Characteristics and dynamics of microbial communities in Dermatophagoides farinae: Insights Across Developmental Stages

Characteristics and dynamics of microbial communities in Dermatophagoides farinae: Insights Across Developmental Stages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characteristics and dynamics of microbial communities in Dermatophagoides farinae: Insights Across Developmental Stages Zhewei Fan, Yujie Hong, Shuya Zhou, Huijie Zhang, Mo Zhuo, Xinyan Yang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5860420/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Dermatophagoides farinae causes human allergic diseases and contains a large number of microbes. The structure of the microbial community is an essential prerequisite for understanding the intricate symbiotic relationships between microbes and hosts. The characteristics and dynamics of symbiotic microbes at different developmental stages of D. farinae , however, are not well understood. Methods We performed high-throughput amplicon sequencing to investigate microbial community in D. farinae at different developmental stages. Results The results showed that microbial communities were diverse and dynamic during D. farinae development. Bacterial communities were generally richer than fungi in each developmental stage. The species richness and diversity of the bacterial community declined significantly from immature stages to adults. The highest species richness of the fungal community existed in nymphs. Eggs had the lowest fungal diversity. At 97% similarity, we assigned 40 phyla and 616 genera of bacteria and annotated 11 fungal phyla composed of 276 genera. The dominant bacterial and fungal phyla in all stages were Proteobacteria and Ascomycota , respectively. Staphylococcus was more abundant in eggs than in other stages, Bordetella , Pseudomonas , and Stenotrophomonas were dominant in both larvae and nymphs, and Burkholderia-Caballeronia-Paraburkholderia and Ralstonia were abundant in adults. Vibrionimonas was dominant in both eggs and adults. Aspergillus was the dominant fungal genus at all stages. Xeromyces was abundant in eggs, and Penicillium and Sarocladium were abundant in other stages. Correlation analysis showed the existence of strong and complex correlations in the dominant microbial genera, and most of these correlations were positive. The functional analysis showed that microbes participate in various life activities in D. farinae. Bacteria tend to have a higher functional abundance than fungi, such as substance metabolism. The functions of bacteria gradually enriched in adults. We observed similar fungal functional abundance in all stages. Conclusion this study has enriched our knowledge of the microbial communities associated with D. farinae and has provided clues for discovering microbes that play important functions in D. farinae . amplicon sequencing characteristics Dermatophagoides farinae diversity dynamics microbe Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Dermatophagoides farinae belongs to the class Acari, the order Astigmata, the family Pyroglyphidae, and the genus Dermatophagoides . The life cycle of D. farinae consists of four stages (i.e., egg, larvae, nymph, and adult), and they can quickly grow from eggs to adults when the external conditions are suitable [1]. In the laboratory, D. farinae can complete its life cycle in about 30 days. D. farinae can lead to the destruction of storage and human allergic diseases, so the the ability to effectively control the number of D. farinae in the environment holds great significance [2–5]. Although repeated use of the chemicals has led to resistance as a result of D. farinae ’s high reproductive potential and short life cycle [6]. Treatment with symbiotic microbes has been successfully used in insects, but the role of commensal microbes in D. farinae remains unclear [7]. Microbial communities are widely present in arthropods, and hosts interact with microbes in vivo and co-evolve. The host provides places and nutrients for microbes, which influence the biological functions of microbes in many aspects [8]. Commensal microbes play an important role in the survival of the host, including but not limited to the following: (1) enhance the host’s digestive ability and provide nutrients lacking in the food required, such as essential amino acids and vitamins [9–11]; (2) shorten the development time of the host and improve the survival rate and influence the lifetime [12–13]; (3) regulate the host’s mating and reproductive systems [14]; (4) avoid or relieve the invasion of pathogens to the host [15–16]; and (5) improve the host’s tolerance to adverse conditions and thus increase adaptation to the environment [17]. It is necessary to consider the role and impact of microbes to obtain a comprehensive understanding of D. farinae. With the rapid development of next-generation sequencing (NGS), amplicon sequencing has been widely used in the studies of D. farinae microbiomes. Previous reports have detailed descriptions of bacterial communities in D. farinae populations [18–20]. Hubert’s research revealed significant differences in the species and abundance of symbiotic microorganisms during the growth and decline phases of the population. It has been speculated that this pattern of change is influenced by the composition of microbes in D. farinae at different developmental stages [21]. A previous study also showed significant diversity differences between the bacterial communities of larvae and adults [22]. The composition of changes in the symbiotic microbial communities of D. farinae may have adapted to the nutritional and physiological needs of their host development, which deserves further investigation. Fungi have been reported to shape the microbiomes of mites, but little research has been conducted on the composition and dynamic changes in the symbiotic fungal community in different developmental stages of D. farinae [23]. The premise and key point of this study was to clarify the diversity of symbiotic microbial communities, identify key symbiotic microbes, and speculate their biological functions for the study of the cooperative evolution and interaction mechanism of D. farinae and symbiotic microbes. We planned to use high-throughput sequencing (16S rRNA gene V4 hypervariable region and ITS1-ITS2 sequence) to systematically analyze the diversity and dynamic changes in symbiotic microbes at different developmental stages of D. farinae and to explore the potential function of species and the interactive relationships in microbial species during growth and development. This study has provided basic data and new ideas for the further study of key microorganisms regulating the growth and development of D. farinae , as well as an analysis of their biological functions. Materials and methods Cultivation and Sample Collection of D.farinae Sample Collection Mites were obtained from laboratory in Wannan Medical College (N31°36′63″, E118°41′05″). The populations were cultured for more than 15 generations and identified as D.farinae by both morphological and molecular characteristics. We had taken the mixture of D.farinae and growth medium, sieved jointly with two flour sifter (aperture 0.15mm and 0.10mm) and separated eggs from the intermediate mixture. Using an oil painting brush, eggs were collected from the intermediate mixture. Some of these eggs were washed with detergent to eliminate surface contaminants and subsequently preserved in EP tubes filled with absolute ethanol. The remaining eggs were incubated in a laboratory setting to develop mites, under controlled conditions of 25 ± 1°C temperature and 70 ± 5% relative humidity, with a photoperiod consisting of 16 hours of light and 8 hours of darkness. These mites were provided with a diet consisting of flour, corn flour, dried fish meal, and yeast powder at a ratio of 4:3:1:1 (w/w). Based on the life cycle of D.farinae and microscopic examination of their morphology, the larvae, nymphs, male and female adults were individually collected. Subsequently, these mites were washed with detergent and fixed in EP tubes filled with absolute ethanol. Four replicate samples were collected for each developmental stage and then stored in a -80℃ cryogenic freezer for the preparation of genomic DNA. Eggs, larvae nymphs, and adult males and adult females labeled E, L, N, M, and F respectively. DNA Extraction and PCR The total DNA required for the high-throughput sequencing was extracted using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, USA) kit. The quality and quantity of DNA samples were determined by the agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) respectively. The V4 region of the bacterial 16S rRNA gene was amplified using PCR with the primers 515F (5'-GTGCCAGCMGCCGCGG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Similarly, the ITS1-ITS2 region of the fungal genome was amplified with the primers ITS1F (5'-AGAGTTTGATCCTGGCTCAG-3') and ITS2R (5'-AGAGTTTGATCCTGGCTCAG-3'). The PCR reaction system comprised a total volume of 20 µL, which included 0.8 µL (5 µM) of both forward and reverse primers, along with 10 ng of template DNA. The PCR reaction was performed as initial denaturation (95℃ for 5 min), 35 reaction cycles (each at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 45 s), and a final extension (72℃ for 10 min). The amplified products were electrophoresis on a 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.), following the manufacturer's protocol. The sequencing task was conducted by Shanghai Lingen Biotechnology Co., Ltd. Raw sequences are available at the GenBank SRA under the project identifier PRJNA1136690. Bioinformatics Analysis and Statistical Analysis The identification and elimination of chimeric sequences were performed using the FLASH (v1.2.11, https://ccb.jhu.edu/software/FLASH/index.shtml ), Trimmomatic (v0.36, http://www.usadellab.org/cms/index.php?page=trimmomatic ), and UCHIME (v4.1, http://drive5.com/uchime/uchime_download.html).Clea n sequences exhibiting 97% similarity were grouped into operational taxonomic units (OTUs) by means of the UPARSE software (version 7.1, http://drive5.com/uparse/ ). The RDP classifier Bayesian algorithm was used to analyze the taxonomic representation sequences of OTUs, and the confidence threshold was 0.7. Compared with the Silva138rRNA and Unite7 databases, and the microbial composition of each sample at each taxonomic level was counted. Alpha diversity index analysis was performed using Mothur software (v1.30.2, https://www.mothur.org/wiki/Download_mothur ) [24]. The Kruskal-Wallis test was employed to analyze the difference of alpha diversity microbial communities at different developmental stages of D.farinae . Beta diversity analysis was completed using Qiime software (v1.9.1, http://qiime.org/install/index.html ). The Correlation analysis plot was generated using R software (v.4.2.2) package ggplot2 (v.3.4.2) through Hiplot Pro ( https://hiplot.com.cn/ ). The pearson correlation coefficient was used to measure the abundance correlations among bacteria–bacteria, fungi–fungi, and bacteria–fungi during the growth of D. farinae , and the correlation coefficient c value and P value were calculated, with P < 0.05 as the significant correlation. The genome in the predicted sample was predicted by reconstructing the unobserved state of the Community Phylogenetic Survey 2 (PICRUSt 2) analysis ( https://github.com/picrust/picrust2 ), and then the genome function was predicted. Results General Characteristics of the Sequence Data Using Illumina high-throughput sequencing technology, we obtained a total of 719,595 