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. 2012;7(7):e36978.
doi: 10.1371/journal.pone.0036978. Epub 2012 Jul 17.

Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae

Affiliations

Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae

Xiaoshu Tang et al. PLoS One. 2012.

Abstract

Background: 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.

Methods/principal findings: To determine the composition of the gut microbiota of two lepidopteran pests, Spodoptera littoralis and Helicoverpa armigera, 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, etc. 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.

Conclusions: 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic tree of bacterial divisions retrieved from S. littoralis larval gut based on sequence similarity.
The 16S rRNA gene sequence of the cyanobacterium Synechococcus elongatus PCC 6301 (NC_006576.1) and the Armatimonadetes Chthonomonas calidirosea T49 (AM749780.1) were used as the out groups. A detailed description of the phylotypes and accession numbers of the most closely related reference sequences can be found in Table S1. The accession number of the other reference sequences are: Enterococcus durans Ed-02 (HM130537.1), Lactobacillus brevis T9 (JQ301799.1), Staphylococcus aureus subsp. aureus JH1(CP000736.1), Micrococcus luteus NCTC2665 (CP001628.1), Corynebacterium diphtheriae 31A (CP003206.1), Burkholderia pseudomallei K96243 (NC_006350.1), Rhizobium etli CFN 42 (CP000133.1), Desulfobacterium autotrophicum HRM2 (CP001087.1). The two digit bootstrap number and the three decimal posterior probabilities are shown on major nodes. The bottom bar represents substitution rate per site.
Figure 2
Figure 2. Change of bacterial composition along the digestive tract of 5th-instar larvae of artificial food-feeding S. littoralis.
(a), The structure of the alimentary canal. The digestive tract was cut into three segments (I, II, and III) for sampling as indicated by the dotted lines. (b), Relative abundance of bacteria in the three segments revealed by cloning and sequencing. (c), Rarefaction curves of the bacterial diversity in gut section I and section III.
Figure 3
Figure 3. Bacterial localization in the gut of S. littoralis larvae with Fluorescent In Situ Hybridization.
Scale bar equals 10 µm. A, Detection of Clostridium sp. In the midgut. The three images shown are TIC image, fluorescent image of universal probe (EUB, red) and of specific probe (SPE, green). B to G are merged images of TIC, EUB and SPE. The bacteria detected only with universal probe are red, and the bacterial with both probes are green. B, a large aggregate of Clostridium sp. deep in the gut lumen. C, Detection of E. mundtii. D, Detection of E. casseliflavus. E, P. acnes in the midgut. F, E. coli detected in the midgut; G, K. pneumonia detected in the midgut. Bacteria detected only by universal probe are highlighted with white arrows; Bacteria stained by sequence-specific probes are pointed by open arrows. Insect tissue is indicated by arrow heads.
Figure 4
Figure 4. Different gut bacterial community structures in S. littoralis larvae of different instars feeding on artificial diet.
A, The bacterial community compositions detected by cloning and sequencing from insects that are 2-days (n = 33), 6-days (n = 104), 10-days (n = 232), and 14-days (n = 490). The arrow represents the life span of an S. littoralis larva. The developmental stages, hatch, pupation, and larval instars are represented by bars. The inset shows the relative abundance of bacteria detected on the epithelium of 10-day old larvae (n = 94). B, The rarefaction curves of the richness indices Chao1 and ACE, and the diversity indices Shannnon and Simpson based on sequences retrieved from larvae. Indices were calculated using 95% confidence level and 0.03 distance cutoff for OUT clustering.
Figure 5
Figure 5. The impact of diet on the gut community in S. littoralis larvae revealed by cloning and sequencing.
A, Growth curve of the insects. Black dots indicate where the insect gut was sampled. Af, artificial food; Ba, barley; Lb, Lima bean. B, Gut bacterial composition of 6-day-old larvae feeding on Lima bean for 4 days (n = 283, L-6), in 10-day-old larvae feeding on Lima bean (n = 139, L-10), and in the gut of 10-day-old larvae feeding on barley (n = 192, B-10). Case-specific species are shadowed. Singletons are black. C, The rarefaction curves of the richness indices Chao1 and ACE, and the diversity indices Shannnon and Simpson. Indices were calculated using 95% confidence level and 0.03 distance cutoff for OUT clustering.
Figure 6
Figure 6. The phylogeny-based β-diversity values between bacterial communities detected in the gut of S. littoralis larvae at different instars and after feeding on different diets by cloning and sequencing.
The upper values are the parsimony scores and the lower values are the weighted UniFrac scores. Higher score indicates that the two samples are more different on bacterial composition. All significance are lower than 0.001. Artificial food was depicted as cubes; Lima bean as a single leaf; barley as a whole plant.

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