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Front. Microbiol., 26 April 2017 | https://doi.org/10.3389/fmicb.2017.00663
Metagenomic Sequencing of Diamondback Moth Gut Microbiome Unveils Key Holobiont Adaptations for Herbivory
Xiaofeng Xia1,2,3,4, Geoff M. Gurr1,2,3,5, Liette Vasseur1,2,3,6, Dandan Zheng1,2,3,4, Huanzi Zhong7, Bingcai Qin7, Junhan Lin1,2,3,4, Yue Wang1,2,3,4, FengQin Song1,2,3,4, Yong Li1,2,3,4, Hailan Lin1,2,3,4 and Minsheng You1,2,3,4*†
1State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, China
2Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, China
3Key Laboratory of Integrated Pest Management for Fujian-Taiwan Crops, Ministry of Agriculture, Fuzhou, China
4Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou, China
5Graham Centre, Charles Sturt University, Orange, NSW, Australia
6Department of Biological Sciences, Brock University, St. Catharines, ON, Canada
7BGI-Shenzhen, Shenzhen, China
Herbivore specialists adapt to feed on a specific group of host plants by evolving various mechanisms to respond to plant defenses. Insects also possess complex gut microbiotas but their potential role in adaptation is poorly understood. Our previous study of the genome of diamondback moth, Plutella xylostella, revealed an intrinsic capacity to detoxify plant defense compounds, which is an important factor in its success as a pest. Here we expand on that work with a complete taxonomic and functional profile of the P. xylostella gut microbiota obtained by metagenomic sequencing. Gene enrichment in the metagenome, accompanied by functional identification, revealed an important role of specific gut bacteria in the breakdown of plant cell walls, detoxification of plant phenolics, and synthesis of amino acids. Microbes participating in these pathways mainly belonged to three highly abundant bacteria: Enterobacter cloacae, Enterobacter asburiae, and Carnobacterium maltaromaticum. Results show that while the gut microbial community may be complex, a small number of functionally active species can be disproportionally important. The presence of specific enzymes in the microbiota community, such as supporting amino acid synthesis, digestion and detoxification functions, demonstrates the beneficial interactions between P. xylostella and its gut microbiota. These interactions can be potential targets for manipulation to provide novel pest management approaches.
The eukaryote gut co-evolves with a microbiome that plays important roles in digestion, nutrition, development, and immune responses (Warnecke et al., 2007; Dong et al., 2009; Engel et al., 2012; Engel and Moran, 2013b). Animals are usually deficient in genes necessary for some key metabolic functions, such as the synthesis of essential amino acids and degradation of cellulose (Ley et al., 2008; Hansen and Moran, 2011; Zhu et al., 2011). These functions can be accomplished by their symbionts; such as the case for cellulose breakdown by the termite (Nasutitermes) gut bacteria (Warnecke et al., 2007), vitamin B12 biosynthesis and pectin degradation by honey bee (Apis mellifera) gut bacteria (Engel et al., 2012; Engel and Moran, 2013a), the production of amino acids by the pea aphid (Acyrthosiphon pisum) symbiont Buchnera aphidicola (Hansen and Moran, 2011), and fenitrothion insecticide degradation by the bean bug (Riptortus pedestris) symbiont Burkholderia (Kikuchi et al., 2012). The significance of these microbes within the gut has prompted the intestinal microbiota to be considered a separate organ of the host (Possemiers et al., 2011). Others have proposed that an animal with its microbiota forms a holobiont, which is subject to common selection pressures (Zilber-Rosenberg and Rosenberg, 2008).
The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the world's most destructive pests that has co-evolved with the cruciferous plants, such as cabbage, broccoli, and cauliflower (Talekar and Shelton, 1993). This lepidopteran is difficult to control because it has developed resistance to most classes of insecticides (Baxter et al., 2005; Zalucki et al., 2012). Many studies have focused on the bacterial diversity of the P. xylostella gut based on culturing and denaturing gradient gel electrophoresis (DGGE) methods (Indiragandhi et al., 2007; Lin et al., 2015a,b) but these only partially reveal the gut microbial diversity. Our previous study based on the sequencing of V6 region of 16S rRNA supplied a more complete understanding of the P. xylostella gut microbiota than earlier culturing-based studies (Xia et al., 2013). Yet a full understanding of bacterial community structure coupled with functional studies has been lacking.
Metagenomics is a powerful tool for comprehensive characterization of the gut microbial diversity as it avoids the need for culturing (Tringe et al., 2005). Studies based on metagenomics can provide full taxonomic profiles of the holobiont. The present study extended our previous research on the P. xylostella genome (You et al., 2013) by examining the hologenome, providing insights into the co-evolution of this herbivore's gut microbiota. In this work, we also have complemented the hologenome with functional information that underpins our understanding on the key holobiont adaptations for herbivory. We revealed that, despite the complexity of this microbiome, a small number of bacterial species of particular importance to key digestive and detoxification processes underpin the success of this globally significant pest.
Insect Rearing
A susceptible P. xylostella strain (hereafter, SS) was collected in July 2004 from a vegetable field in Fuzhou (26.08°N, 119.28°E), Fujian province, south eastern China. All necessary permits were obtained from the Institute of Plant Protection of the Fujian Academy of Agricultural Sciences. The larvae were fed on radish seedlings at 25 ± 2°C, 70–80% relative humidity and a 16 h light/8 h dark photoperiod without exposure to insecticides. Pupae were collected and transferred to a 500 mL plastic bottle where emerging adults were fed 10% honey solution to mate and lay eggs. Neonates were able to pass through perforations in these bottles and drop onto radish plants below.
Collecting P. xylostella Gut Contents and DNA Extraction
To collect the gut contents, 200 individuals each of 3rd-instar larvae, pupae, and adults were randomly sampled regardless of sex. The samples were surface-sterilized with 75% ethanol for 90 s and rinsed with sterilized-deionized water. After dissection, the gut contents were homogenized with 1 ml sterile deionized water and frozen at −80°C for DNA extraction. Total DNA from the larvae, pupae and adults samples was extracted using the QIAamp® DNA Stool Mini Kit (Gene Company Limited, Lee Chung Street, Hong Kong, P.R.C.) following the manufacturer's protocol. The DNA products were run on 1.0% agarose gels and recovered for library construction and sequencing.
DNA Library Construction and Sequencing
DNA library construction was performed following the manufacturer's instructions (Illumina, San Diego, California, U.S.). We constructed one paired-end (PE) library with insert size of 350 bp for each life stage sample. Then, high-throughput sequencing was performed using Illumina Hiseq 2,000 to obtain the metagenome data. The read length for each PE end was 100 bp. The raw reads that contained three or more "N" or adapter contamination were discarded. If there were continuous low-quality (Phred quality score <20) bases at the end of one read, these bases were trimmed from the read. The trimmed reads with lengths below 30 bp were discarded, and host contaminated reads were removed; the remaining effective reads were used for further analysis. For each sample, the effective reads were assembled to obtain long contigs by using SOAP de novo assembler (Li et al., 2010). Then, the contigs longer than 500 bp were used to predict ORFs by the Meta Gene program (Noguchi et al., 2006). Using ORFs longer than 100 bp from all samples, we constructed a non-redundant gene catalog and effective reads from each sample were aligned against it by SOAP2 (Li et al., 2009) using the criterion ≥90% identity. We translated the aligned results to gene relative abundance by counting the number of reads that mapped to the gene. Each gene in the gene catalog was aligned against NCBI NR database for taxonomy classification and against KEGG database for functional annotation by the BLASTP program with the criterion of expect value <10−5. Based on the annotation information of the gene catalog, we summed the relative abundances of the genes from the same taxon or same KO (KEGG ortholog group) to generate the taxonomic profile or KO functional profile. The raw sequences of gut microbiota of P. xylostella larvae, pupae, and adults were deposited to the SRA (Sequence Read Archive) database under accession number of SRP018285.
Biomass Degradation Experiments
Fifteen 4th-instar larvae were randomly sampled regardless of sex. The samples were surface-sterilized with 75% ethanol for 90 s and rinsed with sterilized-deionized water. After dissection with a sterilized scalpel, the gut contents were homogenized with 1 mL sterile deionized water. The gut content suspension was diluted to 10−4, 10−5, and 10−6, and 50 μL of the suspension was spotted onto the caboxymethyl cellulose (CMC), xylan, and pectin degrading enzyme isolation medium. The media for CMC, xylan, and pectin degrading enzyme isolations were: CPS1 (g/L): CMC-Na 10 g, peptone 1 g, (NH4)2SO4 4 g, NaCl 1 g, K2HPO4 2 g, MgSO4·7H2O 0.4 g, agar 20 g, pH 7.0; XPS1 (g/L): xylan 8 g, yeast powder 1 g, (NH4)2SO4 4 g, K2HPO4 2 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, agar 20 g, pH 7.0, and PPS1 (g/L): pectin 10 g, yeast powder 1 g, (NH4)2SO4 4 g, K2HPO4 1 g, KCl 0.5 g, MgSO4·7H2O 0.4 g, FeSO4 0.01 g, agar 20 g, pH 7.0. The plates were cultured at 30°C for 3 days. Bacteria grown on the plates were purified and identified by another identification medium [CPS2 (g/L): CMC-Na 5 g, (NH4)2SO4 2 g, NaCl 1 g, K2HPO4 2 g, MgSO4·7H2O 0.2 g, CaCl2 0.1 g, agar 20 g, pH 7.0; XPS2 (g/L): xylan 5 g, (NH4)2SO4 2 g, K2HPO4 2 g, NaCl 1 g, MgSO4·7H2O 0.2 g, agar 20 g, pH 7.0, and; PPS2 (g/L): pectin 5 g, (NH4)2SO4 2 g, K2HPO4 1 g, NaNO3 3 g, KCl 0.5 g, MgSO4·7H2O 0.2 g, CaCl2 0.15 g, FeSO4 0.01 g, agar 20 g, pH 7.0], which contained less substrate than the isolation medium. The potentially degrading bacteria grown on the plates (CPS2, XPS2, and PPS2) at 30°C for 7 days were used for enzyme activity identification. The CPS2 plates were dyed with 0.1% Congo red for 10 min, the plates were floated with 1 mol/L NaCl for 5 min, and CMC degrading enzyme activity was determined by the visible clearance zones. The XPS2 and PPS2 plates were dyed by 0.1% Lugel solution and Bromophenol blue for 5 min, respectively, to see whether clearance zones became visible.
Bacteria Identification
