Complementary Microscopic and Metabarcoding Studies Allow for a Better Understanding of the Symbiotic Microbiome of Leafhopper Species Iassus lanio (Hemiptera, Cicadellidae).

Leafhoppers' microbiome patterns were shaped by deep co-evolutionary adaptation driven by dietary specialization. Their microbiome is dominated by obligate symbionts that supplement their nutrient-poor phloem-sap diet, as well as facultative symbionts, including both bacterial and fungal microorganisms. In this study, NGS metabarcoding techniques were performed, supplemented by confocal and electron microscopy, to thoroughly investigate the symbiotic system of the Auchenorrhyncha species Iassus lanio, a representative of the poorly studied leafhopper subfamily Iassinae. The obtained results include descriptions of the composition, distribution, and ultrastructure of microorganisms, as well as the phylogeny of ancient symbionts. Two obligate symbionts were found: the ancient Auchenorrhyncha symbiont Karelsulcia bacterium and the yeast-like symbiont Ophiocordyceps. Karelsulcia bacteria occur exclusively in specialized organs called bacteriomes, while fungal microorganisms inhabit mycetocytes within the fat body. Both symbionts are transmitted transovarially from mother to offspring. The presence of Wolbachia, Sodalis and Cardinium was detected. Sodalis and Cardinium were observed in the fat body. The ultrastructure of Cardinium showed a characteristic microtubule crest inside. The obtained phylogeny of Karelsulcia bacteria indicates Iassinae affinity with the Coelidiinae and Deltocephalinae subfamily symbionts. Taxonomic profiling revealed that both sequencing methods detected the same range of bacterial taxa, while ONT exhibited improved resolution for dominant species. Differential abundance analysis emphasized platform-specific biases. These studies highlight the complementary roles of different microscopy and metabarcoding techniques, demonstrating the complexity of symbiotic systems in leafhoppers and thereby improving our understanding of the host-symbiont relationship and expanding our knowledge of the structure and localization of insect microorganisms.