Pan W , Wang X , Ren C , Jiang X , Gong S , Xie Z , Wong NK , Li X , Huang J , Fan D , Luo P , Yang Y , Ren X , Yu S , Qin Z , Wu X , Huo D , Ma B , Liu Y , Zhang X , E Z , Liang J , Sun H , Yuan L , Liu X , Cheng C , Long H , Li J , Wang Y , Hu C , Chen T .

42176132 National Natural Science Foundation of China (National Science Foundation of China), 41906101 National Natural Science Foundation of China (National Science Foundation of China), 2022YFD2401301 Ministry of Science and Technology of the People & apos;s Republic of China (Chinese Ministry of Science and Technology), 2024A1515010899 Guangdong Science and Technology Department (Science and Technology Department, Guangdong Province), 2024A1515011418 Guangdong Science and Technology Department (Science and Technology Department, Guangdong Province), HJWK-2021-21 Shanghai Educational Development Foundation, 2024-MRB-00-001 Administration of Ocean and Fisheries of Guangdong Province

	Fig. 1. The landscape of feeding habits and digestive enzymes of the tropical sea cucumber H. leucospilota.a Scene of H. leucospilota feeding on seabed sediments. b Relative abundance of the 10 most abundant eukaryotic phyla in the digestive tract contents determined by 18S sequencing (Supplementary Data 1). The digestive tract regions include foregut (Fg, n = 5), midgut (Mg, n = 5) and hindgut (Hg, n = 5) collected from different individuals. Each color represents one specific eukaryotic phylum. Source data are provided as a Source Data file. c Alpha-diversity index of microbiota in the environment (En, n = 10 in total), feces (Fc, n = 5) and digestive tract (DT, n = 19 in total) determined by 16S sequencing (Supplementary Data 2), displayed by the Chao1 estimator and Shannon diversity index. The environment samples include seawater (Sw, n = 5) and seabed sediments (SS, n = 5). The first quartile forms the bottom and the third quartile forms the top of the box, in which the line represents the median value. The whiskers range from 2.5th to 97.5th percentile, and points below and above the whiskers are drawn as individual dots. P values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test, where only the P values between SS and Fc groups are shown (****P < 0.001). d The numbers of digestive enzyme amylase (AMY), maltase-glucoamylase (MGA), sucrase-isomaltase (SUIS), lactase (LPH), trehalase (TREA), chymotrypsins (CTR), chymotrypsin-like elastase (CLE), pancreatic triacylglycerol lipase (LIPP), gastric triacylglycerol lipase (LIPG), hepatic triacylglycerol lipase (LIPC), chitinase (CHIA) and chitinase domain-containing protein (CHIP) genes in 23 Deuterostomia species. The size of the circles represents the number of digestive enzyme genes of a particular category (Supplementary Data 3). The squares indicate the feeding habits of different species, while the clusters indicate their evolutionary status. Arrows and bubbles on the right indicate whether a certain digestive enzyme group has undergone gene expansion or contraction within a certain taxonomic group.
	Fig. 2. The dietary shift and digestive enzyme expression patterns during the embryonic and larval development of H. leucospilota.a Developmental stages include the fertilized egg (FE), 2-cells (2C), 4-cells (4C), 8-cells (8C), 16-cells (16C), morula (Mr), blastula (Bs), rotated-blastula (RB), early-gastrula (EG), late-gastrula (LG), early-auricularia (EA), mid-auricularia (MA), auricularia (Ar), doliolaria (Dl), pentactula (Pt), 1-mm juvenile (J1), 20-mm juvenile (J20) and adult (A). The orange text represents different developmental stages, including embryos, larvae, juveniles, and adults. The blue text represents the different locomotion models, including non-swimming, swimming, attaching and bottom-crawling. The green text represents the sources of nutrition, including endogenous nutrition, planktotrophic feeding, and deposit feeding. b The expressional presence and absence of digestive enzyme amylase (AMY), maltase-glucoamylase (MGA), sucrase-isomaltase (SUIS), lactase (LPH), trehalase (TREA), chymotrypsins (CTR), chymotrypsin-like elastase (CLE), pancreatic triacylglycerol lipase (LIPP), gastric triacylglycerol lipase (LIPG) and chitinase domain-containing protein (CHIP) genes during the fertilized egg (FE), embryonic (E), larval (L), juvenile (J) and adult (A) stages (Supplementary Data 4). Absence or presence in expression of a certain digestive enzyme gene during a certain developmental stage is marked in yellow or blue. c Heatmap illustrating digestive enzyme expression among different developmental stages. The red box indicates three sets of digestive enzyme expression patterns corresponding to the dietary shift (Supplementary Data 4). Blue and red colors represent relatively low and high expression levels, respectively, as scaled by the rows. Clusters in different colors represent different expression patterns for digestive enzyme genes during development. The gene expression level at each stage is derived from the average of samples (n = 3) taken from different individuals. Source data are provided as a Source Data file. d Spatial distribution of CHIP (Hl-20219), SUIS (Hl-27148), and CTR (Hl-19115) mRNA in the fertilized egg (FE), embryos (E), larvae (L) detected by WMISH, and in the juveniles (J) detected by FISH. The intestines (In) in the juvenile sections are indicated. The black and white scale bars for WMISH and FISH are 100 μm and 200 μm, respectively. Each experiment was performed for one time.