The representative bacteria that contained cellulose, xylan and pectin degrading activities were purified and named as DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6. Strains were cultured in LB medium (g/L): peptone 10 g, yeast powder 5 g, NaCl 10 g, pH 7.0. Genomic DNA of each strain was isolated by Rapid Bacterial Genomic DNA Isolation Kit (Sango biotech, China). To amplify 16S rRNA of each strain for bacterial identification, universal primers targeting the 16S rRNA, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were designed as previously described (Weisburg et al., 1991). The PCR was carried out in a total volume of 15 μL: H2O 10.5 μL, 10 × PCR ExTaq Buffer 1.5 μL, DNA template (100 ng/μL) 1.0 μL, 27F (10 mmol/L) 0.5 μL, 1492R (10 mmol/L) 0.5 μL, dNTP 1.0 μL, ExTaq (5 U/μL) 0.15 μL. After initial denaturation at 95°C for 5 min, amplification was performed using 30 cycles of 30 s at 95°C, 30 s at 56°C, 60 s at 72°C, followed by a final extension at 72°C for 10 min. Amplification products were then run on 1.0% agarose gels and purified, and the products were sequenced by ABI PRISM 3730. The 16S rRNA sequences were submitted to the NCBI GenBank (GenBank Accession Numbers: KT957438, KT957439, KT957440, KT957441, KT957442, and KT957443 which corresponding to the above strains of DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6, respectively).
The 16S rRNA sequences of DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6 were used as queries to do homolog search using the NCBI blast program. The homolog sequences were downloaded and aligned by clustalX1.83 with the default settings, phylogenetic analysis was performed by MEGA5.0 based on the Neighbor-Joining method, and 1,000 bootstrap replications were used to assess the phylogenetic tree.
Phenol Biodegradation Experiments
The P. xylostella gut microbiota were cultured at 30°C in sterilized mineral salt medium (MSM) (g/L): (NH4)2SO4, 0.4; K2HPO4, 0.4; KH2PO4, 0.2; MgSO4, 0.1; NaCl, 0.1; Fe2(SO4).H2O, 0.01; MnSO4.H2O, 0.01; for a 100 mg/L final concentration of filter-sterilized phenol as a carbon source, pH 7.5. The 100 mL phenol medium in a 250 mL conical flask with the bacteria was incubated in a rotary shaker at 150 rpm. Phenol degradation was monitored in the colorimetric assay according to established method (Ahmad et al., 2012). Phenol concentration was measured every 12 h for the total 24 h. The experiments were performed in triplicate. Data were analyzed by one-way ANOVA followed by LSD post-hoc test using IBM SPSS Statistics 19.
Compositional Diversity of the P. xylostella Gut Microbiota
Gut microbiota of P. xylostella larvae, pupae, and adults were sequenced. De novo assembly of 3.46 Gb effective reads resulted in 30,282 contigs, and 50,724 ORFs were predicted from the contigs, with a total length of 34.18 Mb (Supplementary Tables 1–3). Taxonomic profiling of the metagenome indicated that the P. xylostella gut microbiota was dominated by Proteobacteria (comprising 94.8, 99.1, and 99.7% in the gut of larva, pupa, and adult, respectively), followed by Firmicutes (2.35, 0.93, and 0.31% in larva, pupa, and adult), and to lesser extent, Cyanobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, and the archaea species Euryarchaeota (Figure 1A, Supplementary Table 4). The gut microbiota also contained a relatively small proportion of Eukaryota of the species of Basidiomycota (0.18% in larva, 0.0004% in pupa, and 0.001% in adult) and Ascomycota (1.31% in larva, 0.002% in pupa, and 0.01% in adult). Bacteria represented ~98% of the entire microbiota (Figure 1A, Supplementary Table 4).
Figure 1. Proportional composition of microbiota in the P. xylostella gut generated from the metagenomics data. Composition at the phylum level (A), and composition at the species level (B).
The present metagenomic study provided a complete taxonomic profile of the P. xylostella gut microbiota (Figure 1, Supplementary Figures 1–5). Overall, 10 phyla and 148 species of microorganisms were identified (Supplementary Table 4). The species diversity is consistent with our previous study of larvae, with 150 OTUs identified at the order level of the P. xylostella midgut microbiota based on the V6 region sequencing (Xia et al., 2013). The Enterobacteriales from Proteobacteria (88.5% in larva, 98.7% in pupa, and 99.0% in adult) dominated at the order level followed by Lactobacillales from Firmicutes (1.68% in larva, 0.85% in pupa, and 0.27% in adult). The most abundant species in Enterobacteriales were E. cloacae (39.2, 48.2, and 35.5% in larva, pupa, and adult, respectively) and E. asburiae (14.3, 12.6, and 25.0% in larva, pupa, and adult; Figure 1B, Supplementary Table 4). Although, the Proteobacteria were dominated by E. cloacae and E. asburiae, this phylum was represented in the microbiome by 100 species (Supplementary Table 4).
The comparison among the three developmental stages indicated that adults and pupa had similar diversity of gut microbiota, which was lower than the microbiota of larva (Figure 1, Supplementary Figures 1–5). The greater diversity of microbes in the larva compared with the pupa/adult may be the result of differences in the usual food sources among these life stages (plant tissue for larva vs. simple carbohydrates for the adults) and the need of larva to metabolize structurally complex carbohydrates and plant allelochemicals. Previous study based on bacterial culture and PCR-DGGE analysis also suggest that the abundance and diversity of bacteria in the P. xylostella gut are much higher in larva than that in pupa/adult (Lin et al., 2015b). Proteobacteria however were more abundant in the pupa and adult than in larva, corresponding with lower abundance of Firmicutes. At the species level, the abundance of E. asburiae was greater in the adult stage while E. cloacae was more abundant at the larval stage (Figure 1B). Anopheles gambiae also shows variation in gut bacterial communities with larva and pupa being similar but different from adults reflecting the change from detritus to blood feeding (Wang et al., 2011).
In order to validate the results of the composition of P. xylostella gut microbiota, a bar-coded pyrosequencing on the V3~V5 of 16S rRNA region in different P. xylostella developmental stages of the gut microbiota was carried out (see Supplementary Materials and Methods). A total of 47,872 effective reads from 9 samples with an average of 5,319 reads per sample were produced (Supplementary Table 5). Sixty-two OTUs were calculated based on the 97% cut-off (Supplementary Table 6), which was much less than the metagenomic sequencing and previous sequencing based on the V6 region by Illumina. The rarefaction curves suggested that the pupa and adult reached a plateau. However, the larva was far from reaching the plateau, which might be a reason of the low abundance of OTUs (Supplementary Figure 6). Alpha diversity analysis indicated that the composition of larval gut microbiota was much more complex than that of pupa and adult (Supplementary Table 7). Although, the OTUs were much fewer than in the metagenomics analysis, the main composition and structure were very similar with the metagenomics results. The phylum of Proteobacteria was the dominant taxon in the P. xylostella gut microbiota (from 70.42 to 97.44% in larva, 97.44 to 99.58% in pupa, and 99.64 to 100% in adult), followed by Firmicutes (from 2.45 to 29.48% in larva, 0.42 to 1% in pupa, and 0 to 0.36% in adult; Figure 2). More than 99% of long reads based on the bar-coded pyrosequencing could be identified to the family level (Supplementary Table 8). The Enterobacteriaceae (Phylum: Proteobacteria, Order: Enterobacteriales) dominated the gut ecology (from 57.89 to 97.29% in larva, 97.33 to 99.58% in pupa, and 99.61 to 99.86% in adult). Carnobacteriaceae (Phylum: Firmicutes, Order: Lactobacillales) was the second most abundant family (from 0.42 to 29.12% in larva, 0.42 to 1% in pupa, and 0 to 0.36% in adult; Supplementary Table 6). These results were consistent with the metagenomic analysis.
Figure 2. Proportional composition of microbiota in the P. xylostella gut generated from the 454 pyrosequencing data. Composition at the phylum level (A), and composition at the family level (B), Neighbor-Joining method phylogeny of OTUs (C). The phylogenetic tree revealed that the microbiota of P. xylostella gut were divided into six clusters corresponding with the phylum of Proteobacteria (Gammaproteobacteria was shown in blue, Betaproteobacteria with light yellow, and the alphaproteobacteria with green), Firmicutes (shown in purple), Actinobacteria (shown in dark yellow), and Bacteroidetes (shown in pink). Most of the sequences from the P. xylostella gut fell into the clusters of Proteobacteria and Firmicutes. Bootstrap values >50 were shown in the tree.
Results showing dominance of Proteobacteria and to a lower extend Firmicutes were consistent among the three methods we used in the study. Yun et al. (2014) report in a study of 218 insect species, a similar pattern where Proteobacteria and Firmicutes represent 82.8% of the total sequences. However, Lactobacillales and Vibrionales (Phylum Proteobacteria) were 10 times more abundant in our previous study based on the sequencing of V6 region of 16S rRNA (Xia et al., 2013) than the other two methods. Variation in abundance among the different sequencing methods may be due to differences in developmental stages and physiological conditions of the individuals (Yun et al., 2014; Mandal et al., 2015). The low abundance of Vibrionales detected in metagenomics, and even not detected by 454 pyrosequencing may be due to a very low abundance in P. xylostella gut environment compared to its high abundance in the previous study (Xia et al., 2013) caused by the enrichment of this order by the V6 amplification. It is possible that insect collection in different seasons or physiological health of the samples may result in different gut ecology (Wong et al., 2011; Yun et al., 2014). The method of bacterial DNA extraction also may have affected the structure of gut symbiont (Wagner Mackenzie et al., 2015), different sequencing strategies targeted different genes or different regions can also affect the result (Sun et al., 2013; Filippidou et al., 2015). In summary, there is a need to examine which research methods give more objective and real evaluation of gut microbial diversity.
Comparative analyses of the diversity of different gut microbiota have revealed that different insects host different gut microbiota. The honey bee midgut is dominated by Proteobacteria and Actinobacteria, and Snodgrassella is important (Engel et al., 2012). Termites contain Proteobacteria, Spirochaetes, and Fibrobacteres, the latter of which functioned in cellulose digestion (Hongoh et al., 2006; Warnecke et al., 2007), while the mosquito gut microbiome is dominated by Proteobacteria and Cyanobacteria (Wang et al., 2011). The environmental habitat, phylogeny of host, diet, and developmental stages all determined the insect gut bacterial diversity (Yun et al., 2014). Our finding underlined the importance of gut microbiota identity and composition in the evolutionary adaptation of insects to differing diets including difficult-to-digest and well-defended plant material (Hongoh et al., 2005).
Functional Diversity of the P. xylostella Gut Microbiota
