Larvae of an invasive scarab increase greenhouse gas emissions from soils and recruit gut mycobiota involved in C and N transformations

Abstract

Background: Soil-derived prokaryotic gut communities of the Japanese beetle Popillia japonica Newman (JB) larval gut include heterotrophic, ammonia-oxidizing, and methanogenic microbes potentially capable of promoting greenhouse gas (GHG) emissions. However, no research has directly explored GHG emissions or the eukaryotic microbiota associated with the larval gut of this invasive species. In particular, fungi are frequently associated with the insect gut where they produce digestive enzymes and aid in nutrient acquisition. Using a series of laboratory and field experiments, this study aimed to (1) assess the impact of JB larvae on soil GHG emissions; (2) characterize gut mycobiota associated with these larvae; and (3) examine how soil biological and physicochemical characteristics influence variation in both GHG emissions and the composition of larval gut mycobiota.

Methods: Manipulative laboratory experiments consisted of microcosms containing increasing densities of JB larvae alone or in clean (uninfested) soil. Field experiments included 10 locations across Indiana and Wisconsin where gas samples from soils, as well as JB and their associated soil were collected to analyze soil GHG emissions, and mycobiota (ITS survey), respectively.

Results: In laboratory trials, emission rates of CO₂, CH₄, and N₂O from infested soil were ≥ 6.3× higher per larva than emissions from JB larvae alone whereas CO₂ emission rates from soils previously infested by JB larvae were 1.3× higher than emissions from JB larvae alone. In the field, JB larval density was a significant predictor of CO₂ emissions from infested soils, and both CO₂ and CH₄ emissions were higher in previously infested soils. We found that geographic location had the greatest influence on variation in larval gut mycobiota, although the effects of compartment (i.e., soil, midgut and hindgut) were also significant. There was substantial overlap in the composition and prevalence of the core fungal mycobiota across compartments with prominent fungal taxa being associated with cellulose degradation and prokaryotic methane production/consumption. Soil physicochemical characteristics such as organic matter, cation exchange capacity, sand, and water holding capacity, were also correlated with both soil GHG emission, and fungal a-diversity within the JB larval gut. Conclusions: Results indicate JB larvae promote GHG emissions from the soil directly through metabolic activities, and indirectly by creating soil conditions that favor GHG-associated microbial activity. Fungal communities associated with the JB larval gut are primarily influenced by adaptation to local soils, with many prominent members of that consortium potentially contributing to C and N transformations capable of influencing GHG emissions from infested soil.

Keywords: Japanese beetles; carbon dioxide; core mycobiota; fungal ITS1; methane; nitrous oxide.

Figure 1

(A–C) Carbon dioxide (CO₂), (D–F) methane (CH₄), and (G–I) nitrous oxide (N₂O) gas emissions from isolated third instar Japanese beetle (Popillia japonica Newman) larvae (no soil) (A,D,G), infested soil (B,E,H), and previously infested soil (C,F,I) in laboratory microcosms (n = 5). Microcosms consisted of 473 mL clear glass wide-mouth canning jars, and 0.1 Kg of soil when applicable. Linear regression estimates and performance parameters derived from these data can be found in Supplementary Table S1.

Figure 2

(A) Carbon dioxide (CO₂), (B) methane (CH₄), and (C) nitrous oxide (N₂O) emissions from field soils infested with Japanese beetle (Popillia japonica Newman) larvae. Sampling was performed under field conditions at 8 locations across Indiana and Wisconsin, United States, in 2019. Larval density and soil physicochemical characteristics were used as predictors for the linear mixed models presented in Supplementary Table S2.

Figure 3

Core fungal microbiota at different detection thresholds of relative abundance across compartments: midgut and hindgut from third instar larvae of the Japanese beetle (Popillia japonica Newman), and associated soil. Taxa with sample prevalence > 19% (>4 out of 21 samples) are presented. Colors show the presence of each taxon in different compartments at ≥0.01 relative abundance. Taxa are presented at Order level when available, otherwise Class or Phylum level are presented as indicated by Class/Phylum name followed by one or two underscore symbols, respectively. For complete taxonomic affiliation at Phylum, Class, or Order level refer to Supplementary Table S5.

Figure 4

Alpha diversity for fungal communities in guts from third instar Japanese beetle (Popillia japonica) larvae and associated soil. Boxplots show median, interquartile range, and 1.5× the interquartile range per location. Regardless of the α-diversity metric, the influence of compartment on α-diversity varied consistently with location (ART, compartment × location, p < 0.001). n = 3 for each compartment at each location. Refer to Supplementary Table S7 for statistical significance.

Figure 5

Compositional biplot portraying beta diversity of fungal communities in midgut and hindgut of third instar Japanese beetle (Popillia japonica Newman) larvae and associated soil. The biplot was generated using DEICODE (Robust Aitchison PCA; Martino et al., 2019) and visualized in EMPeror (Vázquez-Baeza et al., 2013). Data points represent individual samples where symbol shape denotes location while symbol color denotes compartment (i.e., midgut, hindgut, or soil). Top 20 taxa driving differences in ordination space at the order rank, when available, are illustrated by the arrows. The phyla within fungi are represented by specific hues, where Ascomycota (10) = green, Basidiomycota (2) = gray, and Mortierellomycota (5) = purple. AMPtk’s ‘hybrid’ taxonomy assignment (Palmer et al., 2018) was used for taxonomic classification. Refer to Supplementary Table S10 for statistical significance.

Figure 6

Canonical correspondence analysis (CCA) calculated based on a Chi-square dissimilarity matrix of fungal communities at the OTU rank in the midgut (A) and hindgut (B) of third instar Japanese beetle (Popillia japonica) larvae and host soil physicochemical parameters. Vectors show soil variables: cation exchange capacity (CEC), % organic matter (OM), pH, % sand (Sand), and water holding capacity (WHC). Significant soil variables are presented with two (p ≤ 0.01) or one (p ≤ 0.05) asterisks.