Biodiversity and Activity of the Gut Microbiota across the Life History of the Insect Herbivore Spodoptera littoralis

Abstract

Microbes that live inside insects play critical roles in host nutrition, physiology, and behavior. Although Lepidoptera (butterflies and moths) are one of the most diverse insect taxa, their microbial symbionts are little-studied, particularly during metamorphosis. Here, using ribosomal tag pyrosequencing of DNA and RNA, we investigated biodiversity and activity of gut microbiotas across the holometabolous life cycle of Spodoptera littoralis, a notorious agricultural pest worldwide. Proteobacteria and Firmicutes dominate but undergo a structural "metamorphosis" in tandem with its host. Enterococcus, Pantoea and Citrobacter were abundant and active in early-instar, while Clostridia increased in late-instar. Interestingly, only enterococci persisted through metamorphosis. Female adults harbored high proportions of Enterococcus, Klebsiella and Pantoea, whereas males largely shifted to Klebsiella. Comparative functional analysis with PICRUSt indicated that early-instar larval microbiome was more enriched for genes involved in cell motility and carbohydrate metabolism, whereas in late-instar amino acid, cofactor and vitamin metabolism increased. Genes involved in energy and nucleotide metabolism were abundant in pupae. Female adult microbiome was enriched for genes relevant to energy metabolism, while an increase in the replication and repair pathway was observed in male. Understanding the metabolic activity of these herbivore-associated microbial symbionts may assist the development of novel pest-management strategies.

Figure 1. Changes in bacterial community diversity across life stages of S. littoralis.

(a) Overview of development stages of the host. (b) Rarefaction curves depicted from randomly subsampled data sets with the same number of 16S sequences. The near saturated rarefaction curve indicates that the vastness of microbial diversity was retrieved from each sample. (c) Overview of the microbiota change during host development. Abundance of the 16S rRNA gene at each developmental stage at the phylum level. Relative abundances of the most dominant phyla including Proteobacteria, Firmicutes and Actinobacteria changed significantly (p < 0.001) across the life cycle.

Figure 2. Similarity analysis of microbial communities.

(a) UPGMA clustering of samples at different developmental stages according to community composition and structure. (b) Principal coordinates analysis (PCoA) plot visualizing the data based on β-diversity metrics of UniFrac.

Figure 3. DGGE profiles of PCR-amplified 16S rRNA gene fragments of bacterial communities from S. littoralis larva and adult samples.

(a) DGGE profile of the mature larval gut microbiota of different individuals (L = larva). (b) DGGE profile of the adult gut microbiota of different individuals (M = male adult and F = female adult). (c) Cluster analysis of the DGGE patterns of the male (M) and female (F) samples.

Figure 4. The microbiota associated with the egg mass of S. littoralis.

(a) Relative abundance of major taxa (to genera level) in the DNA and RNA data sets. (b) Venn diagram showing overlaps of OTUs (at 97% similarity) between the DNA (purple circle) and RNA (red circle) data sets. Values are the numbers of OTUs calculated using the total data set.

Figure 5. The larval gut microbiota of S. littoralis.

Relative abundance of major taxa (to genera level) in the DNA and RNA data sets of early-instar larvae (E-instar) and late-instar larvae (L-instar).

Figure 6. The microbiota associated with the pupae of S. littoralis.

(a) Relative abundance of major taxa in the DNA and RNA data sets. (b) Venn diagram showing overlaps of OTUs (at 97% similarity) between the DNA (purple circle) and RNA (red circle) data sets.

Figure 7. The adult gut microbiota of S. littoralis.

(a) Relative abundance of major taxa (to genera level) in the DNA and RNA data sets of female and male adults. (b) Venn diagram showing overlaps of OTUs (at 97% similarity) between the DNA (purple circle) and RNA (red circle) data sets of male adult and female adult (c).

Figure 8. Phylogenetic analysis of dominant taxa identified from microbiotas associated with S. littoralis.

Maximum-likelihood tree constructed on the basis of 16S rRNA gene sequences. Bootstrap values were obtained from a search with 500 replicates. Strain and accession numbers are given behind the species names.

Figure 9. Inferred functions of bacterial communities associated with S. littoralis.

All of the predicted KEGG metabolic pathways are shown at the second hierarchical level and grouped by major functional categories.