MetaGene analysis revealed 15,046, 18,324, and 18,197 non-redundant protein-coding genes in the gut microbiota of the P. xylostella larva, pupa, and adult, respectively (Supplementary Table 3). The gene functions annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that metabolic function was the most abundant in the metagenome (representing 48.1, 47.9, and 47.9% of the total KO function in gut microbiota of the P. xylostella larva, pupa, and adult, respectively; Figure 3A). Further analysis showed the participation of gut bacteria in metabolic activities associated with glycans, carbohydrates, amino acids, vitamins, xenobiotics, and terpenoids, which are linked to digestion, nutrition, and detoxification (Figure 3B). The most enriched functions within these activities were carbohydrate metabolism (representing 10.5, 10.4, and 10.4% of the total KO function in larva, pupa, and adult, respectively) and amino acid metabolism (9.6% in larva, 9.4% in pupa, and 9.4%, in adult; Figure 3B). This indicated that gut microbiota might be involved in food digestion and nutrition. The Anoplophora glabripennis metagenome also shows enriched pathways involved in the metabolism of carbohydrates and amino acids, which play key roles on woody tissue digestion and nutrient synthesis (Scully et al., 2013). Interestingly, the different categories of KEGG ortholog in larva, pupa, and adult were very similar. This could be due to the large abundance of Proteobacteria in all developmental stages.
Figure 3. KEGG ortholog (KO) group annotation of the P. xylostella gut microbiota metagenome. Functional composition of the gut microbiota metagenome (A), and function of metabolism of the gut microbiota metagenome, the "Enzyme Families" in the figure represent the enzymes of "Peptidases" and "Protein kinases" that were detected in the DBM gut metagenome (B).
Brassicaceae, the exclusive food of P. xylostella, contains defense compounds such as glucosinolates, which are a deterrent for most insect species (Ratzka et al., 2002). P. xylostella has adapted to overcome the toxicity of these compounds (You et al., 2013). Xenobiotic biodegradation (representing 3.8, 3.7, and 3.7% of the total KO functions in larva, pupa, and adult, respectively) and terpenoid metabolism (2.0% in larva, and 1.9 in both pupa and adult; Figure 3B) suggested that the gut microbiota had adapted to the heavily defended diet of P. xylostella. KEGG ipath2 analysis showed that the gut microbiota metagenome exhibited more metabolic pathways than the P. xylostella genome with 18,071 genes (You et al., 2013; Supplementary Figure 7), potentially providing the host insect with advantages in digestion, nutrition, and overcoming toxicity of plant defense compounds.
Carbohydrate Breakdown Activity of the P. xylostella Gut Microbiota
The enzymatic degradation of cellulose and hemicellulose by animals can be difficult so its presence in the diet hinders digestion (Morrison et al., 2009; Suen et al., 2010; Zhu et al., 2011). Previous studies have revealed that some microbes possess a subset of enzymatic systems for hydrolysis of plant biomass (Morrison et al., 2009; Suen et al., 2010; Hess et al., 2011). Based on the Carbohydrate-Active Enzymes (CAZy) database (http://www.cazy.org; Lombard et al., 2014), a total of 2,469 genes from six families, which contained modules for catalyzing the breakdown or modification of carbohydrates and glycoconjugates, were detected in the P. xylostella gut microbiota. These were glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activities (AAs), and carbohydrate binding modules (CBMs) (Supplementary Tables 9, 10). GHs are a widespread group of enzymes important in the degradation of plant cell wall that hydrolyse the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety (Henrissat and Davies, 1997). Fifty-five different GH families were found in the P. xylostella metagenome. Among them, GH5 is linked to the activity of endoglucanase, and GH9 to the activities of endoglucanase and β-glucosidase (Lombard et al., 2014). The P. xylostella gut microbiota lacked debranching enzymes but had abundant oligosaccharide-degrading enzymes (Supplementary Tables 9, 10).
Comparative analysis of the profiles of GH families detected from the metagenomes of the termite hindgut (Warnecke et al., 2007), honey bee gut (Engel et al., 2012), beetle (A. glabripennis) gut (Scully et al., 2013), leaf-cutter ant garden (Suen et al., 2010), and panda gut (Zhu et al., 2011) revealed that GH5 cellulases were most abundant in the termite hindgut and the least in guts of honey bee and panda (Supplementary Table 10). Honey bees consume only pollen and do not require powerful cellulose degradation (Engel and Moran, 2013a). Though bamboo is rich in cellulose, the panda has a very low digestion efficiency, reflecting the low abundance and diversity of cellulose and hemicellulose digestion enzymes in its metagenome (Zhu et al., 2011). In contrast, cellulases were much more diverse in wood-feeding termites and A. glabripennis where endohemicellulases and debranching enzymes were also diverse and highly abundant. The high abundance and diversity of biomass digestion enzymes in the gut microbiota of these two species not only suggest their function in plant digestion but also indicate the possible co-evolution of the insect gut microbiome with diet (Warnecke et al., 2007; Scully et al., 2013). The overall abundance and diversity of the cellulases and endohemicellulases in the P. xylostella gut microbiota were at intermediate levels compared to those found in termites and honey bees (Supplementary Table 10). The Brassicaceae, exclusive food of P. xylostella, contains over 3,000 species (Hall et al., 2002) and is rich in cellulose and hemicellulose in cell walls. The high abundance of GH5 cellulases in the P. xylostella gut microbiota may contribute to its adaptive capacity to feed on these plants, specifically to maximize the extent to which these complex substrates are digested.
A series of enzymes associated with cellulose and hemicellulose hydrolysis was found to be encoded by C. maltaromaticum and Enterobacter living in the P. xylostella gut, including 1,4-beta-cellobiosidase, endoglucanase, and beta-glucosidase. Involved in xylan hydrolysis, 1,4-beta-xylosidase was encoded by Enterobacter, whilst pectinesterase and polygalacturonase for pectin biodegradation were encoded by both C. maltaromaticum and Enterobacter (Figures 4A–C, Supplementary Table 11). According to P. xylostella genomic data (You et al., 2013), none of these enzymes are encoded by the host insect genome. KEGG analysis also suggested the capacity of the P. xylostella gut microbiota to influence the starch and sucrose metabolic pathways (Supplementary Figure 8). These enzymes were widely involved in the metabolism of carbohydrate, which indicated their potential roles in the digestion of Brassicaceae tissue.
Figure 4. P. xylostella gut bacteria-mediated metabolism of biomass. The schematic of biodegradation of caboxymethyl cellulose (CMC) (A), xylan (B), and pectin (C) with red words beside the arrow lines representing the degradation enzymes identified from the metagenome, and experimentally validated degradation of CMC (D), pectin (E), and xylan (F) with Escherichia coli used as negative control (CK). CMC, pectin and xylan degradation is detected by the formation of clearance zones around bacterial lawns, respectively, the Escherichia coli as negative control was cultured in the center of plates. The numbers of 1–6 in (D–F) represented that the six strains (DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6) of CMC-, xylan-, and pectin-biodegrading bacteria isolated from P. xylostella gut. All these six strains were subjected to the genus of Enterobacter spp. The GenBank Accession Numbers: KT957438, KT957439, KT957440, KT957441, KT957442, and KT957443 were corresponding to the above strains of DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6, respectively. The clearance zones in the plates (D–F) produced by the six strains indicated that the ability of biomass degrading.
The ability of the six strains (DBMG1, DBMG2, DBMG3, DBMG4, DBMG5, and DBMG6) isolated from P. xylostella gut to degrade CMC, xylan, and pectin was confirmed by the formation of clearance zones around bacterial lawns (Figures 4D–F). The CMC is a cellulose derivative obtained by chemical modification of natural cellulose, the degradation ability of CMC indicated the activity of endoglucanase, which would be important for carbohydrate degradation, even cellulolytic activity (Hägerdal et al., 1978). The six strains were identified as genus Enterobacter by whole-length 16S rRNA and phylogenetic analysis (Supplementary Figure 9). The phylogenetic tree suggested that the six Enterobacter spp. exhibited different 16S rRNA identities and performed different biodegradation activities (Figures 4D–F, Supplementary Table 12). DBMG1 and DBMG3 formed an independent clade, and were more effective at biodegradation, especially for CMC and xylan, than the other clade containing DBMG2, DBMG4, DBMG5, and DBMG6 (Figures 4D–F, Supplementary Figure 9). In honey bee, genetic variation among different species of bacteria may reflect functional divergence within its gut (Engel et al., 2012). Our results suggest that within the P. xylostella gut, the most abundant species (Enterobacter spp.) contribute to plant cell wall degradation. This capacity enhances food utilization efficiency for the insect host (Warnecke et al., 2007).
Plant Defense Compound Detoxification Activity in the Gut Microbiota of P. xylostella
Herbivory requires the evolution of a broad range of mechanisms to not only digest dietary components such as plant cell walls but also to detoxify a range of plant defense compounds (Ratzka et al., 2002; Whiteman and Jander, 2010). Our previous study of the P. xylostella genome reveals the mechanism of detoxification of some plant defense compounds, particularly glucosinolates (You et al., 2013). Other compounds including phenolics are, however, abundant in Brassicacea species (Cartea et al., 2010) and comprise an important part of the plant's defense against herbivores. Phenolics are harmful to insects as toxic or anti-digestive compounds (Bi and Felton, 1995; Duffey and Stout, 1996; Mazid et al., 2011). It has been suggested that microorganisms can degrade phenol and catechol by both aerobic and anaerobic pathways (Basha et al., 2010).
In this study, we were able to identify the entire aerobic pathway for catechol degradation in the P. xylostella gut bacteria symbiont metagenome, including catechol 1,2-dioxygenase, muconate cycloisomerase, muconolactone D-isomerase, 3-oxoadipate enol-lactonase, 3-oxoadipate CoA-transferase, acetyl-CoA acyltransferase, 3-oxoadipyl-CoA thiolase, and 3-oxoadipate enol-lactonase/4-carboxymuconolactone decarboxylase (Figure 5A, Supplementary Table 13). All of these genes were encoded by Enterobacter (especially E. asburiae and E. cloacae). We believe that these two species confer protection to the host insect against certain plant defense compounds of Brassica plants. Further functional analytical experiments indicated that the P. xylostella gut bacteria could degrade the phenol significantly in vitro (Figure 5B).
Figure 5. P. xylostella gut bacteria-mediated biodegradation of phenols. Aerobe pathway of phenol biodegradation, enzymes beside the arrow lines are identified from the metagenome (A), and degradation efficiency at 12 and 24 h after treatment with colored histograms representing different treatments (B). Yellow column indicated that the phenol media without the presence of bacteria, and the purple indicated that the phenol media with the presence of P. xylostella gut bacteria, the stars ** over the columns within a graph denote significant differences (p < 0.01) between the different treatments.
