Intracellular Localization of the Bacterial Endosymbiont Cardinium in the Ostracod Heterocypris spadix

Symbiosis is a key driver of evolution in life-history traits and reproductive strategies. Some symbiotic microorganisms manipulate host reproduction to enhance their own transmission, a phenomenon well studied in insects but less understood in crustaceans. Among these microorganisms, Cardinium manipulates host reproductive systems, such as parthenogenesis, cytoplasmic incompatibility, and male killing in arthropods. However, its role in ostracods, small bivalve-shelled crustaceans, remains unclear. Some ostracod species reproduce via parthenogenesis, and high Cardinium infection rates in these lineages suggest a potential link between the symbiont and asexual reproduction. To investigate this relationship, we examined Cardinium localization in the parthenogenetic ostracod Heterocypris spadix from Japan. Using tissue clearing and fluorescence in situ hybridization (FISH), we visualized Cardinium within the ovaries. FISH observations revealed a widespread infection across the germarium, nurse cells, and oocytes. In early-stage oocytes, bacteria were evenly dispersed throughout the cytoplasm, whereas in more-developed oocytes, they clustered around the nucleus. Additionally, Cardinium was also detected in the hepatopancreas, indicating infection of both the reproductive and digestive systems. The presence of Cardinium in host reproductive structures, particularly the germarium, nurse cells, and developing oocytes, suggests its role in reproductive manipulation. To our knowledge, this study provides the first detailed localization of Cardinium in ostracods, reinforcing its potential influence on reproduction. Future research using antibiotics and genomic analysis will be crucial to confirm Cardinium's role in parthenogenesis induction.