<i>In Vivo</i> Pyro-SIP Assessing Active Gut Microbiota of the Cotton Leafworm, <i>Spodoptera littoralis</i>

<div><p>The gut microbiota is of crucial importance for the host with considerable metabolic activity. Although great efforts have been made toward characterizing microbial diversity, measuring components' metabolic activity surprisingly hasn't kept pace. Here we combined pyrosequencing of amplified 16S rRNA genes with <i>in vivo</i> stable isotope probing (Pyro-SIP) to unmask metabolically active bacteria in the gut of cotton leafworm (<i>Spodoptera littoralis</i>), a polyphagous insect herbivore that consumes large amounts of plant material in a short time, liberating abundant glucose in the alimentary canal as a most important carbon and energy source for both host and active gut bacteria. With ¹³C glucose as the trophic link, Pyro-SIP revealed that a relatively simple but distinctive gut microbiota co-developed with the host, both metabolic activity and composition shifting throughout larval stages. <i>Pantoea</i>, <i>Citrobacter</i> and <i>Clostridium</i> were particularly active in early-instar, likely the core functional populations linked to nutritional upgrading. <i>Enterococcus</i> was the single predominant genus in the community, and it was essentially stable and metabolically active in the larval lifespan. Based on that <i>Enterococci</i> formed biofilm-like layers on the gut epithelium and that the isolated strains showed antimicrobial properties, <i>Enterococcus</i> may be able to establish a colonization resistance effect in the gut against potentially harmful microbes from outside. Not only does this establish the first in-depth inventory of the gut microbiota of a model organism from the mostly phytophagous Lepidoptera, but this pilot study shows that Pyro-SIP can rapidly gain insight into the gut microbiota's metabolic activity with high resolution and high precision.</p></div

Frequency of 16S rRNA sequences in the microbiota obtained from the native-glucose control (bacterial relative abundance) and [¹³C]-glucose treatment (bacterial metabolic activity), represented as a heatmap.

<p>Left panel displays dynamic changes of taxa in early-instar larvae and right panel for late-instar. Warm colors indicate higher and cold colors lower abundance, calculated according to the formula in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085948#pone-0085948-g002" target="_blank">Fig. 2A</a> (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085948#pone-0085948-g005" target="_blank">Fig. 5</a> for the percentage of taxa).</p

Bacterial diversity and relative abundance in the gut microbiota of early-instar larvae.

<p>(<b>A</b>) Rarefaction curves of 16S rDNA sequences were obtained from representative SIP fractions of the control ([¹²C]) and labeling treatment ([¹³C]). (<b>B</b>) Relative abundance of bacterial taxa in different SIP fractions, represented in a relative area graph as revealed by pyrosequencing. Abbreviations: [¹²C] Light, light fractions (fractions 9–11, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085948#pone-0085948-g002" target="_blank">Fig. 2A</a>) of native-glucose amendment; [¹²C] Middle, middle fraction (fraction 7) of that; [¹²C] Heavy, heavy fractions (fractions 4–5) of that; [¹³C] Light, light fractions of ¹³C-glucose amendment; [¹³C] Middle, middle fraction of that; [¹³C] Heavy, heavy fractions of that.</p

Phylogenetic analysis of (a) Firmicutes and (b) Proteobacteria identified from the gut microbiota of cotton leafworm.

<p>(<b>A</b>) Maximum Likelihood tree was derived from partial 16S rDNA sequence data for members of Firmicutes. (<b>B</b>) Neighbor-Joining tree was derived from partial 16S rDNA sequence data for members of Proteobacteria. Representative pyrosequences from this work and near full-length 16S rDNA sequences retrieved from previous clone-library-based studies are indicated by black circles (•) and blue circles, respectively. Labeled taxa are marked with triangles (▴). Reference sequences are downloaded from GenBank (accession numbers are in parentheses.). <i>Methanosarcina barkeri</i> (AF028692) is used as an outgroup. Family-level clusters are indicated by different colors. Bootstrap values (in percent) are based on 1000 replications. Bar represents 2% sequence divergence. Right section denotes percentage of representative bacterial 16S rRNA sequences in the total dataset of each sample. Abbreviations: +¹²C, native-glucose amendment; +¹³C, ¹³C-glucose amendment; L, light fractions; H, heavy fractions.</p

Bacterial diversity and relative abundance in the gut microbiota of late-instar larvae.

<p>(<b>A</b>) Rarefaction curves of 16S rDNA sequences were obtained from representative SIP fractions of early-instar and late-instar larvae. Abbreviations: E, representative fractions from early-instar larvae fed on ¹³C-glucose; L, fractions from late-instar larvae fed on ¹³C-glucose. (<b>B</b>) Relative abundance of bacterial taxa in different SIP fractions, represented in a relative area graph as revealed by pyrosequencing. Abbreviations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085948#pone-0085948-g003" target="_blank">Fig. 3</a>.</p

Sugar composition in the gut content of cotton leafworm.

<p>(<b>A</b>) Sugars in the gut content of larvae fed on cotton or (<b>B</b>) artificial diet by GC-MS characterization after aldononitrile acetate derivatization. The larval alimentary canal is divided into three regions: foregut, midgut and hindgut, shown in diagram (<b>C</b>). (<b>D</b>) Quantification of dominant glucose reveals a significant decrease in average content along the gut. However, cotton-feeding larvae exhibit higher amount of glucose in all gut regions. <b>*</b> and <b>^a</b> indicate significant difference: P (<b>*</b>¹) = 0.0020, P (<b>*</b>²) = 0.0366, P (^a) = 0.0017. Error bars indicate standard errors. 1, Ribose; 2, Arabinose; 3, Mannose; 4, Glucose; 5, Galactose.</p

DataSheet1.DOCX

<p>The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with ¹³C cellulose and ¹⁵N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In ¹³C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In ¹⁵N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, ¹³C cellulose- and ¹⁵N urea labeled bacteria. The incorporation of ¹⁵N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.</p

Complexity and Variability of Gut Commensal Microbiota in Polyphagous Lepidopteran Larvae

<div><h3>Background</h3><p>The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood.</p> <h3>Methods/Principal Findings</h3><p>To determine the composition of the gut microbiota of two lepidopteran pests, <em>Spodoptera littoralis</em> and <em>Helicoverpa armigera</em>, we applied cultivation-independent techniques based on 16S rRNA gene sequencing and microarray. The two insect species were very similar regarding high abundant bacterial families. Different bacteria colonize different niches within the gut. A core community, consisting of Enterococci, Lactobacilli, Clostridia, <em>etc</em>. was revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that developmental stage and diet had a great impact on shaping the bacterial communities. Some low-abundant species might become dominant upon loading external disturbances; the core community, however, did not change significantly. Clearly the insect gut selects for particular bacterial phylotypes.</p> <h3>Conclusions</h3><p>Because of their importance as agricultural pests, phytophagous Lepidopterans are widely used as experimental models in ecological and physiological studies. Our results demonstrated that a core microbial community exists in the insect gut, which may contribute to the host physiology. Host physiology and food, nevertheless, significantly influence some fringe bacterial species in the gut. The gut microbiota might also serve as a reservoir of microorganisms for ever-changing environments. Understanding these interactions might pave the way for developing novel pest control strategies.</p> </div