From plant metabolites to the active core of the gut microbiota of cotton leafworm, Spodoptera littoralis
The herbivorous insect gut is a unique biochemical environment, which provides constant and controlled nutrient fluxes, principally abundant polysaccharides, amino acids and a diverse array of plant secondary metabolites (phytotoxins, carotenoids etc.) for the host and microbial metabolism. Through efficient digestion, the insect host gains carbon and energy. But phytotoxins commonly make plants unpalatable to herbivores and lead to a decreased fitness after ingestion. Carotenoids, not just another group of plant secondary metabolites, have a wide distribution in insects with various and fundamental functions. Microorganisms are actively involved in all those metabolic processes due to their fast generation cycle and the ease of adaptation. In the present work, I first investigate an unusual carotenoid uptake phenomenon in Spodoptera, which involves the crystallization of carotenes in the foregut and the accumulation of a single bacterial species. Next, the indigenous gut microbiota is characterized, which probably plays an important role in the host physiology and in the multitrophic interaction between insects and plants. a. Carotenoids in the gut Carotenoids are currently being intensely investigated regarding their very wide distribution in nature and critical function in all living organisms for light detection, oxidation control or coloration. In the leaf chewing herbivores such as cotton leafworm, carotenoids are usually absorbed from their host plants which contain a rich source of those organic pigments; and the ingested carotenoids are often accumulated in various host organs, either unaltered or with some metabolic modifications. Despite carotenoids’ general importance, the uptake mechanism is still poorly understood. Here, I investigated the “red crop” phenomenon, an accumulation of carotenes in crystalline inclusions in the enlarged foregut of the polyphagous Spodoptera larvae fed on some toxic plants. This pigmentation has survival value to the host. Caterpillars which fail to develop this “red crop” structure exhibit a high mortality. A combination of chemical characterization methods, especially the Raman microscopic analysis of the crystals in situ, revealed that beta carotene, not the most abundant and ubiquitous lutein in the foliage of host plant, is selectively sequestered in the crop and finally crystallizes there due to the exceptionally high concentrations. The carotene crystals give the insect foregut a distinctive orange-red color. Notably, the crystals are embedded in a homogenous lawn of the bacterium Enterococcus casseliflavus. However, 13C-IRMS data clearly indicated that carotenes are selectively taken from the food plant. The physicochemical conditions inside the foregut lumen (e.g. the consistent oxidative stress and high concentrations of amphiphilic compounds) and/or some specific carotene binding proteins are likely to play a role in the carotene absorption and transport process. Further investigations, such as the gene expression analysis in the crop tissue, are needed to characterize candidates responsible for this extraordinary beta carotene selectivity. Considering that cotton leafworms experience a greater oxidative stress during foraging, it has been hypothesized that high concentrations of beta carotene can efficiently prevent oxidative damage in this tissue by enhancing the non-enzymatic detoxification. Bioassay with the pro-oxidant partially restored the red crop phenomenon in larvae fed on carotene-fortified artificial diet, suggesting that ROS, but may be not the sole one, could stimulate the carotene sequestration in the crop. Since carotenoids have various functions in animals, other effects of this selective accumulation of beta carotene should be considered as well. Although huge amounts of Enterococci thrive in the crop, no evidence indicated their contribution to the formation of carotene crystals but they may benefit from this phenomenon by using carotenes as their own antioxidant. This red crop is not specific to Spodoptera littoralis; I also observed the formation of pigments in other lepidopteran species, such as Spodoptera exigua and Helicoverpa armigera. Therefore, this phenomenon may be ubiquitous in such lepidopteran herbivores living in the wild. And this also suggests an important role of carotenoids in the host biology, probably as the frontline of defense against oxidative stress during foraging. b. Bacteria in the gut The gut microbiota is of crucial importance for the host with considerable metabolic activity. Although Lepidoptera is one of the largest insect orders and a primary group of phytophagous agricultural pests, little is known about the microbes associated with them. This study has made efforts to comprehensively characterize the gut microbiota in different lepidopteran model organisms and provide some light into the potential metabolic functions of the core components inside the host. The gut microbiota profile of two lepidopteran species, Spodoptera littoralis and Helicoverpa armigera, is very similar regarding high abundant bacterial families that are dominated by Firmicutes and Proteobacteria. Different bacteria colonize specialized niches within the gut. A core community, consisting of Pantoea, Enterococci, Lactobacilli and Clostridia, is revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that the developmental stage and the diet have some impacts 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 as the indigenous community, which may contribute to the host fitness. Not only examined the composition and diversity of the gut microbiota, the components’ metabolic activity was also measured in Spodoptera littoralis by using a refined Pyro-SIP approach. With 13C glucose as the trophic link, Pyro-SIP revealed that the gut microbiota co-develops with the host, both metabolic activity and composition shifting throughout larval stages. Bacteria from the Clostridiaceae and Enterobacteriaceae families are particularly active in the early instar, which are well-known plant biomass degraders and likely the core functional populations linked to nutritional upgrading. Enterococcaceae is highly active in the late instar. On the grounds that Enterococci are maintained in a biofilm-like structure on the gut epithelium and that the isolated strains efficiently produce a mixture of antibiotic regents, Enterococcus is suggested to be a defensive mutualist which helps the host resist potentially harmful bacteria from outside. Notably, Enterococcus is vertically transmitted from eggs. This pilot study shows that Pyro-SIP can rapidly gain insight into the microbiota’s metabolic activity with high resolution and high precision, which sets the stage for future studies on the targeted metabolic pathway, for instance, labeling plant defense compounds to assess active bacteria involved in the host detoxification process. With the development of such new approaches, the role of lepidopteran gut microbiota will become more apparent than currently. Because of the simplicity of gut and the well-defined gut microbiota, cotton leafworm provides an excellent naturally-occurring model in which to study the complex digestive-tract microbial symbiosis and furthermore the multitrophic herbivore-microbe-plant interaction. A better understanding of the insect microbiology and transforming this knowledge to manage herbivorous pests in general will secure our food supply and economics.