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. 2022 Nov 2;25(12):105478.
doi: 10.1016/j.isci.2022.105478. eCollection 2022 Dec 22.

Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica

Shunta Yorimoto  1   2 , Mitsuru Hattori  3 , Maki Kondo  4 , Shuji Shigenobu  1   2
Affiliations

Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica

Shunta Yorimoto et al. iScience. .

Abstract

Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera-Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.

Keywords: Evolutionary biology; Genetics; Molecular biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Symbiont composition determined by deep sequencing of 16S rRNA amplicons of natural populations of Ceratovacuna japonica (A) A colony of Ce. japonica on the bamboo grass P. chino. Red arrowheads indicate soldiers. (B) Locations and host plant species of collected Ce. japonica. (C) Relative abundance of bacterial symbionts in Ce. japonica. The V1–V2 region amplified from sample 16 was excluded owing to sequencing failure. “Others” includes unclassified sequences or those with low abundance. (D) Summary of diagnostic PCR detection of Buchnera, Arsenophonus, and Hamiltonella in Ce. japonica. See also Figure S1 for agarose gel electrophoresis data. Each bacterial symbiont was detected using specific primers targeting a single-copy gene, dnaK. Each sample ID corresponds to those in Figures 1B, 1C, 1D, and S1. Detected bacterial symbionts are shown by the “+” character.
Figure 2
Figure 2
Phylogenetic analysis of Buchnera and Arsenophonus (A) Maximum likelihood (ML) tree of Buchnera aphidicola based on the length of 1,337 bp of 16S rRNA sequences. Escherichia coli and Ishikawaella capsulata were used as outgroups. The labels indicate the name of the host species. (B) ML tree of Arsenophonus based on concatenated 204 single-copy orthologous protein sequences composed of 54,777 amino acid positions. E. coli and Proteus mirabilis were used as outgroups. Target symbionts in this study are highlighted in red on each tree. Bootstrap values no less than 70% are indicated on each node. Scale bars represent 0.02 and 0.1 substitutions per site.
Figure 3
Figure 3
Localization and morphology of Buchnera and Arsenophonus symbionts (A and B) Light microscopic images of tissue sections stained with hematoxylin and eosin. The dorsal sections of an adult individual are shown. The whole body (A) and the magnified image of the bacteriome (B). (C) Light microscopic image of dissected bacteriomes. (D) Confocal image of the bacteriome structure. Nuclei (red) and F-actin (green) were stained by DAPI and phalloidin. (E–H) Confocal images of Buchnera, Arsenophonus, and Hamiltonella stained with fluorescent probes specific to each bacterium. Whole bodies of adult individuals (E and F) and dissected bacteriomes (G and H). In (E and G), gray (DAPI), green (Cy5), and magenta (Cy3) signals indicate nuclei, Buchnera, and Arsenophonus, respectively. In (F and H), gray (DAPI), green (Cy5), and magenta (Cy3) signals indicate nuclei, Hamiltonella, and Arsenophonus, respectively. Cyan arrowheads indicate Hamiltonella in (F and H). (I–O) Electron microscopy of a bacteriome dissected from an adult individual. (I) Low-magnification image of three types of symbionts in the bacteriome. Red and blue arrowheads indicate Arsenophonus and Hamiltonella in the space between bacteriocytes, respectively. (J and K) Cytoplasm of a bacteriocyte harboring Buchnera (J) and Arsenophonus (K). Cyan arrowheads indicate mitochondria. (L) Boundary of bacteriocytes harboring Buchnera and bacteriocytes harboring Arsenophonus. Black arrowheads indicate membranes of bacteriocytes. (M−O) High magnification images of membranes of Buchnera (M), Arsenophonus (N), and Hamiltonella (O) In (M−O), black, yellow, and white arrowheads indicate the symbiosomal membrane, outer membrane, and inner membrane, respectively. (P) Schematic diagram of the bacteriome morphology. Scale bars show 200 μm in (A and C), 100 μm in (E and F), 50 μm in (B, D, and G), 20 μm in (H), 2 μm in (I), 1 μm in (J and K), and 100 nm in (L–O). a, Arsenophonus; b, Buchnera; bc, bacteriocyte; bo, bacteriome; emb, embryo; h, Hamiltonella; n, host nucleus; sc, sheath cell; t, trachea.
