Data processing solutions to render metabolomics more quantitative: case studies in food and clinical metabolomics using Metabox 2.0.

In classic semiquantitative metabolomics, metabolite intensities are affected by biological factors and other unwanted variations. A systematic evaluation of the data processing methods is crucial to identify adequate processing procedures for a given experimental setup. Current comparative studies are mostly focused on peak area data but not on absolute concentrations. In this study, we evaluated data processing methods to produce outputs that were most similar to the corresponding absolute quantified data. We examined the data distribution characteristics, fold difference patterns between 2 metabolites, and sample variance. We used 2 metabolomic datasets from a retail milk study and a lupus nephritis cohort as test cases. When studying the impact of data normalization, transformation, scaling, and combinations of these methods, we found that the cross-contribution compensating multiple standard normalization (ccmn) method, followed by square root data transformation, was most appropriate for a well-controlled study such as the milk study dataset. Regarding the lupus nephritis cohort study, only ccmn normalization could slightly improve the data quality of the noisy cohort. Since the assessment accounted for the resemblance between processed data and the corresponding absolute quantified data, our results denote a helpful guideline for processing metabolomic datasets within a similar context (food and clinical metabolomics). Finally, we introduce Metabox 2.0, which enables thorough analysis of metabolomic data, including data processing, biomarker analysis, integrative analysis, and data interpretation. It was successfully used to process and analyze the data in this study. An online web version is available at http://metsysbio.com/metabox.

Microbial community profiling and culturing reveal functional groups of bacteria associated with Thai commercial stingless worker bees (Tetragonula pagdeni).

Stingless bees play a crucial role in the environment and agriculture as they are effective pollinators. Furthermore, they can produce various products that can be exploited economically, such as propolis and honey. Despite their economic value, the knowledge of microbial community of stingless bees, and their roles on the bees' health, especially in Thailand, are in its infancy. This study aimed to investigate the composition and the functions of bacterial community associated with Tetragonula pagdeni stingless bees using culture-independent and culture-dependent approaches with emphasis on lactic acid bacteria. The culture-independent results showed that the dominant bacterial phyla were Firmicutes, Proteobacteria and Actinobacteria. The most abundant families were Lactobacillaceae and Halomonadaceae. Functional prediction indicated that the prevalent functions of bacterial communities were chemoheterotrophy and fermentation. In addition, the bacterial community might be able to biosynthesize amino acid and antimicrobial compounds. Further isolation and characterization resulted in isolates that belonged to the dominant taxa of the community and possessed potentially beneficial metabolic activity. This suggested that they are parts of the nutrient acquisition and host defense bacterial functional groups in Thai commercial stingless bees.

Bacterial Communities in Lanna Fermented Soybeans from Three Different Ethnolinguistic Groups in Northern Thailand.

Northern Thailand, the main part of the Lanna region, is home to a diverse range of ethnic groups, each with their own food and cultural heritage. The bacterial compositions in fermented soybean (FSB) products indigenous to three Lanna ethnolinguistic groups, including Karen, Lawa, and Shan, were investigated in this study. Bacterial DNA was extracted from the FSB samples and subjected to 16S rRNA gene sequencing using the Illumina sequencing platform. Metagenomic data showed that the predominant bacteria in all FSBs were members of the genus Bacillus (49.5-86.8%), and the Lawa FSB had the greatest bacterial diversity. The presence of genera Ignatzschineria, Yaniella, Atopostipes in the Karen and Lawa FSBs and Proteus in the Shan FSB might be indicators of food hygiene problems during processing. The network analysis predicted antagonistic effects of Bacillus against some indicator and pathogenic bacteria. The functional prediction revealed some potential functional properties of these FSBs. The presence of Bacillus in all FSBs and Vagococcus in the Shan FSB suggests that these FSBs could potentially be good sources of beneficial bacteria, and they should be conserved and promoted for health and food security reasons. However, food processing hygiene measures should be introduced and monitored to warrant their properties as health foods.

Construction of Light-Responsive Gene Regulatory Network for Growth, Development and Secondary Metabolite Production in Cordyceps militaris.

Cordyceps militaris is an edible fungus that produces many beneficial compounds, including cordycepin and carotenoid. In many fungi, growth, development and secondary metabolite production are controlled by crosstalk between light-signaling pathways and other regulatory cascades. However, little is known about the gene regulation upon light exposure in C. militaris. This study aims to construct a gene regulatory network (GRN) that responds to light in C. militaris. First, a genome-scale GRN was built based on transcription factor (TF)-target gene interactions predicted from the Regulatory Sequence Analysis Tools (RSAT). Then, a light-responsive GRN was extracted by integrating the transcriptomic data onto the genome-scale GRN. The light-responsive network contains 2689 genes and 6837 interactions. From the network, five TFs, Snf21 (CCM_04586), an AT-hook DNA-binding motif TF (CCM_08536), a homeobox TF (CCM_07504), a forkhead box protein L2 (CCM_02646) and a heat shock factor Hsf1 (CCM_05142), were identified as key regulators that co-regulate a large group of growth and developmental genes. The identified regulatory network and expression profiles from our analysis suggested how light may induce the growth and development of C. militaris into a sexual cycle. The light-mediated regulation also couples fungal development with cordycepin and carotenoid production. This study leads to an enhanced understanding of the light-responsive regulation of growth, development and secondary metabolite production in the fungi.

Impact of Nosema Disease and American Foulbrood on Gut Bacterial Communities of Honeybees Apis mellifera.

