Complementary microscopic and metabarcoding (Illumina, Oxford Nanopore) studies allow for a better understanding of the microbiome pattern of leafhopper species Iassus lanio (Cicadellidae, Hemiptera)

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 bacteria and fungi microorganisms. In this study, we compared two metabarcoding techniques, supplemented by microscopic studies (confocal and electron microscopy), to thoroughly investigate the microbiome of the Auchenorrhyncha species Iassus lanio , a representative of the poorly studied leafhopper subfamily Iassinae. In particular, to describe the composition, distribution and ultrastructure of microorganisms and to investigate the phylogeny of its ancient symbionts. We found two obligate symbionts, 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. We detected the presence of Sodalis , Wolbachia , and Cardinium bacteria in the fat body. The obtained phylogeny of Karelsulcia bacteria indicates an affinity with the Deltocephalinae subfamily symbionts. Taxonomic profiling revealed that both methods detected the same range of taxa, while ONT exhibited improved resolution for dominant bacterial species. Karelsulcia and Cardinium differential abundance analysis highlighted platform-specific biases, with ONT overrepresenting Karelsulcia taxa while underrepresenting Cardinium . These studies highlight the complementary roles of different microscopy and metabarcoding techniques, demonstrating the complexity of bacterial and fungal 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.