Figure 4
Figure 4
Genomic features of Buchnera CJ and Arsenophonus CJ (A) Circular Buchnera CJ genome. (B) Circular Arsenophonus CJ genome. (C) Linear Hamiltonella CJ genome. Outer to innermost rings correspond to (i) genome coordinates in kilobases; (ii) predicted protein-coding genes on the plus strand (red); (iii) predicted protein-coding genes on the minus strand (blue); (iv) transfer RNAs (green); (v) ribosomal RNAs (purple); (vi) pseudogenes (black) in (A–C). (D) Gene orders of plasmids of Buchnera CJ pLeu and pTrp. Arrows indicate the direction of transcription. White arrows indicate pseudogenes. (E) COG classification of protein-coding genes of Buchnera and Arsenophonus. (F) Comparison of gene repertoires responsible for nutrient synthesis by Buchnera and Arsenophonus. E. coli is shown as an example of a free-living bacterium. Color blocks indicate the completeness of the minimal gene set for metabolic pathways: red, orange, yellow, light blue, and blue mean 0–24%, 25–49%, 50–74%, 75%–99%, and 100% of the completeness, respectively. In the aphid-Buchnera symbiosis, a metabolic collaboration is known for amino acid syntheses, where the host complements the steps missing from Buchnera as observed in the pathways of leucine, isoleucine, valine, and methionine syntheses (Shigenobu and Wilson, 2011; The International Aphid Genomics Consortium, 2010).
Figure 5
Figure 5
Infection and developmental integration of Buchnera and Arsenophonus symbionts during host embryogenesis (A) Embryo during anatrepsis. Neither Buchnera nor Arsenophonus are observed at this stage. (B) S-shape embryo. Both Buchnera and Arsenophonus begin to infect the embryo from the posterior part. (C) Twisting embryo. Infection with both Buchnera and Arsenophonus is continuing. New bacteriocytes harboring Buchnera are formed. (D and E) Only Cy5 (D) and Cy3 (E) signals in C are shown. (F) Limb bud formation. Limb buds are formed in the thorax region and the germband is elongating. Symbiont transmission has finished at this stage. (G and H) Germband retraction is completed after katatrepsis. G and H show the lateral view and dorsal view of the same individual. The germband is retracted to the posterior tip. One huge bacteriome exists in the abdomen. (I) An embryo prior to larviposition. The bacteriome is divided and forms a pair of bacteriomes. (A–I) Blue (DAPI), green (Cy5), and red (Cy3) indicate nuclei, Buchnera, and Arsenophonus, respectively. (A′–I′) Schematic diagram of images in (A–I). Cyan, green, and magenta indicate nuclei of bacteriocytes, Buchnera, and Arsenophonus, respectively. (A–G): Lateral view, (H and I): Dorsal view. Scale bar: 20 μm for (D and E); 50 μm for (A–C and F–H); 100 μm for I. gb, germband; hd, head.
Figure 6
Figure 6
Comparison of symbiosis status between soldier and reproductive castes (A and B) Light microscopic images of first-instar nymphs of reproductive (A) and soldier (B) castes. (C and D) Comparison of symbiont titers of first instar nymphs between castes. (C) Buchnera and (D) Arsenophonus titers. Buchnera and Arsenophonus were measured by qPCR using the bacterial symbiont dnaK gene standardized by the host RpL7 gene. Asterisks indicate statistically significant differences (Welch’s t-test p< 0.001 in A, p< 0.05 in B). (E and F) Confocal images of the localization of both symbionts in the first instar nymphs of reproductive (E) and soldier (F) castes. Blue (DAPI), green (Cy5), and red (Cy3) indicate nuclei, Buchnera, and Arsenophonus, respectively. Scale bars indicate 500 μm in (A and B) and 100 μm in (E and F). hd, head.
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