Honeybees, Apis mellifera, are important pollinators of many economically important crops. However, one of the reasons for their decline is pathogenic infection. Nosema disease and American foulbrood (AFB) disease are the most common bee pathogens that propagate in the gut of honeybees. This study investigated the impact of gut-propagating pathogens, including Nosema ceranae and Paenibacillus larvae, on bacterial communities in the gut of A. mellifera using 454-pyrosequencing. Pyrosequencing results showed that N. ceranae was implicated in the elimination of Serratia and the dramatic increase in Snodgrassella and Bartonella in adult bees' guts, while bacterial communities of P. larvae-infected larvae were not affected by the infection. The results indicated that only N. ceranae had an impact on some core bacteria in the gut of A. mellifera through increasing core gut bacteria, therefore leading to the induction of dysbiosis in the bees' gut.

Soil Metabarcoding Offers a New Tool for the Investigation and Hunting of Truffles in Northern Thailand.

Truffles (Tuber spp.) are well-known as edible ectomycorrhizal mushrooms, and some species are one of the most expensive foods in the world. During the fruiting process, truffles produce hypogeous ascocarps; a trained pig or dog is needed to locate the ascocarps under the ground. Truffles in northern Thailand have been recorded in association with Betulaalnoides and Carpinus poilanei. In this study, we investigated the soil mycobiota diversity of soil samples from both of these truffle host plants in native forests using environmental DNA metabarcoding to target the internal transcribed spacer 1 (ITS1) region of the rDNA gene for the purposes of investigation of truffle diversity and locating truffles during the non-fruiting phase. In this study, a total of 38 soil samples were collected from different locations. Of these, truffles had been found at three of these locations. Subsequently, a total of 1341 putative taxonomic units (OTUs) were obtained. The overall fungal community was dominated by phylum-level sequences assigned to Ascomycota (57.63%), Basidiomycota (37.26%), Blastocladiomycota (0.007%), Chytridiomycota (0.21%), Glomeromycota (0.01%), Kickxellomycota (0.01%), Mortierellomycota (2.08%), Mucoromycota (0.24%), Rozellomycota (0.01%), Zoopagomycota (0.003%), and unidentified (2.54%). The results revealed that six OTUs were determined to be representative and belonged to the genus Tuber. OTU162, OTU187, OTU447, and OTU530 belonged to T. thailandicum, T. lannaense, T. bomiense, and T. magnatum, whereas OTU105 and OTU720 were acknowledged as unrecognized Tuber species. From 38 locations, OTUs of truffles were found in 33 locations (including three previously known truffle locations). Thus, 30 collection sites were considered new locations for T. thailandicum, T. bomiense, and other unrecognized Tuber species. Interestingly, at 16 new locations, mature ascocarps of truffles that were undergoing the fruiting phase were located underground. All 16 truffle samples were identified as T. thailandicum based on morphological characteristics and molecular phylogenetic analysis. However, ascocarps of other truffle species were not found at the new OTUs representative locations. The knowledge gained from this study can be used to lead researchers to a better understanding of the occurrence of truffles using soil mycobiota diversity investigation. The outcomes of this study will be particularly beneficial with respect to the search and hunt for truffles without the need for trained animals. In addition, the findings of this study will be useful for the management and conservation of truffle habitats in northern Thailand.

Bacterial Communities in Three Parts of Intestinal Tracts of Carpenter Bees (Xylocopa tenuiscapa).

This study investigated different bacterial communities in three intestinal parts (foregut, midgut and hindgut) of Xylocopatenuiscapa to understand the roles of gut bacteria. Our phylogenetic analysis revealed that X. tenuiscapa is closely related to Xylocopa latipes. The 16S rRNA gene in the genomic DNA samples from the gut was examined by illumina (Solexa) and a total of 998 operational taxonomic unit (OTUs) clusters were found. Taxonomic classification identified 16 bacterial phyla and unclassified bacteria. The dominant bacteria taxa in the three parts of X. tenuiscapa gut were Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. In the foregut, Lactobacillales and Enterobacteriaceae were predominantly found. The population in the midgut was similar to that in the foregut, with the addition of Gilliamella, which was also abundant. The most dominant bacteria identified in the hindgut were similar to those in the midgut and Lactobacillales, Enterobacteriaceae, Gilliamella, Bifidobacteriaceae and Flavobacteriaceae appeared in abundance. Moreover, our results suggest that a community structure of bacteria in different parts of X. tenuiscapa's gut may be an important indicator of carpenter bees' health. This functional study of bacterial communities revealed significant differences among the three intestinal parts and is the first report of the gut bacteria structure in solitary bees.

Impact of Sacbrood Virus on Larval Microbiome of Apis mellifera and Apis cerana.

In this study, we examined the impact of Sacbrood virus (SBV), the cause of larval honeybee (Apis mellifera) death, producing a liquefied a larva sac, on the gut bacterial communities on two larval honeybee species, Apis mellifera and Apis cerana. SBV was added into a worker jelly food mixture and bee larvae were grafted into each of the treatment groups for 24 h before DNA/RNA extraction. Confirmation of SBV infection was achieved using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and visual symptomology. The 16S rDNA was sequenced by Illumina sequencing. The results showed the larvae were infected with SBV. The gut communities of infected A. cerana larvae exhibited a dramatic change compared with A. mellifera. In A. mellifera larvae, the Illumina sequencing revealed the proportion of Gilliamella, Snodgrassella and Fructobacillus was not significantly different, whereas in A. cerana, Gilliamella was significantly decreased (from 35.54% to 2.96%), however, with significant increase in Snodgrassella and Fructobacillus. The possibility of cross-infection should be further investigated.