Bacterial symbionts support larval sap feeding and adult folivory in (semi-)aquatic reed beetles

Abstract

Symbiotic microbes can enable their host to access untapped nutritional resources but may also constrain niche space by promoting specialization. Here, we reconstruct functional changes in the evolutionary history of the symbiosis between a group of (semi-)aquatic herbivorous insects and mutualistic bacteria. Sequencing the symbiont genomes across 26 species of reed beetles (Chrysomelidae, Donaciinae) spanning four genera indicates that the genome-eroded mutualists provide life stage-specific benefits to larvae and adults, respectively. In the plant sap-feeding larvae, the symbionts are inferred to synthesize most of the essential amino acids as well as the B vitamin riboflavin. The adult reed beetles' folivory is likely supported by symbiont-encoded pectinases that complement the host-encoded set of cellulases, as revealed by transcriptome sequencing. However, mapping the occurrence of the symbionts' pectinase genes and the hosts' food plant preferences onto the beetles' phylogeny reveals multiple independent losses of pectinase genes in lineages that switched to feeding on pectin-poor plants, presumably constraining their hosts' subsequent adaptive potential.

Fig. 1. Reed beetle symbionts show strongly reduced and perfectly syntenic genomes.

a–c Representative images of Donaciinae beetles, a Donacia thalassina, b Plateumaris braccata, c Donacia versicolorea (picture kindly provided by Rebekka Janke). d Localization of symbiotic organs (white arrowhead) at the midgut/hindgut junction. Scale bar 0.5 mm. e Fluorescence in situ hybridization micrograph showing a cross-section of the symbiotic organs of a female Donacia vulgaris. Fluorescently labeled symbionts (yellow) are visible in the cells and the lumen of the enlarged Malpighian tubules. General DNA counterstaining was done with DAPI (blue). Scale bar 40 µm. f Genome of “Candidatus Macropleicola muticae”, the symbiont of Macroplea mutica. Picture of M. mutica kindly provided by Lech Borowiec. g Hive plot depicting perfect synteny across the symbiont genomes of five representative Donaciinae spanning the phylogenetic diversity of the subfamily. Coloring of genes in f and g: environmental information processing (green); genetic information processing (violet); metabolism (peach); RNA (yellow); cysteine and methionine metabolism (blue); phenylalanine, tyrosine, and tryptophan metabolism (pink); other amino acids metabolism (brown); other (gray).

Fig. 2. Evolution of (semi-)essential amino acid (AA) biosynthesis pathways in Donaciinae symbionts.

a Comparison of symbiont genomes across 26 species of Donaciinae. Phylogenomic tree represents the relationships among symbionts, based on an alignment of 49 marker genes. Blue and magenta arrowheads indicate methionine and tryptophan biosynthesis genes, respectively, that have been lost in the symbionts of particular host taxa. Coloring of genes is the same as in Fig. 1f, g. b Schematic AA biosynthesis pathways as well as glycolysis and TCA cycle in reed beetle symbionts, with important intermediates and enzymes highlighted. Enzymatic steps in green are present across all symbiont genomes, those in gray are absent from all genomes. Colored steps indicate loss of enzymatic steps in particular taxa (see legend). Amino acids are colored according to the inferred capacity of the symbionts to produce them. Note that the loss of ilvE in Macroplea is assumed to be compensated for by alternative symbiont or host enzymes.

Fig. 3. Plant cell wall degrading enzymes (PCWDEs) encoded by reed beetles and their symbionts.

Numbers of PCDWE-encoding genes detected in the host gut transcriptomes and symbiont genomes, respectively, are indicated for Donaciinae species across four genera, and their congruence with the host plant preferences of the adult beetles and the presence of symbiotic bacteria in adult males are highlighted. The dendrogram on the left side depicts the phylogenetic relationships among the host species, reconstructed based on all 13 protein-coding genes in the mitochondrial genome. For the PCWDEs, the number of copies in the host transcriptome and the presence/absence of GH28 on the symbiont chromosome and plasmid is given, respectively. The presence of symbiotic bacteria in adult males was deduced from previous reports as well as the results of dissections and FISH in the present study. Host plant preferences are based on published reports^,,,,.

Fig. 4. Phylogeny of Donaciinae symbiont pectinases perfectly mirrors that of the entire symbiont genomes.

a Phylogenomic tree of Donaciinae symbionts based on 49 marker genes (FastTree analysis implemented in Kbase; local support values in percent are given at the nodes). Colored lines denote the reconstructed presences (solid lines) and losses (dashed lines) of chromosome-encoded (red lines) and plasmid-encoded polygalacturonases (green lines); b Phylogeny of pectinases encoded by the Donaciinae symbionts (FastTree analysis; local support values are given at the nodes). Branches leading to chromosome-encoded copies are highlighted in red, those leading to plasmid-encoded copies in green. The phylogenies of both pectinases are perfectly congruent with the phylogenomic tree of the symbionts.

