) additions. The bacteria Kocuria rhizophila and Bacillus cereus decreased population growth in D. pteronyssinus, whereas the yeasts Hyphopichia pseudoburtonii, Hyphopichia burtonii, and Candida ciferrii decreased population growth in D. farinae. These results indicate that some microorganisms are an important food source for HDM.

• Klíčová slova
• bacteria, feeding, fungi, mites, respiration,
• MeSH
• Bacteria * MeSH
• Dermatophagoides farinae fyziologie   MeSH
• Dermatophagoides pteronyssinus fyziologie   MeSH
• dieta MeSH
• houby * MeSH
• krmivo pro zvířata analýza   MeSH
• populační růst MeSH
• stravovací zvyklosti MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

UNLABELLED: Major domestic mite allergens are present in feces. We present a detailed 2D-E-MS/MS proteomic analysis of the Dermatophagoides pteronyssinus feces. Precise cultivation yielded a pure fecal extract. We detected differences in fecal allergens/digestive enzymes between D. pteronyssinus and D. farinae using 2D-E fingerprinting, including unique information on species-specific protease isoforms. Proteomic analysis was performed by 2D-E coupled with MALDI-TOF/TOF identification. The species-specific differences in the fecal extracts of the mites were attributed to trypsin-like proteases known as group 3 allergens. In D. farinae, Der f 3 exhibited high abundance with a pI similar (acidic) to that of the cysteine protease Der f 1 and the chymotrypsin protease Der f 6, whereas in D. pteronyssinus, Der p 3 was rarely detected and exhibited low abundance only at basic pI. Moreover, Der p 9 was detected at a pI of ~ 10, in contrast to Der p 1 and Der p 6, suggesting different compartmentalization in the body. Overall, in D. pteronyssinus feces, allergens of groups 1, 2, 6, and 15 were quantitatively similar to those of D. farinae with the exception of the group 3 and 9 allergens. This work provides novel insights into mite-defecated proteins/digestive enzymes, which are important allergens. SIGNIFICANCE: Millions of people are affected by allergy and asthma, and their number is growing. In homes, the major triggers of allergy and asthma are the house dust mites Dermatophagoides farinae and D. pteronyssinus, and a clear understanding of the development of diseases caused by these mites is needed. The major sources of mite allergens are their feces, which are deposited in the environment and are easily inhaled as part of aeroplankton. However, descriptions of and comparisons between the major fecal allergens of these two mites are lacking. This study shows that similar group 1 (cysteine protease), 2 (NPC2 family), 6 (chymotrypsin) and 15 (chitinase-like) allergens are present in the feces of these two mite species, as determined by 2D-E mapping, whereas group 3 (trypsin) and 9 (collagenolytic protease) allergens in the feces of the two species are different. The results provide unique MS/MS mapped fingerprints of mite species-specific isoforms in feces. The presence of ubiquitin in mite feces suggests that these proteins participate in the post-translational modification of fecal proteins. The findings are essential for understanding differences between D. farinae and D. pteronyssinus with respect to immunoreactivity, protease activation mechanisms, association with microbes, and food utilization.

• Klíčová slova
• Chitinase, Cysteine protease, Digestion, NPC2, Ubiquitin,
• MeSH
• 2D gelová elektroforéza MeSH
• alergeny analýza   MeSH
• Dermatophagoides farinae chemie   MeSH
• Dermatophagoides pteronyssinus chemie   MeSH
• druhová specificita MeSH
• feces chemie   MeSH
• protein - isoformy MeSH
• proteomika metody   MeSH
• tandemová hmotnostní spektrometrie MeSH
• trypsin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• alergeny MeSH
• protein - isoformy MeSH
• trypsin MeSH

Dermatophagoides farinae fecal allergens are a major source of immunogens in home environments; however, as the source of mite fecal allergen is considered spent growth medium extract that can only mimic the pure fecal extract. In this study, we prepared and using proteomic methods analyzed a D. farinae fecal extract for the first time. The preparation approach used D. farinae feces that were produced within 8 weeks of initiating cultivation in minimized growth media. The feces were collected via adhesion to the tissue culture flask surfaces after removing the SGM and mites. This study contains in-depth proteomic mapping of the allergenic isoforms from the D. farinae fecal extract. Despite extensive analysis, MALDI TOF/TOF spectrometry showed that only six proteins/allergens, Der f1, Der f2, Der f3, Der f6, Der f15 and ferritin, originated from D. farinae. No other analyzed proteins were exactly assigned to Dermatophagoides or to similar invertebrate species by sequence similarity. The remaining proteins were assigned mostly to yeasts or cereals (originally dietary proteins); however, many of the proteins were not successfully identified in the current NCBInr. The numerous dietary proteins identified in the feces suggest that these proteins remained highly stable after passing through the gut. Isoforms of the allergens Der f1, Der f3 and Der f15 were identified in more MWs indicating the presence of zymogens and active-enzyme forms. The identified fecal allergens accumulate in the environment during the life of the mite and represent quantitatively greater amounts of mite immunogens than those that were missed in the 2D-E. The results contribute to our understanding of D. farinae digestive physiology with regard to the enzymes/proteins present in the feces.

• MeSH
• 2D gelová elektroforéza metody   MeSH
• antigeny roztočů domácího prachu chemie   MeSH
• Dermatophagoides farinae chemie   MeSH
• feces chemie   MeSH
• protein - isoformy MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny roztočů domácího prachu MeSH
• protein - isoformy MeSH

House dust mites produce antibacterial proteins suppressing bacterial growth. The 14.5-kDa bacteriolytic protein (UniProtKB Q8MWR6) has been known in Dermatophagoides pteronyssinus Trouessart. We have applied polymerase chain reaction and reverse transcription-PCR to detect a homologous gene sequence coding for a Q8MWR6-related protein in Dermatophagoides farinae (Hughes) using genomic DNA and total RNA, respectively. The resulting PCR product of expected size, 243 bp, was obtained from both Dermatophagoides spp., while no amplification was achieved from stored product mite samples. Sequence of the gene fragment from D. farinae showed 83% similarity to the previously described one in D. pteronyssinus. Successful amplification of the expected product from cDNA generated with oligo-dT primer implies that the NlpC/P60-like protein in Dermatophagoides mites is of eukaryotic or mite origin.

• MeSH
• Dermatophagoides farinae chemie   genetika   metabolismus   MeSH
• Dermatophagoides pteronyssinus chemie   genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• polymerázová řetězová reakce MeSH
• proteiny členovců chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny členovců MeSH