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

Biodiversity and activity of the gut microbiota across the life history of the insect herbivore Spodoptera littoralis

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

Quantitative proteomic analysis of germination of Nosema bombycis spores under extremely alkaline conditions

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH >10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for Microsporidia. Here we show, using an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated respectively by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analyses in vitro and in vivo. Furthermore the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape of N. bombycis spores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general

CRISPR/Cas9–Mediated Genome Editing for <i>Pseudomonas fulva</i>, a Novel <i>Pseudomonas</i> Species with Clinical, Animal, and Plant–Associated Isolates

As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time–consuming and laborious endeavor. Here, by using the CRISPR–Cas system, a versatile gene–editing tool with a two–plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic–related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked–in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species

Data-driven prediction of contract failure of public-private partnership projects

The public-private partnership (PPP) has been adopted by many governments in developing countries to provide better public services. However, PPP projects have a high risk of contract failure. To proactively predict PPP contract failure and obtain the most significant failure factors from a quantitative perspective, this research compared the performance of different combinations of machine learning models and data-balancing techniques. Forty-three project-specific and country-specific factors were examined, and the top 15 were chosen for the transportation, water and sewer, and energy sectors. The results show that the selected model can forecast contract failure with a recall of 75.9%, 73.3%, and 76.2%, respectively. This study showed the effectiveness and applicability of machine learning in predicting PPP contract failure. The results can facilitate decision making by forecasting the probability of PPP contract failure in the early planning stage

Environmental mitigation of sludge combustion via two opposite modifying strategies: kinetics and stabilization effect

Nowadays, environmental mitigation remains a key issue challenging sludge combustion toward its clean utilization. Herein, two opposite additives, CaO and kaolin, representing two different strategies, were employed for environmental mitigation of sludge combustion. An advanced TG-MS (thermogravimetric-mass spectrometer) system was used. The online MS analysis indicated that kaolin remarkably decreased the release of NO whereas the fixation effect of CaO on SO was more pronounced. Furthermore, the experiments using a fixed tube furnace proved that overall kaolin had a better role controlling volatility of trace elements (Cr, Ni, Cu, Zn, Pb, As and Ba) than CaO and we found that the fixation mechanism of CaO changed at 750 °C, whereas that of kaolin changed at 500 °C. Then the kinetics of the various stages during sludge combustion were investigated. We found that low temperature stages were along with the formation of new phases, whereas, at high temperatures, the diffusion could be rate-controlling step. The present study thus provides not only a deep understanding of sludge combustion mechanisms but also two effective modifying strategies toward the environmental mitigation of sludge combustion

Colonization of the Intestinal Tract of the Polyphagous Pest Spodoptera littoralis with the GFP-Tagged Indigenous Gut Bacterium Enterococcus mundtii

The alkaline gut of Lepidopterans plays a crucial role in shaping communities of bacteria. Enterococcus mundtii has emerged as one of the predominant gut microorganisms in the gastrointestinal tract of the major agricultural pest, Spodoptera littoralis. Therefore, it was selected as a model bacterium to study its adaptation to harsh alkaline gut conditions in its host insect throughout different stages of development (larvae, pupae, adults, and eggs). To date, the mechanism of bacterial survival in insects' intestinal tract has been unknown. Therefore, we have engineered a GFP-tagged species of bacteria, E. mundtii, to track how it colonizes the intestine of S. littoralis. Three promoters of different strengths were used to control the expression of GFP in E. mundtii. The promoter ermB was the most effective, exhibiting the highest GFP fluorescence intensity, and hence was chosen as our main construct. Our data show that the engineered fluorescent bacteria survived and proliferated in the intestinal tract of the insect at all life stages for up to the second generation following ingestion

Distributed robust operation strategy of multi‐microgrid based on peer‐to‐peer multi‐energy trading

Abstract In microgrid (MG) systems, traditional centralised energy trading models can lead to issues such as low energy efficiency due to unstable energy supply and lack of flexibility. Peer‐to‐peer (P2P) trading models have been widely used due to their advantages in promoting the sustainable development of renewable energy and reducing energy trading costs. However, P2P multi‐energy trading requires mutual agreements between two microgrids (MGs), and the uncertainties of renewable energy and load affects energy supply security. To address these issues, this article proposed a distributed robust operation strategy based on P2P multi‐energy trading for multi‐microgrid (MMG) systems. Firstly, a two‐stage robust optimisation (TRO) method was adopted to consider the uncertainties of P2P multi‐energy trading between MGs, which reduced the conservatism of robust optimisation (RO). Secondly, a TRO model for P2P multi‐energy trading among MGs was established based on the Nash bargaining theory, where each MG negotiates with others based on their energy contributions in the cooperation. Additionally, a distributed algorithm was used to protect the privacy of each MG. Finally, the simulation results based on three MGs showed that the proposed approach can achieve a fair distribution of cooperative interests and effectively promote cooperation among MGs