Fig. 5. In vitro pectinase activity assays with gut extracts from different reed beetles.

a Agar diffusion assays for polygalacturonase activity; gut extracts exhibiting clearly visible enzymatic effects are highlighted in dark green, those with weak activity in light green (Dcla Donacia clavipes, Dvul Donacia vulgaris, Dcin Donacia cinerea, Dtha Donacia thalassina, Dsim Donacia simplex, Pser Plateumaris sericea, Prus Plateumaris rustica, DcraF Donacia crassipes female, DcraM Donacia crassipes male, Dsem Donacia semicuprea, Dbic Donacia bicolor, Dmar Donacia marginata, Pbra Plateumaris braccata, Dprx Donacia proxima, Dcct Donacia cincticornis, Dful Donacia fulgens, DdenM Donacia dentata male, DdenF Donacia dentata female, Dtom Donacia tomentosa, DverM Donacia versicolorea male, DverF Donacia versicolorea female, negC negative control (no sample), posC positive control (Phaedon cochleariae)). Note that none of the Poales-feeding species showed polygalacturonase activity. b Thin-layer chromatography comparing the efficiency of D. crassipes (Dcra; symbiont genome encodes two pectinases) and D. versicolorea (Dver; symbiont genome encodes one pectinase) female gut extracts in breaking down different pectins (PGA polygalacturonic acid, deesterified (no methylation); Pcit pectin from citrus (60% of galacturonic acid residues are methylated); Pest pectin from citrus, esterified (85% of galacturonic acid residues are methylated); TGA trigalacturonic acid, DGA digalacturonic acid, GA galacturonic acid). Source data are provided as a Source Data file.

Fig. 6. Phylogenomic analyses of Donaciinae and their symbiotic bacteria resolve the evolution of pectinolytic enzymes and their congruence with host plant preferences.

a Phylogenomic tree of Donaciinae symbionts based on 49 marker genes (FastTree analysis implemented in Kbase; local support values in percent are given at the nodes). Colored lines denote the reconstructed presences (solid lines) and losses (dashed lines) of chromosome-encoded (red lines) and plasmid-encoded polygalacturonases (green lines). Next to the phylogeny, images of representative host taxa are given (from top to bottom: Cassida rubiginosa (outgroup), Plateumaris braccata, Macroplea appendiculata, Donacia versicolorea, Donacia crassipes, Donacia cinerea, Donacia marginata, pictures kindly provided by Lech Borowiec); b representative genomes of major symbiont clades, with range of genome sizes within the clade given inside each genome; a red triangle indicates the presence of a polygalacturonase (GH28) gene in the genome; c schematic alignment of plasmid sequences (linearized) across the 26 reed beetle symbionts; the presence of a polygalacturonase (GH28) gene on the plasmid is highlighted in green; d host plant preferences in the major Donaciinae clades, colored on the order level (blue: Poales; green: Alismatales; orange: Nymphaeales); background shading of Donaciinae clades corresponds to host plant preferences; e phylogenetic tree of reed beetles based on all 13 protein-coding genes in the mitochondrial genome; local support values in percent are given at the nodes; dashed lines connect the host taxa to their corresponding symbiont in a, and asterisks denote discrepancies between host and symbiont phylogenies.

Fig. 7. Fluorescence in situ hybridization micrographs of symbiotic organs in Donaciinae.

Females (left panel) and males (right panel) of four representative species feeding on different host plants are shown (for fluorescence micrographs of 11 different species, see Supplementary Fig. 8). Note that different probes were used (see Supplementary Table 2), so the symbionts of different species are labeled in red (Cy3, a, b), green (Cy5, c–e), or yellow (Cy3 and Cy5, g). DAPI (blue) was used for general DNA counterstaining. Filled white arrowheads highlight symbiont-filled Malpighian tubules (symbiotic organs), empty arrowheads point to Malpighian tubules without symbionts. The following species are shown (host plant order given in brackets): a, b Donacia crassipes (Nymphaeales), c, d Donacia dentata (Alismatales), e, f Donacia semicuprea (Poales), g, h Plateumaris sericea (Poales). Note that only the Alismatales-feeding and Nymphaeales-feeding species show symbiont-bearing organs in adult males (b, d), whereas the males of Poales-feeding species are symbiont-free (f, h). By contrast, females carry symbionts in all species (a, c, e, g). Scale bars 50 µm.