high-quality bacterial sequences and 710,966 fungal sequences after we merged reads and filtration. The average read length of bacterial and fungal samples was 252 and 248 bp, respectively. The sequence coverage rates of both bacterial and fungal communities were more than 0.9905. At a 97% sequence similarity level, these reads from bacteria and fungi were classified into 2449 and 982 operational taxonomic units (OTUs), respectively. We obtained 765, 1064, 1381, 521, and 324 bacterial OTUs in eggs, larvae, nymphs, male adults, and female adults, respectively, and the number of fungal OTUs from the corresponding stages was 251, 455, 507, 252, and 262, respectively. Among D. farinae at different developmental stages, we found the highest total amount of both bacterial and fungal OTUs in nymphs and the lowest amount in adults. The number of bacterial OTUs was significantly higher than that of fungal OTUs at all developmental stages (Tables 1 and 2 ). Table 1 Sequence and diversity indices of bacterial samples in D. farinae at different developmental stages. Sample Clean Sequences Average Length OTUs Ace Chao Shannon Simpson Coverage E 150906 250 765 490.0 ± 53.4 436.2 ± 41.5 4.16 ± 0.33 0.054 ± 0.023 0.9991 L 135851 252 1064 460.3 ± 126.4 381 ± 56.34 4.01 ± 0.15 0.037 ± 0.004 0.9905 N 128918 252 1381 651.4 ± 441.8 466.2 ± 222.2 4.11 ± 0.24 0.036 ± 0.005 0.9988 M 153775 252 521 257.5 ± 139.6 176.7 ± 72.7 2.94 ± 0.34 0.121 ± 0.049 0.9924 F 150145 252 324 157.5 ± 39.6 133.4 ± 29.4 2.49 ± 0.54 0.189 ± 0.091 0.9977 Total 719,595 – 2449 – – – – – Average – 252 – – – – – – †Clean sequences is the number of high-quality sequences. E, L, N, M, and F represent eggs, larvae, nymphs, male adults, and female adults, respectively. Table 2 Sequence and taxonomic information of fungal samples in D. farinae at different developmental stages. Sample Clean Sequences Average Length OTUs Ace Chao Shannon Simpson Coverage E 149622 261 251 179.1 ± 13.3 173.9 ± 11.14 1.40 ± 0.13 0.467 ± 0.068 0.9988 L 152244 245 455 287.9 ± 6.63 271.8 ± 10.7 2.10 ± 0.16 0.237 ± 0.010 0.9981 N 149801 246 507 337.6 ± 34.7 321.9 ± 39.2 2.17 ± 0.24 0.231 ± 0.029 0.9977 M 130213 245 252 172.5 ± 35.1 151.3 ± 30.0 2.35 ± 0.08 0.158 ± 0.011 0.9991 F 129086 243 262 254.3 ± 112.7 208.9 ± 88.1 2.32 ± 0.18 0.166 ± 0.020 0.9986 Total 710966 – 982 – – – – – Average – 248 – – – – – – †Clean sequences is the number of high-quality sequences. E, L, N, M, and F represent eggs, larvae, nymphs, male adults, and female adults, respectively. Diversity of Microbial Communities Throughout the Life Cycle of D. farinae According to the results of diversity indices, dilution curves of these indices tended to be flat on bacterial and fungal communities in each sample, indicating adequate sequencing depth (see Supplementary Fig. 1A–1H). The Ace and Chao indices demonstrated that the highest community richness of bacteria and fungi was exhibited in nymphs and the lowest community richness of bacteria and fungi was exhibited in adults. The richness of the bacterial community in eggs and larvae was significantly higher than that of male and female adults (P < 0.05). The richness of the bacterial community slightly decreased from eggs to larvae, increased to the highest level in nymphs, but dropped to the lowest level in adults. The fungal community richness of nymphs was significantly higher than that of eggs and adults (P < 0.05) and was higher than that of larvae. The richness of the fungal community increased gradually from eggs to nymphs and then decreased in adults. The Shannon and Simpson indices showed that the diversity of bacteria was highest in the eggs and was roughly reduced in adults, which was lower than in the other stages (P < 0.05). Fungal diversity was lowest in the eggs and gradually increased in the other stages (Tables 1 and 2 ). Neither the bacterial nor fungal community richness and diversity differed significantly between male and female adults (Fig. 1 ). We compared bacterial and fungal community β-diversity in D. farinae samples grouped by developmental stage according to principal coordinates analysis (PCoA). We detected a similar association between the bacterial and fungal communities. The samples in the same developmental stage had a similar structure and thus were clustered into a group. We identified an important factor in bacterial and fungal differences in the developmental stage. In both bacterial and fungal communities, the microbial community in larvae and nymphs was similar, as was the microbial composition of male and female adults. The egg samples were clustered into one group (Fig. 2 ). Composition of Microbial Communities at Different Developmental Stages in D. farinae According to the OTU classification based on the Silve138.1 and Unite7 databases, we annotated the bacteria in all samples into 40 phyla, 111 classes, 222 orders, 364 families, and 616 genera, and the fungi were annotated into 11 phyla, 35 classes, 77 orders, 166 families, and 276 genera. At the phylum and genus taxonomic levels, we conducted in-depth analyses of bacterial and fungal communities with a relative abundance of more than 1.00% (Supplementary Tables 1 and 2). Proteobacteria was the dominant bacterial phylum at all stages (43.02–87.84% in relative abundance), followed by Bacteroidota (10.91–23.29%) and Firmicutes (0.61–14.10%). The dominant phylum of fungi was Ascomycota , which constituted more than 96.40% at all stages. Furthermore, at the level of bacterial genera, Vibrionimonas (15.46%), Staphylococcus (10.89%), and Chitinophaga (5.36%) were dominant in eggs. In larvae and nymphs, Bordetella (9.79% and 9.96% relative abundance in larvae and nymphs, respectively), Pseudomonas (8.98% and 9.40%), Methyloversatilis (8.97% and 8.36%), and Stenotrophomonas (6.65% and 6.70%) were abundant. An unclassified genus of Rhizobiaceae became the dominant genus in male and female adults (24.73% and 29.95% relative abundance in male and female adults, respectively), followed by Vibrionimonas (13.61% and 7.73%), Burkholderia-Caballeronia-Paraburkholderia (7.16% and 7.73%), Ralstonia (9.34% and 6.37%), and Ramlibacter (6.50% and 5.02%) (Table 2 A). Aspergillus was the abundant genus of fungi at all stages of D. farinae , accounting for 71.29%, 18.14%, 18.67%, 33.45%, and 32.35% in eggs, larvae, nymphs, male adults, and female adults, respectively. Xeromyces (19.60%) was abundant in eggs, and Penicillium and Sarocladium accounted for 18.80% and 36.74% in the samples at each developmental stage, except for eggs (Fig. 3 and Supplementary Tables 3 and 4). Species Differences of D. farinae at Different Developmental Stages As shown in Fig. 4 , at the genus level, we identified 63 genera of bacteria throughout all developmental stages and 69 shared genera of bacteria in eggs, male adults, and female adults, which highly overlapped and may have been related to the vertical transmission of commensal bacteria. The 30 genera were shared by all stages in fungi, and the 33 genera shared by eggs, male adults, and female adults were also identical (Fig. 4 ). As shown in the heat map in Fig. 5 , we observed differences in the abundance of shared bacteria and fungi in different developmental stages; nonetheless, they were always present with D. farinae (Fig. 5 ).We also observed specific bacteria and fungi at each developmental stage. We conducted a linear discriminant analysis effect size (LEfSe) analysis to explore the differential microorganisms among the groups. In the egg stage, except for Staphylococcus , Chitinophaga , Xeromyces , and Aspergillus as the dominant genera, we observed that Bradyrhizobium , Variovorax , Phyllobacterium , Gibberella , Wallemia , and Fusarium were significantly more abundant than in other stages (from phylum to genus, LDA > 4.0, P < 0.01). In the larvae, Methyloversatilis , Brucella , Azospirillum , Brevundimonas , Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium , Pseudoflavitalea , Sphingomonas , and Sarocladium were significantly abundant (from phylum to genus, LDA > 4.0, P < 0.01). In nymphs, Bordetella , Pseudomonas , Stenotrophomonas , and Penicillium were the dominant genera, which was the same as in Flavobacterium , Pyrenochaetopsis , and Trichoderma , which were significantly more abundant than in other stages (from phylum to genus, LDA > 4.0, P 4.0, P 4.0, P < 0.01) (Supplementary Fig. 2). The significantly enriched bacteria and fungi in some of these stages could play a specific role in the development and growth of D. farinae. Correlation Analysis of Microbial Species in the D. farinae We conducted a correlation analysis to reveal the relationships between bacterial and fungal genera during the development of D. farinae . We focused on an analysis of 20 dominant bacterial genera and 15 dominant fungal genera (Fig. 6 ). The abundance correlations among bacteria–bacteria, fungi–fungi, and bacteria–fungi during the growth of D. farinae were complex, and the ratio of significantly positive and negative correlations was 79:40, 20:16, and 83:56, respectively. The number of positive correlations was greater than the number of negative correlations between bacteria and bacteria, bacteria and fungi, and fungi and fungi. The relationship between the dominant bacteria at the same developmental stage was mainly a positive correlation, like the larvae and nymph stages between Pseudomonas and Stenotrophomonas , Bordetella , and Methyloversatilis . In adults, Ralstonia was positively connected with unclassified genera, including Rhizobiaceae , Burkholderia-Caballeronia-Paraburkholderia , Methyloversatilis , Ramlibacter , and Vibrionimonas . The negative correlations between bacteria and fungi were associated primarily with Aspergillus , Talaromyces , Oidiodendron , and Wallemia. The negative correlations between fungi and fungi were primarily associated with Talaromyces , Penicillium , Sarocladium , and Purpureocillium. Correlation in microbial species may be a significant factor influencing the dynamics of bacterial and fungal communities. Functional Analysis of Microbial Communities at Different Developmental Stages of D. farinae We used PICRUSt2 software to predict the relative abundance of microbiome-related Clusters of Orthologous Groups (COG)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functions in all samples and analyzed the results. According to both COG and KEGG databases, the abundance of various functions in these bacteria was greater than in fungi. The bacterial functional abundance was greater in adults than in larvae, nymphs, and eggs. We observed little difference in the functional abundance of fungi at each stage; larvae and nymphs had greater fungal functional abundance than eggs and adults. The COG functional classification revealed that bacteria had a higher abundance in amino acid transport and metabolism, carbohydrate transport and metabolism, cell membrane biogenesis, and energy production and conversion. Fungi had a higher abundance in translation, ribosomal structure, and biogenesis than in other pathways (Fig. 7 ). The KEGG functions included metabolism, genetic information processing, environmental information processing, cellular processes, organism systems, and human diseases. The KEGG functional classification showed that during the development and growth of D. farinae , some functions were enriched in both bacteria and fungi compared with other pathways, such as carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins. Fungi had a higher abundance in translation compared with other pathways (Fig. 8 ). This result suggested that commensal bacteria may have played a lead role in D. farinae , but the