cm dii  (Fisher Scientific)

Journal: Nature Communications

Article Title: Gut microbiota-derived butyrate primes systemic immunity in honey bees by mediating lipid metabolic reprogramming

doi: 10.1038/s41467-026-69073-0

Figure Lengend Snippet: a Experimental design for ( b – e ). Newly emerged worker bees were divided into GF (germ-free), HK (inoculated with heat-killed gut homogenate), and CV (inoculated with nurse bee gut homogenate) groups. Gut bacterial inoculation was conducted within one day after emergence. Bees were maintained for 5 days prior to Hafnia injection, and separate groups of bees were used for the survival assay, gene expression analysis, and confocal microscopy. Created in BioRender. Liu, L. (2025) https://BioRender.com/en83bag . b Survival rates for GF, HK, and CV bees post Hafnia injection. Bars represent mean ± s.d. from three biological replicates (20 individuals in each replicate). The results are from one of two independent experiments (Supplementary Fig. for replicate data). A two-sided Mantel-Cox test was used. Different letters above bars indicate statistically significant differences among treatments ( P < 0.05). c Antimicrobial peptide (AMP) gene expression in fat bodies of GF, HK, and CV bees, measured by qPCR (SYBR Green, Actin -normalized). Bars represent mean ± s.e.m. n = 6 individuals for each group from 3 cup cages. d Ventral view of bee abdominal tergum showing the dorsal vessel. Yellow arrows indicate the ostia. e Hemocyte aggregation post-GFP- Hafnia injection (green). Hemocytes were stained with CM-DiI (red), and dorsal vessels were outlined by white dashed lines. Scale bars: 250 μm. Representative images from one of two independent experiments (Supplementary Fig. – for replicate data). f Quantitative analysis of CM-DiI staining in hemocytes (see Fig. 1e and Supplementary Fig. ). Bars represent mean ± s.e.m. n = 5 individuals per group from 3 cup cages. ( c , f ) Two - way ANOVA with Tukey’s multiple comparisons test. Different letters above bars indicate statistically significant differences among treatments ( P < 0.05). ns: not significant. All P values are listed in Supplementary Dataset . Source data are provided as a Source Data file.

Article Snippet: At 3, 6, 12, and 24 h after Hafnia infection, bees were injected thoracically with 2 μL of a solution containing 67 μM Celltracker CM-DiI (Cat#40718ES50, Yeasen, China) and 1.08 mM Hoechst 33342 (Cat#C0031, Solarbio, China) in PBS.

Techniques: Injection, Clonogenic Cell Survival Assay, Gene Expression, Confocal Microscopy, SYBR Green Assay, Staining

Journal: Nature Communications

Article Title: Gut microbiota-derived butyrate primes systemic immunity in honey bees by mediating lipid metabolic reprogramming

doi: 10.1038/s41467-026-69073-0

Figure Lengend Snippet: a Survival rates of GF, CL (inoculated with a set of five core bacteria mixed at equal numbers), acetate-supplemented, and butyrate-supplemented bees following Hafnia injection. Bars represent mean ± s.d. n = 3 replicates for each group (30 individuals per replicate). The results are from one of two independent experiments (Supplementary Fig. for replicate data). A two-sided Mantel-Cox test was used. Different letters above bars indicate statistically significant differences among treatments ( P < 0.05). b Abdominal butyrate concentrations for GF, CL, and single bacterium-inoculated bees 5 days post-colonization. Ga: Gilliamella ; Sa: Snodgrassella ; Ba: Bifidobacterium ; F4: Bombilactobacillus ; F5: Lactobacillus . The isolates for each strain are listed in Supplementary Table . Bars represent mean ± s.e.m. n = 3 replicates for each group, each replicate pooled from 3 individuals from 3 cup cages. The experiment was repeated twice with similar results. One-way ANOVA with the Tukey post hoc test. Different letters above bars indicate statistically significant differences among treatments ( P < 0.05). c AMP expression in the fat bodies of GF, CV, CL, and butyrate-supplemented bees, before Hafnia infection and 3 h after infection (GF as control). Bars represent mean ± s.e.m. n = 8 individuals for each group from 3 cup cages. Gene expression was quantified using qPCR as shown in Fig. . Two-sided Mann–Whitney test and two-sided unpaired Student’s t test, depending on data normality. ns: not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001. Created in BioRender. Liu, L. (2025) https://BioRender.com/hdvmsxq . d CM-DiI-stained hemocytes (red) of GF, HK, CL, and butyrate-supplemented bees before infection and at 3 h post-GFP- Hafnia infection (green). Dorsal vessels were outlined with white dashed lines. Scale bars: 250 μm. Representative images from one of two independent experiments (see Supplementary Figs. , for replicate data). All P values are listed in Supplementary Dataset . Source data are provided as a Source Data file.