Benzoate is a simple aromatic carboxylic acid that occurs naturally in many plants and serves as an intermediary for many plant secondary metabolites (Qualley et al., 2012). A series of enzymes participating in benzoate degradation were identified in the metagenome including benzoate 1,2-dioxygenase alpha subunit, benzoate 1,2-dioxygenase beta subunit, and benzoate-CoA ligase (Supplementary Figure 10, Supplementary Table 14). A major pathway for benzoate degradation had catechol as an intermediate step (Supplementary Figure 10). In addition, enzymes participating in the degradation of aromatic compounds, such as 2-[hydroxy(phenyl)methyl]-succinyl-CoA dehydrogenase BbsD subunit for toluene biodegradation, p-cumic alcohol dehydrogenase, p-cumate dioxygenase, and p-cumic aldehyde dehydrogenase for xylene, citronellol/citronellal dehydrogenase for geraniol, salicylate hydroxylase, and 2,6-dioxo-6-phenylhexa-3-enoate hydrolase for dioxin, and N-ethylmaleimide reductase, nitroreductase/dihydropteridine reductase, and nitroreductase for nitrotoluene, were detected in the P. xylostella gut microbiota (Supplementary Table 14). These aromatic compounds may affect the gut environment and modulate the microbiota (Lee et al., 2006; Xu et al., 2013). Thus, we speculate that the abundance of aromatic compound degradation enzymes in the P. xylostella gut microbiota may have led to co-benefits for P. xylostella and its gut microbiota.
Some plant phenolics, such as phenol, catechol, chlorogenic acid, and caffeic acid, are directly or indirectly toxic to herbivores by stimulating DNA degradation via the induction of reactive oxygen species (ROS) when they are oxidized (Moran et al., 1997; Schweigert et al., 2000, 2001). Plants wounded by insects can increase production of ROS, resulting in oxidative damage to the herbivore midgut (Bi and Felton, 1995). ROS have been shown to be toxic to larvae of several species of lepidopteran and their expressions must be carefully controlled (Ahmad et al., 1987; Ahmad and Pardini, 1990; Duffey and Stout, 1996). The concentration and conversion of ROS to nontoxic compounds can be regulated by superoxide dismutases (SODs), peroxidase, and catalases (Bandyopadhyay et al., 1999; DeJong et al., 2007; Mittapalli et al., 2007). Our study identified 11 SOD genes, 14 catalase genes and 29 peroxidase genes in P. xylostella gut bacterial symbionts (Supplementary Table 15). These genes were encoded by C. maltaromaticum and Enterobacter spp., especially E. asburiae and E. cloacae. These abundant species participate in the detoxification of ROS and can provide strong protection for the host insect. In addition, Enterobacter spp., especially E. asburiae and E. cloacae, also contained genes encoding carboxylesterase (COE) and glutathione S-transferase (GST) (Supplementary Table 16). These enzymes induce xenobiotic detoxification (Li et al., 2007; You et al., 2013). The GST family of Drosophila melanogaster can be induced under phenol exposure (Shen et al., 2003). Our results support the hypothesis that these microbiota enzymes have a potential ability to enhance resistance of P. xylostella gut to ROS.
Amino Acid Synthesis in the Gut Microbiota of P. xylostella
Animals cannot synthesize some essential amino acids and must therefore obtain them from food (Hansen and Moran, 2011). To compensate for host deficiency, host symbiont cooperation in producing essential amino acids is a common phenomenon in phloem-feeding insects (Baumann, 2005), for example, symbiotic bacteria of the pea aphid synthesize arginine (Hansen and Moran, 2011). Based on P. xylostella genome database (Tang et al., 2014), P. xylostella lacks the entire pathway for the synthesis of histidine (His) from phosphoribosyl pyrophosphate (PRPP) as well as the pathway for the synthesis of threonine (Thr) from aspartate (Asp). We found that the P. xylostella gut bacteria metagenome possessed the whole pathway for synthesis of His and Thr (Figure 6). Our results are significant as they suggest that P. xylostella-symbiont cooperates with its host to produce these lacking amino acids, thus having an important impact on host nutrition. The genes participating in the synthesis of those amino acids were found mainly in E. asburiae, E. cloacae, and C. maltaromaticum (Supplementary Table 17). Whilst the symbiotic bacteria of pea aphid are intracellular (Hansen and Moran, 2011), in P. xylostella, it is unclear whether these synthesized amino acids are secreted into the intestine for direct use by the host or are retained by the symbiotic bacteria and available to the host only on decomposition of the bacteria. Further research is needed to identify these pathways; nevertheless, our study opens an avenue for the study of nutrition interaction between P. xylostella and its gut bacteria.
Figure 6. Biosynthetic pathways of histidine and threonine by P. xylostella gut bacteria. The blue words above the arrow lines representing the enzymes of amino acid biosynthesis that identified from the metagenome.
Our study based on metagenomic analysis of P. xylostella gut microbiota using next-generation sequencing provides a detailed profile of taxonomic and functional information regarding the P. xylostella gut microbiota. We identified a series of enzymes encoded by bacteria that participate in food digestion, nutrition, and the detoxification of plant defense compounds, especially of phenolic and ROS detoxification. The results suggest that while the bacterial community in the P. xylostella gut is complex, the main species participating in those functions are few, with E. asburiae, E. cloacae, and C. maltaromaticum being the three most abundant species. Comparative analysis of the profiles of plant tissue degradation enzymes detected in different insects has revealed that the abundance and diversity of the digestion enzymes are linked to the diet of these insects. This highlights the complexity of the possible co-evolution of gut microbiota and the host insect to cope with the challenges posed by its diet. Although, there were some differences of the gut microbiota composition between larvae and pupae/adult in P. xylostella gut, the extent was much lower compared with some other lepidopteran insects, such as Heliconius erato (Hammer et al., 2014), Heliothis virescens (Staudacher et al., 2016), and Spodoptera littoralis (Chen et al., 2016). In these three lepidopteran insects, the gut microbiota greatly varies across different developmental stages. In contrast, we found that P. xylostella gut microbiota were much consistent through life stages and further the functions classified by KEGG were identical. This may be a unique phenomenon to P. xylostella leading to a new question: does the same set of bacteria perform different functions during host development? Future studies should include metatranscriptomics and metaproteomics to validate this phenomenon and thus clarify the expressions of functional genes of the gut in vivo.
Our research, combined with the previous P. xylostella genome studies, indicates that its gut microbe complements its own genome allowing the resulting holobiont to function as an evolutionarily adaptive herbivore and assuming the status as one of the world's most serious pests. Accordingly, information on the gut microbiota, particularly the bacterial species and functional groups that play a particularly important role for the host's success, offers scope for future studies to develop new pest management approaches that exploit novel targets of chemical, genetic and biological control for this and other insect pests.
XX and MY designed the research; XX, DZ, JL, HL, FS, and YL performed the research; XX, HZ, BQ, and YW analyzed the data; MY and XX contributed reagents/materials/analysis tools; XX, MY, GG, and LV wrote the paper; and MY, XX, GG, and LV revised the paper.
This work, including the efforts of MY was funded by the projects of the National Natural Science Foundation of China (Nos. 31230061 and 31320103922), XX was supported by the project of the National Natural Science Foundation of China (Nos. 31501639).
We are grateful for the Fellowship of Outstanding Ph.D. Students at Fujian Agriculture and Forestry University (1122Yb010). LV is supported by a Minjiang Scholar Fellowship and GG by a Thousand Talents Fellowship. We thank Qing Sun for assistance with preparing figures.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fmicb.2017.00663/full#supplementary-material
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Xia, X., Zheng, D., Zhong, H., Qin, B., Gurr, G. M., Vasseur, L., et al. (2013). DNA sequencing reveals the midgut microbiota of diamondback moth, Plutella xylostella (L.) and a possible relationship with insecticide resistance. PLoS ONE 8:e68852. doi: 10.1371/journal.pone.0068852
Xu, B., Xu, W., Yang, F., Li, J., Yang, Y., Tang, X., et al. (2013). Metagenomic analysis of the Pygmy loris fecal microbiome reveals unique functional capacity related to metabolism of aromatic compounds. PLoS ONE 8:e56565. doi: 10.1371/journal.pone.0056565
You, M., Yue, Z., He, W., Yang, X., Yang, G., Xie, M., et al. (2013). A heterozygous moth genome provides insights into herbivory and detoxification. Nat. Genet. 45, 220–225. doi: 10.1038/ng.2524
Yun, J. H., Roh, S. W., Whon, T. W., Jung, M. J., Kim, M. S., Park, D. S., et al. (2014). Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host. Appl. Environ. Microbiol. 80, 5254–5264. doi: 10.1128/AEM.01226-14
Zalucki, M. P., Shabbir, A., Silva, R., Adamson, D., Shu-Sheng, L., and Furlong, M. J. (2012). Estimating the economic cost of one of the world's major insect pests, Plutella xylostella (Lepidoptera: Plutellidae): just how long is a piece of string? J. Econ. Entomol. 105, 1115–1129. doi: 10.1603/EC12107
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Keywords: diamondback moth, bacteria diversity, gut symbiosis, carbohydrate biodegradation, plant defense, phenolic compounds, amino acids, reactive oxygen species
Citation: Xia X, Gurr GM, Vasseur L, Zheng D, Zhong H, Qin B, Lin J, Wang Y, Song F, Li Y, Lin H and You M (2017) Metagenomic Sequencing of Diamondback Moth Gut Microbiome Unveils Key Holobiont Adaptations for Herbivory. Front. Microbiol. 8:663. doi: 10.3389/fmicb.2017.00663
Received: 06 February 2017; Accepted: 31 March 2017;
Published: 26 April 2017.
Michael Thomas-Poulsen, University of Copenhagen, Denmark
Hong Yang, Central China Normal University, China
Yongqi Shao, Zhejiang University, China
Peter Kamp Busk, Technical University of Denmark, Denmark
Copyright © 2017 Xia, Gurr, Vasseur, Zheng, Zhong, Qin, Lin, Wang, Song, Li, Lin and You. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Minsheng You, msyou@iae.fjau.edu.cn
†Present Address: Minsheng You, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, China
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Inspirational and spiritual signs are an elegant way to bring more positivity to your home décor. This collection showcases a wide range of styles and color schemes, including pink, green, blue and many more. From whimsical fantasy scenes to butterflies, stars and books, there's something for every taste and occasion. Browse our spiritual and inspirational sign category to find something for your living space or to search for a unique motivational gift!