role of fungi should not be ignored. The role of fungi was relatively more stable than the role of bacteria. Discussion Symbiotic microbes recently became a key focus of many studies because of the important roles they play in the growth, development, adaptability, and reproduction of hosts. Numerous studies have focused on D. farinae , which is known to cause human allergic disease. The microbial communities of D. farinae have been characterized using high-throughput sequencing techniques, which have largely enriched knowledge of the microbial composition in D. farinae. Few studies, however, have examined the community characteristics and dynamics of symbiotic bacteria and fungi across the different developmental stages of D. farinae , thus lacking exploration of function and species interaction. We used high-throughput sequencing of the 16S rRNA and ITS amplicon amplification to report on the bacterial and fungal communities in D. farinae across different stages. We found that the microbial structure of D. farinae was significantly influenced by the developmental stage, which was consistent with many previous studies on mites, such as Panonychus citri , Dermanyssus gallinae , and Tetranychus truncatus [25–27]. The composition of microbial communities in D. farinae , however, was less different by sex, which was consistent with the results of previous research [22,28]. The richness and diversity of bacteria and fungi during the growth and development of D. farinae changed dynamically in different patterns. The diversity and richness of bacterial communities in eggs, larvae, and nymphs were significantly higher than those of male and female adults. The highest species richness of the fungal community was observed in nymphs. This was similar to the higher bacterial diversity and richness in the immature stage than in the mature stage, which was found in some insects such as psyllids, fruit flies, planthoppers, and ladybugs [29–32]. Previous studies on insects have shown that the higher richness and diversity of microbes in the immature stage are related to the microbial richness and diversity from eggs or diet [30,32]. All shared bacteria in each stage came from those shared by eggs and adults (Supplementary Tables 5 and 6). The shared bacteria may have been due to vertical transmission and could be a factor that has caused the high diversity of the immature stage of D. farinae . The dietary preference of D. farinae for bacteria and fungi was also an important factor for the higher richness and diversity of bacteria and for the higher richness of fungi in the immature stage of D. farinae [33]. Diet may be a source for some stage-specific species. In the immature stage, more bacteria and fungi may have promoted the digestion and absorption of substances in the underdeveloped gut. However, with the gradual improvement of intestinal structure and function in adults, because of the selectivity of the gut, partial exogenous species were discharged along with feces [34]. The molting process in the immature stage of D. farinae may also lead to the loss of some species along with the exoskeleton [35–37]. These reasons likely caused the decrease in richness and diversity in adults. Further analysis showed that the bacteria in D. farinae at different developmental stages were distributed in 40 phyla, with Proteobacteria serving as the main phylum, possibly because of its involvement in host adaptation, digestion, nutrient supply, and energy metabolism [38,39]. The changes in the dominant bacterial genera may have better adapted to the needs of the different developmental stages of D. farinae. Vibrionimonas was the most abundant bacterial genus in eggs, which probably came from the vertical transmission of adults. Staphylococcus was significantly enriched in eggs, which decreased significantly in the next stages. The cell wall of Staphylococcus is a suitable target for lysozyme-like enzymes in mites. The hydrolysis of Staphylococcus cell walls can provide nutrients for mites [40–42]. We speculate that it hydrolyzed to provide nutrients to eggs. Chitinophaga , which is a differential genus and is significantly enriched in eggs, likely degraded the eggshell to ensure the developmental process from eggs to larvae [43,44]. After becoming larvae, D. farinae began to eat, and the laboratory feed was mainly composed of flour. Pseudomonas and Stenotrophomonas were abundant in the larvae and nymphs, especially in nymphs, and both demonstrated cellulose-degrading activity and were beneficial for food digestion [45–48]. Pseudomonas contributed to promoting host lipid and vitamin metabolism, which may have facilitated the absorption and utilization of food [49,50]. The increase in Pseudomonas and Stenotrophomonas was conducive to the enhancement of the digestive ability of larvae and nymphs. The correlation between Pseudomonas and Stenotrophomonas was significantly positive, which probably was related to their similar functional roles. Adults had more functions, such as mating and reproduction, although the richness and diversity of bacteria were lower than in the immature stage. Bacteria could play more diverse and stronger functions in D. farinae . Vibrionimonas , which is a Gram-negative bacterium that has been reported as a gut bacterium in Sirex noctilio (Hymenoptera: Siricidae) and Dactylispa xanthospila (Gestro) (Coleoptera: Chrysomelidae: Cassidinae) [51,52], was abundant in eggs and adults. Its function may be related to the utilization of nutrients as gut bacteria of mites. Burkholderia-Caballeronia-Paraburkholderia not only fixed nitrogen, degraded carbohydrates, and provided essential amino acids and vitamin B in nutrition [53], but also enhanced host resistance in ecology, thus affecting the growth and reproduction of the host [54–57]. Ralstonia , as symbiotic bacteria, existed in numerous insects and played an important role in the development of the host, which may have been beneficial for aerobic respiration [58,59]. Ralstonia also utilized a series of aromatic compounds as energy to detoxify, which suggested that it may be beneficial for D. farinae to resist adverse external environments and enhance adaptability [60]. The correlation between Burkholderia-Caballeronia-Paraburkholderia and Ralstonia was significantly positive. A previous study found that the cooccurrence of Burkholderia-Caballeronia-Paraburkholderia and Ralstonia may be related to their carbon source utilization patterns and fatty acid methyl-ester production [61]. The dominant fungal phylum of D. farinae was Ascomycota , which also plays a dominant role throughout nature. Unlike the dynamic changes in bacteria, the structure of the fungal communities of D. farinae at different developmental stages have been relatively stable. Aspergillus was the dominant fungus at all developmental stages, which was consistent with the dominant fungal genus of D. farinae cultivation in Hubert’s study [21]. Xeromyces had high abundance in eggs. Other studies have shown that Xeromyces is closely related to cofactor and vitamin metabolism, energy metabolism, methane metabolism, carbohydrate metabolism, and glycosaminoglycan degradation [62]. Except for the eggs, both Penicillium and Sarocladium had high abundance in larvae, nymphs, and adults. Penicillium , as a dominant fungal genus, could degrade cellulose that enhanced the host’s digestion and immune system, thus causing defense disorders and influencing its behavior and health [63]. The relatively stable fungal genera may have been instrumental at different developmental stages of D. farinae and thus should be further studied. The COG and KEGG functional classification revealed that the abundance of various functions in these bacteria was greater than that of fungi, which was likely associated with the higher numbers of bacteria than fungi. Several reports have shown that D. farinae had more abundant bacteria than fungi, and bacteria may play a leading role in assisting the growth and development of D. farinae [20,21]. Male and female adults’ functional abundance was greater than that of larvae, nymphs, and eggs in bacteria. However, we observed little difference in the functional abundance of fungi at each stage, and eggs, larvae, and adults had similar fungal compositions. The functional abundance of bacteria was positively correlated with the developmental time of D. farinae . In bacteria, we found similarities in the developmental time and functional abundance of larvae and nymphs. Male and female adults had the longest developmental time, and the most enriched functions of bacteria were also found in adults [64]. The abundance correlations between bacteria–bacteria, fungi–fungi, and bacteria–fungi during the growth of D. farinae were strong and complex. Notably, the number of positive correlations was greater than the number of negative correlations in microbial genera, which may have contributed to the synergistic function. For instance, Pseudomonas and Stenotrophomonas had similar functions in digestion. Burkholderia-Caballeronia-Paraburkholderia and Ralstonia had similar synergistic functions in substance metabolism. Species with negative correlations also deserve further investigation. Previous studies on insects have shown that symbionts may compete with others within the host for limited space and resources, which would result in the exclusion of less competitive symbionts. Another hypothesis is that symbionts may negatively affect the density of several microbes, resulting in the absence of certain microbes [65,66]. The negative correlations were primarily associated with the fungal genera Aspergillus , Penicillium , Talaromyces , Sarocladium , and Purpureocillium. Aspergillus and Penicillium produced a variety of antimicrobial secondary metabolites [67,68]. Multiple reports have shown that fungi influence the structure of the microbial community in mites. The decrease in bacterial and fungal species and abundance during the developmental stages of D. farinae could be attributed to fungi and their metabolites. The impact of fungi on the entire microbial spectrum is equally significant. The complex abundance correlation in species may be one of the reasons for these dynamic bacterial and fungal communities. We also made a discovery regarding the symbiotic microbes of D. farinae that may contribute to human diseases. D. pteronyssinus at different developmental stages produced different levels of allergens, and nymphs produced more allergens[69]. We speculate that the nymphs of D. farinae could also produce more allergens, which could easily lead to allergic diseases. Nymphs had more bacteria than other stages, including the dominant bacteria Bordetella , which produced allergens. The number of bacteria and related molecules was positively correlated with the inflammatory response [20,21]. In addition, Ralstonia produces lipopolysaccharides, which could bind to certain receptors involved in allergic reactions to induce asthma. The severity of asthma in nonatopic children is related to the concentration of total fungi. Aspergillus is the most common pathogen causing allergic bronchopulmonary aspergillosis, and the Wallemia genus can cause human health problems, including allergies and asthma. Declarations Acknowledgments We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript. Funding This work was supported by the National Natural Science Foundation of China (No.31870352). Availability of data and materials Data are provided within the manuscript. Authors’ contributions ZheWei Fan: Methodology, Software, Investigation, Formal analysis, Resources,Writing - Original Draft. Yujie Hong: Methodology, Investigation, Resources, Formal analysis, Writing - Original Draft. Shuya Zhou: Investigation, Resources, Visualization, Writing - Original Draft.Huijie Zhang& Mo Zhuo &Xinyan Yang& Yawen Yang & Min Ling & Ziyan Wang: Formal analysis, Resources, Visualization. Xianglin Tao & EntaoSun : Conceptualization, Methodology, Data curation, Writing - Review&Editing, Funding acquisition, Resources, Supervision, Validation, Project administration. