Small, Smaller, Smallest: The Origins and Evolution of Ancient Dual Symbioses in a Phloem-Feeding Insect

Abstract

Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients lacking in host diets. Xylem sap and phloem sap are both deficient as insect diets, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola, of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia-ALF has the smallest bacterial genome yet sequenced (112 kb), and that the Sulcia-ALF genome (190 kb) is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 essential amino acids, as observed for several symbiont pairs from xylem-feeding Auchenorrhyncha. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a consequence of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to tryptophan, and phylogenetic results suggest that Nasuia-ALF is most closely related to Zinderia, the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia/Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of leafhoppers and spittlebugs >200 Ma, and possibly since the origin of the Auchenorrhyncha, >260 Ma.

Swapping symbionts in spittlebugs: evolutionary replacement of a reduced genome symbiont

“…Sulcia was retained in a large proportion of descendant lineages, whereas the betaproteobacterial symbiont was more often lost and replaced by new symbionts, including Hodgkinia cicadicola (Alphaproteobacteria) in cicadas (Cicadoidea), Baumannia cicadellinicola (Gammaproteobacteria) in sharpshooters (Cicadellidae: Cicadellinae) and SLs in P. spumarius and close relatives. Where it has been retained, in some planthoppers (Fulgoridae), leafhoppers (Cicadellidae: Deltocephalinae), and spittlebugs (Urban and Cryan, 2012;Bennett and Moran, 2013;Koga et al, 2013), the betaproteobacterial symbiont appears to have a highly degenerate genome. For example, the Zinderia-CARI genome contains only 202 protein-coding genes, exhibits extreme divergence of protein and rRNA sequences, the most extreme AT nucleotide bias (13.5% G þ C) yet reported, and an altered genetic code in which TGA encodes tryptophan rather than Stop (McCutcheon et al, 2009).…”

“…Recently the betaproteobacterial symbiont of deltocephaline leafhoppers, Nasuia deltocephalinicola, was shown to have the smallest genome (112 kb) sequenced to date with the same altered genetic code . Based on molecular phylogenies, Nasuia and Zinderia belong to the same symbiont clade, and they are proposed to have descended from an ancestral symbiont infecting a shared ancestor of leafhoppers (Cicadellidae) and spittlebugs (Cercopoidea) (Urban and Cryan, 2012;Bennett and Moran, 2013;Koga et al, 2013). Both lack many genes for energy production , supporting elimination of these genes before the divergence of Nasuia and Zinderia, and before the acquisition of SLs by an ancestor of Philaenini (Figure 4).…”

“…The small population sizes and asexuality of maternally transmitted symbionts result in the accumulation of mildly deleterious mutations, each of small effect but cumulatively resulting in lowered functionality and fitness (Moran, 1996). Among the most extreme examples of deteriorating genomes are those found in insect symbionts (Moran, 2007;Moran, 2010, 2012;Bennett and Moran, 2013). Up to 90% of ancestral genes and associated metabolic capabilities may be lost, and retained genes show extensive sequence changes that reflect fixation of mutations deleterious to gene function.…”

“…For example, symbiont gene products exhibit thermal instability (Lambert and Moran, 1998;van Ham et al, 2003;Baumann, 2005;Moran, 2007), and the resulting heat sensitivity of symbionts can affect hosts, in which reproduction fails when symbionts are eliminated by heat (Moran, 1996;Dunbar et al, 2007). Furthermore, obligate symbiont lineages cannot regain lost functions as they do not incorporate foreign genes through the lateral gene transfer processes that are frequent in most bacteria (Tamas et al, 2002;Moran, 2007;Moran et al, 2008;Moran, 2010, 2012;Bennett and Moran, 2013).…”

“…Most sap-feeding insects of suborder Auchenorrhyncha host two symbionts, Sulcia muelleri (Bacteroidetes) and a partner that varies among host groups (Moran, 2007;McCutcheon and Moran, 2010;Bennett and Moran, 2013). In each case, the paired symbionts show strikingly complementary sets of genes related to production of essential amino acids (the 10 protein amino acids that are required nutrients for animals).…”