Dynamic Acquisition and Loss of Dual-Obligate Symbionts in the Plant-Sap-Feeding Adelgidae (Hemiptera: Sternorrhyncha: Aphidoidea)

Abstract

Sap-sucking insects typically engage in obligate relationships with symbiotic bacteria that play nutritional roles in synthesizing nutrients unavailable or in scarce supply from the plant-sap diets of their hosts. Adelgids are sap-sucking insects with complex life cycles that involve alternation between conifer tree species. While all adelgid species feed on spruce during the sexual phase of their life cycle, each adelgid species belongs to a major lineage that feeds on a distinct genus of conifers as their alternate host. Previous work on adelgid symbionts had discovered pairs of symbionts within each host species, and unusual diversity across the insect family, but left several open questions regarding the status of bacterial associates. Here, we explored the consistency of symbionts within and across adelgid lineages, and sought evidence for facultative vs. obligate symbiont status. Representative species were surveyed for symbionts using 16S ribosomal DNA gene sequencing, confirming that different symbiont pairs were consistently present within each major adelgid lineage. Several approaches were used to establish whether symbionts exhibited characteristics of long-term, obligate mutualists. Patterns of symbiont presence across adelgid species and diversification with host insects suggested obligate relationships. Fluorescent in situ hybridization and electron microscopy localized symbionts to bacteriocyte cells within the bacteriome of each species (with one previously known exception), and detection of symbionts in eggs indicated their vertical transmission. Common characteristics of long-term obligate symbionts, such as nucleotide compositional bias and pleomorphic symbiont cell shape were also observed. Superimposing microbial symbionts on the adelgid phylogeny revealed a dynamic pattern of symbiont gains and losses over a relatively short period of time compared to other symbionts associated with sap-sucking insects, with each adelgid species possessing an older, “senior” symbiont and a younger “junior” symbiont. A hypothesis relating adelgid life cycles to relaxed constraints on symbionts is proposed, with the degradation of senior symbionts and repeated acquisition of more junior symbionts creating opportunities for repeated colonization of new alternate-conifer hosts by adelgids.

Host relatedness influences the composition of aphid microbiomes

“…The bacterial phylogeny is pruned to include only lineages that cluster with known or putative genera of facultative symbionts (see Supporting Information Figs. S1 and S3), although we note that Profftia and Gilettelia in adelgids likely represent co‐obligate associations (von Dohlen et al ., ). Similarly, the aphid genus Cinara has an apparently dynamic system of co‐obligate associations, with many species hosting Serratia as a co‐obligate symbiont (Lamelas et al ., ), but with some examples in which other bacterial species including Sodalis and Erwinia play the co‐obligate role (Meseguer et al ., ).…”

Evolutionary loss and replacement of Buchnera, the obligate endosymbiont of aphids

“…The secondary symbiont, Serratia symbiotica, can replace Buchnera within A. pisum following experimental elimination of Buchnera; S. symbiotica colonizes the cytoplasm of bacteriocytes where it can fulfill the essential role of Buchnera [57]. Likewise, multiple symbiont types can colonize bacteriocytes of some adelgids [58]. But, novel co-obligate symbionts are sometimes restricted to novel types of bacteriocytes distinct from those harboring the more ancient partners [10,59].…”

“…In contrast to these cases, the symbiont found in Geopemphigus aphids represents a case in which a new functional symbiont became established without persistence of the ancient partner. In adelgids, which are relatives of aphids feeding on sap of conifers, each host species contains two symbiont types, but a phylogenetic analysis shows a succession, in which a more ancient symbiont is replaced by a more recent one, which in turn is replaced by yet a more recent symbiont [10,58,61,62]. Symbiont succession shows a somewhat different pattern in the sap-feeding Auchenorrhyncha (which includes cicadas, spittlebugs, leafhoppers, treehoppers, and planthoppers).…”

Evolutionary recent dual obligatory symbiosis among adelgids indicates a transition between fungus and insect associated lifestyles

“…Interestingly, two types of bacteriocyte-associated symbionts have been identified in all populations and life stages of most adelgid species (9)(10)(11)(12)(13)(14). These symbionts belong to at least six different lineages within the Gammaproteobacteria or the Betaproteobacteria.…”

“…However, the Vallotia -Profftia system differs from the Annandia -Pseudomonas system in A.tsugae where functions of the symbionts in essential amino acid synthesis are more balanced and redundant. It has been suggested that repeated replacement of symbionts among adelgids might be a consequence of periods with relaxed selection on symbiont functions due to different feeding behavior of adelgids on primary and secondary host trees -that is feeding on nutrient-rich parenchyma cells on spruce versus nutrient-poor phloem sap on alternate hosts -and multiple origins of hostalternating lifestyles(10). Annandia, the ancient symbiont of adelgids has lost many functions in essential amino acid biosynthesis, which could support this hypothesis(25), however the Vallotia -Profftia system does not follow this pattern.One of the most remarkable findings of our study is the evolutionary link between the betaproteobacterial insect symbiont, Vallotia, and endosymbionts of Rhizopus fungi supported by their close phylogenetic relationships and a high-level of genomic synteny.…”