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Hibberson, C. E. and Vogelnest, L. J. (2014) Storage mite contamination of commercial dry dog food in south‐eastern A ustralia[J]. Australian veterinary journal,92(6):219-224. Fernández-Caldas, E., Puerta,L. and Caraballo, L.(2014) Mites and allergy. Chem Immunol Allergy.100:234–42. Tang, V. H., Stewart, G. A. and Chang, B. J. (2015) House dust mites possess a polymorphic, single domain putative peptidoglycan d, l endopeptidase belonging to the NlpC/P60 Superfamily[J]. FEBS Open bio,5:813-823. Hubert, J., Nesvorna, M., Kopecky, J., Erban, T. and Klimov, P. 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Microb Lett ,44(174):77–81 Nicoletti, R., Andolfi, A., Becchimanzi, A. and Salvatore, M.M. (2023) Anti-Insect Properties of Penicillium Secondary Metabolites. Microorganisms,11(5):1302. Calzada, D., Martín-López, L. and Carnés, J.(2023) Growth, allergen profile and microbiome studies in Dermatophagoides pteronyssinus cultures. Sci Rep. 13(1):10633. Supplementary Tables and Figures Supplementary tables and figures are not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5860420","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408651657,"identity":"d2a271aa-7ac1-4d18-8787-16d40202a8f8","order_by":0,"name":"Zhewei Fan","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhewei","middleName":"","lastName":"Fan","suffix":""},{"id":408651659,"identity":"31934d0e-a2ab-4769-975b-7b386835492b","order_by":1,"name":"Yujie Hong","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Hong","suffix":""},{"id":408651662,"identity":"92b5c750-b864-4280-8847-a8db42fbf0aa","order_by":2,"name":"Shuya Zhou","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shuya","middleName":"","lastName":"Zhou","suffix":""},{"id":408651663,"identity":"b6a54c51-99ce-4c21-a109-281b6db7f089","order_by":3,"name":"Huijie Zhang","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Huijie","middleName":"","lastName":"Zhang","suffix":""},{"id":408651664,"identity":"6cb59233-62cc-4283-a7d8-e5f2181d7e5e","order_by":4,"name":"Mo Zhuo","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Mo","middleName":"","lastName":"Zhuo","suffix":""},{"id":408651665,"identity":"860f234f-1c6b-4192-aeeb-1b80b408cf63","order_by":5,"name":"Xinyan Yang","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xinyan","middleName":"","lastName":"Yang","suffix":""},{"id":408651666,"identity":"fe30082f-0634-4679-a4ff-2f1d1e589da9","order_by":6,"name":"Yawen Yang","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Yang","suffix":""},{"id":408651668,"identity":"06c0abe0-2083-41de-9b95-1f0bb2f522e2","order_by":7,"name":"Min Ling","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Ling","suffix":""},{"id":408651670,"identity":"6650cea2-69c7-407f-bd21-0b885d28647d","order_by":8,"name":"Ziyan Wang","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ziyan","middleName":"","lastName":"Wang","suffix":""},{"id":408651671,"identity":"f5c068e4-8858-493a-b0c1-ace8074a7292","order_by":9,"name":"Feng Yang","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Yang","suffix":""},{"id":408651672,"identity":"4fac1c43-d939-4f8d-a361-50e6a4cd64ef","order_by":10,"name":"Xianglin Tao","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xianglin","middleName":"","lastName":"Tao","suffix":""},{"id":408651673,"identity":"3089a9d2-cfed-44aa-ab42-18094bc6bf15","order_by":11,"name":"Entao Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYHACAzDJz8x88AEJWhIYGCTb2ZINSNNicJ7HTIAo9fz9hzc+LvxhJ2d8mMGMgaHGJpqgFokbacXGMxKSjc0OM6Q9YDiWlttAUM8NHjNpngTmxG2HGY4bMDYcJqxF/vwZ8988CfWJm5sZ2ySI0mJwIMeMmSfhcOIGZmY24rQYAv0izZN23FjiMBuzQQIxfpE7f3jjZx6bajn+/vMfH3yosSHC+ygggTTlo2AUjIJRMApwAQDXfjrYcC9tdAAAAABJRU5ErkJggg==","orcid":"","institution":"Wannan Medical College","correspondingAuthor":true,"prefix":"","firstName":"Entao","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-01-19 16:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5860420/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5860420/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75482688,"identity":"53374160-8612-44d6-8ee6-ec49ce122552","added_by":"auto","created_at":"2025-02-05 05:41:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":465308,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity among bacteria and fungi of\u003cem\u003e D. farinae \u003c/em\u003eat different developmental stages: (A) Differences between richness index for bacteria; (B) differences between diversity index for bacteria; (C) differences between richness index for fungi; and (D) differences between diversity index for fungi.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/111b5ffa69622b9b869c704a.png"},{"id":75481794,"identity":"d6cfeac8-e502-43ce-8e82-8b86f826f343","added_by":"auto","created_at":"2025-02-05 05:33:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292868,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of bacterial and fungal communities. PCoA of bacterial communities(A). PCoA of fungal communities(B).The values of axes 1 and 2 are the percentages that can be explained by the corresponding axis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/4eb5905e56a68e8d84228044.png"},{"id":75481796,"identity":"a25c81db-7f9a-47ce-ab85-5bb7282f880e","added_by":"auto","created_at":"2025-02-05 05:33:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":733310,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance plots of the bacteria and fungi in the samples from different developmental stages of\u003cem\u003e D.farinae\u003c/em\u003e at the levels of phylum and genus. Composition of bacteria at phylum level (A) and genus level (B). Composition of fungi at phylum level (C) and genus level (D).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/b63b228df5f9e19726c62977.png"},{"id":75481799,"identity":"90779a83-0db0-43a1-8f9d-aa7fde498ef9","added_by":"auto","created_at":"2025-02-05 05:33:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":753485,"visible":true,"origin":"","legend":"\u003cp\u003eThe Veen plot diagram on the number of shared genus and specificial genus to each development stage of \u003cem\u003eD.farinae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/883df33966d46430cc751df3.png"},{"id":75482687,"identity":"e307366b-1b36-40a1-9db8-dd1f844277a7","added_by":"auto","created_at":"2025-02-05 05:41:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":497444,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap on relative abundance of the dominant taxa in each genus and sample.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/dbe7532145b7359fdd1b2e07.png"},{"id":75481801,"identity":"399d1de5-caa7-4c3a-8a12-5d46d4e8b350","added_by":"auto","created_at":"2025-02-05 05:33:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":867483,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis heatmap of bacterial and fungal genus in \u003cem\u003eD.farinae\u003c/em\u003eat different developmental stages. Correlation plot showing the relationship among top 20 predominant bacteria genus(A), the relationship among top 15 predominant fungi genus(B), and the relationship between the top 20 bacteria genus and the top 15 fungi genus(C).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/9d57d8f809c6a074bee91441.png"},{"id":75481822,"identity":"2840cd13-5efa-4e9d-8646-e7284233bb21","added_by":"auto","created_at":"2025-02-05 05:33:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":468500,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in bacterial and fungal COG function profiles during development of \u003cem\u003eD.farinae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/0eafe755070e0a62a25d9e17.png"},{"id":75481798,"identity":"6267b120-6c90-47e5-9028-75e143cabc5c","added_by":"auto","created_at":"2025-02-05 05:33:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":686496,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in bacterial and fungal KEGG function profiles during development of\u003cem\u003e D.farinae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/ced4bfc7206e2b22845ecdf3.png"},{"id":75925224,"identity":"487988eb-b08a-4c43-ba2d-c1cdacbc1a10","added_by":"auto","created_at":"2025-02-10 15:09:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5965798,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5860420/v1/07585a38-55a6-43f1-8997-a9b5c4552862.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characteristics and dynamics of microbial communities in Dermatophagoides farinae: Insights Across Developmental Stages","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eDermatophagoides farinae\u003c/em\u003e belongs to the class Acari, the order Astigmata, the family Pyroglyphidae, and the genus \u003cem\u003eDermatophagoides\u003c/em\u003e. The life cycle of \u003cem\u003eD. farinae\u003c/em\u003e consists of four stages (i.e., egg, larvae, nymph, and adult), and they can quickly grow from eggs to adults when the external conditions are suitable [1]. In the laboratory, \u003cem\u003eD. farinae\u003c/em\u003e can complete its life cycle in about 30 days. \u003cem\u003eD. farinae\u003c/em\u003e can lead to the destruction of storage and human allergic diseases, so the the ability to effectively control the number of \u003cem\u003eD. farinae\u003c/em\u003e in the environment holds great significance [2\u0026ndash;5]. Although repeated use of the chemicals has led to resistance as a result of \u003cem\u003eD. farinae\u003c/em\u003e\u0026rsquo;s high reproductive potential and short life cycle [6]. Treatment with symbiotic microbes has been successfully used in insects, but the role of commensal microbes in \u003cem\u003eD. farinae\u003c/em\u003e remains unclear [7].\u003c/p\u003e \u003cp\u003eMicrobial communities are widely present in arthropods, and hosts interact with microbes in vivo and co-evolve. The host provides places and nutrients for microbes, which influence the biological functions of microbes in many aspects [8]. Commensal microbes play an important role in the survival of the host, including but not limited to the following: (1) enhance the host\u0026rsquo;s digestive ability and provide nutrients lacking in the food required, such as essential amino acids and vitamins [9\u0026ndash;11]; (2) shorten the development time of the host and improve the survival rate and influence the lifetime [12\u0026ndash;13]; (3) regulate the host\u0026rsquo;s mating and reproductive systems [14]; (4) avoid or relieve the invasion of pathogens to the host [15\u0026ndash;16]; and (5) improve the host\u0026rsquo;s tolerance to adverse conditions and thus increase adaptation to the environment [17]. It is necessary to consider the role and impact of microbes to obtain a comprehensive understanding of \u003cem\u003eD. farinae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eWith the rapid development of next-generation sequencing (NGS), amplicon sequencing has been widely used in the studies of \u003cem\u003eD. farinae\u003c/em\u003e microbiomes. Previous reports have detailed descriptions of bacterial communities in \u003cem\u003eD. farinae\u003c/em\u003e populations [18\u0026ndash;20]. Hubert\u0026rsquo;s research revealed significant differences in the species and abundance of symbiotic microorganisms during the growth and decline phases of the population. It has been speculated that this pattern of change is influenced by the composition of microbes in \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages [21]. A previous study also showed significant diversity differences between the bacterial communities of larvae and adults [22]. The composition of changes in the symbiotic microbial communities of \u003cem\u003eD. farinae\u003c/em\u003e may have adapted to the nutritional and physiological needs of their host development, which deserves further investigation. Fungi have been reported to shape the microbiomes of mites, but little research has been conducted on the composition and dynamic changes in the symbiotic fungal community in different developmental stages of \u003cem\u003eD. farinae\u003c/em\u003e [23].