UNLABELLED: The intracellular parasite Cardinium influences the bacterial microbiome composition of arthropod hosts; however, the mechanisms involved remain poorly understood. We sought to evaluate the interactions between Cardinium (cTPut) and SOL in Tyrophagus putrescentiae cultures based on relative abundance and gene expression data. First, we assembled the genome of Candidatus Krakonobacterium acarorum (formerly the Soliltalea-like symbiont SOL), a novel lineage of the Bacteroidota symbiont of mites. The assemblage SOL genome (1.2 Mb) contained complete pathways for the biosynthesis of lipoic acids, pantothenate, and menaquinone from futalosine. SOL is considered a facultative inhabitant (with prevalences ranging from 36% to 80% among individuals) of the gut (from 102 to 104 copies/mite) that is not detected in eggs, suggesting an extracellular location in the gut of mites. Second, gene expression was analyzed in SOL-inhabited cultures, including two cultures with cTPut and two cultures without cTPut. Correlation-based evidence for competition between cTPut and SOL was found mainly in the expression of transporter proteins. The presence of cTPut decreased interactions between SOL and the mite host; however, SOL is under greater control by mites in the presence of cTPut than in the absence of cTPut. Mite KEGG gene expression levels in the peroxisome, autophagy, sphingolipid, apoptosis, PI3K-Akt, and lysozyme pathways were more strongly correlated with SOL gene expression in cultures without cTPut than in those with cTPut. In contrast, mite KEGG gene expression levels in the proteasome, NF-κB, TNF, calcium, and Rap1 signaling pathways were more strongly correlated with SOL in the presence of cTPut. The explanation for these results is that cTPut mostly interacts with the mite host, resulting in changes in the host's immunity-related/regulatory pathways, indirectly affecting the symbiont SOL. IMPORTANCE: Here, we describe the novel Bacteroidetes symbiont (SOL) of mites. The analysis of gene expression in meta-transcriptomic samples from cultures with and without the intracellular parasite Cardinium revealed the effect of Cardinium on SOL as a model facultative symbiont of mites. Our findings suggest that there is competition between these two symbionts for nutrients. In addition, Cardinium can influence other bacterial symbionts via mite host immunity-related and regulatory pathways. Tyrophagus putrescentiae is a cosmopolitan pest mite that contaminates the home environment, including stored food and feed, with allergens. The interactions between intracellular bacteria and other members of the microbiome influence host physiology and indirectly affect allergen production.

• Keywords
• Bacteroidetes, Bacteroidota, Cardinium, gene expression, interaction, mite, symbionts,
• MeSH
• Acaridae * microbiology   MeSH
• Bacteroidetes * genetics   physiology   classification   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Mites * microbiology   MeSH
• Gene Expression Profiling MeSH
• Symbiosis * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

UNLABELLED: We investigated the tripartite interactions between two intracellular bacterial symbionts, Cardinium and Wolbachia in Tyrophagus putrescentiae. Cultures of Tyrophagus putrescentiae are typically single-infected by one intracellular symbiont. However, co-infection can be experimentally induced by mixing single-infected cultures, resulting in 10% of mite individuals being double-infected (Cardinium + Wolbachia) and a corresponding reduction in host fitness. Here, we assembled the genomes of Cardinium and Wolbachia and analyzed their gene expression in parental single-infected and mixed mite cultures using population-level samples (ranging from 7,500 to 10,000 mites). Wolbachia interacts more extensively with its mite host than Cardinium in single-infected cultures. However, in mixed cultures, (i) Wolbachia exhibited reduced regulation of the host compared with Cardinium; (ii) the gene expression profile of Cardinium shifted, increasing its interactions with the host, whereas the gene expression profile of Wolbachia remained unchanged; and (iii) Wolbachia genes exhibited a loss of interactions with mite gene expression, as indicated by reduced correlations (for example with host MAPK, endocytosis, and calcium signaling pathways). The experiments show that at the mite population level, symbiont infection disrupts gene expression interaction between the two symbionts and their host in different ways. Wolbachia was more influenced by Cardinium gene expression than vice versa. Cardinium can inhibit the growth of Wolbachia by disrupting its interaction with the host, leading to a loss of Wolbachia's influence on mite immune and regulatory pathways. The reasons for responses are due to co-infection or the reduced frequency of Wolbachia single-infected individuals due to the analyses of population-level samples. IMPORTANCE: We found that Cardinium disrupts the interaction between Wolbachia and mite host. In Wolbachia single-infected cultures, strong correlations exist between symbiont and host gene expressions. Interestingly, although Cardinium can also interact with the host, this interaction appears weaker compared with Wolbachia in single-infected cultures. These results suggest that both symbionts affect mite host gene expression, particularly in immune and regulatory pathways. In mixed samples, Cardinium appears to outcompete Wolbachia by disrupting its host interaction. It indicates competition between these two intracellular symbionts in mite populations. Wolbachia belongs to a mite-specific supergroup Q, distinct from the more commonly studied Wolbachia supergroups. As these mite-specific bacteria exhibit pathogen-blocking effects, our findings may have relevance for other systems, such as ticks and tick-borne diseases. The study sheds light on intracellular symbiont interaction within a novel mite-symbiont model.

