Diet breadth and exploitation of exotic plants shift the core microbiome of Cephaloleia, a group of tropical herbivorous beetles

Abstract

The beetle genus Cephaloleia has evolved in association with tropical ginger plants and for many species their specific host plant associations are known. Here we show that the core microbiome of six closely related Costa Rican Cephaloleia species comprises only eight bacterial groups, including members of the Acinetobacter, Enterobacteriacea, Pseudomonas, Lactococcus, and Comamonas. The Acinetobacter and Enterobacteriacea together accounted for 35% of the total average 16S rRNA ribotypes recovered from all specimens. Further, microbiome diversity and community structure was significantly linked to beetle diet breadth, between those foraging on less than two plant types (specialists) versus over nine plant types (generalists). Moraxellaceae, Enterobacteriaceae, and Pseudomonadaceae were highly prevalent in specialist species, and also present in eggs, while Rickettsiaceae associated exclusively with generalist beetles. Bacteria isolated from Cephaloleia digestive systems had distinct capabilities and suggested a possible beneficial role in both digestion of plant-based compounds, including xylose, mannitol, and pectin, and possible detoxification, via lipases. Cephaloleia species are currently expanding their diets to include exotic invasive plants, yet it is unknown whether their microbial community plays a role in this transition. In this study, colonization of invasive plants was correlated with a dysbiosis of the microbiome, suggesting a possible relationship between gut bacteria and niche adaptation.

Keywords: Acinetobacter; Costa Rica; Diet; Enterobacteriacea; Herbivory; Insect; Invasive species; Microbiome; Tropical beetles.

Figure 1. Costa Rican Cephaloleia species in this study.

Costa Rican Cephaloleia species in this study include (A) C. dilaticollis, (B) C. placida, (C) C. reventazonica, (D) C. dorsalis, (E) C. fenestrata, and (F) C. belti. (G) A mating pair of C. erichsonii on a young rolled leaf of Calathea lutea. All life stages and most behavior, including mating, take place on plants within the order Zingiberales. Scale bars = 1 mm for all images, except far right where the scale bar is 1 cm. Photo credits: S. Goffredi.

Figure 2. Relative abundance of bacterial families.

Relative abundance of bacterial families from (A) beetles foraging on native plants, including Cephaloleia placida, C. dilaticollis, C. fenestrata, C. doralis, C. belti, and C. reventazonica, and (B) beetles foraging on invasive plants C. placida, C. dilaticollis, and C. belti compared to those species foraging on native plants. Each color group on the graph represents a distinct genus-level OTU or lowest level available. Families that constituted <1% of sequences from individual specimens were grouped as “Other.”

Figure 3. Prevalence versus relative abundance of bacterial OTUs.

Prevalence versus relative abundance of bacterial OTUs associated with six Cephaloleia species (n = 29 specimens, collected on native diets). Certain dominant OTU groups are indicated separately by color (e.g., Acinetobacter), with 12 shown by OTU# based on ≥60% prevalence or ≥10% relative abundance (including four noted in green that matched bacterial groups typically thought of as pathogens).

Figure 4. Metabolic capabilities of bacteria isolated from the digestive system of Cephaloleia beetles.

Metabolic capabilities of bacteria isolated from the digestive system of Cephaloleia beetles, including the ability to use lactose/glucose, pectin, xylose, and mannitol, as well as the presence of proteases, esterases, and lipases. White shading and the number “1” indicate the ability to digest the specified compound. Dark gray shading and the number “0” indicate an inability to digest the specified compound, “0.5” indicates a partial ability. At left, a phylogenetic tree, based on 767 bp 16S rRNA sequences, built with Tamura–Nei distance model and UPGMA method, of beetle digestive system isolates shown to the left. Scale bar, 0.1 divergence.

Figure 5. Box plots of beetle-associated average diversity (Shannon index) and relative percent abundance of four key bacterial families.

Box plots of beetle-associated average diversity (Shannon index) and relative percent abundance of four key bacterial families (identified by SIMPER to be responsible for up to 46% of the cumulative (dis)similarity among six Cephaloleia species examined in this study (n = 29 specimens, collected on native diets)). Any data points outside of the 25–75% range are identified by open symbols. Species abbreviations are as follows: plac = C. placida, dilat = C. dilaticollis, fen = C. fenestrata, dor = C. dorsalis, belti = C. belti, rev = C. reventazonica.

Figure 6. Non-metric multidimensional scaling (NMDS) ordination of microbial communities associated with Cephaloleia beetles.

Each point represents all 16S rRNA sequences recovered from a single specimen. Displayed data was square root transformed, which minimizes errors in the ordination due to PCR bias while also not sacrificing genuine differences between samples. Samples with similar microbial communities plot closer together. ANOSIM p values are shown. Lower stress values indicate better representation of the intersample (dis)similarities in two dimensions. (A) Ordination comparing four specialist species and two generalist species, the latter designated by triangles. p = 0.001, suggesting a distinct difference between the two feeding strategies. (B) Ordination showing the eggs of three species (outlined), compared to adults. p = 0.060 for all samples, suggesting a similarity to the communities found in specialist adults. p = 0.022 for the generalist species C. belti eggs versus adults, suggesting a significant difference between the two. (C) Ordination comparing three species, found on both native (filled symbols) and invasive plant species (open symbols). p = 0.001 for the two specialist species combined; p = 0.05 for C. belti, suggesting a significant difference in both cases.

Figure 7. Dominant bacterial 16S rRNA sequences, recovered from eggs and adults of Cephaloleia species.

Relative abundance of the six most dominant bacterial genera, based on 16S rRNA sequences, recovered from eggs (left) and adults (right) of three Cephaloleia species. Species abbreviations are as follows (n egg, n adult, respectively): Cb = C. belti (2, 8), Cdil = C. dilaticollis (2, 3), Cdor = C. dorsalis (1, 3). Photo credits: S. Goffredi.