A select number of recordings from the Archive of Literature on Tape (AHLOT) are available for streaming online. The AHLOT is a collection of nearly 700 recordings of critically acclaimed writers from Iberia and Latin America together with Hispanic/Latino writers reading selections from their work.
about current Hispanic Members, essays, and images of each individual Member, including rare photos.
the uniforms of the Army, Navy, Air Force, and Marines.
The exhibition 1492: An Ongoing Voyage describes both pre- and post-contact America, as well as the Mediterranean world at the same time.
preparation of a spectacular and ornate map of what was then referred to as the 4th part of the world, America.
which can be found in the Library's collections.
and goods found in the New Word during his voyage of 1514.
artifacts that make up the Jay I. Kislak Collection at the Library of Congress.
of Hispanic Americans who have positively influenced and enriched our nation and society.
Mexico, ten years after the Spanish conquest in 1521.
Drake's voyages throughout the then Spanish territory of the Americas.
the colonial period, 1492 to 1819.
Mexico and the United States during the Mexican Revolution.
including the coast of present-day California, were discovered and mapped.
first-person accounts, political writings, and histories drawn from the LC's General Collections.
Multi-format English-Spanish digital library site that explores the interactions between Spain and the United States in America from the fifteenth to the early nineteenth centuries.
A presentation of resources and documents about the Spanish-American War, the period before the war, and some of the fascinating people who participated in the fighting or commented about it.