\u003c/p\u003e \u003cp\u003eThe premise and key point of this study was to clarify the diversity of symbiotic microbial communities, identify key symbiotic microbes, and speculate their biological functions for the study of the cooperative evolution and interaction mechanism of \u003cem\u003eD. farinae\u003c/em\u003e and symbiotic microbes. We planned to use high-throughput sequencing (16S rRNA gene V4 hypervariable region and ITS1-ITS2 sequence) to systematically analyze the diversity and dynamic changes in symbiotic microbes at different developmental stages of \u003cem\u003eD. farinae\u003c/em\u003e and to explore the potential function of species and the interactive relationships in microbial species during growth and development. This study has provided basic data and new ideas for the further study of key microorganisms regulating the growth and development of \u003cem\u003eD. farinae\u003c/em\u003e, as well as an analysis of their biological functions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCultivation and Sample Collection of D.farinae Sample Collection\u003c/h2\u003e \u003cp\u003eMites were obtained from laboratory in Wannan Medical College (N31\u0026deg;36\u0026prime;63\u0026Prime;, E118\u0026deg;41\u0026prime;05\u0026Prime;). The populations were cultured for more than 15 generations and identified as \u003cem\u003eD.farinae\u003c/em\u003e by both morphological and molecular characteristics. We had taken the mixture of \u003cem\u003eD.farinae\u003c/em\u003e and growth medium, sieved jointly with two flour sifter (aperture 0.15mm and 0.10mm) and separated eggs from the intermediate mixture. Using an oil painting brush, eggs were collected from the intermediate mixture. Some of these eggs were washed with detergent to eliminate surface contaminants and subsequently preserved in EP tubes filled with absolute ethanol. The remaining eggs were incubated in a laboratory setting to develop mites, under controlled conditions of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C temperature and 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, with a photoperiod consisting of 16 hours of light and 8 hours of darkness. These mites were provided with a diet consisting of flour, corn flour, dried fish meal, and yeast powder at a ratio of 4:3:1:1 (w/w). Based on the life cycle of \u003cem\u003eD.farinae\u003c/em\u003e and microscopic examination of their morphology, the larvae, nymphs, male and female adults were individually collected. Subsequently, these mites were washed with detergent and fixed in EP tubes filled with absolute ethanol. Four replicate samples were collected for each developmental stage and then stored in a -80℃ cryogenic freezer for the preparation of genomic DNA. Eggs, larvae nymphs, and adult males and adult females labeled E, L, N, M, and F respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA Extraction and PCR\u003c/h3\u003e\n\u003cp\u003eThe total DNA required for the high-throughput sequencing was extracted using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, USA) kit. The quality and quantity of DNA samples were determined by the agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) respectively. The V4 region of the bacterial 16S rRNA gene was amplified using PCR with the primers 515F (5'-GTGCCAGCMGCCGCGG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Similarly, the ITS1-ITS2 region of the fungal genome was amplified with the primers ITS1F (5'-AGAGTTTGATCCTGGCTCAG-3') and ITS2R (5'-AGAGTTTGATCCTGGCTCAG-3'). The PCR reaction system comprised a total volume of 20 \u0026micro;L, which included 0.8 \u0026micro;L (5 \u0026micro;M) of both forward and reverse primers, along with 10 ng of template DNA. The PCR reaction was performed as initial denaturation (95℃ for 5 min), 35 reaction cycles (each at 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 45 s), and a final extension (72℃ for 10 min). The amplified products were electrophoresis on a 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.), following the manufacturer's protocol. The sequencing task was conducted by Shanghai Lingen Biotechnology Co., Ltd. Raw sequences are available at the GenBank SRA under the project identifier PRJNA1136690.\u003c/p\u003e\n\u003ch3\u003eBioinformatics Analysis and Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eThe identification and elimination of chimeric sequences were performed using the FLASH (v1.2.11,\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccb.jhu.edu/software/FLASH/index.shtml\u003c/span\u003e\u003cspan address=\"https://ccb.jhu.edu/software/FLASH/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Trimmomatic (v0.36,\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.usadellab.org/cms/index.php?page=trimmomatic\u003c/span\u003e\u003cspan address=\"http://www.usadellab.org/cms/index.php?page=trimmomatic\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and UCHIME (v4.1,\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://drive5.com/uchime/uchime_download.html).Clea\u003c/span\u003e\u003cspan address=\"http://drive5.com/uchime/uchime_download.html).Clea\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003en sequences exhibiting 97% similarity were grouped into operational taxonomic units (OTUs) by means of the UPARSE software (version 7.1, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://drive5.com/uparse/\u003c/span\u003e\u003cspan address=\"http://drive5.com/uparse/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The RDP classifier Bayesian algorithm was used to analyze the taxonomic representation sequences of OTUs, and the confidence threshold was 0.7. Compared with the Silva138rRNA and Unite7 databases, and the microbial composition of each sample at each taxonomic level was counted. Alpha diversity index analysis was performed using Mothur software (v1.30.2, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mothur.org/wiki/Download_mothur\u003c/span\u003e\u003cspan address=\"https://www.mothur.org/wiki/Download_mothur\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [24]. The Kruskal-Wallis test was employed to analyze the difference of alpha diversity microbial communities at different developmental stages of \u003cem\u003eD.farinae\u003c/em\u003e. Beta diversity analysis was completed using Qiime software (v1.9.1, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://qiime.org/install/index.html\u003c/span\u003e\u003cspan address=\"http://qiime.org/install/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The Correlation analysis plot was generated using R software (v.4.2.2) package ggplot2 (v.3.4.2) through Hiplot Pro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hiplot.com.cn/\u003c/span\u003e\u003cspan address=\"https://hiplot.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The pearson correlation coefficient was used to measure the abundance correlations among bacteria\u0026ndash;bacteria, fungi\u0026ndash;fungi, and bacteria\u0026ndash;fungi during the growth of \u003cem\u003eD. farinae\u003c/em\u003e, and the correlation coefficient c value and P value were calculated, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the significant correlation. The genome in the predicted sample was predicted by reconstructing the unobserved state of the Community Phylogenetic Survey 2 (PICRUSt 2) analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/picrust/picrust2\u003c/span\u003e\u003cspan address=\"https://github.com/picrust/picrust2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and then the genome function was predicted.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Characteristics of the Sequence Data\u003c/h2\u003e \u003cp\u003eUsing Illumina high-throughput sequencing technology, we obtained a total of 719,595 high-quality bacterial sequences and 710,966 fungal sequences after we merged reads and filtration. The average read length of bacterial and fungal samples was 252 and 248 bp, respectively. The sequence coverage rates of both bacterial and fungal communities were more than 0.9905. At a 97% sequence similarity level, these reads from bacteria and fungi were classified into 2449 and 982 operational taxonomic units (OTUs), respectively. We obtained 765, 1064, 1381, 521, and 324 bacterial OTUs in eggs, larvae, nymphs, male adults, and female adults, respectively, and the number of fungal OTUs from the corresponding stages was 251, 455, 507, 252, and 262, respectively. Among \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages, we found the highest total amount of both bacterial and fungal OTUs in nymphs and the lowest amount in adults. The number of bacterial OTUs was significantly higher than that of fungal OTUs at all developmental stages (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequence and diversity indices of bacterial samples in \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eClean Sequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOTUs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAce\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChao\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShannon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSimpson\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCoverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e150906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e490.0\u0026thinsp;\u0026plusmn;\u0026thinsp;53.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e436.2\u0026thinsp;\u0026plusmn;\u0026thinsp;41.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.054\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.9991\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e135851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e460.3\u0026thinsp;\u0026plusmn;\u0026thinsp;126.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e381\u0026thinsp;\u0026plusmn;\u0026thinsp;56.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.037\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.9905\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e128918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e651.4\u0026thinsp;\u0026plusmn;\u0026thinsp;441.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e466.2\u0026thinsp;\u0026plusmn;\u0026thinsp;222.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.036\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.9988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e153775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e257.5\u0026thinsp;\u0026plusmn;\u0026thinsp;139.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e176.7\u0026thinsp;\u0026plusmn;\u0026thinsp;72.