Vitamin supplementation by gut symbionts ensures metabolic homeostasis in an insect host

Abstract

Despite the demonstrated functional importance of gut microbes, our understanding of how animals regulate their metabolism in response to nutritionally beneficial symbionts remains limited. Here, we elucidate the functional importance of the African cotton stainer's (Dysdercus fasciatus) association with two actinobacterial gut symbionts and subsequently examine the insect's transcriptional response following symbiont elimination. In line with bioassays demonstrating the symbionts' contribution towards host fitness through the supplementation of B vitamins, comparative transcriptomic analyses of genes involved in import and processing of B vitamins revealed an upregulation of gene expression in aposymbiotic (symbiont-free) compared with symbiotic individuals; an expression pattern that is indicative of B vitamin deficiency in animals. Normal expression levels of these genes, however, can be restored by either artificial supplementation of B vitamins into the insect's diet or reinfection with the actinobacterial symbionts. Furthermore, the functional characterization of the differentially expressed thiamine transporter 2 through heterologous expression in Xenopus laevis oocytes confirms its role in cellular uptake of vitamin B1. These findings demonstrate that despite an extracellular localization, beneficial gut microbes can be integral to the host's metabolic homeostasis, reminiscent of bacteriome-localized intracellular mutualists.

Keywords: host–microbe metabolic integration; mutualism; nutritional symbiosis; symbiont transmission; vitamin supplementation.

Figure 1.

Fitness of control and aposymbiotic D. fasciatus on an artificial diet with and without B vitamins, respectively. (a,b) Survivorship and developmental time from egg hatching to adulthood, respectively. Shading of boxes signifies the experimental treatments. Lines represent medians, boxes comprise the 25–75 percentiles and whiskers denote the range. Significant differences (Friedman test with Wilcoxon–Wilcox post hoc measures, p < 0.05) are marked by an asterisk.

Figure 2.

Pairwise MA plots for expressed genes among aposymbiotic and symbiotic D. fasciatus reared on their natural diet of cottonseeds. The scatter plot depicts the distribution of aposymbiotic/symbiotic log² intensity ratio (M-value) versus the log² average intensity (A-value). Each dot represents a single gene in comparison. The red line indicates M = 0. The upper and lower blue lines represent expression fold changes of 2 and 0.5, respectively. Grey dots represent constitutively expressed genes, black dots depict differentially expressed genes (more than twofold). B vitamin genes are designated by red dots. The lines of dots on the left side signify genes that were only observed in a single sample.

Figure 3.

Differential expression of B-vitamin metabolism and stress-related host genes by qPCR for symbiotic and aposymbiotic D. fasciatus reared on cottonseeds (normalized to the 60S ribosomal protein L13a). (a) Intracellular B vitamin processing genes: thiamine pyrophosphokinase (TPK), riboflavin kinase (RFK), nicotinamide mononucleotide adenylyltransferase (NMNAT), pantothenate kinase (PANK), pyridoxal kinase (PK), biotin-protein lyase (BPL), dihydrofolate reductase (DHFR), and transcobalamine 2 (TCII). (b) B vitamin transport and extracellular processing genes: thiamine transporter 2 (THTR2), proton-coupled folate transporter (PCFT) and thiamine alkaline phosphatase (ALKP). (c) A stress indicator; heat shock protein (Hsp) 70 and a glucose transporter (GLUT8). Shading of boxes signifies the experimental treatments (see legend). Lines represent medians, boxes comprise the 25–75 percentiles and whiskers denote the range. Significant differences were assessed based on the normalized expression in reference to the 60S ribosomal protein L13a with Mann–Whitney U-tests. Differentially expressed transcripts at p < 0.05 are marked by asterisks (*).

Figure 4.

Differential expression of B-vitamin metabolism and stress-related host genes by qPCR for symbiotic and aposymbiotic D. fasciatus reared on a complete or vitamin-deficient artificial diet (normalized to the 60S ribosomal protein L13a). (a) Intracellular B vitamin processing genes: TPK, RFK, NMNAT, PANK, PK, BPL, DHFR and TCII. (b) B vitamin transport and extracellular processing genes: THTR2, PCFT and ALKP. (c) A stress indicator Hsp70 and a glucose transporter GLUT8. For gene abbreviations, see figure 2. Shading of boxes signifies the experimental treatments (see legend). Lines represent medians, boxes comprise the 25–75 percentiles and whiskers denote the range. Significant differences were assessed based on the normalized expression in reference to the 60S ribosomal protein L13a with the Kruskal–Wallis test and Dunn post hoc tests. Differentially expressed transcripts at p < 0.05 and p < 0.01 are marked by single (*) and double (**) asterisks, respectively.

Figure 5.

THTR2 mediated uptake of (a) 50 pM Thiamin and (b) 100 µM 2-deoxy-d-glucose into Xenopus oocytes. Transport activity was determined by quantifying the radiolabelled substrate uptake in oocytes injected with THTR2-cRNA, water or hGLUT1-cRNA, respectively. Lines represent medians, boxes comprise the 25–75 percentiles and whiskers denote the range. Significant differences at p < 0.01 are marked by double asterisks (**) (ANOVA).

Figure 6.

Metabolic pathways for B vitamin biosynthesis in the genome of C. glomerans. Each arrow represents one step in the biosynthetic pathway. Enzyme names in green font indicate that a candidate gene for this step was detected in the annotated genome sequence. Enzyme names in blue indicate that a candidate gene for this step was detected in the host's genome (see figures 3a and 4a).