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.

Keywords: bacterial symbionts; complex life cycles; dual symbionts; host alternation; insects; symbiont replacements.

FIGURE 1

Cospeciation of ‘Ca. Pseudomonas adelgestsugas’ symbionts and Adelges tsugae hosts from five genetically divergent populations [genetic divergence among A. tsugae suggests some populations may be distinct species (Havill et al., 2006, 2016)]. Phylogeny of ‘Ca. Pseudomonas adelgestsugas’ symbionts (left) was estimated from 16S rRNA sequences. A. tsugae phylogeny (right) was simplified from Havill et al. (2006) and Havill et al. (2016), in which all population-level nodes were significantly supported. Sample from Taiwan is new to this study. WNA, western North America.

FIGURE 2

Localization and ultrastructure of bacteriocyte-associated endosymbionts in A. cooleyi. (A) General detection of symbionts in paired bacteriomes of second-instar stage from spruce galls (transverse section) by fluorescence in situ hybridization (FISH), using general eubacterial probe 1507r labeled with Alexa-568-5-dUTP (red). Inset: general detection of symbionts, as above, in an egg. (B) Detection of ‘Ca. Vallotia cooleyia’ in second-instar stage from spruce galls (transverse section) by FISH, using the beta probe mix of b187, b442, and b1025 labeled with Alexa-568-5-dUTP (red). (C) Simultaneous detection of both symbionts in second-instar stage from spruce galls (transverse section) by FISH, using the beta mix (as above) labeled with Bodipy-650-14-dUTP for ‘Ca. Vallotia cooleyia’ (blue), and gamma probe mix of G-69, G-439, and G-1128 labeled with Alexa-568-5-dUTP for ‘Ca. Gillettellia cooleyia’ (red). (D) TEM micrograph of bacteriocyte containing two bacterial morphotypes. Green, autofluorescence of insect cuticle; n, bacteriocyte nucleus.

FIGURE 3

Localization of bacteriocyte-associated endosymbionts in A. laricis generations from larch by FISH. (A) Detection of ‘Ca. Vallotia tarda’ in first-instar stage from larch generations, using specific probe b1027 labeled with Alexa-568-5-dUTP (red). (B) Detection of ‘Ca. Profftia tarda’ in first-instar stage from larch generations, using specific probe g1023 labeled with Alexa-568-5-dUTP (red). (C) Detection of both symbionts in paired bacteriomes of first-instar stage from larch generations (transverse section), using beta mix Al-b70 + Al-b152 + Al-b1256 labeled with Alexa-568-5-dUTP for ‘Ca. Vallotia tarda’ (red), and gamma mix Al-g1023 + Al-g1128 labeled with Bodipy-650-14-dUTP for ‘Ca. Profftia tarda’ (blue). (D) Adult generation with eggs, probed with general eubacterial probe 1507r labeled with Alexa-568-5-dUTP (red); individual bacteriocytes surround the egg, which contains a cluster of transmitted symbionts at the posterior pole (left). Inset: ‘Ca. Vallotia tarda’ clustered at the posterior pole of an egg, detected with specific probe b1027 labeled with Alexa-568-5-dUTP (red). Green, autofluorescence from insect cuticle; e, egg.

FIGURE 4

Localization of bacteriocyte-associated endosymbionts in P. similis by FISH. (A) General detection of both symbionts of second-instar stage (frontal section) using eubacterial probe 1507r labeled with Alexa-568-5-dUTP (red). Central bacteriocytes containing ‘Ca. Hartigia pinicola’ fluoresce more brightly with this probe. (B) Detection of ‘Ca. Hartigia pinicola’ symbionts within central bacteriocytes of a second-instar stage using a mix of PinGam2-470 and PinGam2-828 labeled with Alexa-568-5-dUTP (red). (C) Simultaneous detection of ‘Ca. Annandia pinicola’ using GamC-440 labeled with Bodipy-650-14-dUTP (blue), and ‘Ca. Hartigia pinicola’ using PinGam2-470 and PinGam2-828 labeled with Alexa-568-5-dUTP (red). (D) General detection of both symbionts in an egg using eubacterial probe 1507r labeled with Alexa-568-5-dUTP (red). A cluster of symbionts is located at the posterior pole (upper right); other symbionts from the disintegrated bacteriome are clustered on the lower left. Green, autofluorescence of insect cuticle; n, bacteriocyte nucleus.

FIGURE 5

Ultrastructure of Pineus pini endosymbionts residing in bacteriocytes. (A) Ultrathin section of bacteriome in whole-mount insects, showing two distinct bacteriocytes containing ‘Ca. Annandia pinicola’ (left) and ‘Ca. Hartigia pinicola’ (right). (B) High magnification of ‘Ca. Annandia pinicola’ cell envelope, comprising three membrane layers, presumably corresponding to inner and outer membranes and a symbiosome membrane. (C) High magnification of ‘Ca. Hartigia pinicola’ cell envelope, comprising three membrane layers, presumably corresponding to inner and outer membranes and a symbiosome membrane; no peptidoglycan layer is apparent. Ap, ‘Ca. Annandia pinicola’; Hp, ‘Ca. Hartigia pinicola’; n, bacteriocyte nucleus.

FIGURE 6

Hypothesis of symbiont acquisitions and replacements during Adelgidae evolutionary history (dated phylogram based on Havill et al., 2007). This scenario posits ‘Ca. Annandia’ as the original, ancestral “senior” symbiont. ‘Ca. Annandia’ was joined by the “junior” symbiont ‘Ca. Hartigia’ in the Pineus lineage (feeding on alternate-host pine), and by the junior symbiont ‘Ca. Pseudomonas’ in the A. tsugae species complex (on alternate-host hemlock). ‘Ca. Annandia’ was replaced by the Serratia-type ancestor of ‘Ca. Ecksteinia’ and ‘Ca. Gillettellia’ (these symbionts are sister taxa within the gammaproteobacterial Serratia lineage) to become the new senior symbiont in the fir + Douglas-fir + larch lineage. ‘Ca. Ecksteinia’ was joined by junior symbiont ‘Ca. Steffania’ in the fir lineage, and ‘Ca. Gillettellia’ was joined by junior symbiont ‘Ca. Vallotia’ in the ancestor of the Douglas-fir + larch lineage. In the ancestor of the larch lineage, the Serratia-type symbiont was lost; ‘Ca. Vallotia’ remained to become the new senior symbiont, and was joined by junior symbiont ‘Ca. Profftia.’