Diversity and genomics of bacteriome-associated symbionts in treehopper <i>Darthula hardwickii</i> (Hemiptera: Aetalionidae) and implications of their nutritional functions

Symbionts play important roles in insect nutritional ecology, and the phylogenies of some vertically transmitted symbionts mirror the host phylogeny. Here we report the diversity, distribution, transmission, and potential functions of symbionts harbored in the aetalionid treehopper <i>Darthula hardwickii</i> (Aetalionidae) using multiple methods and compare the potential functions of its obligate symbiont <i>Karelsulcia</i> with that of the related aetalionid <i>Aetalion reticulatum. D. hardwickii</i> harbors <i>Karelsulcia</i> in bacteriomes, a yeast-like fungal symbiont (YLS) in fat bodies, and <i>Tisiphia</i> in both the bacteriomes and fat bodies. <i>Karelsulcia</i> and YLS are vertically transmitted to the ovaries but do not cluster to form a "symbiont ball" in terminal oocytes, as is the case in other auchenorrhynchan insects. YLS harbored in <i>D. hardwickii</i> represents the first known instance of a fungal symbiont being associated with treehoppers. Phylogenetic analysis revealed that Aetalionidae are derived from within Membracidae. Gene truncation and absence were revealed in the tryptophan biosynthetic pathway of <i>Karelsulcia</i> from <i>D. hardwickii</i>, suggesting this symbiont is no longer capable of providing this essential amino acid (EAA) to its host. Tryptophan is presumed to be supplied to <i>D. hardwickii</i> by YLS since tryptophan-related genes are either absent or degraded in <i>Karelsulcia</i> and <i>Tisiphia</i>. No truncated genes were found in <i>Karelsulcia</i> from <i>A. reticulatum</i>, but it has lost genes related to the synthesis of other EAAs, as in some leafhoppers. This study sheds new light on the diversity and functions of the nutritional endosymbionts of Membracoidea and processes that may have precipitated symbiont replacement in this diverse insect lineage.IMPORTANCESymbionts in sap-feeding insects play important roles related to nutrition of their hosts, which may change through evolutionary time and vary across host and symbiont lineages. This comparative genomic study indicates that, compared to the related symbionts of other leaf- and treehoppers, the <i>Karelsulcia</i> symbiont of the treehopper <i>Darthula hardwickii</i> has lost the ability to provide the EAA tryptophan to its host. This function is apparently being performed by a coexisting yeast-like symbiont (YLS). This is the first report of a YLS in a species of treehopper, which suggests that the processes involved in symbiont replacement in treehoppers are similar to those observed in other sap-sucking auchenorrhynchan insects. Phylogenetic analyses of <i>Karelsulcia</i> lineages of Membracoidea largely mirror the host insect phylogeny but suggest that Aetalionidae may have originated from Membracidae, in contrast to some recent phylogenies based on the genomic data from the host insects.