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.121\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.9924\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e150145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e157.5\u0026thinsp;\u0026plusmn;\u0026thinsp;39.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e133.4\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.189\u0026thinsp;\u0026plusmn;\u0026thinsp;0.091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.9977\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e719,595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u0026dagger;Clean sequences is the number of high-quality sequences. E, L, N, M, and F represent eggs, larvae, nymphs, male adults, and female adults, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequence and taxonomic information of fungal samples in \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eClean Sequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003cp\u003eLength\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOTUs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAce\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChao\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eShannon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSimpson\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003eCoverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e149622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e179.1\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e173.9\u0026thinsp;\u0026plusmn;\u0026thinsp;11.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.467\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.9988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e152244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e287.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e271.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.9981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e149801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e337.6\u0026thinsp;\u0026plusmn;\u0026thinsp;34.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e321.9\u0026thinsp;\u0026plusmn;\u0026thinsp;39.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.231\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.9977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e130213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e172.5\u0026thinsp;\u0026plusmn;\u0026thinsp;35.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e151.3\u0026thinsp;\u0026plusmn;\u0026thinsp;30.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.158\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.9991\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e129086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e254.3\u0026thinsp;\u0026plusmn;\u0026thinsp;112.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e208.9\u0026thinsp;\u0026plusmn;\u0026thinsp;88.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.166\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.9986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e710966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u0026dagger;Clean sequences is the number of high-quality sequences. E, L, N, M, and F represent eggs, larvae, nymphs, male adults, and female adults, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDiversity of Microbial Communities Throughout the Life Cycle of D. farinae\u003c/h2\u003e \u003cp\u003eAccording to the results of diversity indices, dilution curves of these indices tended to be flat on bacterial and fungal communities in each sample, indicating adequate sequencing depth (see Supplementary Fig.\u0026nbsp;1A\u0026ndash;1H). The Ace and Chao indices demonstrated that the highest community richness of bacteria and fungi was exhibited in nymphs and the lowest community richness of bacteria and fungi was exhibited in adults. The richness of the bacterial community in eggs and larvae was significantly higher than that of male and female adults (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The richness of the bacterial community slightly decreased from eggs to larvae, increased to the highest level in nymphs, but dropped to the lowest level in adults. The fungal community richness of nymphs was significantly higher than that of eggs and adults (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and was higher than that of larvae. The richness of the fungal community increased gradually from eggs to nymphs and then decreased in adults. The Shannon and Simpson indices showed that the diversity of bacteria was highest in the eggs and was roughly reduced in adults, which was lower than in the other stages (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Fungal diversity was lowest in the eggs and gradually increased in the other stages (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Neither the bacterial nor fungal community richness and diversity differed significantly between male and female adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe compared bacterial and fungal community β-diversity in \u003cem\u003eD. farinae\u003c/em\u003e samples grouped by developmental stage according to principal coordinates analysis (PCoA). We detected a similar association between the bacterial and fungal communities. The samples in the same developmental stage had a similar structure and thus were clustered into a group. We identified an important factor in bacterial and fungal differences in the developmental stage. In both bacterial and fungal communities, the microbial community in larvae and nymphs was similar, as was the microbial composition of male and female adults. The egg samples were clustered into one group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComposition of Microbial Communities at Different Developmental Stages in D. farinae\u003c/h3\u003e\n\u003cp\u003eAccording to the OTU classification based on the Silve138.1 and Unite7 databases, we annotated the bacteria in all samples into 40 phyla, 111 classes, 222 orders, 364 families, and 616 genera, and the fungi were annotated into 11 phyla, 35 classes, 77 orders, 166 families, and 276 genera. At the phylum and genus taxonomic levels, we conducted in-depth analyses of bacterial and fungal communities with a relative abundance of more than 1.00% (Supplementary Tables\u0026nbsp;1 and 2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eProteobacteria\u003c/em\u003e was the dominant bacterial phylum at all stages (43.02\u0026ndash;87.84% in relative abundance), followed by \u003cem\u003eBacteroidota\u003c/em\u003e (10.91\u0026ndash;23.29%) and \u003cem\u003eFirmicutes\u003c/em\u003e (0.61\u0026ndash;14.10%). The dominant phylum of fungi was \u003cem\u003eAscomycota\u003c/em\u003e, which constituted more than 96.40% at all stages. Furthermore, at the level of bacterial genera, \u003cem\u003eVibrionimonas\u003c/em\u003e (15.46%), \u003cem\u003eStaphylococcus\u003c/em\u003e (10.89%), and \u003cem\u003eChitinophaga\u003c/em\u003e (5.36%) were dominant in eggs. In larvae and nymphs, \u003cem\u003eBordetella\u003c/em\u003e (9.79% and 9.96% relative abundance in larvae and nymphs, respectively), \u003cem\u003ePseudomonas\u003c/em\u003e (8.98% and 9.40%), \u003cem\u003eMethyloversatilis\u003c/em\u003e (8.97% and 8.36%), and \u003cem\u003eStenotrophomonas\u003c/em\u003e (6.65% and 6.70%) were abundant. An unclassified genus of \u003cem\u003eRhizobiaceae\u003c/em\u003e became the dominant genus in male and female adults (24.73% and 29.95% relative abundance in male and female adults, respectively), followed by \u003cem\u003eVibrionimonas\u003c/em\u003e (13.61% and 7.73%), \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (7.16% and 7.73%), \u003cem\u003eRalstonia\u003c/em\u003e (9.34% and 6.37%), and \u003cem\u003eRamlibacter\u003c/em\u003e (6.50% and 5.02%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). \u003cem\u003eAspergillus\u003c/em\u003e was the abundant genus of fungi at all stages of \u003cem\u003eD. farinae\u003c/em\u003e, accounting for 71.29%, 18.14%, 18.67%, 33.45%, and 32.35% in eggs, larvae, nymphs, male adults, and female adults, respectively. \u003cem\u003eXeromyces\u003c/em\u003e (19.60%) was abundant in eggs, and \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eSarocladium\u003c/em\u003e accounted for 18.80% and 36.74% in the samples at each developmental stage, except for eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Tables\u0026nbsp;3 and 4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSpecies Differences of D. farinae at Different Developmental Stages\u003c/h3\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, at the genus level, we identified 63 genera of bacteria throughout all developmental stages and 69 shared genera of bacteria in eggs, male adults, and female adults, which highly overlapped and may have been related to the vertical transmission of commensal bacteria. The 30 genera were shared by all stages in fungi, and the 33 genera shared by eggs, male adults, and female adults were also identical (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As shown in the heat map in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, we observed differences in the abundance of shared bacteria and fungi in different developmental stages; nonetheless, they were always present with \u003cem\u003eD. farinae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).We also observed specific bacteria and fungi at each developmental stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe conducted a linear discriminant analysis effect size (LEfSe) analysis to explore the differential microorganisms among the groups. In the egg stage, except for \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eChitinophaga\u003c/em\u003e, \u003cem\u003eXeromyces\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e as the dominant genera, we observed that \u003cem\u003eBradyrhizobium\u003c/em\u003e, \u003cem\u003eVariovorax\u003c/em\u003e, \u003cem\u003ePhyllobacterium\u003c/em\u003e, \u003cem\u003eGibberella\u003c/em\u003e, \u003cem\u003eWallemia\u003c/em\u003e, and \u003cem\u003eFusarium\u003c/em\u003e were significantly more abundant than in other stages (from phylum to genus, LDA\u0026thinsp;\u0026gt;\u0026thinsp;4.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the larvae, \u003cem\u003eMethyloversatilis\u003c/em\u003e, \u003cem\u003eBrucella\u003c/em\u003e, \u003cem\u003eAzospirillum\u003c/em\u003e, \u003cem\u003eBrevundimonas\u003c/em\u003e, \u003cem\u003eAllorhizobium-Neorhizobium-Pararhizobium-Rhizobium\u003c/em\u003e, \u003cem\u003ePseudoflavitalea\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, and \u003cem\u003eSarocladium\u003c/em\u003e were significantly abundant (from phylum to genus, LDA\u0026thinsp;\u0026gt;\u0026thinsp;4.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In nymphs, \u003cem\u003eBordetella\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e were the dominant genera, which was the same as in \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003ePyrenochaetopsis\u003c/em\u003e, and \u003cem\u003eTrichoderma\u003c/em\u003e, which were significantly more abundant than in other stages (from phylum to genus, LDA\u0026thinsp;\u0026gt;\u0026thinsp;4.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In adult males, \u003cem\u003eRalstonia\u003c/em\u003e, \u003cem\u003eMethylobacterium-Methylorubrum\u003c/em\u003e, \u003cem\u003eCurvibacter\u003c/em\u003e, \u003cem\u003eRamlibacter\u003c/em\u003e, and \u003cem\u003ePurpureocillium\u003c/em\u003e were significantly abundant (LDA\u0026thinsp;\u0026gt;\u0026thinsp;4.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In adult females, \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e was the only significantly abundant genus (from phylum to genus, LDA\u0026thinsp;\u0026gt;\u0026thinsp;4.