The Cardinium wins on Wolbachia in double-infected mite cultures
Authors: Tresnakova Eliska , Glowska Eliza , Hubert Jan 
Published: January 2024

Abstract
The different cultures of stored product mite Tyrophagus putrescentiae are single-infected by intracellular bacteria Cardinium or Wolbachia. No natural double-infected Cardinium/Wolbachia-infected mites are known. Under the experiment, single-infected mite (Wolbachia 5N, 5P and Cardinium 5L, 5S) cultures were mixed to double-infected cultures (5LP, 5LN, 5SP, 5SN). The mite fitness and symbionts' presence were analyzed during 5-month-long experiment. Cardinium, Wolbachia and mite genomes were assembled and gene expression in single and double-infected cultures was analyzed. In double-infected cultures, Cardinium infection increased with the time of the experiment from 50 to 95% of infected mites. Cardinium + Wolbachia-infected mite individuals proportion ranged from 0 to 20% of mites in double-infected cultures. Wolbachia infection disappeared in all double-infected cultures up to 5 months of the experiment duration. The double-infected cultures had lower fitness than single-infected cultures. After a month of experiment, the fitness of originally double-infected cultures increased to the level of parental cultures. The correlation analyses of gene expression showed that Wolbachia had well-established interactions with mite predicted KEGG gene expression in a single-infected population. The expression of mite protein was strongly influenced by the presence of Wolbachia, but not by Cardinium. The total numbers of Cardinium-expressed genes did not change, while there was a ten-fold decrease in Wolbachia in double-infected cultures. Cardinium and Wolbachia gene expression showed 30% negative and 70% positive (N = 3793) correlations. The number of correlations between Wolbachia and mite gene expression 5 times decreased in double-infected cultures. The Cardinium had a 6-fold higher number of genes than Wolbachia with significantly higher expression in the multiple infected samples. The gene expression analysis provides a suggestion that the presence of Cardinium inhibits the growth of Wolbachia by the disruption of the Wolbachia interaction with mite host. However, we cannot eliminate stochastic processes resulting in the increase of Wolbachia abundance and symbiont change. Importance We sought insight into the intracellular symbionts’ competition in the novel mite host model. The manipulative experiments established double-infected Wolbachia Cardinium cultures, which were unstable due to their low fitness. Cardinium prevailed during five months in all 4 double-infected cultures. The competition disrupted Wolbachia's interaction with its host on the level of gene expression. The genome expression is highly correlated between Wolbachia and mite hosts in single Wolbachia-infected cultures. These correlations disappeared in multi-infected cultures. Differently, the interaction among host and Cardinium genes showed low differences in the gene expression level. Although Cardinium/Wolbachia-infested individuals are rare, the gene expression of Cardinium and Wolbachia had a high number of positive correlations. It indicates that the symbionts reacted to each other. The data indicates that we have established a new model to study Wolbachia and Cardinium interactions.