One of my late Fall traditions of the last few years has been planting Paperwhites for the upcoming Holidays. Each year I look forward to their wonderful pristine white blooms filling the darkening days of Fall and Winter. I've collected quite a few vintage "containers" in the last few years, so part of the fun of planting the bulbs is digging out my stash and deciding which ones to use.
I unbagged my bulbs and have them in an old strawberry flat with boxes that I've whitewashed. I just enjoy looking at them, while they wait their turn to be planted.
so these will be just the first of that inspiration coming up.
As you can see, my helper is here.
Now if I can just keep her away from them.
A McCoy matte vase, an old chipped sugar bowl, and a new find, which is a French measuring pitcher, are readied for my first planting.
I use pea gravel as a base, then just a little potting mix. Just enough to cover the bottom 1/3 of the bulb. Too much water or soil will cause them to rot. In a day or two I'll sprinkle moss on top, which helps hold in the moisture.
so they can have some good light to get started.
the kiddos love them too.
and I love to see them grow each day!
Oh, I need to get some!
I have never planted paperwhites. Not sure if we have enough light in Minnesota in November and December.
On my blog I like to talk about seasonal and monthly traditions. I would love to start this tradition myself. I think I will do it this week! It will be the start to many years.
these will be amazing, I love your choice of containers as well.I don't like the smell of paper whites so I planted some daffodils, mini daffs, and should be blooming by christmas.
I love paperwhites too! I'll be using them in a centerpiece this year for my Christmas Charity House. Your vintage pots are great too.
Every year I say that I am going to do this and never have. I'm going out tomorrow and buy some and plant them. Thanks for the instructions and the inspiration.
Are you sure we were not separated at birth! I'm posting about paperwhites tomorrow! I love how they smell. Your white planters and pots are going to be real stunners when the flowers bloom!
Every year I plan on doing this and every year I miss the chance. I do have Star of Bethlehem in pots maybe I can force them indoors. I love the planters.
Debra, now you've inspired me to try again. I got some at a "big box store" last year and they bloomed but had an AWFUL smell..sort of plastic-y. I looked at one of your older posts and saw that you use the Ziva variety. I'll see if I can find some locally. Thanks for the inspiration.
I planted some last year a bit too late and they bloomed after New Years! Thanks for the reminder to start now. I love your pretty containers!
Thank you! I just bought 8 bulbs to try--now I need a few more! I didn't realize they would be that easy to plant--going to have my little one help.
I just finished planting my paperwhites for the holidays a couple weeks ago and they are sprouting nicely! Love your French measuring pitcher as a planter.
I don't have a green thumb when it comes to houseplants, but you've made it look so easy I'm going to get me some today and give it a try. Lots of fab inspiration on your Pinterest board. Thanks!!
Thanks so much for sharing this. Love your choice of containers.
Thanks for the reminder that I need to get mine started as well!!!
Great idea Debra! I think some paperwhites are just what I need. I love the gorgeous containers you used.
I was just thinking of doing some paperwhites. I'm so glad you shared your vintage containers. I'm going to make a few for family to take as they leave on Thanksgiving night. Miss you! Trying to get back into the swing of things again. Hope all is well.
Wanting to try my hand at planting "paperwhites" this year. Most I've seen seem to be in water, but I think your idea of potting soil sounds more correct! Tomorrow is "errand day" On Crooked Creek...so, guess I'd better get them added to my list!!! Thanks for the tips for planting!
I have some planted by the back gate that I get to enjoy around my December birthday. I look forward to trying the alcohol water to make some shorter indoor blooms! I look forward to following your future posts.
Ok, if you promise I can't mess this up I will give it a try. My thumb is not that green and often things don't grow well for me. I would love for you to link this up to my Fall into Fall party going on right now. Hope to see you there.
OK! You have convinced me! I am definitely doing this! I adore Paperwhites, and have wanted to grow some for awhile!! Thanks for the encouragement, Debra! You rock! Sending you an email!
OH! And thank you so much for joining the Blissful Whites Wednesday party!!
is this the right time to plant them?? I thought maybe you would have to do it earlier??
going to your pin board to look around.
I can't wait to see them bloom Debra! I also always love seeing your helper! Thanx for coming to THT. Hope your week is going well.
Thanks for the reminder Debra. I always intend to plant them for Christmas, but usually remember too late to actually do it! Now...where on earth did I put those bulbs?
I'm planting Sunday! Your containers are beautiful! I'm your newest follower!
Jody introduced me to you!
This is something I have been wanting do. Thanks for the inspiration and for the nudge! Love that French measuring cup too.
I really should do some planting. Great photos Debra.
I was just thinking today that I needed to get my Paperwhites. I plant them every year. Have for as long as I can remember. Although, the older I get the more I can't remember. lol! Planning on planting them in my different pieces of vintage milk glass this year. Love all of your fun containers. I had a gorgeous Amaryllis last year. I cut it off and stuck in in my cold storage just to see what would happen. The other day I noticed that it was growing beautifully. Thanks for sharing with Share Your Cup.
They will look lovely! Thanks for linking up with HSH!
Can't wait to see them in full bloom Debra. I'm sure they will be very pretty in their gorgeous containers.