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Supplementary Fig.\u0026nbsp;2). The significantly enriched bacteria and fungi in some of these stages could play a specific role in the development and growth of \u003cem\u003eD. farinae.\u003c/em\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation Analysis of Microbial Species in the D. farinae\u003c/h2\u003e \u003cp\u003eWe conducted a correlation analysis to reveal the relationships between bacterial and fungal genera during the development of \u003cem\u003eD. farinae\u003c/em\u003e. We focused on an analysis of 20 dominant bacterial genera and 15 dominant fungal genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The abundance correlations among bacteria\u0026ndash;bacteria, fungi\u0026ndash;fungi, and bacteria\u0026ndash;fungi during the growth of \u003cem\u003eD. farinae\u003c/em\u003e were complex, and the ratio of significantly positive and negative correlations was 79:40, 20:16, and 83:56, respectively. The number of positive correlations was greater than the number of negative correlations between bacteria and bacteria, bacteria and fungi, and fungi and fungi. The relationship between the dominant bacteria at the same developmental stage was mainly a positive correlation, like the larvae and nymph stages between \u003cem\u003ePseudomonas and Stenotrophomonas\u003c/em\u003e, \u003cem\u003eBordetella\u003c/em\u003e, and \u003cem\u003eMethyloversatilis\u003c/em\u003e. In adults, \u003cem\u003eRalstonia\u003c/em\u003e was positively connected with unclassified genera, including \u003cem\u003eRhizobiaceae\u003c/em\u003e, \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e, \u003cem\u003eMethyloversatilis\u003c/em\u003e, \u003cem\u003eRamlibacter\u003c/em\u003e, and \u003cem\u003eVibrionimonas\u003c/em\u003e. The negative correlations between bacteria and fungi were associated primarily with \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e, \u003cem\u003eOidiodendron\u003c/em\u003e, \u003cem\u003eand Wallemia.\u003c/em\u003e The negative correlations between fungi and fungi were primarily associated with \u003cem\u003eTalaromyces\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003eSarocladium\u003c/em\u003e, and \u003cem\u003ePurpureocillium.\u003c/em\u003e Correlation in microbial species may be a significant factor influencing the dynamics of bacterial and fungal communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Analysis of Microbial Communities at Different Developmental Stages of D. farinae\u003c/h2\u003e \u003cp\u003eWe used PICRUSt2 software to predict the relative abundance of microbiome-related Clusters of Orthologous Groups (COG)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functions in all samples and analyzed the results. According to both COG and KEGG databases, the abundance of various functions in these bacteria was greater than in fungi. The bacterial functional abundance was greater in adults than in larvae, nymphs, and eggs. We observed little difference in the functional abundance of fungi at each stage; larvae and nymphs had greater fungal functional abundance than eggs and adults. The COG functional classification revealed that bacteria had a higher abundance in amino acid transport and metabolism, carbohydrate transport and metabolism, cell membrane biogenesis, and energy production and conversion. Fungi had a higher abundance in translation, ribosomal structure, and biogenesis than in other pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The KEGG functions included metabolism, genetic information processing, environmental information processing, cellular processes, organism systems, and human diseases. The KEGG functional classification showed that during the development and growth of \u003cem\u003eD. farinae\u003c/em\u003e, some functions were enriched in both bacteria and fungi compared with other pathways, such as carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins. Fungi had a higher abundance in translation compared with other pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This result suggested that commensal bacteria may have played a lead role in \u003cem\u003eD. farinae\u003c/em\u003e, but the role of fungi should not be ignored. The role of fungi was relatively more stable than the role of bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSymbiotic microbes recently became a key focus of many studies because of the important roles they play in the growth, development, adaptability, and reproduction of hosts. Numerous studies have focused on \u003cem\u003eD. farinae\u003c/em\u003e, which is known to cause human allergic disease. The microbial communities of \u003cem\u003eD. farinae\u003c/em\u003e have been characterized using high-throughput sequencing techniques, which have largely enriched knowledge of the microbial composition in \u003cem\u003eD. farinae.\u003c/em\u003e Few studies, however, have examined the community characteristics and dynamics of symbiotic bacteria and fungi across the different developmental stages of \u003cem\u003eD. farinae\u003c/em\u003e, thus lacking exploration of function and species interaction. We used high-throughput sequencing of the 16S rRNA and ITS amplicon amplification to report on the bacterial and fungal communities in \u003cem\u003eD. farinae\u003c/em\u003e across different stages. We found that the microbial structure of \u003cem\u003eD. farinae\u003c/em\u003e was significantly influenced by the developmental stage, which was consistent with many previous studies on mites, such as \u003cem\u003ePanonychus citri\u003c/em\u003e, \u003cem\u003eDermanyssus gallinae\u003c/em\u003e, and \u003cem\u003eTetranychus truncatus\u003c/em\u003e [25\u0026ndash;27]. The composition of microbial communities in \u003cem\u003eD. farinae\u003c/em\u003e, however, was less different by sex, which was consistent with the results of previous research [22,28].\u003c/p\u003e \u003cp\u003eThe richness and diversity of bacteria and fungi during the growth and development of \u003cem\u003eD. farinae\u003c/em\u003e changed dynamically in different patterns. The diversity and richness of bacterial communities in eggs, larvae, and nymphs were significantly higher than those of male and female adults. The highest species richness of the fungal community was observed in nymphs. This was similar to the higher bacterial diversity and richness in the immature stage than in the mature stage, which was found in some insects such as psyllids, fruit flies, planthoppers, and ladybugs [29\u0026ndash;32]. Previous studies on insects have shown that the higher richness and diversity of microbes in the immature stage are related to the microbial richness and diversity from eggs or diet [30,32]. All shared bacteria in each stage came from those shared by eggs and adults (Supplementary Tables\u0026nbsp;5 and 6). The shared bacteria may have been due to vertical transmission and could be a factor that has caused the high diversity of the immature stage of \u003cem\u003eD. farinae\u003c/em\u003e. The dietary preference of \u003cem\u003eD. farinae\u003c/em\u003e for bacteria and fungi was also an important factor for the higher richness and diversity of bacteria and for the higher richness of fungi in the immature stage of \u003cem\u003eD. farinae\u003c/em\u003e [33]. Diet may be a source for some stage-specific species. In the immature stage, more bacteria and fungi may have promoted the digestion and absorption of substances in the underdeveloped gut. However, with the gradual improvement of intestinal structure and function in adults, because of the selectivity of the gut, partial exogenous species were discharged along with feces [34]. The molting process in the immature stage of \u003cem\u003eD. farinae\u003c/em\u003e may also lead to the loss of some species along with the exoskeleton [35\u0026ndash;37]. These reasons likely caused the decrease in richness and diversity in adults.\u003c/p\u003e \u003cp\u003eFurther analysis showed that the bacteria in \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages were distributed in 40 phyla, with \u003cem\u003eProteobacteria\u003c/em\u003e serving as the main phylum, possibly because of its involvement in host adaptation, digestion, nutrient supply, and energy metabolism [38,39]. The changes in the dominant bacterial genera may have better adapted to the needs of the different developmental stages of \u003cem\u003eD. farinae. Vibrionimonas\u003c/em\u003e was the most abundant bacterial genus in eggs, which probably came from the vertical transmission of adults. \u003cem\u003eStaphylococcus\u003c/em\u003e was significantly enriched in eggs, which decreased significantly in the next stages. The cell wall of \u003cem\u003eStaphylococcus\u003c/em\u003e is a suitable target for lysozyme-like enzymes in mites. The hydrolysis of \u003cem\u003eStaphylococcus\u003c/em\u003e cell walls can provide nutrients for mites [40\u0026ndash;42]. We speculate that it hydrolyzed to provide nutrients to eggs. \u003cem\u003eChitinophaga\u003c/em\u003e, which is a differential genus and is significantly enriched in eggs, likely degraded the eggshell to ensure the developmental process from eggs to larvae [43,44]. After becoming larvae, \u003cem\u003eD. farinae\u003c/em\u003e began to eat, and the laboratory feed was mainly composed of flour. \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e were abundant in the larvae and nymphs, especially in nymphs, and both demonstrated cellulose-degrading activity and were beneficial for food digestion [45\u0026ndash;48]. \u003cem\u003ePseudomonas\u003c/em\u003e contributed to promoting host lipid and vitamin metabolism, which may have facilitated the absorption and utilization of food [49,50]. The increase in \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e was conducive to the enhancement of the digestive ability of larvae and nymphs. The correlation between \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e was significantly positive, which probably was related to their similar functional roles. Adults had more functions, such as mating and reproduction, although the richness and diversity of bacteria were lower than in the immature stage. Bacteria could play more diverse and stronger functions in \u003cem\u003eD. farinae\u003c/em\u003e. \u003cem\u003eVibrionimonas\u003c/em\u003e, which is a Gram-negative bacterium that has been reported as a gut bacterium in Sirex noctilio (Hymenoptera: Siricidae) and Dactylispa xanthospila (Gestro) (Coleoptera: Chrysomelidae: Cassidinae) [51,52], was abundant in eggs and adults. Its function may be related to the utilization of nutrients as gut bacteria of mites. \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e not only fixed nitrogen, degraded carbohydrates, and provided essential amino acids and vitamin B in nutrition [53], but also enhanced host resistance in ecology, thus affecting the growth and reproduction of the host [54\u0026ndash;57]. \u003cem\u003eRalstonia\u003c/em\u003e, as symbiotic bacteria, existed in numerous insects and played an important role in the development of the host, which may have been beneficial for aerobic respiration [58,59]. \u003cem\u003eRalstonia\u003c/em\u003e also utilized a series of aromatic compounds as energy to detoxify, which suggested that it may be beneficial for \u003cem\u003eD. farinae\u003c/em\u003e to resist adverse external environments and enhance adaptability [60]. The correlation between \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e and \u003cem\u003eRalstonia\u003c/em\u003e was significantly positive. A previous study found that the cooccurrence of \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e and \u003cem\u003eRalstonia\u003c/em\u003e may be related to their carbon source utilization patterns and fatty acid methyl-ester production [61].