Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics

Abstract

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Keywords: KEGG; MG-RAST; RefSeq; bioinformatics; echinoderm; gut microbiome; high-throughput sequencing.

Figure 1

Relative abundance stacked column bar graphs illustrating the taxa distribution assigned to domain Bacteria through RefSeq as implemented in MG-RAST (v4.0.3). (A) The phylum level (class for Proteobacteria) distribution is shown, and phyla with an average abundance of <1% were listed as “Other”. (B) The relative abundance of the top 50 genera across all samples was plotted. The low represented taxa were categorized as “Other”. Bar graphs generated using Microsoft Excel software (Microsoft, Seattle, WA, USA), and samples are indicated as follows: LV.GD = green sea urchin Lytechinus variegatus gut digesta; SP.GD = purple sea urchin Strongylocentrotus purpuratus gut digesta.

Figure 2

Heatmap analysis of the genera comprising domain Bacteria assigned through RefSeq using MG-RAST (v4.0.3). The analysis was performed using R (v3.3.2) with the heatmap.2 function from the gplots (v3.0.1) package. The sample dendrogram was constructed based on the Bray–Curtis similarity value through Vegan (v2.4.3), and the gradient of relative abundance was illustrated using RColorBrewer package as “blue” for more abundant and “sky blue” for less abundant. The trace lines (black) were generated to further elaborate on the relative abundance of taxa. Samples are indicated as follows: LV.GD = green sea urchin Lytechinus variegatus gut digesta; SP.GD = purple sea urchin Strongylocentrotus purpuratus gut digesta.

Figure 3

Relative abundance bar graphs were generated for each sample based on the KEGG-Level-2 categories determined through KEGG Orthology (KO) data following the MG-RAST (v4.0.3) workflow. KEGG-Level-2 categories were binned into their respective KEGG-Level-1 broad hierarchical functional category and ranked in decreasing order from top to bottom based on their average abundance. Each KEGG-Level-1 category was color-coded as follows: cellular processing = blue; environmental processing = brown; genetic information = orange; metabolism = red; and “other” = gray. The bar graphs for each sample have been color-coded and indicated as follows: SP.GD = purple sea urchin Strongylocentrotus purpuratus gut digesta; SP.GD1 = purple and SP.GD2 = light purple; LV.GD = green sea urchin Lytechinus variegatus gut digesta; LV.GD1 = green and LV.GD2 = light green.

Figure 4

Relative abundance scatter plot analysis of the KEGG map Ids derived from the KEGG-Level-1 category of “metabolism”. KEGG map Id data was retrieved from the KEGG-Level-3 functional categories from MG-RAST (v4.0.3) and was uploaded into STAMP (v2.1.3). Technical replicates were grouped, and count data were normalized as the relative proportion of each KEGG map Id per group from the KEGG-Level-1 category of metabolism. The X-axis and Y-axis show each KEGG map Id relative abundance per group, including the histograms to show the number of functional entries falling at the specified abundance on the scatter plot. Those KEGG map Ids that were noticeably enriched in one group were indicated by their KEGG pathway name and Id number. The regression statistic (R²) was also shown in the plot. Sample groups and color codes are indicated as LV.GD = green sea urchin Lytechinus variegatus gut digesta (green); SP.GD = purple sea urchin Strongylocentrotus purpuratus gut digesta (purple).

Figure 5

(A) The KEGG Orthology (KO) functional categories comprising the KEGG-Level-3 pathway of nitrogen metabolism (00910), including the direction of the reaction, were mapped. Inorganic nitrogen metabolism was mapped to show the substrate and product of each indicated reaction as it pertains to nitrate (NO₃⁻) reduction, nitrite (NO₂⁻) ammonification, denitrification to nitric oxide (NO), nitrous oxide (N₂O), and nitrogen gas (N₂), and nitrogen fixation into ammonia (NH₃). (B) The KEGG Orthology (KO) functional categories comprising the KEGG-Level-3 pathway of nitrogen metabolism (00910), including the direction of the reaction, were mapped. The assimilation of ammonia into the amino acids glutamine and asparagine are also shown, including the subsequent enzyme-catalyzed amino acid transitions. The count data associated with the specified gene are shown as column bar graphs for each sample, including the KO category (KO number) and the gene name assigned through the KEGG database. Each category was color-coded according to their sample indicated in the color key as follows: SP.GD = purple sea urchin Strongylocentrotus purpuratus gut digesta; SP.GD1 = purple and SP.GD2 = light purple; LV.GD = green sea urchin Lytechinus variegatus gut digesta; LV.GD1 = green and LV.GD2 = light green. The metabolic pathways were elaborated using the information provided through KEGG Mapper as implemented in MG-RAST (v4.0.3). * The KEGG number K01953 corresponding to the asnB gene is listed multiple times due to its function in the generation of glutamate and asparagine from glutamine and aspartate.

Figure 5

(A) The KEGG Orthology (KO) functional categories comprising the KEGG-Level-3 pathway of nitrogen metabolism (00910), including the direction of the reaction, were mapped. Inorganic nitrogen metabolism was mapped to show the substrate and product of each indicated reaction as it pertains to nitrate (NO₃⁻) reduction, nitrite (NO₂⁻) ammonification, denitrification to nitric oxide (NO), nitrous oxide (N₂O), and nitrogen gas (N₂), and nitrogen fixation into ammonia (NH₃). (B) The KEGG Orthology (KO) functional categories comprising the KEGG-Level-3 pathway of nitrogen metabolism (00910), including the direction of the reaction, were mapped. The assimilation of ammonia into the amino acids glutamine and asparagine are also shown, including the subsequent enzyme-catalyzed amino acid transitions. The count data associated with the specified gene are shown as column bar graphs for each sample, including the KO category (KO number) and the gene name assigned through the KEGG database. Each category was color-coded according to their sample indicated in the color key as follows: SP.GD = purple sea urchin Strongylocentrotus purpuratus gut digesta; SP.GD1 = purple and SP.GD2 = light purple; LV.GD = green sea urchin Lytechinus variegatus gut digesta; LV.GD1 = green and LV.GD2 = light green. The metabolic pathways were elaborated using the information provided through KEGG Mapper as implemented in MG-RAST (v4.0.3). * The KEGG number K01953 corresponding to the asnB gene is listed multiple times due to its function in the generation of glutamate and asparagine from glutamine and aspartate.