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All About Crops
Water & the Environment
For more Info on CA Ag Today Radio
Author: Patrick Cavanaugh
CaWater4All Petition at World Ag Expo
Aubrey Bettencourt, executive director of the California Water Alliance, was at the 2016 World Ag Expo collecting signatures for the CaWater4All Water Priorities Ballot Initiative petition to get on the November ballot. The ballot initiative would reallocate High-Speed Rail funds for water storage projects in the state of California. Video produced and edited by Charmayne Hefley.
Leave a comment on CaWater4All Petition at World Ag Expo
Youth in California Cattle
By Charmayne Hefley, Associate Editor
Statistically, it appears millennials aren't considering agriculture as a career path. The USDA's "2007 Census of Agriculture: Farmers by Age," reports the average age of cattle ranchers was 57.8 years old. Malorie Bankhead, director of communications for the California Cattlemen's Association (CCA) and a millennial herself, said, "Young people in the beef industry have a really unique opportunity to get involved in something called the Young Cattlemen's Committee (YCC), the young affiliate of our California Cattlemen's Association.
Young Cattlemen's Committee (YCC)
Bankhead explained, "There are four college chapters: Fresno State, Chico State, Cal Poly and UC Davis. We don't discourage membership from high school students or even folks younger than that who are interested in getting involved. The membership is $25 per year, and with that, you're afforded a wealth of opportunity to become involved. We have a fairly robust scholarship program available to YCC members where we interview up-and-coming leaders in the beef industry who are focusing academically and extracurricularly on the beef industry, with the career goal to reenter the beef industry."
Bankhead said the Young Cattlemen on the Capitol event, set for April 5, "is another opportunity for young people in the industry—specifically YCC members—to come to the CCA office in Sacramento to to learn from our staff about the current hot topics in the beef industry affecting ranchers. We turn to discussions with those folks and each other and build your network in the beef industry. We also tour the Capitol, visit some legislators and network with them on issues impacting the beef industry." Registration information is forthcoming on the CCA website under the YCC tab.
Author Patrick CavanaughPosted on February 15, 2016 May 31, 2016 Categories Know the issues, Livestock & Poultry, Dairy and Cattle, Trade and Marketing, Law/Politics, University of California, Davis, United States Department of Agriculture (USDA), Events and Training, Government and Agencies, Livestock & Poultry, California State University, Fresno, California Polytechnic State University (Cal Poly), Education, Politics and Law, California Cattlemen's Association, California Agriculture News, newsTags: Chico State Malorie Bankhead Young Cattlemen's Committee (YCC) USDA 2007 Census of Agriculture: Farmers by Age Young Cattlemen on the Capitol Sacramento CA Youth in California CattleLeave a comment on Youth in California Cattle
Valley's Gold Prepares for Fourth Season
Currently in the midst of season three, Valley's Gold, a weekly television series focusing on agriculture in the Central Valley, is scheduled to continue for a fourth season.
Ryan Jacobsen, the series' host and Fresno County Farm Bureau CEO, is surprised but pleased by the show's success. "I guess, just by the blessings of being able to do what we do and show this incredible industry, it's resonating with folks," Jacobsen said. "They love the show and the opportunities this show has given us to be able to show how our food is produced—not the romanticized view—but the view of truly how it's done and the people who come together to make this industry drive. It's just been a phenomenal opportunity to share Ag's story."
Jacobsen said California's diversity has allowed the show, brought to viewers by Fresno County Farm Bureau and ValleyPBS, to continue with consistent, original content. "You would think after that many seasons we would run out of crops," Jacobsen said, "but because of this phenomenal Valley and this phenomenal state, we're finding more and more stories. The more we dig, the more we find and the more we're able to share the wealth of what we produce here."
Sponsored by GAR Tootelian, BRANDT and Meyers Water Bank, the show airs on Wednesdays at 7pm PST and is re-broadcasted on the weekends, Saturday at 6:30pm and Sunday at 10:30am.
Author Patrick CavanaughPosted on February 11, 2016 June 3, 2016 Categories Get Educated, Know the issues, Meet the People, Health, Safety & Nutrition, Data, Statistics, Events and Training, Science and Research, Education, Produce, California Agriculture News, Media Promotions, news, San Joaquin ValleyTags: Ryan Jacobsen Gar Tootelian Fresno County Farm Bureau Valley's Gold ValleyPBS BRANDT Meyers Water BankLeave a comment on Valley's Gold Prepares for Fourth Season
Vertical Integration and Grape Flavors
Vertical integration, the combination in one company of two or more stages of production normally operated by separate companies, has helped lead to success in many facets of the agriculture industry. Harold McClarty, who founded HMC Farms with Mike Jensen, said without vertical integration, his farm would not be able to survive or have nearly the same level of success. "We would not be in stone fruit if we were not vertically integrated," McClarty explained. "There are three profit centers: the growing of it, the packing of it and the selling of it. We have all three of those profit centers. Without them we would not be able to survive the tough years."
Cotton Candy grape clamshell (Photo source: Grapery)
McClarty also said the grape industry has made significant strides in flavor profiles in the marketplace. One popular flavor is Cotton Candy, which is grown and sold by Grapery, a company founded by Jack Pandol in 1996. Grapery also grows and sells Gum Drop grapes, Moon Drop grapes, Flavor Pops grapes and more.
The increase in unique flavors, according to McClarty, has helped grapes become a popular snack. "There's been a revolution in the varieties of grapes grown just over the last five to seven years," McClarty said. "These new varieties have revolutionized grapes and enabled us to produce more on less.
To evaluate a grape, McClarty suggests you "look at a berry's color, crispness and sweetness; these are the things that make a grape taste delicious. It really is a revolution, and we haven't seen it in the California marketplace as much as we have in the rest of the United States. The retail chains in California just haven't embraced it as well as some of the really good supermarkets on the East Coast and in the Southeast," noted McClarty.
Author Patrick CavanaughPosted on February 9, 2016 May 31, 2016 Categories All About Crops, Know the issues, Health, Safety & Nutrition, Grapes, Trade and Marketing, Table Grapes, Data, Statistics, Science and Research, Floriculture/Horticulture, California Table Grape Commission, California Agriculture News, newsTags: HMC Farms Harold McClarty Mike Jensen profit center Jack Pandol cotton candy grape Gum Drop grapes Moon Drop grapes Flavor Pops grapes Vertical Integration and Grape FlavorsLeave a comment on Vertical Integration and Grape Flavors
Joel Nelsen on New Cases of HLB in San Gabriel
On Wednesday of last week, two more trees, an orange and a kumquat, were found infected with the citrus-deadly Huanglongbing (HLB) disease in the core of San Gabriel. The trees are located on separate properties. One has been removed, and the other is awaiting the owner's permission for removal. Last summer, 10 diseased trees were confirmed in the San Gabriel area, and a quarantine, already in place since then, will not need to be expanded.
Joel Nelsen, president of California Citrus Mutual, commented on the HLB discovery, "The two trees were found in the core area, pretty adjacent to the others that were found in San Gabriel," Nelsen said. The finding "is a result of the program and its intensity in continuing to look for positive HLB finds. Unfortunately, these two trees were positive. The Asian Citrus Psyllids (ACP) in the area have been treated. One tree has already been pulled. The other homeowner wasn't home so we're hoping for cooperation there. It's not positive, but again it shows that the program is trying to find HLB before it finds the commercial citrus industry."
Author Patrick CavanaughPosted on February 8, 2016 May 31, 2016 Categories Know the issues, Meet the People, Pests & Diseases, Invasive Species, Citrus, Data, Statistics, Asian Citrus Psyllid (ACP), Oranges, California Department of Food and Agriculture, Crop Protection, Huanglongbing (HLB), Quarantine, Kumquats, Regulations, National Agriculture News, Pests and Diseases, Science and Research, California Department of Pesticide Regulation, California Citrus Mutual, California Association of Pest Control Advisors (CAPCA), California Agriculture News, news, San Joaquin ValleyTags: Joel NelsenLeave a comment on Joel Nelsen on New Cases of HLB in San Gabriel
Agriculture Detector K-9 on Duty!
California Ag Today's associate broadcaster Charmayne Hefley recently asked Soya, a mixed-breed agriculture detector K-9 (canine) with big responsibilities, and Samantha Tomlinson, Soya's handler with the Fresno County Ag Commissioner's office, what type of dog Soya is.
ST: We don't know exactly what she is for sure, but we're thinking about getting her DNA-tested someday. We're thinking she's a lab-border-collie.
Samantha Tomlinson, Soya and Charmayne Hefley
CAT: How long have you and Soya been together?
ST: We have been matched up since mid-July when we went through the training program in Georgia through the National Detector Dog Training Center.
CAT: What is Soya able to do for the Ag Commissioner's office?
ST: Soya smells out parcels for potential plant material and she alerts us [to suspicious ones] by scratching. We check to see if [the material] has been properly certified and if it's good to go.
CAT: What are some of her recent detections?
ST: She can detect a number of things. She was initially trained on five scents: mango, stone fruit, apple, guava, and citrus. From there, through scent association, she's been able to find a number of additional agricultural materials, including avocados, blueberries, nuts, soil, cut flowers; anything that is plant material, Soya can find.
CAT: What region does Soya cover?
ST: Right now we've been covering only Fresno County because we still are in what we call the "acclimation phase," as she's still new. We've been working at FedEx and UPS, but we'll broaden our horizons eventually and we'll be in the post office, GSO, OnTrac, any service that ships parcels."
CAT: How important is it for the agricultural industry to have dogs like Soya working for it?
ST: Well Soya and I are considered a "first line of defense" for California's multi-billion dollar ag industry. She is in the facilities checking boxes sent from potentially quarantined areas from within the state and from outside the state for materials that may contain any pests or diseases that could prove detrimental to California agriculture.
CAT: People may not know when they're shipping certain items—certain plant materials—from one county to the next that the destination county may have a quarantine in place. How do people properly ship plant material?
ST: Every county is actually different. If you are thinking of shipping some of your backyard fruits to your nephew or grandson, I would contact your local ag commissioner and make sure there are not any quarantines in place for both the county you're shipping from and the county you're shipping to. In these facilities, we look for boxes to be properly labeled with the growing origin and we inspect thye contents inside. Depending on what is inside, where it's grown and where it's going, we act accordingly.
Author Patrick CavanaughPosted on February 5, 2016 May 31, 2016 Categories Know the issues, Meet the People, Ag Crime, Law/Politics, Citrus, Avocados, Quarantine, Regulations, Food Safety Modernization Act, Apples, Blueberries, Education, Floriculture/Horticulture, Specialty Crops, Tree Fruit, Tree Nuts, California Agriculture NewsTags: soil dogs Soya Samantha Tomlinson Soya's handler with the Fresno County Ag Commissioner canine handler National Detector Dog Training Center mango guava agriculture detector K-9 agriculture detectiveLeave a comment on Agriculture Detector K-9 on Duty!
California Weed Science Society Meeting
The California Weed Science Society (CWSS) held their annual meeting in Sacramento from January 13-15, 2016. The meeting fostered collaboration between Pest Control Advisors (PCAs) and farmers as they gathered to learn the newest innovations in weed science.
John Roncoroni, weed science farm advisor for the UC Cooperative Extension in Napa County, as well as the outgoing CWSS president, said four Fresno State students gave presentations at the meeting on their research. "We've had really great student participation—the amount of student scholarships we've given is up this year, the posters, the students and our attendance this year is up," Roncoroni said. Pre-registration was about 530 people.
Roncoroni suspects that rain pushed people to attend, "because—with that rain—we're looking at more weeds this year. So people are looking for the newest information on weeds. This year's conference really has done a really good job of putting that information together. Kate Walker, our program chair, has really done a fine job of putting together a great program."
Kate Walker, technical service representative for BASF Corporation, is also the new, incoming CWSS president.
California Weed Science Society (CWSS)
The CWSS recently updated its published textbook, Principles of Weed Control, 4th Edition, that focuses on the applied aspects of weed control. The purpose of this textbook is to provide access to the fundamental principles and concepts of weed management in California. The book is designed for use at the college level by students who have an interest in pursuing a plant science or associated background of course work. It is also a useful resource for individuals studying to become PCAs and applicators or for consultants who work in weed science. For more information, go to the CWSS website Publications page.
Author Patrick CavanaughPosted on February 5, 2016 May 31, 2016 Categories Get Educated, Know the issues, Pests & Diseases, Health, Safety & Nutrition, UC Cooperative Extension, Data, Statistics, Weather, Crop Protection, Climate, Environment, Events and Training, California State University, Fresno, Pests and Diseases, Science and Research, Education, California Department of Pesticide Regulation, Weeds, California Association of Pest Control Advisors (CAPCA), California Agriculture News, newsTags: Napa County Kate Walker Pest Control Advisors (PCAs) John Roncoroni weed science farm advisor California Weed Science Society (CWSS)Leave a comment on California Weed Science Society Meeting
Dogs Keep Agriculture Safe
Meet Soya, an agriculture detector K-9, and her handler, Samantha Tomlinson, who works for the Fresno County Ag Commissioner's office. Tomlinson and Soya work as the first line of defense to protect California's agriculture industry from potential pests and diseases that can be shipped in packages. Charmayne Hefley reports. Story by Charmayne Hefley and Patrick Cavanaugh.
Leave a comment on Dogs Keep Agriculture Safe
California Water Priorities Ballot Measure
Petitioning for California Water Priorities Ballot Measure Kicks off Friday in Fresno
California Water Alliance (CalWA) executive director Aubrey Bettencourt announced TODAY that a petition drive for the California Water Priorities Ballot Measure will take place Friday, January 29, 2016, at KMJ Studios, 1071 W. Shaw Avenue in Fresno, from 2-6 pm.
Earlier this week, the proposed ballot measure was approved by Attorney General Kamala Harris, opening the door to begin signature-gathering efforts with initiative supporters. Since the measure's approval, CalWA has received an overwhelming amount of support and requests for petitions to circulate.
Electronic copies of the petition are not available, as use of them would disqualify the signatures gathered. Requests for petitions may be made online at www.CaWater4All.com. CalWA will begin mailing petitions on Friday, January 29.
A second signature drive is being scheduled February 9-11, 2016, at the World Ag Expo in Tulare. More information on this event will be available on the CaWater4All website.
The California Water Alliance is a statewide nonprofit organization dedicated to raising awareness about the nature of water and promoting long-term, sustainable solutions that meet the health and security needs of families, cities, businesses, farmers and the environment.
The California Water Alliance Initiative Fund Committee (FPPC ID#1381113), sponsored by the California Water Alliance, a non-profit IRC §501(c)(4) organization, is a state primarily formed ballot measure recipient committee organized to qualify two or more state ballot measures for the November 2016 ballot. It is permitted to accept unlimited, non-tax-deductible donations from individuals, corporations, partnerships, nonprofit organizations, and any other lawfully permissible sources. For more information and restrictions, please visit http://cawater4all.com/.
The Water Priorities Constitutional Amendment and Bond Act Fact Sheet
California Water Explained
California Water Use Facts
California Water Polling Infographic
Author Patrick CavanaughPosted on January 28, 2016 May 31, 2016 Categories Know the issues, Water & the Environment, Law/Politics, Irrigation, Weather, Water Bond, Climate, Economics, Education, Politics and Law, California Water Alliance, Water, California Agriculture News, press releaseTags: Aubrey Bettencourt Kamala Harris KMJ Studios California Water Priorities Ballot Measure About the California Water Alliance Initiative Fund CommitteeLeave a comment on California Water Priorities Ballot Measure
Dogs Detect Citrus Diseases
Animals have been known to be able to use their senses to detect things that humans require extensive technology to detect. Mary Palm, USDA Animal and Plant Health Inspection Service (APHIS) national coordinator for citrus pest programs, said dogs have been successfully trained to detect canker disease, and now Huanglongbing disease (HLB), in citrus.
"Over the past four to five years," Palm said, "a researcher in Florida first determined dogs could actually detect canker, began training dogs," Palm said, "and then trained different sets of dogs to detect it. It turned out that they were very good at it. In a demonstration there, none of the people could find any symptoms in a particular citrus tree, but the dogs came through and found the symptoms. Once the people came back and looked very closely, sure enough, it was there."
Palm said the USDA Huanglongbing Multi Agency Coordination (MAC) Group funded research to determine if dogs could detect HLB in citrus as well as they detected canker. There are now five dogs being trained and tested daily. If grower demand increases, the use of canines in pest management could increase.
Palm said they will probably come up in the next year or two with certification criteria for other companies to train dogs and certify them as detectors. Palm said, "They would be able to get a certificate to show they had conducted all of the tests necessary with the [proper] degree of accuracy." Palm said the dogs in this program have a 99 percent success rate at detecting HLB disease.
Palm said approximately 80 trees are put inside every night, the positive ones in one greenhouse and the negative ones in another. The trees are taken out the next day and positioned in different patterns for dogs to inspect throughout the day. When the dogs are brought out, even their trainers don't know which trees are positive or negative. The trained dogs are more than 99 percent accurate."
USDA Huanglongbing Multi Agency Coordination (MAC) Group Funded Projects: List for the Control and Mitigation of Huanglongbing FY 2015
Author Patrick CavanaughPosted on January 25, 2016 May 31, 2016 Categories All About Crops, Know the issues, Pests & Diseases, Crop Protection, Huanglongbing (HLB), Quarantine, United States Department of Agriculture (USDA), Economics, National Agriculture News, Pests and Diseases, California Agriculture News, newsTags: APHIS citrus canker Mary Palm citrus pest programs dogs USDA Huanglongbing Multi Agency Coordination (MAC) Group certified dog detectorsLeave a comment on Dogs Detect Citrus Diseases
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redpajama_set_name: RedPajamaCommonCrawl


