Gut microbiome insights from 16S rRNA analysis of 17-year periodical cicadas (Hemiptera: Magicicada spp.) Broods II, VI, and X

Abstract

Periodical cicadas (Hemiptera: Magicicada) have coevolved with obligate bacteriome-inhabiting microbial symbionts, yet little is known about gut microbial symbiont composition or differences in composition among allochronic Magicicada broods (year classes) which emerge parapatrically or allopatrically in the eastern United States. Here, 16S rRNA amplicon sequencing was performed to determine gut bacterial community profiles of three periodical broods, including II (Connecticut and Virginia, 2013), VI (North Carolina, 2017), and X (Maryland, 2021, and an early emerging nymph collected in Ohio, 2017). Results showed similarities among all nymphal gut microbiomes and between morphologically distinct 17-year Magicicada, namely Magicicada septendecim (Broods II and VI) and 17-year Magicicada cassini (Brood X) providing evidence of a core microbiome, distinct from the microbiome of burrow soil inhabited by the nymphs. Generally, phyla Bacteroidetes [Bacteroidota] (> 50% relative abundance), Actinobacteria [Actinomycetota], or Proteobacteria [Pseudomonadota] represented the core. Acidobacteria and genera Cupriavidus, Mesorhizobium, and Delftia were prevalent in nymphs but less frequent in adults. The primary obligate endosymbiont, Sulcia (Bacteroidetes), was dominant amongst core genera detected. Chryseobacterium were common in Broods VI and X. Chitinophaga, Arthrobacter, and Renibacterium were common in Brood X, and Pedobacter were common to nymphs of Broods II and VI. Further taxonomic assignment of unclassified Alphaproteobacteria sequencing reads allowed for detection of multiple copies of the Hodgkinia 16S rRNA gene, distinguishable as separate operational taxonomic units present simultaneously. As major emergences of the broods examined here occur at 17-year intervals, this study will provide a valuable comparative baseline in this era of a changing climate.

Figure 1

Map of brood emergences and sampling locations. Shown are regions where and when different broods of periodical cicadas (Broods II, VI, and X) are likely to emerge. Map was created using ARCgis Online (Environmental Systems Research Institute). Cicada brood emergence was compiled by Simon and colleagues.

Figure 2

Violin plots showing alpha diversity comparison between MTP sets of cicada samples.

Figure 3

Beta diversity indices. NMDS plot of nymph gut microbiomes and microbiomes of (A) soil inhabited by nymphs in CT (Brood II) and NC (Brood VI), (B) Brood X gut microbiomes and microbiomes of soil in MD, and (C) cicada gut microbiomes showing life stage, brood, collection location, and sex of Brood X cicadas. Ellipses represents 95% confidence interval based on Bray–Curtis dissimilarity index. (D) Venn diagram of bacterial communities showing the number of shared and exclusive bacterial taxa is shown relative to MTP set. (E) Relative abundance of 20 most common core bacterial genera detected across all MTP sets.

Figure 4

Bacterial community composition. (A) Stacked bar plot showing relative sequencing read abundance of 10 most abundant bacterial phyla. (B) Heatmap showing log₁₀(relative abundance) of 30 most abundant bacterial genera. Dendrogram shows k-means clustering of samples.

Figure 5

Average bacterial community compositions. Stacked bar plot showing relative abundance of (A) ten most abundant bacterial phyla detected across MTP set and (B) twenty most abundant bacterial genera detected across MTP set, ordered from most abundant to least abundant.

Figure 6

Taxonomic biomarkers calculated using LEfSe. Heatmap (left) provides log10₁₀ (relative abundance) of taxonomic biomarkers at the genus level. Bar plot (right) shows linear discriminant analysis (LDA) effect size used to support high-dimensional class comparisons of (A) Brood II nymphs (CT) and Brood VI nymphs (NC) versus soil inhabited by nymphs (CT and NC), (B) Adults (Brood X, MD, and Brood VI, NC) versus soil (MD and NC), (C) Brood II (CT) versus Brood II (VA), (D) Brood II nymphs (CT and VA) versus Brood VI nymphs (NC), (E) Brood X male nymphs (MD) versus Brood X female nymphs (MD), and (F) Adults (Brood X, MD, and Brood VI, NC) versus nymphs (Brood X, MD, and Brood VI, NC).

Figure 7

Detection and identification of Hodgkinia OTUs. (A) Stacked bar plot showing relative abundance of Hodgkinia OTUs classified by cicada host species. Characterization of Hodgkinina and proposed nomenclature is described elsewhere. MAGNEO, Magicicada neotredecim; MAGTRE, Magicicada tredecim; MAGCAS, Magicicada cassini; MAGTCS, Magicicada tredecassini; MAGSEP, Magicicada septendecim. (B) Violin plot showing number of observed Hodgkinia OTUs between brood and MTP set. (C) Heatmap showing Log₁₀ (relative abundance) of Hodgkinia OTUs. Dendrogram shows k-means clustering of samples.