	Fig. 3. The structure and digestive enzyme system of the H. leucospilota digestive tract.a Anatomical structure of the digestive tract, which is further divided into regions including the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc). Foregut, midgut and hindgut make up the intestine (In). Other tissues shown include the body wall (Bw), muscle (Ms), mouth (Mt), anus (An) and rete mirabile (RM). b Histological structures of different regions of the digestive tract, including the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg), and rectum (Rc), as revealed by HE staining. The black scale bars are 100 μm. c Heatmap illustrating the expression of digestive enzyme genes amylase (AMY), maltase-glucoamylase (MGA), sucrase-isomaltase (SUIS), lactase (LPH), trehalase (TREA), chymotrypsins (CTR), chymotrypsin-like elastase (CLE), pancreatic triacylglycerol lipase (LIPP), gastric triacylglycerol lipase (LIPG) and chitinase domain-containing protein (CHIP) in different tissues, including the body wall (BW), muscle (Ms), oral tentacles (OT), Cuvierian organ (CO), respiratory tree (RT), Polian vesicle (PV), coelomocytes (Cc), ovary (Ov), testis (Ts), rete mirabile (RM), transverse vessel (TV) and intestine (In) (Supplementary Data 5). The digestive tract is further divided into five regions, including the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc) (Supplementary Data 6). Blue and red colors represent relatively low and high expression levels, respectively, as scaled by digestive enzyme classifications. The gene expression level in each tissue is derived from the average of tissue samples (n = 3 or 4) taken from different individuals. Source data are provided as a Source Data file. d FISH of MGA (Hl-27400), CTR (Hl-19115), LIPG (Hl-25219), and CHIP (Hl-20219) mRNA in the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc) of the digestive tract. The white scale bars are 400 μm. Each experiment was performed for one time. e Enzyme activity assay of amylase, protease, lipase, chitinase, and cellulase in the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc) of the digestive tract. Data presented here are expressed as mean±SEM (n = 5 from 5 individuals). Source data are provided as a Source Data file. f Diagram illustrating the functional compartmentalization of the H. leucospilota digestive tract, showing different expression and activity patterns for digestive enzymes across the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc).
	Fig. 4. Microbiome within the H. leucospilota digestive tract and its contribution to digestive activities.a Relative abundance of the 10 most abundant phyla along the seawater (Sw, n = 5) and seabed sediments (SS, n = 5) in environments, feces (Fc, n = 5), and different regions of the digestive tract, including the esophagus (Es, n = 4), foregut (Fg, n = 4), midgut (Mg, n = 5), hindgut (Hg, n = 3) and rectum (Rc, n = 3) (Supplementary Data 2). Each color represents a specific microbial group. Source data are provided as a Source Data file. b Phylogenetic relationships and taxonomic classifications of the 1922 microbial species from the digestive tract with active transcription (Supplementary Data 7). c Heat plots displaying the values for mRNA expression of microbial genes involved in protein, carbohydrate, and lipid metabolism in the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc) of the digestive tract (Supplementary Data 8). The size and color of the plots represent relative expression level, with redder and larger plots indicating higher expression levels. Digestive tract region samples were collected from 3 individuals then mixed into 1 for sequencing. Source data are provided as a Source Data file. d Enzyme activity assay of amylase, protease, and lipase in the intestine after antibiotic exposure for 0- (control), 3- and 7-days. Data presented here are expressed as mean±SEM (n = 5 from 5 individuals; P values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test, where **P < 0.01, ***P < 0.001).
