Wolbachia and Spiroplasma are intracellular bacteria that are of great interest to entomologists,because of their ability to alter insect host biology in multiple ways.In the spider mite Tetranychus truncatus,co-infection of Wolbachia and Spiroplasma can induce cytoplasmic incompatibility (CI) and fitness costs;however,little is known about the effect of co-infection at the genetic level and the molecular mechanisms underlying CI.In this study,we explored the influence of the two symbionts on male mite host fitness and used RNA sequencing to generate the transcriptomes of T.truncatus with four different types of infection.In total,we found symbiont-infected lines had a higher hatch proportion than the uninfected line,and the development time of the uninfected line was longer than that of the other lines.Co-infection changed the expression of many genes related to digestion detoxification,reproduction,immunity and oxidation reduction.Our results indicate that co-infection of Wolbachia and Spiroplasma confers multiple effects on their hosts,and helps illuminate the complex interactions between endosymbionts and arthropods.