Article Snippet: At 3, 6, 12, and 24 h after Hafnia infection, bees were injected thoracically with 2 μL of a solution containing 67 μM Celltracker CM-DiI (Cat#40718ES50, Yeasen, China) and 1.08 mM Hoechst 33342 (Cat#C0031, Solarbio, China) in PBS.

Techniques: Bacteria, Injection, Expressing, Infection, Control, Gene Expression, MANN-WHITNEY, Staining

Journal: Nature Communications

Article Title: Gut microbiota-derived butyrate primes systemic immunity in honey bees by mediating lipid metabolic reprogramming

doi: 10.1038/s41467-026-69073-0

Figure Lengend Snippet: a PCA of abdominal metabolomes from GF and butyrate-supplemented bees at day 5 post-supplementation (n = 6 per group, each point represents one biological replicate). b Heatmap of the discriminating metabolites in the arachidonic acid pathway in the abdomen of GF and butyrate-supplemented bees. c Prostaglandin E 2 (PGE 2 ) levels in abdomen, hindgut and hemolymph in GF, CV, CL, and butyrate-supplemented bees at day 5 post-colonization (abdomen: n = 6; hindgut and hemolymph: n = 4; each pooled from 5 individuals from 3 cup cages). One of two independent experiments (Supplementary Fig. ). Two-sided Mann–Whitney and two-sided unpaired Student’s t test, depending on data normality. d Knockdown of fat body Pla2 in GF bees following dsRNA injection (GFP dsRNA as control). n = 8 per group from 3 cup cages. P = 0.0394, two-sided Welch’s t test. e Effect of Pla2 RNAi on PGE 2 levels in fat bodies of GF and butyrate-supplemented bees ( n = 5 replicates per group, each pooled from 5 individuals from 3 cup cages). P = 0.0039, two-sided Student’s t test. f Survival of GF, GF + PGE 2 , CL, and CL+acetylsalicylic acid (ASA) bees post- Hafnia infection. Bars represent mean ± s.d. from three replicates per group (30 individuals per replicate) from one of two independent experiments (Supplementary Fig. ). Two-sided Mantel–Cox test; different letters indicate significant differences ( P < 0.05). g AMP expression in fat bodies before and 3 h post- Hafnia infection (GF as control; n = 8 per group from 3 cup cages. Two-sided Mann–Whitney and two-sided unpaired Student’s t test, depending on data normality. h In vivo hemocyte staining using CM-DiI (red) before infection (3 h after PGE 2 supplementation) and at 3 h post-infection with GFP- Hafnia (green). Dorsal vessels were outlined with white dotted lines. Scale bars: 250 μm. Representative images from two independent experiments (Supplementary Figs. , ). In ( c – e , g ), bars represent mean ± s.e.m. * P < 0.05, ** P < 0.01, and *** P < 0.001. All P values are listed in Supplementary Dataset . Source data are provided as a Source Data file.

Article Snippet: At 3, 6, 12, and 24 h after Hafnia infection, bees were injected thoracically with 2 μL of a solution containing 67 μM Celltracker CM-DiI (Cat#40718ES50, Yeasen, China) and 1.08 mM Hoechst 33342 (Cat#C0031, Solarbio, China) in PBS.

Techniques: MANN-WHITNEY, Knockdown, Injection, Control, Infection, Expressing, In Vivo, Staining