text:
Q: JSON in MySQL : get data from an object within a range of keys What I have:

*

*A mysql table, say table1

*table1 contains two columns viz. id and data

*id is int but the twist is data is JSON type and its keys are comparable

*the table1 contains only one row (for the sake of this question)
table1

id
data

1
{'1': 'content1', '2': 'content2',    '3':'content3',.......,'10000':'content10000' }

What I want to have:
I want a query such that it returns me key-value pairs within a range of keys, say, 100 to 200.
What I'm getting on searching on internet:
Everywhere I got only the answers where one can get rows which have the values within a range, but here the case is I want values of keys within a range.

A: Ok, I start with this data:
MariaDB [test]> select * from table1;
+------+---------------------------------------------------------------------------+
| id   | data                                                                      |
+------+---------------------------------------------------------------------------+
|    1 | {"1": "content1", "2": "content2", "3":"content3","10000":"content10000"} |
+------+---------------------------------------------------------------------------+

Query:
WITH RECURSIVE data AS (
  SELECT
     JSON_VALUE(JSON_KEYS(data), '$[0]') AS k,
     JSON_EXTRACT(data, CONCAT('$.',JSON_VALUE(JSON_KEYS(data), '$[0]'))) AS v,
     0 AS idx
  FROM table1
  UNION
  SELECT
     JSON_VALUE(JSON_KEYS(data), CONCAT('$[', d.idx + 1, ']')) AS k,
     JSON_EXTRACT(data, CONCAT('$.',JSON_VALUE(JSON_KEYS(data), CONCAT('$[', d.idx + 1, ']')))) AS v,
     d.idx + 1 AS idx
  FROM data AS d, table1
  WHERE d.idx < JSON_LENGTH(JSON_KEYS(data)) - 1
) SELECT * FROM data;

sample output (because if only have limited data):
+-------+----------------+------+
| k     | v              | idx  |
+-------+----------------+------+
| 1     | "content1"     |    0 |
| 2     | "content2"     |    1 |
| 3     | "content3"     |    2 |
| 10000 | "content10000" |    3 |
+-------+----------------+------+

Adding a WHERE-clause should not be a problem.

redpajama_set_name: RedPajamaStackExchange


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If you're a real estate agent, you're probably accustomed to selling things. You sell condos, lots, single-family homes, mansions, and potentially even the idea of certain neighborhoods. But, are you selling yourself? It is absolutely imperative that you learn to sell yourself as an agent if you'd like to continue to grow your client base and your income.
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As always, please write me a comment if you enjoyed this article– I love hearing your feedback. You can also check out my other blogs and YouTube channel.
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