	Fig. 5. The roles of Holothuroidea-specific intestinal i-type lysozyme in bacterial digestion.a Heatmap illustrating the expression of lysozyme genes (Hl-18105, Hl-181095, Hl-18110, Hl-36988 and Hl-36992) in different tissues (Supplementary Data 5) and digestive tract regions (Supplementary Data 6), as well as embryonic and larval development stages (Supplementary Data 4). Tissue samples include the body wall (BW), muscle (Ms), oral tentacles (OT), Cuvierian organ (CO), respiratory tree (RT), Polian vesicle (PV), coelomocytes (Cc), ovary (Ov), testis (Ts), rete mirabile (RM), transverse vessel (TV) and intestine (In). Digestive tract regions include the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc). Embryonic and larval development stages include the fertilized egg (EF), 2-cells (2C), 4-cells (4C), 8-cells (8C), 16-cells (16C), morula (Mr), blastula (Bs), rotated-blastula (RB), early-gastrula (EG), late-gastrula (LG), early-auricularia (EA), mid-auricularia (MA), auricularia (Ar), doliolaria (Dl), pentactula (Pt), 1-mm juvenile (J1), 20-mm juvenile (J20) and adult (A). Blue and red colors represent relatively low and high expression levels, respectively. The gene expression level in each tissue or developmental stage is derived from the average of samples (n = 3 or 4) taken from different individuals. Source data are provided as a Source Data file. b FISH of in-iLyz (Hl-36988 and Hl-36992) mRNA in the esophagus (Es), foregut (Fg), midgut (Mg), hindgut (Hg) and rectum (Rc) of the digestive tract. The white scale bars are 400 μm. c Phylogenetic tree of lysozyme genes in typical vertebrate, ecdysozoan, lophotrochozoan, and echinoderm species (Supplementary Fig. 7). Different dot colors represent different taxonomic groups and different line colors represent different lysozyme types. d Phylogenetic analysis and heatmap comparison for tissue expression of the lysozyme genes in H. leucospilota and H. scabra (Supplementary Data 5). Selected tissues include body wall (BW), coelomocytes (Cc), rete mirabile (RM) and intestine (In). Blue and red colors represent relatively low and high expression levels, respectively, as scaled by the rows. The gene expression level at each stage is derived from the average of samples (n = 3) taken from different individuals. Source data are provided as a Source Data file. e Enzyme activity assay of lysozyme in the body wall (BW), coelomic fluid (CF), rete mirabile (RM), and intestine (In) of H. leucospilota. Data presented here are expressed as mean±SEM (n = 4 from 4 individuals). Source data are provided as a Source Data file. f The mRNA expression of cc-iLyz (Hl-18105) in the coelomocytes and in-iLyz (Hl-36988 and Hl-36992) in the intestine after V. harveyi injection for 0 (control), 24, 48 and 72 hours. Data presented here are expressed as mean±SEM (n = 10 from 10 individuals; P values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test, where * P < 0.05, ***P < 0.001). Source data are provided as a Source Data file. g The mRNA expression of cc-iLyz (Hl-18105) in the coelomocytes and in-iLyz (Hl-36988 and Hl-36992) in the intestine after starvation for 0 (control), 7, 14 and 30 days. Data presented here are expressed as mean±SEM (n = 10 from 10 individuals; P values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test, where *P < 0.05, ***P < 0.001, ****P < 0.0001). Source data are provided as a Source Data file. h The relative abundances of the resident bacteria and digested bacteria in the seabed sediments (SS, n = 5) and the feces (Fc, n = 5) and digestive tract (DT, n = 19) of H. leucospilota (Supplementary Data 2). Data presented here are expressed as mean±SEM (P values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test, where *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Source data are provided as a Source Data file. The corresponding lysoplate assay of the recombinant H. leucospilota in-iLyz (rHl-in-iLyz) protein or hen egg white lysozyme (HEWL) was performed against seedbed sediment bacteria P. marcusii (Rhodobacteraceae), O. marina (Verrucomicrobiaceae), B. cremea (Planctomycetaceae), B. aggregatus (Geodermatophilaceae), F. oceanosedimentum (Flavobacteriaceae), P. aurantiacus (Halomonadaceae), probiotic bacteria B. subtilis (Bacillaceae), pathogenic bacteria V. harveyi (Vibrionaceae) and standard bacteria M. lysodeik. The PBS group and the HEWL group were used as the negative and positive control, respectively. Each experiment was repeated four times.