• Keywords
• Cardinium, Wolbachia, gene expression, genome, interaction, mite,
• MeSH
• Bacteroidetes * physiology   genetics   MeSH
• Mites * microbiology   MeSH
• Symbiosis MeSH
• Wolbachia * genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

We examined host and bacterial gene expression profiles in the stored product mite Tyrophagus putrescentiae co-infected with Wolbachia (wTPut) and Cardinium (cTPut) while varying the presence of the Erwiniaceae symbiont (SLS). SLS, a novel symbiont in the family Erwiniaceae, with a genome size of 1.7 Mb, is found in 16% of mite species in infected cultures. In addition, SLS was detected in mite feces but not in their eggs. Although Wolbachia expression remained unchanged, the presence or absence of SLS significantly affected Cardinium expression. It indicated that the effect of Wolbachia on SLS was neutral. In SLS-positive samples, Cardinium exhibited 29 upregulated and 48 downregulated genes compared to SLS-negative samples. Furthermore, Cardinium gene expression strongly correlated with mite KEGG gene expression in SLS-positive samples. Positive Spearman's correlations between Cardinium gene expression and mite KEGG immune and regulatory pathways were doubled in SLS-positive compared to SLS-negative samples. The diversity of expressed genes in the mite host decreased in the presence of SLS. Cardinium had more interacting genes to mite host in SLS-positive samples than without SLS. Transposases are the most affected Cardinium genes, showing upregulation in the presence of SLS. Correlation analyses revealed interactions between Cardinium and SLS via mite immune and regulatory pathways, including lysosome, ubiquitin-mediated proteolysis, PIK3_Akt, and cGMP-PKG. The results showed that Cardinium indirectly affects the gut symbionts of mites.IMPORTANCEThis study introduces a new model to analyze interactions between intracellular bacterial symbionts, gut bacterial symbionts, and their mite hosts. Using gene expression correlations, we investigated how the intracellular Cardinium responds to the novel Erwiniaceae gut symbiont in the mold mite Tyrophagus putrescentiae. The data showed that both mite and Cardinium gene expression are different in the samples with and without Erwiniaceae symbionts. In the presence of Erwiniaceae symbionts, Cardinium increased the interaction with the mite host in terms of changes in gene expression. The mite immune and regulatory pathway gene expression is differently correlated to Cardinium genes in relation to Erwiniaceae symbionts. As a well-known producer of allergens, T. putrescentiae physiology and thus its allergen production are influenced by both symbionts, potentially affecting the release of allergens into human environments.

• Keywords
• Cardinium, Erwiniaceae, Sodalis, Tyrophagus putrescentiae, Wolbachia, allergens, bacterial symbionts, gene expression, stored product mite,
• MeSH
• Acaridae * microbiology   MeSH
• Bacteroidetes * genetics   physiology   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Mites * microbiology   MeSH
• Gastrointestinal Microbiome * MeSH
• Symbiosis * MeSH
• Wolbachia genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH