Caterpillars lack a resident gut microbiome

Abstract

Many animals are inhabited by microbial symbionts that influence their hosts' development, physiology, ecological interactions, and evolutionary diversification. However, firm evidence for the existence and functional importance of resident microbiomes in larval Lepidoptera (caterpillars) is lacking, despite the fact that these insects are enormously diverse, major agricultural pests, and dominant herbivores in many ecosystems. Using 16S rRNA gene sequencing and quantitative PCR, we characterized the gut microbiomes of wild leaf-feeding caterpillars in the United States and Costa Rica, representing 124 species from 15 families. Compared with other insects and vertebrates assayed using the same methods, the microbes that we detected in caterpillar guts were unusually low-density and variable among individuals. Furthermore, the abundance and composition of leaf-associated microbes were reflected in the feces of caterpillars consuming the same plants. Thus, microbes ingested with food are present (although possibly dead or dormant) in the caterpillar gut, but host-specific, resident symbionts are largely absent. To test whether transient microbes might still contribute to feeding and development, we conducted an experiment on field-collected caterpillars of the model species Manduca sexta Antibiotic suppression of gut bacterial activity did not significantly affect caterpillar weight gain, development, or survival. The high pH, simple gut structure, and fast transit times that typify caterpillar digestive physiology may prevent microbial colonization. Moreover, host-encoded digestive and detoxification mechanisms likely render microbes unnecessary for caterpillar herbivory. Caterpillars illustrate the potential ecological and evolutionary benefits of independence from symbionts, a lifestyle that may be widespread among animals.

Keywords: Lepidoptera; herbivory; insects; mutualism; symbiosis.

Fig. 1.

Comparisons of bacterial density, relative abundance of plant DNA, and intraspecific variability between caterpillars and other animals expected to host functional microbiomes. Medians are indicated by black dashed lines, and points are horizontally jittered. Data for each species are listed in SI Appendix, Table S1. One caterpillar species yielding <100 total sequences was excluded. For species with multiple replicates, the median is plotted. (A) The density of bacterial 16S rRNA gene copies in caterpillar feces versus fecal (vertebrates) or whole-body homogenate (other insect) samples of other animals (n = 121 caterpillar species, 24 other species). Two caterpillar species with lower amplification than DNA extraction blanks are not shown. (B) The proportion of sequence libraries assigned to plant chloroplast or mitochondrial rRNA (n = 123 caterpillars, 21 other herbivores). (C) The proportion of bacterial sequences belonging to core phylotypes, defined for each species as those present in the majority of conspecific individuals analyzed. Included are species with at least three replicates with >100 bacterial sequences each (n = 7 caterpillars, 19 other animals). For species with more than three replicates, points show the median core size across all combinations of three individuals, and error bars show the interquartile range.

Fig. 2.

The abundance and composition of caterpillar fecal bacteria compared with paired diet (leaf) samples. (A) The density of bacterial 16S rRNA gene copies in ground leaves versus feces for 16 caterpillars collected in Colorado. Parallel lines indicate an association between plant and fecal bacterial abundances across pairs. (B) The correlation between beta diversity (Bray–Curtis dissimilarity) across caterpillar fecal samples collected in Costa Rica and paired leaf-surface samples (n = 24 caterpillar species, 19 plant species; 26 individuals each). Here, only samples with >2,000 sequences are shown to facilitate visualization.

Fig. 3.

An increasing concentration of an antibiotic cocktail, applied to leaves before feeding, does not reduce M. sexta larval growth (n = 62). Males and females are plotted separately, as they were expected to differ in size. Fresh weight was measured 6 d after pupation. Pupal weight correlates with adult fecundity (50) and is often used as a proxy of insect fitness.