Fig 1.
Weight (left panel) and mean faecal scores (right panel) during the experimental periods for Groups C (green) and IF (red). In the right panel, white dots and vertical bars in the box plots shows the mean and the median, respectively. In both panels, error bars show the standard deviation of the mean from three subjects. The weight was recorded individually once during each period, and the faecal scores were recorded daily.
Fig 2.
Relative abundance of microbes at the phylum (a), family (b), and genus (c) levels for Groups C and IF (left and right in each panel, respectively) under the sampling timings (Pre, Post, and Follow_up).
Fig 3.
Shannon (left) and Chao1 (right) indices used to visualize the diversity within the samples of each group for the three sampling timings (left for Group C and right for Group IF in each panel, respectively). Vertical lines in the box plots show the medians.
Fig 4.
NMDS of the microbiota (a) and transcripts (b) data under the Pre (circle), Post (triangle), and Follow_up (square) conditions for Group C (red) and IF (blue), using Bray-Curtis (left) and Jaccard (right) indices.
Fig 5.
LEfSe characterization of the dominant microbial taxa according to the treatments with (IF: Post and Follow_up for Group IF) or without (NoIF: all conditions for Group C and Pre for Group IF) insect feeding.
(a) LDA scores, above the threshold 2 for the microbiota on various taxonomic levels ranked according to the effect size (alpha = 0.05) for the treatments with (red, IF: Post and Follow_up for Group IF) and without (green, NoIF: all conditions for Group C and Pre for Group IF) insect feeding. (b) Cladogram based on the ranked list in (a) to visualize the relationships between the treatments and the phylogenetic relationships among the microbes. Phylogenetic clades are ordered from the centre of the circle, with narrower to broader taxonomic levels. Diameters of outer circles correspond to the relative abundance in the microbial community. Red and green points in the circles show the most abundant classes under the IF and NoIF treatments, respectively. Points in light green show the clades that are not significant.
Fig 6.
Distribution of DEG categories found in microbiota.
(a) Categories of the DEG analysis. (b) Relative distribution of the DEG categories. (c) Relative distribution of microbes in each DEG category at the phylum level, showing upregulated changes. Categories under the insect-feeding treatment were G5>others, G5>anywhere, G6>others, and G6>anywhere. (d) Relative distribution of microbes in each DEG category at the phylum level, showing downregulated changes. Categories under the insect-feeding treatment were G5>others, G5>anywhere, and G6>others.
Fig 7.
Normalized read counts of the microbes identified by the differentially expressed gene (DEG) analysis of faecal SSU rRNA at the phylum level.
In each panel, the left and the right three bars show Pre, Post, and Follow_up conditions for Group C (left) and Group IF (right), respectively. Black arrows in the panels indicate the groups which have DEGs compared with the other groups. (a-g) Upregulated DEGs, with insect-feeding nonrelevant (a, b, c) and insect-feeding relevant (d, e, f, g) conditions. (h-l) Downregulated DEGs with insect-feeding nonrelevant (h, i, j) and insect-feeding relevant (k, l) conditions. No DEG was found under the Follow_up condition in Group C (G3). For f, and g, data for each microbiota were compared with those of the other groups separately. In other graphs, data for each microbiota were compared with the total of the other groups.
Fig 8.
Heatmap of the SSU rRNA data (genus level) with DEGs.
For microbes unable to be annotated at the genus level, upper taxonomies (i.e., order, family) were assigned. Groups C and IF are presented on left and right of the panel, respectively. For each group, the upper and lower parts present the data from upregulated and downregulated genera, respectively. G1: Pre condition for Group C; G2: Post condition for Group C; G4: Pre condition for Group IF, G5: Post condition for Group IF; G6: Follow_up condition for Group IF; AW: DEGs from any comparisons among the conditions. G3 is not presented because DEGs were not found under this condition (Follow_up condition of Group C). The heatmap with skyblue squares on the right indicates the changes in microbiota in accordance with the experimental treatments (i.e., G5 and G6 of Group IF).
Fig 9.
Results overlapped from the LEfSe and DEG analysis, showing the microbes specifically expressed just after the insect feeding treatment (a, b) and two weeks after the treatment (c, d), indicated by the black arrows.
(a) Upregulation found under the Post condition in three microbes of the genus Bacteroides. (b) Downregulation found under the Post condition in four microbes of the genus Bacteroides. (c) Downregulation found under the Follow_up condition in two microbes of the genus Olsenella. (d) Downregulation found under the Follow_up condition in a microbe of the genus Oribacterium.
Fig 10.
Relative distribution of DEG categories found in transcripts.
(a) Relative distribution of transcript functions of DEGs. (b) Relative distribution of transcripts in each DEG category at the phylum level, showing upregulated changes. Categories under the insect-feeding treatments were G5>others, G5>anywhere, and G6>others. (c) Relative distribution of transcripts in each DEG category at the phylum level, showing downregulated changes. Categories under the insect-feeding treatment were G5>others and G6>others.
Fig 11.
Heatmap with clustering among the SSU rRNA (genus level) and annotated transcripts with DEGs, according to the experimental period (indicated on the bottom of the heatmap) in Groups C (left) and IF (right). Transcript data are presented with T with the genera name of the originating microbes. Increases and decreases are shown in magenta and green, respectively. Hierarchical clustering for Group IF enabled items from the microbiota and transcriptome data to be listed together, where items with similar characteristics were listed close to each other. The data relevant to the experimental treatment (insect feeding) are emphasized by the skyblue squares. Upregulated and downregulated DEGs are located upper and lower panels of the figure, respectively.
Fig 12.
Microbial profiles of fed insects.
(a) Relative abundance of microbes at the phylum level for house cricket (Ad: left) and giant mealworm (Za: right). (b) Shannon (left) and Chao1 diversity indices of the fed insects. (c) NMDS using Bray-Curtis index of crickets (Ad: pink) and worm (Za: skyblue).