\u003c/p\u003e \u003cp\u003eThe dominant fungal phylum of \u003cem\u003eD. farinae\u003c/em\u003e was \u003cem\u003eAscomycota\u003c/em\u003e, which also plays a dominant role throughout nature. Unlike the dynamic changes in bacteria, the structure of the fungal communities of \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages have been relatively stable. \u003cem\u003eAspergillus\u003c/em\u003e was the dominant fungus at all developmental stages, which was consistent with the dominant fungal genus of \u003cem\u003eD. farinae\u003c/em\u003e cultivation in Hubert\u0026rsquo;s study [21]. \u003cem\u003eXeromyces\u003c/em\u003e had high abundance in eggs. Other studies have shown that \u003cem\u003eXeromyces\u003c/em\u003e is closely related to cofactor and vitamin metabolism, energy metabolism, methane metabolism, carbohydrate metabolism, and glycosaminoglycan degradation [62]. Except for the eggs, both \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eSarocladium\u003c/em\u003e had high abundance in larvae, nymphs, and adults. \u003cem\u003ePenicillium\u003c/em\u003e, as a dominant fungal genus, could degrade cellulose that enhanced the host\u0026rsquo;s digestion and immune system, thus causing defense disorders and influencing its behavior and health [63]. The relatively stable fungal genera may have been instrumental at different developmental stages of \u003cem\u003eD. farinae\u003c/em\u003e and thus should be further studied.\u003c/p\u003e \u003cp\u003eThe COG and KEGG functional classification revealed that the abundance of various functions in these bacteria was greater than that of fungi, which was likely associated with the higher numbers of bacteria than fungi. Several reports have shown that \u003cem\u003eD. farinae\u003c/em\u003e had more abundant bacteria than fungi, and bacteria may play a leading role in assisting the growth and development of \u003cem\u003eD. farinae\u003c/em\u003e [20,21]. Male and female adults\u0026rsquo; functional abundance was greater than that of larvae, nymphs, and eggs in bacteria. However, we observed little difference in the functional abundance of fungi at each stage, and eggs, larvae, and adults had similar fungal compositions. The functional abundance of bacteria was positively correlated with the developmental time of \u003cem\u003eD. farinae\u003c/em\u003e. In bacteria, we found similarities in the developmental time and functional abundance of larvae and nymphs. Male and female adults had the longest developmental time, and the most enriched functions of bacteria were also found in adults [64].\u003c/p\u003e \u003cp\u003eThe abundance correlations between bacteria\u0026ndash;bacteria, fungi\u0026ndash;fungi, and bacteria\u0026ndash;fungi during the growth of \u003cem\u003eD. farinae\u003c/em\u003e were strong and complex. Notably, the number of positive correlations was greater than the number of negative correlations in microbial genera, which may have contributed to the synergistic function. For instance, \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e had similar functions in digestion. \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e and \u003cem\u003eRalstonia\u003c/em\u003e had similar synergistic functions in substance metabolism. Species with negative correlations also deserve further investigation. Previous studies on insects have shown that symbionts may compete with others within the host for limited space and resources, which would result in the exclusion of less competitive symbionts. Another hypothesis is that symbionts may negatively affect the density of several microbes, resulting in the absence of certain microbes [65,66]. The negative correlations were primarily associated with the fungal genera \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003eTalaromyces\u003c/em\u003e, \u003cem\u003eSarocladium\u003c/em\u003e, and \u003cem\u003ePurpureocillium. Aspergillus\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e produced a variety of antimicrobial secondary metabolites [67,68]. Multiple reports have shown that fungi influence the structure of the microbial community in mites. The decrease in bacterial and fungal species and abundance during the developmental stages of \u003cem\u003eD. farinae\u003c/em\u003e could be attributed to fungi and their metabolites. The impact of fungi on the entire microbial spectrum is equally significant. The complex abundance correlation in species may be one of the reasons for these dynamic bacterial and fungal communities.\u003c/p\u003e \u003cp\u003eWe also made a discovery regarding the symbiotic microbes of \u003cem\u003eD. farinae\u003c/em\u003e that may contribute to human diseases. \u003cem\u003eD. pteronyssinus\u003c/em\u003e at different developmental stages produced different levels of allergens, and nymphs produced more allergens[69]. We speculate that the nymphs of \u003cem\u003eD. farinae\u003c/em\u003e could also produce more allergens, which could easily lead to allergic diseases. Nymphs had more bacteria than other stages, including the dominant bacteria \u003cem\u003eBordetella\u003c/em\u003e, which produced allergens. The number of bacteria and related molecules was positively correlated with the inflammatory response [20,21]. In addition, \u003cem\u003eRalstonia\u003c/em\u003e produces lipopolysaccharides, which could bind to certain receptors involved in allergic reactions to induce asthma. The severity of asthma in nonatopic children is related to the concentration of total fungi. \u003cem\u003eAspergillus\u003c/em\u003e is the most common pathogen causing allergic bronchopulmonary aspergillosis, and the \u003cem\u003eWallemia\u003c/em\u003e genus can cause human health problems, including allergies and asthma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No.31870352).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZheWei Fan: Methodology, Software, Investigation, Formal analysis, Resources,Writing - Original Draft. Yujie Hong: Methodology, Investigation, Resources, Formal analysis, Writing - Original Draft. Shuya Zhou: Investigation, Resources, Visualization, Writing - Original Draft.Huijie Zhang\u0026amp; Mo Zhuo \u0026amp;Xinyan Yang\u0026amp; Yawen Yang \u0026amp; Min Ling \u0026amp; Ziyan Wang: Formal analysis, Resources, Visualization. Xianglin Tao \u0026amp; EntaoSun : Conceptualization, Methodology, Data curation, Writing - Review\u0026amp;Editing, Funding acquisition, Resources, Supervision, Validation, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHibberson, C. E. and Vogelnest, L. J. 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(2024) Microbial changes and associated metabolic responses modify host plant adaptation in Stephanitis nashi. Insect Science,10.1111/1744-7917.13340. \u003c/li\u003e\n\u003cli\u003eLarrondo, Veliz. J. and Calvo, M. (1990) The antimicrobial capacity of Penicillium and Aspergillus strains isolated from vineyard soils. Microb Lett ,44(174):77\u0026ndash;81\u003c/li\u003e\n\u003cli\u003eNicoletti, R., Andolfi, A., Becchimanzi, A. and Salvatore, M.M. (2023) Anti-Insect Properties of Penicillium Secondary Metabolites. Microorganisms,11(5):1302.\u003c/li\u003e\n\u003cli\u003eCalzada, D., Mart\u0026iacute;n-L\u0026oacute;pez, L. and Carn\u0026eacute;s, J.(2023) Growth, allergen profile and microbiome studies in Dermatophagoides pteronyssinus cultures. Sci Rep. 13(1):10633.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Tables and Figures","content":"\u003cp\u003eSupplementary tables and figures are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"amplicon sequencing, characteristics, Dermatophagoides farinae, diversity, dynamics, microbe","lastPublishedDoi":"10.21203/rs.3.rs-5860420/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5860420/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDermatophagoides farinae causes human allergic diseases and contains a large number of microbes. The structure of the microbial community is an essential prerequisite for understanding the intricate symbiotic relationships between microbes and hosts. The characteristics and dynamics of symbiotic microbes at different developmental stages of \u003cem\u003eD. farinae\u003c/em\u003e, however, are not well understood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed high-throughput amplicon sequencing to investigate microbial community in \u003cem\u003eD. farinae\u003c/em\u003e at different developmental stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that microbial communities were diverse and dynamic during \u003cem\u003eD. farinae\u003c/em\u003e development. Bacterial communities were generally richer than fungi in each developmental stage. The species richness and diversity of the bacterial community declined significantly from immature stages to adults. The highest species richness of the fungal community existed in nymphs. Eggs had the lowest fungal diversity. At 97% similarity, we assigned 40 phyla and 616 genera of bacteria and annotated 11 fungal phyla composed of 276 genera. The dominant bacterial and fungal phyla in all stages were \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eAscomycota\u003c/em\u003e, respectively. \u003cem\u003eStaphylococcus\u003c/em\u003e was more abundant in eggs than in other stages, \u003cem\u003eBordetella\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eStenotrophomonas\u003c/em\u003e were dominant in both larvae and nymphs, and \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e and \u003cem\u003eRalstonia\u003c/em\u003e were abundant in adults. \u003cem\u003eVibrionimonas\u003c/em\u003e was dominant in both eggs and adults. \u003cem\u003eAspergillus\u003c/em\u003e was the dominant fungal genus at all stages. \u003cem\u003eXeromyces\u003c/em\u003e was abundant in eggs, and \u003cem\u003ePenicillium\u003c/em\u003e and \u003cem\u003eSarocladium\u003c/em\u003e were abundant in other stages. Correlation analysis showed the existence of strong and complex correlations in the dominant microbial genera, and most of these correlations were positive. The functional analysis showed that microbes participate in various life activities in \u003cem\u003eD. farinae.\u003c/em\u003e Bacteria tend to have a higher functional abundance than fungi, such as substance metabolism. The functions of bacteria gradually enriched in adults. We observed similar fungal functional abundance in all stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ethis study has enriched our knowledge of the microbial communities associated with \u003cem\u003eD. farinae\u003c/em\u003e and has provided clues for discovering microbes that play important functions in \u003cem\u003eD. farinae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Characteristics and dynamics of microbial communities in Dermatophagoides farinae: Insights Across Developmental Stages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 05:33:27","doi":"10.21203/rs.3.rs-5860420/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0de67556-419c-4c63-8154-7015c73f7957","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-05T05:33:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-05 05:33:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5860420","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5860420","identity":"rs-5860420","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}